TM 8-227 WAR DEPARTMENT TECHNICAL MANUAL METHODS FOR LABORATORY TECHNICIANS October 17, 1941 IM 8—337 TECHNICAL MANUAL ] No. 8-227 WAR DEPARTMENT, Washington, October 17, 1941. METHODS FOR LABORATORY TECHNICIANS Prepared under direction of The Surgeon General Chapter 1. General. Paragraphs Section I. General 1-2 II. Medical supplies 3-5 III. Laboratory reports 6-8 IV, Compound microscope- 9-11 V. Miscellaneous procedures 12-20 Chapter 2. Examination of blood—clinical procedures 21—40 Chapter 3. General chemical technic. Section I. Care and use of laboratory apparatus 41-46 II. Indicators 47-49 III, Volumetric solutions 50-51 IV. Useful laboratory arts and recipes 52-56 Chapter 4. Examination of urine. Section I. Collection and preservation of specimens 57-58 II. Physical examination 59-64 III. Routine chemical examination 65-72 IV- Microscopic examination of urinary sediments- 73-76 V. Quantitative chemical examination 77-83 Chapter 5. Examination of gastric contents. Section I. General 84 II. Physical examination 85-86 III. Test meals 87-89 IV. Chemical analysis 90-94 Chapter 6. Blood chemistry. Section I. Colorimetry 95-96 II. Specimens for analysis - 1 97-99 III. Chemical examination 100-118 Chapter 7. General bacteriological methods. Section I. General characteristics of bacteria 119-127 II. Preparation of glassware 128-129 III. Sterilization 130-135 IV. Preparation of stains and solutions 136-151 V. Determination of biological activity-- 1 152-162 VI. Preparation of culture media 163-214 VII. Care of laboratory animals... 215-222 Chapter 8. Methods o± studying bacteria. Section I. General 223 II. Direct methods 224 228 406303°--41 1 TM 8-237 Chapter 8. Methods of studying bacteria—Continued. Paragraphs Section III. Staining methods 229-230 IV. Culture methods 231-243 V. Serological methods 244-247 VI. Animal experimentation 248-257 Chapter 9. Pathogenic bacteria. Section I. Classification of bacteria 258-263 II. Bacteria of medical importance 264—318 III. Stock culture maintenance 319-321 IV. Preparation of autogenous vaccines 322-326 y. Production of diagnostic antigens and anti- sera 327-328 VI, Handling of bacteriological specimens 329-347 Chapter 10. Special bacteriological methods. Section I. Bacterial food poisoning 348-350 II. Bacteriology of war wounds 351-365 III. Bacteriological examination of water 366-372 IV. Bacteriological examination of dairy prod- ucts 373-375 V. Rickettsiae 376-384 VI. Filterable viruses 385-395 VII. Fungi 396-400 Chapter 11. Special serological methods. Section I. Human blood groups 401-405 II. Sero-diagnosis of syphilis 406-414 Chapter 12. Protozoological methods. Section I. Protozoa 415 II. Intestinal protozoa 416-425 III. Blood and tissue flagellates 426-429 IV. Malarial parasites 430-439 Chapter 13. Helminthological methods. Section I. Classification 440-442 II. Important pathogenic species-: 443-448 III. Methods of examination 449-455 Chapter 14. Entomological methods. Section I. General 456-457 II. Arthropods of medical importance 458-477 III. Handling and shipment of specimens 478-479 Chapter 15. Pathological methods. Section I. Gross pathology methods 480-486 II. Histologic technic 487-493 Page Appendix. List of references - 416 Index 419 2 METHODS FOR LABORATORY TECHNICIANS TM 8-227 1-2 Chapter 1 GENERAL Paragraphs Section I. General 1-2 II. Medical supplies 3-5 III. Laboratory reports 6-8 IV. Compound microscope 9-11 y. Miscellaneous procedures 12-20 Section I GENERAL P»ragrapb Purpose 1 Scope 2 1. Purpose.—This manual is intended primarily for use in train- ing medical laboratory technicians in Army laboratories. It also provides detailed directions for most of the diagnostic clinical lab- oratory tests required in a hospital, corps area, or field laboratory. Standardization of technical procedure and the use of standard medical supplies and equipment in Army laboratories are encouraged. 2. Scope.—This manual is based upon the fourth (1935) edition of Laboratory Methods of the United States Army, but has been entirely rewritten to meet the different objective. As a training manual for the apprentice technician it has been given detail of tech- nic but abridgment of subject interpretation. Some subjects, not properly approached by the apprentice technician, have been given an entry for general orientation. Due to limitation of space, the less frequently used tests and duplicate or optional tests have been omitted. Where several acceptable tests are available for a single purpose, the one most adaptable to apprentice technician and to Army laboratories has been described. A number of new approved methods have been added. Names of bacteria, protozoa, and insects have been changed to conform to latest accepted nomenclature. Free reference has been made to standard texts on the various subjects. (See app.) 3 TM 8-227 3-4 MEDICAL DEPARTMENT Section II MEDICAL SUPPLIES Paragraph Standard 3 Nonstandard 4 Laboratory supplies 5 3. Standard.—These are entered in current edition of Medical De- partment Supply Catalog and stocked by supply depots. This list is designed to provide for all ordinary requirements. Insofar as practicable only standard items should be used, methods being chosen when available which will utilize standard items. Most laboratory supplies are in classes 1 and 4 of this catalog. An allowance table in the Medical Department Supply Catalog estimates the annual average requirement for laboratories serving the various popula- tion or bed capacity groups. Special requirements, or lack of re- quirement, warrant deviation from these estimates. a. S. GN items.—These are expensive items which are available for issue only after prior specific authority of The Surgeon General. b. Expendable items.—These are items such as glass slides which, when they become unserviceable, can be discarded without further accountability. These are indicated by a capital letter “Xr in column following the item number. It is customary, however, for these items to be assembled within the laboratory for periodic review by the chief of service to determine rates of losses, avoidable factors of breakage, and replacement requirements. c. Nonexpendable items.—These are items which remain charged to the responsible or accountable officer until disposed of by memo- randum receipt, shipping ticket, or survey by a board of officers. Broken items of this group should never be discarded, but the parts preserved and turned in to the accountable officer for credit. d. Deteriorating supplies.—These are items such as rubber goods and biologicals, which require more frequent replacement than do the other items. These are indicated in the supply catalog by the figure “(1)” in column after item number. 4. Nonstandard.—These are items not entered in the supply cata- log, either because they are replaceable by some standard item or because their use is so limited as to not warrant storage at the supply depots. A few items listed in this manual are nonstandard for the latter reason. Requisitions for nonstandard supplies are required to include exact data as to name, grade, cost, and dealers, and to state that no standard supply item will fill the need. 4 METHODS FOR LABORATORY TECHNICIANS TM 8-227 5—7 5. Laboratory supplies.—Requisitions are submitted periodically by the medical supply officer of a unit upon the basis of estimates made by him or specific requests made to him by the officer in charge of the laboratory. a. Medical supply depots fill requisitions from stock or by purchase. b. Corps area laboratories and the Army Medical School supply a number of items, such as specific diagnostic products, so listed in class 1 of supply catalog and some unlisted biological products to meet special needs. Section III Paragraph Purpose 6 Preparation 7 Forms S LABORATORY REPORTS 6. Purpose.—Reports of laboratory examinations are made to give useful information for diagnosis, for guidance to treatment, or for epidemiological or sanitary guidance. Those reports which pertain to a hospital patient are filed with and become a part of the clinical record of that patient. Those reports having epidemiological or sanitary reference become a part of the office file of administrative offices. Duplicate reports become the laboratory office file for future reference. It may be stated as an axiom that a laboratory report, as seen on a patient’s record chart, reflects the acumen, industry, and neatness of the laboratory examiner: a neat, concise, understandable report reflects careful technic in the examination; an untidy, incom- plete, or vague report reflects careless or incomplete technical han- dling. A technician should not impair the value of an examination on which he has spent hours of careful work and thought, by ter- minating it in a few minutes with a hasty, careless report. 7. Preparation.—Reports are prepared by the laboratory in dupli- cate; the original report is forwarded to the ward, office, or officer requesting the examination; the duplicate copy is retained in the lab- oratory for file. Some reports, such as autopsy protocols and reports of milk and water analysis, call for multiple copies on special forms. a. Substance of the report is entered by the technician or officer making the examination and should be exact, understandable, neat, and prompt, giving complete identification as to source, date, special circumstances, result of examination, and date of report. h. Verification of the report is made by the initials of the examin- ing technician or officer and the signature of the laboratory officer. 5 TM 8-237 7-8 MEDICAL DEPARTMENT c. Reports of laboratory work are strictly confidential; informa- tion as to results of tests is to be released only through medical channels. Observe caution in giving out information by telephone. d. Work notes are kept for all procedures other than those sub- ject to immediate report. These contain a record of the day-by-day observations and procedures and serve as the basis for the final report of the examination. At no time should memory be used for guidance of past findings and events. e. Monthly and annual reports are prepared by the laboratory officer giving the kinds and numbers of examinations made during the period. These are prepared from the duplicate report forms and from records of each component part of the laboratory on unreported as well as reported undertakings. 8. Forms.—The following Medical Department blank forms are provided and should be used in reporting results of laboratory work: Blood (W. D., M. D. Form No. 55 L-l). Blood (Chemistry) (W. D., M. D. Form No. 55 L-2). Serology (W. D., M. D. Form No. 55 L-3). Spinal Fluid (W. D., M. D. Form No. 55 L-4). Urinalysis (W. D., M. D. Form No. 55 L-5). Urinalysis (Quantitative) (W. D., M. D. Form No. 55 L-6). Sputum (W. D., M. D. Form No. 55 L-7). Gastric Analysis (W. D., M. D. Form No. 55 L-8). Feces (W. D., M. D. Form No. 55 L-9). Carbohydrate Tolerance (W. D., M. D. Form No. 55 L-10). Renal Function (Concentration and Dilution) (W. D., M. D. Form No. 55 U-ll). Renal Function (P. S. P.) (W. D., M. D. Form No. 55 L-12). Renal Function (Urea Clearance) (W. D., M. D. Form No. 55 L-13). Basal Metabolism (W. D.. M. D, Form No. 55 L-14). Miscellaneous (W. D., M. D. Form No. 55 L-15). Report of Sanitary Chemical Analysis of Water (W. D., M. D. Form No. 94). Report of Bacteriological Analysis of Water (W. D., M. D. Form No. 95). Record of Serological Reactions of Syphilis (W. D., M. D. Form No. 97). Note.—Form No. 55 L-15 is used in reporting most bacteriological examina- tions and for other tests for which no special form is provided. 6 METHODS FOR LABORATORY TECHNICIANS TJVC 8-237 £ Section IV Paragraph Structure 9 Use 10 Care 11 COMPOUND MICROSCOPE 9. Structure.—a. A microscope, the working tool of a microbiolo- gist, consists of four groups of parts, each group composed of a number of units: (1) Framework.— (a) Base, on which the microscope rests. (b) Handle, by which it is carried and which supports the magni- fying and adjusting systems. (e) Stage, a perforated horizontal shelf on which the object rests. (d) Mechanical stage, which moves the object about on the stage. (2) Illumination system.— {a) Mirror, which reflects light upward. (b) Condenser, placed just beneath hole in stage. (—v~r i — factor for 0.1 N dichromate. Cc of dichromate Note.—Reference should he made to the section “Volumetric Solutions” in the U. S. Pharmacopoeia, to Van Nostrand’s “Chemical Annual,” or to “The Hand- book of Chemistry and Physics” of the Chemical Rubber Co., etc., for tables of ecpuvalents of N and 0.1 N solutions. Standardization of acids and bases, etc., may be most accurately done by means of samples of benzoic acid, sodium oxalate, arsenic trioxide, and acid potassium phthalate secured from the Bureau of Standards, Washington, U. C., at small cost. Instructions for use accompany all standard samples. Section IV USEFUL LABORATORY ARTS AND RECIPES Stopcock lubricants 52 Cements and adhesives 58 Label protection 54 Cleaning fluid 55 Iodine stains 56 Paragraph 52. Stopcock lubricants.—a. Glycerol.—For sealing ground glass joints and stopcocks to prevent leakage of ethyl ether, petroleum ether, or any other fluid insoluble in it, glycerol (glycerin) is very satis- factory. It prevents sticking as well. h. Stopcock grease.—Melt 2 parts of paraffin and 4 parts of vaseline together, then add slowty 1 part of pure gum rubber cut into small pieces. Stir while heating until a smooth paste is formed. Be careful not to burn the rubber. c. Stopcock lubricants.—(1) Mix together 500 gm of petrolatum and 50 gm of raw crepe rubber. Keep in an oven at 125° to 150° C. for several days until the paste is smooth. (2) Mix 2 parts of ordinary rubber cement and 1 part of vaseline. Heat on a water bath until the solvent from the rubber cement is driven off. 53. Cements and adhesives.—a. Acid-resisting cement.—Mix sodium silicate and asbestos powder to a thin paste. If allowed to dry for a day this cement will resist the strongest acids. 61 TM 8-227 53-56 MEDICAL DEPARTMENT b. Vacuum wax.—For ordinary vacuum seals and for vacuum dis- tillations where the temperature does not get too high, an excellent wax may be made by melting together equal parts of beeswax and rosin. This wax is pliable and is easily removed by using hot water. 54. Label protection.—a. Collodion for labels.—Soak 3 to 4 gm of pyroxylin in 25 cc of absolute ethyl alcohol, then add 75 cc of ether. b. Vinylite lacquer.-—Dissolve 20 gm of vinyl acetate polymer (Viny- lite A) in 100 cc of solvent made by mixing 75 cc of toluene and 25 cc of 95 percent alcohol. This lacquer gives a transparent, colorless film which resists most reagents well. 55. Cleaning fluid.—To remove grease spots from cloth or leather use a mixture of carbon tetrachloride, 80 cc, ligroin, 16 cc, and tertiary amyl alcohol, 4 cc. 56. Iodine stains.—To remove iodine stains from clothing, wash thoroughly with a 10 percent solution of sodium thiosulfate in water, then rinse with clear water. 62 TM 8-227 57-58 METHODS FOR LABORATORY TECHNICIANS Chapter 4 EXAMINATION OF URINE Paragraphs Section I. Collection and preservation of specimens 57-58 II. Physical examination 59-64 III. Routine chemical examination 65-72 IV. Microscopic examination of urinary sediments 73-76 y. Quantitative chemical examination 77-83 Section I COLLECTION AND PRESERVATION OF SPECIMENS Paragraph Samples .• 57 Preservatives 58 57. Samples.—a. Single samples.—These are used as a matter of convenience but only for qualitative examination. Abnormal find- ings in such a specimen should be checked by the examination of a 24-hour specimen. Of the single samples, one passed 3 hours after a meal is best. The single sample voided on arising is least likely to yield pathological findings, yet it may be at times the most desirable for checking the presence of pus and mucus. h. Day and night samples.—In certain conditions and for some tests it may be desirable to collect the day and night urines separately. Usually all urine voided from 8 AM to 8 PM is collected as the day sample and that from 8 PM to 8 AM as the night sample. c. Twenty-four-hour sample.—This sample is preferred for routine examination and is absolutely essential for accurate study of kidney conditions. No quantitative test should be done except on a repre- sentative portion of a 24-hour sample. For convenience the collec- tion is usually started in the morning at 7 AM. The patient voids and this voiding is discarded. All urine passed subsequently is saved. At 7 AM the following morning the patient voids and this urine is added to the sample. The volume is noted, the total speci- men is well mixed, and 120 to 240 cc sent to the laboratory for analysis. 58. Preservatives.—a. Cold.—If a refrigerator is available, sam- ples may be kept in it until examined. Samples for pregnancy tests must be so preserved. Avoid freezing. h. Chemical preservatives.— (1) Toluene.—This is the best chemical 63 TM 8-237 58-61 MEDICAL DEPARTMENT preservative. Sufficient amount should be added to form a thin layer on the surface. (2) Camphor.—A small piece sufficient to give a saturated solution is used. (3) Formaldehyde.—Two drops of the ordinary commercial solu- tion to an ounce of urine are sufficient. If in excess it interferes with albumin, sugar, and indican tests. It is most satisfactory for the preservation of formed elements. Section II PHYSICAL EXAMINATION Paragraph Color 59 Appearance — 60 Reaction . - - _ _ _ 61 Specific gravity _ _ 62 Quantity 63 Total solids 64 59. Color.—Various shades of yellow and amber are normal, the darker shades being associated with increased concentration and high specific gravities, the lighter with dilution and low specific gravities. Increased amounts of the normal urinary pigments (urochrome, uro- bilin, and uroerythrin), the presence of abnormal pigments, and the renal elimination of drugs may produce a variety of colors. Color is usually recorded as straw, yellow, amber, etc., indicating the shade as light, medium, or dark; that is. light straw, dark amber, etc. 60. Appearance.—Normal, freshly passed urine is clear and sparkling. Concentrated urines, on cooling, may develop a white, pink, or reddish sediment. Pus or blood, if present, produce a cloudy or smoky appearance. During the change to alkalinity in older specimens phosphates may separate out. Marked cloudiness in a freshly passed specimen is significant of a pathological condition. Appearance is usually described as clear, slightly cloudy, very cloudy; the sediment as slight, moderate, or heavy, and its color white, pink, reddish, etc. 61. Reaction.—Determine whether the reaction is acid, alkaline, or neutral by the use of blue and red litmus paper. Freshly voided normal urine is usually acid, averaging about pH 6.0. Properly pre- served 24-hour samples are also slightly acid. The diet is the most important factor in modifying the reaction of the urine. (See also par. 81 for the use of nitrazine paper (phenaphthazine) for deter- mining pH.) 64 METHODS FOR LABORATORY TECHNICIANS TM 8-227 62-65 62. Specific gravity.—This is most conveniently estimated with the urinometer, a special type of hydrometer. Each urinometer is calibrated to give readings at a definite temperature, usually 25° C. (77° F.) ; this calibration is marked on the spindle. If the tempera- ture of the urine is above that at which the urinometer is calibrated, a correction of 0.001 must be added for each 3° C. above the standard temperature and similarly deducted for each 3° C. below this cali- bration temperature. For example, if the urinometer, calibrated at 25° C., reads 1.018 in a urine at a temperature of 31° C.. then 0.002 must be added, giving the corrected specific gravity of 1.020. Nor- mally the specific gravity varies between 1.015 and 1.030; pathologi- cally it may range from 1.000 to 1.060. 63. Quantity.—Normally 1,200 to 1,500 cc are passed in 24 hours. In diabetes mellitus and diabetes insipidus the quantity is much in- creased. In water loss due to diarrhoea, excessive vomiting, or pro- fuse sweating the amount is decreased. Normally, the day volume exceeds the night volume, being three to four times as much. 64. Total solids.—The amount of solids excreted in the urine may be roughly calculated by means of Long’s coefficient. 2.6. The last two figures of the specific gravity taken at 25° C. multiplied by this coefficient give the number of grams of total solids in 1,000 cc of urine. Section III ROUTINE CHEMICAL EXAMINATION Paragraph Albumin . 65 Glucose (Benedict's test) _ 66 Indican (Obermayer’s test) _ __ 67 Acetone 1 68 Aceto-acetic acid (diuretic acid) (ferric chloride test—Gerhardt) 69 Bile pigments 1 70 Urobilinogen 71 Blood I 72 65. Albumin.—All normal urines contain some albumin, but the amount is so small it escapes detection by the methods generally used. The principles employed are either coagulation by heat or precipi- tation by chemical agents. No test is absolutely satisfactory due to interference of other substances precipitated with the albumin. Mucin is a common source of error. When present, it can be removed by adding acetic acid and filtering. All tests for albumin require that the urine be absolutely clear; cloudy samples should be cleared by filtration or centrifugalization, otherwise reactions due to small amounts of albumin will be masked in the general turbidity. 40(5803° 41 5 65 TM 8-227 • 65 MEDICAL DEPARTMENT a. Heat and acetic acid test.— (1) Reagent.—Acetic acid, 5 percent. (2) Procedure.—Fill a test tube two-thirds full of clear urine. Heat the upper portion gently to boiling in an open flame. Pre- cipitates forming at this point may be due to albumin or phosphates. Add 3 to 5 drops of 5 percent acetic acid solution, a drop at a time. (3) Results.—If the precipitate dissolves, it is due to phosphates. If due to albumin, the precipitate will become heavier and more flocculent. Compare the cloudiness produced with the unheated urine in the lower portion of the tube. b. Nitric acid and magnesium sulfate ring test (Roberts'),—(1) Reagent.—To 5 volumes of saturated aqueous solution of magnesium sulfate, U. S. P., add 1 volume of concentrated nitric acid. (2) Procedure.— (a) Place a few cubic centimeters of reagent in a test tube, tilt the tube, and introduce the urine sample with a pipette, allowing it to flow gently down the side of the tube so as to overlay the reagent without mixing. If albumin is present, a flufly, white ring of precipitated albumin forms at the juncture line. (b) Another method uses a pipette made of glass tubing with an inside diameter of about 5 mm. Place a few cubic centimeters of Roberts’ reagent in a test tube. With the pipette take up a small column of urine, about 1 cm long, wipe excess urine from the outside, then place it in the test tube carefully, holding the finger firmly over the upper end until the other end touches the bottom of the tube. Release the finger pressure gradually, allowing the reagent to rise in the pipette, forming a clear, distinct layer with the urine. After standing for a minute or two, read against a dark background. (3) Remits.—A white ring at the junction of the liquids indi- cates albumin, the thickness and density of the ring showing the amount. No confusing colored rings due to indican, iodides, bile pigments, or the oxidation products of organic constituents are formed as is frequently the case when nitric acid alone (Heller’s test) is used. A white ring or cloudiness may form above the contact zone, due to urates or mucus, but such rings are less sharp, broader, and lie above the albumin ring when both are present. c. Sulf6salicylic acid test (Exton).— (1) Reagent.—Dissolve 200 gm of crystalline sodium sulfate (Na2SOt * 10H.O) in 800 cc of water with the aid of heat. Cool to 35° C. and add 50 gm of sulfosalicylic acid. Dissolve and dilute to 1 liter. (2) Procedure.—Mix equal volumes of clear urine and reagent in a test tube. Warm the mixture gently; do not boil. (3) Residts.—If cloudiness does not develop in the cold, albumin is absent. If cloudiness appears and persists or increases on gently heating, albumin is present. 66 TM 8-227 65-67 METHODS FOR LABORATORY TECHNICIANS d. Osgood-Huskins’ test.—(1) Reagents.— (a) Acetic acid, 50 per- cent solution. (b) Sodium chloride, saturated aqueous solution, 30 percent. (2) Procedure.—To 5 volumes of urine in a test tube, add 1 vol- ume of the 50 percent acetic acid, followed by 3 volumes of the satu- rated sodium chloride solution. Heat the mixture gradually to boiling. (3) Results.—A precipitate appearing upon the addition of the acid indicates bile salts, urates, or resin acids, etc., whereas a pre- cipitate appearing after the addition of the salt solution suggests Bence-Jones protein, or globulin in excess of 0.38 gm per liter. As the temperature is raised the precipitate of Bence-Jones protein, if present, will go back into solution; if albumin or globulin are present a precipitate will form. This test has the advantage of indicating the presence of Bence-Jones protein as well as albumin and globulin. 66. Glucose (Benedict’s test).—a. Reagent (Benedict’s qualita- tive solution). Cupric sulfate crystals (CuSG4 • 5H..O) U.S.P IT. 3 gm Sodium carbonate, monohydrated U. S. P 11T. 0 gm (or sodium carbonate, anhydrous U.S.P. 100 gm) Sodium citrate, U. S. P 173. 0 gm Dissolve the copper sulfate in about 100 cc of water. Dissolve the carbonate and citrate in TOO cc of water, with the aid of heat, if necessary. Cool to room temperature and pour in the copper solution slowly with constant stirring. When completely mixed make up the volume to 1.000 cc. h. Procedure.—Place 5 cc of reagent in a test tube. Add 8 drops (0.5 cc), not more, of urine. Boil vigorously over an open flame for 1 to 2 minutes, then allow to cool spontaneously. Do not hasten cool- ing by immersion in cold water. If a large number of tests are to be run, the tubes may be placed in a boiling water bath, or a beaker of boiling water, and heated for 5 minutes, then allowed to cool. c. Results.—In the presence of glucose the entire solution will be filled with a bulky, colloidal precipitate which may be greenish yellow, yellow, or red in color, depending on the amount of glucose present. In the presence of over 0.2 to 0.3 percent of glucose the precipitate will form quickly. If no glucose is present the solution will remain perfectly clear or will show a faint turbidity due to precipitated urates. 67. Indican (Obermayer’s test).—This test depends on the de- composition of indican and the subsequent oxidation of the liberated indoxyl to indigo blue, at times to indigo red. 67 TM 8-227 67-68 MEDICAL DEPARTMENT a. Reagents.—(1) Obermayer's reagent.—Add 2 gm of ferric chloride to 1,000 cc of concentrated hydrochloric acid (sp. gr. 1.19 or 23.6° Baume). (2) Chloroform. b. Procedure.—To 5 cc of urine in a test tube add an equal amount of reagent and 1 to 2 cc of chloroform. Mix by inverting 10 times. Allow the chloroform to settle and examine its color. c. Results.—(1) A pale blue to deep blue to violet color indicates the presence of indican, the intensity of the color being proportional to its concentration. If the oxidation is slow, a red color due to the formation of indigo red may appear. Iodides may give a red-violet color due to the liberation of iodine. The addition of a few drops of a concentrated solution or a small crystal of sodium thiosulfate will discharge this color. Thymol may produce a violet color; the thiosul- fate will destroy this also. Bile pigments interfere with the test and must be removed by adding one-fifth volume of 10 percent calcium or barium chloride solution and filtering. (2) Urotropin (hexamethylene tetramine) and formaldehyde pre- vent the appearance of the indigo blue even when indican is present in large amounts. (3) Report the test as negative, slight excess, or large excess. Normal urines may give a faint blue. 68. Acetone.—a. Sodium nitroprusside test {Lange's).—(1) Rea- gent*.—{a) Acetic acid, glacial, 99 percent. (b) Ammonium hydroxide, 28 percent, stronger ammonia water. (c) Sodium nitroprusside, freshly prepared saturated solution. Dissolve several crystals in 1 to 2 cc of water by gentle heat, having a slight excess of undissolved crystals remaining. (2) Procedure.—Place 5 cc of filtered urine in a test tube, add 0.5 cc of glacial acetic acid and 0.5 cc of the freshly prepared sodium nitroprusside solution and mix. Tilt the tube and carefully overlay the mixture with 1 to 2 cc of strong ammonia water. (3) Results.—A purple or purplish-red ring forms at the contact zone in a few minutes if acetone is present. The ring tends to be more purple or violet in low concentrations, more red-purple in high. Amorphous urates may give a brown or orange ring if present in large amount. b. Ra.ntzman modification.—Aqueous solutions of sodium nitro- prusside decompose rapidly and hence must be freshly prepared. This modification gives a reagent which keeps fairly well. (1) Reagent.—Dissolve 37.5 gm ammonium nitrate crystals and 2.5 gm sodium nitroprusside in distilled water and make up to 100 cc. 68 METHODS FOR LABORATORY TECHNICIANS TM 8-227 68-70 In a brown glass-stoppered bottle this reagent will keep for 2 months. (2) Procedure\—To 3 cc of urine in a test tube add 1 cc of the reagent. Mix and overlay with strong ammonia water. (3) Remit.—If acetone is present a sharply defined purple or bur- gundy-red ring appears at the contact zone. The smaller the amount of acetone present, the longer it takes the ring to appear. c. Ross modification of Rot hero's test.— (1) Reagent.—Mix 1 part of powdered sodium nitroprusside and 100 parts of powdered ammonium sulfate. (2) Procedure.—Place 1 gm of the dry powdered reagent in a test tube and add 5 cc of clear urine. Mix until the powder is dissolved, then overlay with strong ammonia water. (3) Result.—A red-purple permanganate color indicates the presence of acetone. 69. Aceto-acetic acid (diacetic acid) (ferric chloride test— Gerhardt).—a. Reagent.—Ferric chloride, 10 percent aqueous solu- tion. b. Procedure.—To 5 cc of urine in a test tube, add the ferric chloride reagent drop by drop until no more phosphates precipitate. Filter and add more ferric chloride solution. c. Result.—If aceto-acetic acid is present a bordeaux-red color de- velops. A similar color is produced by phenols, coal tar antipyretics, bicarbonates, salicylates, etc. 70. Bile pigments.—a. Rosenbachls modification of Gmeliris test.— (1) Reagent.—Nitric acid, concentrated, containing nitrous acid. This is concentrated nitric acid which is slightly yellow, due to the presence of nitrous acid. Colorless nitric acid will become yellow by exposing it to sunlight in a plain glass container for several days. To hasten this change, heat colorless acid in a beaker with a small sliver of pine wood (piece of match stick) until fumes appear. (2) Procedure.—Filter 10 to 20 cc of urine, acidified with 1 or 2 drops of dilute hydrochloric acid, through a small, heavy filter paper. Introduce 1 drop of the nitric acid into the apex of the paper, then unfold it. (3) Result.—A play of colors appears in the order of green, blue, violet, red, and reddish yellow, the last nearest the center of the paper. h. Flammarsten's test for bilirubin.—(1) Reagents.—(a) Barium chloride, 10 percent aqueous solution. (c) Nitric acid, 1:4.—Dilute 1 part of concentrated acid with 3 parts of water. (b) Ethyl alcohol, absolute. 69 TMC 8-337 70-71 MEDICAL DEPARTMENT [d] Hydrochloric acid, 1:4..—Dilute 1 part of concentrated acid with 3 parts of water. (e) Stock reagent.—Mix 1 part of the dilute nitric acid and 19 parts of the dilute hydrochloric acid. (/) Test reagent.—To 1 part of stock reagent, add 4 parts of the absolute ethyl alcohol. (2) Methods.— {a) With whole urine.—To 2 cc of test reagent in a test tube, add a few drops of urine. (h) With urinary precipitate.—To 5 cc of acid urine (acidify if necessary) add 5 cc of 10 percent barium chloride solution. Mix well and centrifugalize. Decant and discard the supernatant. Mix the precipitate with 2 cc of the test reagent and centrifugalize. (3) Result.—A green color is produced if bile pigments were pres- ent. This test is sensitive to 1 part of bile pigment in 1.000,000 parts of urine. c. Huppert-Nakayama test.— (1) Reagents.—(a) Barium chloride, 5 percent solution. (h) Nakayama reagent.—Dissolve 0.4 gm ferric chloride in a mix- ture of 99 cc of 95 percent ethyl alcohol and 1 cc of concentrated hydrochloric acid. (2) Procedure.—To 5 cc of urine add 5 cc of the barium chloride solution. Mix thoroughly and centrifugalize. Pour off the super- natant fluid. To the sediment add 2 cc of the Nakayama reagent, mix, and bring to a boil. (c) Nitric acid, concentrated. (3) Result.—A brilliant, deep-green color develops if bilirubin is present. On adding a few drops of nitric acid the color changes to violet or red. 71. Urobilinogen.—a. General.—Bilirubin excreted into the in- testine in the bile is decomposed with the production first of the colorless compound urobilinogen, and then urobilin. Normally these compounds are partially absorbed from the intestine, carried to the liver, and reconverted into bilirubin. Some part of the urobilinogen normally finds its way into the general circulation and is excreted in the urine. In disturbances of liver function and in certain toxe- mias, infectious diseases, and hemolytic conditions larger amounts may get into the general circulation and appear in the urine. I). Ehrlich aldehyde test (modification of Wallace and Diamond').— This test is roughly quantitative. (1) Reagent.—Dissolve 2 gm of paradimethylaminobenzaldehyde in 100 cc of 20 percent (by volume) hydrochloric acid. (2) Procedure.—To 10 cc of bile-free, undiluted urine at room 70 TM 8-227 71-73 METHODS FOR LABORATORY TECHNICIANS temperature, or warmed to 21° to 22° C., add 1 cc of the reagent; allow to stand for 3 minutes. If a deep cherry-red color appears, proceed with the test using 10-cc portions of dilutions of the urine. Make 1: 10, 1: 20, 1: 50, 1: 100, and 1: 200 dilutions with tap water at room temperature. Add to the 10-cc portions 1 cc of reagent, let stand 3 to 5 minutes, not longer, and read. (3) Results.—Express the result in terms of the highest dilution giving a faint but definite pink or cherry color; that is, positive in 1: 20 dilution. Normally this is at the 1: 20 dilution. Any greater dilution yielding a definite pink color indicates a pathological amount of urobilinogen. A daily estimation showing positive in greater and greater dilution is especially significant. 72. Blood.—The detection of traces of blood requires microscopic examination of the urinary sediment for red blood cells and chemical examination for hemoglobin. See paragraphs 73 to 76, inclusive, and chapter 5 for the methods. Section IV MICROSCOPIC EXAMINATION OF URINARY SEDIMENTS Paragraph Preparation of specimen 73 Examination of drop 74 Recording of findings 75 Character of urinary sediments 76 73. Preparation of specimen.—a. Urines should be examined within a few hours after voiding, unless kept at a low temperature or preserved with a chemical. Alkaline specimens should be exam- ined as soon as possible. If large amounts of suspended amorphous phosphates in an alkaline urine obscure the field, the specimen should be cleared with dilute acetic acid and reexamined. Heavy urate sedi- ments in an acid urine may be dissolved by gently warming the specimen. h. The sediment for examination is usually secured by centrif- ugalizing a 15-cc portion of the urine at a relatively low speed (1,000 to 2,000 rpm), for 5 minutes. The supernatant urine is poured off as completely as possible and the sediment mixed with the urine remaining by tapping the end of the tube with the finger. A drop of the mixed sediment is removed to a clean glass slide by means of a pipette, or a drop may be poured directly onto the slide from the centrifuge tube. c. If a centrifuge is not available, sedimentation in long, conical urine test glasses may be done. This is not as satisfactory as centrif- 71 TM 8-337 73-76 M E I) [CAL I) K P ART M E NT utilization because of the longer time required and because the various constituents, due to differences in specific gravity, settle out at different rates, giving a less homogeneous sediment. A drop of sediment collected in this manner must be transferred to the slide by means of a pipette. 74. Examination of drop.—Examination is usually made with a low-power objective (16 mm) and a moderately high-power eyepiece (10X). The high-power objective (4 mm) is needed occasionally for detailed examination of casts and to distinguish between pus cells and red blood ceils. For low-power examination it is not necessary to use a cover slip; il may be desirable to use one when the higher power objective is used. The substage condenser should be lowered and the light cut down by the iris diaphragm until the objects stand out clearly. 75. Recording of findings.—The frequency of occurrence of the various objects observed should be noted as well as their mere pres- ence. The terminology used may be: occasional, few, many, very many, etc. A uniform technic of examination and of reporting should be followed so that the results of different examinations may be comparable. The same amount of urine should be centrifugalized at the same speed for the same length of time in each case. The supernatant should be poured off to the same degree of completeness, and approximately the same thickness of drop examined. 76. Character of urinary sediments.—There are two classes of sediments: unorganized chemical compounds; and organized, formed, morphological entities. The latter are by far the more important as the mere presence of some or a superabundance of others indicates pathology somewhere in the urinary tract provided the specimen has not been contaminated. a. Unorganized sediment.—(1) Only rarely has the unorganized sediment clinical significance. Its presence and character depends on metabolic activities of the body which are influenced by innumer- able factors, or upon fermentation and decomposition processes oc- curring in the bladder or in the container after voiding. Unorgan- ized sediments appear as crystals of definite structure that can be readily identified morphologically or as amorphous deposits having a granular, structureless appearance; these latter can be partially identified by solubility or microchemical tests conducted on the slide while under microscopic observation. (2) In acid urines one may find amorphous, pinkish sediments of urates; brownish, wedge-like, “whetstone,” or dumbbell crystals of uric acid; small dumbbell or square “envelope” crystals of calcium 72 TM 8-227 76 METHODS FOR LABORATORY TECHNICIANS 1,S, EPITHELIAL CELLS 2,3, ER Y THRO C Y TES 4, LEUCOCYTES J&2. TP! PL £ PHOSPHATE CRYSTALS I, LEUCOCY TES; 2, S PEP PI A TOZ OA; J, 4, 6, EP/ TPEL /PL CEL L S; s, EPYTHPOCVTES HE STEAL CALC/SAn PHOSPHATE CPYSTALS uric ac/c> crystals .(yellow TO GPEEH/Sp YELLOW). UPLC AC/L> CPYSTALS / YELLOW TO GPEE/YYSH VEl. LOPS). Figukk 8.—Urinary sediments (unorganized). 73 TM 8-227 76 MEDICAL DEPARTMENT oxalate; refractile, colorless, six-sided plates of cystine; yellowish, small spheroids of leucine; fine needles of tyrosine; and brownish needles or prisms of hippuric acid. (3) In neutral urines those already mentioned may occur and in addition slender, pyramidal crystals of neutral calcium phosphate united at their apices to form rosettes. (4) Alkaline urine may contain white amorphous phosphate de- posits; “coffin lid’’ or feathery crystals of ammonium magnesium phosphate (triple phosphates) ; spheres or dumbbells or amorphous deposits of calcium carbonate; and dark yellow to brown “cockle- burr” crystals of ammonium urate. h. Organized sediments.— (1) Casts.—As the name suggests these are molded in the tubules of the kidney and are composed of an albuminous material and various types of cells. They vary greatly in size but in almost all instances their sides are parallel and ends rounded or broken olf squarely. They may be straight or curved, long or short, but the diameter is usually uniform throughout the length. Casts have been classified according to their microscopical characteristics as hyaline, granular, epithelial, blood, pus, fatty, and waxy. The finding of casts in the urine is very important for their presence usually indicates some form of kidney disorder, especially if albumin is also present. (a) Hyaline casts are found more frequently than any others. They are composed of colorless, homogeneous, slightly refractile ma- terial; are usually narrow; cellular elements may be attached to the surface. (h) Granular casts are usually short and thick. The basic hyaline substance is filled with granules which may be fine or coarse. (blue _ yellow—►purple. _ _ _ 6.0 to 7.6 1 cc_ . _ 5.2 to 6.8 152 METHODS FOR LABORATORY TECHNICIAN TM 8-337 153-159 153. Gas formation.—Culture is made in liquid media containing a small inverted tube and indicator as above. Observe daily for the collection of gas in this inverted tube. Record, approximately, the portion of vial filled with gas as percent gas present. 154. Test for indol.—a. Reagent. 1. Para dimethyl-amino benzaldehyde 5 gm (2) Amyl alcohol 75 cc (3) Hydrochloric acid 25 cc Dissolve (1) in (2) and then add the acid. The completed reagent should have a yellow or light-brown color. b. Procedure.—(1) Inoculate rube of tryptone broth. Incubate at 37° C. for 24 hours. (2) Add 0.2 to 0.3 cc of the amyl alcohol indol reagent and shake gently, (3) Allow reagent to rise to surface of medium and observe result. Dark red color is a positive indol test; original color of reagent is a negative test. 155. Nitrate reduction (Ilosvay’s method).—Nitrate broth culture, after 5 days incubation at 37° C. Add 1 cc of solution A and 1 cc of solution B. Positive reaction, red, purple, or maroon; negative reaction, no color change. Solution .4 A-naphthy lamine__- 1 gm 1 ~. . , -r-ir , i- > Dissolve. filter, then add: VV ater, distilled 22 cc J Acetic acid, dilute (sp. gr. 1.04) 180 cc Solution li Sulfanilic acid 0.5 gm Acetic acid, dilute 150 cc 156. Ammonia production.—Peptone water culture, grown for 5 days at 37° C. Add; Nessler’s reagent 0.5 cc. Note color change. Positive, brown; negative, faint yellow. 157. Hydrogen sulfide production.—Lead acetate agar—stab culture, incubated at 87° C. Observe daily for 5 daj’s. Positive reaction, brown or black color; negative reaction, no color change. 158. Reductase test.—Broth culture—24 hours at 37° C. Add 1 drop methylene blue (1 percent aqueous solution). Incubate at 37° C. Note color. Positive (strong reduction), complete decolorization; weak positive, green color; negative, no color change. 159. Methyl red test (M, R.).—Peptone medium (Clark and Bubs’ medium) culture, grown for 4 days at 37° C. Add 5 drops methyl red indicator solution (0.04 percent in 60 percent alcohol). Positive reaction, red color; negative reaction, yellow color. 153 TM 8-327 160-163 MEDICAL DEPARTMENT 160. Voges-Proskauer test (V. P.).—To a 5-cc portion, re- moved asceptically from the peptone medium inoculated for the methyl red test, or to a separately inoculated tube of the same medium, after 24 to 48 hours’ incubation, add an equal volume of 10 percent potassium hydroxide solution. Place in incubator at 37° or 45° C for 6 hours or overnight; observe periodically. A positive test is indicated by an eosin pink color. 161. Citrate utilization test.—Inoculate tube of sodium citrate water or of Simmon’s citrate agar for 4 days at 37° C. Growth indi- cates positive; no growth, negative. 162. Tartrate utilization.—Make deep stab inoculation of tube of Jordan’s tartrate agar. Positive reaction, acid butt; negative reaction, no change in color. Section VI PREPARATION OF CULTURE MEDIA General 163 Adjustment of reaction 164 Clarification . 165 Distribution 166 Storage 167 Meat extract broth 168 Meat extract agar 1 169 Meat infusion broth 170 Meat infusion agar 171 Nutrient gelatin 172 Nutrient broth (for water analysis) 173 Nutrient agar (for water analysis) 174 Lactose broth (for water analysis) ' 175 Levine’s eosin methylene blue agar (for water analysis) 176 Brilliant green lactose bile broth (for water analysis) 177 Tryptone-glucose-extract-milk agar (for milk analysis) 178 McLeod’s gonococcus medium 179 Semisolid media 180 Calcium carbonate broth (for pneumococci) 181 Potato-glycerin-blood agar (for H. pertussis) 182 Robertson’s medium (for anaerobes) __ 183 Dieudonne’s alkaline blood agar (for Vibrio comma) 184 Petroff’s medium (for M. tuberculosis) 185 Cystine blood agar (for P. tularensis) 186 Hiss’ serum-w’ater medium (for fermentation tests) 187 Potato medium 188 Glycerol agar 189 Loeffler’s medium (for O. diphtheriae) 190 Tellurite medium (for C. diphtheriae) 191 Sabouraud’s media (for fungi) 192 Paragraph 154 METHODS FOR LABORATORY TECHNICIANS TM 8-327 162-164 Paragraph Com meal agar 193 Chocolate blood agar 194 Enriched agar media 195 Glucose agar 196 Glucose brain broth (for streptococci and anaerobes) 197 Liver infusion agar (for Brucella) 198 Liver infusion broth (for culturing blood for Brucella) 199 Russell’s double sugar- 200 Eosin methylene blue agar (for typhoid-dysentery isolation) 201 Bismuth sulfite agar (Wilson and Blair) (for typhoid group) 202 Desoxycholate agar and desoxycholate-citrate agar (Liefson) (for typhoid- dysentery group) 203 Selinite-F enrichment medium (for typhoid group isolation from feces and urine) 204 Bile medium (for typhoid group) 205 Simmon’s citrate agar 200 Jordan’s tartrate agar 207 Lead acetate agar (for ILS production) 208 Tryptone broth (for indol test) 209 Clark and Lubs’ medium (for Voges-Proskauer and methyl red tests) 210 Dunham’s peptone solution (for “cholera red” reaction and as base for carbohydrate media) 211 Nitrate broth (for nitrate reduction test) 212 Carbohydrate broth (for fermentation tests) 213 Bromcresol purple milk 214 163. General.—Bacteria may be cultivated upon a great many different kinds of artificial culture media, all of which have the com- mon characteristics of supplying food for the bacteria, having a naturally or artificially adjusted reaction (hydrogen ion concentration or pH) and being of such a nature that they can be readily sterilized. They must meet certain requirements as to surface tension and osmotic pressure and they must be stable. 164. Adjustment of reaction.—The hydrogen ion concentration of media can be determined and adjusted by the electrometric or colorimetric methods. The colorimetric method is the one most frequently used. a. Equipment, colonmetnc method.—For the titration and adjust- ment of media, a set of color standards are used. Color standard sets may be prepared with several different indicators, such as phenol red with a color range from pH 6.8 to 8.4 and brom-thymol blue with a range from pH 6.0 to 7,6. These sets are prepared at the Army Medical School for distribution to Army laboratories. The set of phenol red indicator standards, which are generally used, consists of nine tubes whose pH range is from 6.8 (yellow) to 8.4 (red) ; each tube increasing by 0.2. TM 8-327 164-168 MEDICAL DEPARTMENT h. Technic.—(1) Select the standard tube of the desired pH (most culture media are adjusted to pH 7.2 to 7.6) and place it in the right front hole of a “comparator block.” (2) Place immediately behind it a tube of medium to which no indicator is added. (3) Place a tube containing 10 cc of medium to which has been added 0.5 cc of a 0.02 percent phenol red solution in the left front hole. (4) Place immediately behind it a tube of distilled water. (5) Hold the comparator block toward the daylight and determine whether the medium plus indicator solution has the same color as the pH standard plus medium without indicator. (6) If the medium is acid, as most freshly prepared media are, add measured quantities of N/10 NaOH until the color of the medium matches the standard tube. (If the medium is very alkaline, add measured quantities of N/10 HC1 until the medium matches the standard tube.) (7) From the volume of N/10 NaOH (or HC1) used to adjust 10 cc of medium to the desired pH, the amount of N/l NaOH (or HC1) required for 1,000 cc may be estimated by multiplying the number of cubic centimeters of N/10 solution used by 10. (8) After the final addition of NaOH (or HCl), recheck the pH and readjust if necessary. Note.—As some change in reaction may take place during sterilization, it is desirable to make suitable corrections for this. Broth media with an initial pH of 7.0 changes very little, but if the initial pH is over 7.0 it may become about 0.2 more acid. 165. Clarification.—Media may be clarified by filtration through cotton, gauze, filter paper, or by use of a Berkefeld filter. 166. Distribution.—The media should be placed in sterile glass flasks or tubes before sterilization. This can be done by placing the fluid medium in a large container or funnel from which it is delivered through a rubber tube controlled by a pinchcock. 167. Storage.—After sterilization, label media and store in refrig- erator or cold room. Cotton-stoppered flasks and tubes should be protected with a cap of paper or lead foil. 168. Meat extract broth. Beef extract 3 gm Peptone 10 gm Sodium chloride 5 gm Distilled water 1, 000 cc 3 gm 5 gm '156* METHODS FOR LABORATORY TECHNICIANS TM 8-227 168-173 Add the weighed ingredients to distilled water and heat slowly to 65° C., stirring until dissolved. Adjust loss of volume with distilled water, clarify by filtration through paper, and titrate to pH 7.2 to 7.6. Autoclave 15 minutes at 15 pounds. 169. Meat extract agar. Meat extract broth 1,000 cc Agar 20 to 30 gm Add agar to broth and autoclave 15 minutes at 15 pounds. Filter through cotton, adjust pH to 7.2 to 7.6, and resterilize. 170. Meat infusion broth. Beef or veal round, free from fat, ground 500 gm Distilled water 1,000 cc Mix and infuse in ice box 18 to 24 hours. Remove and heat over low flame 1 hour. Squeeze through gauze until 1,000 cc are obtained. Sodium chloride 5 gm Peptone 10 gm Add to infusion and dissolve over flame. Titrate to pH 8.0. Filter through paper. Cook in autoclave 45 minutes at 15 pounds. Refilter through paper while hot and make up to volume with distilled water. Autoclave one 10-cc portion 15 minutes at 15 pounds, titrate for acid drift, adjust pH of lot, tube, or flask and autoclave 15 minutes at 15 pounds. 171. Meat infusion agar. Meat infusion broth 1,000 cc Agar 20 gm Add agar and proceed as in preparation of meat extract agar. Adjust to pH 7.4. 173. Nutrient gelatin. Meat extract broth 1, 000 cc Sheet gelatin (purified) 120 gm Place the broth in double boiler and add the gelatin. Dissolve gelatin by heating and adjust reaction to pH 7.4, Make up to original volume with distilled water. Add one egg, mixed with small amount of water, to clarify; reheat slowly until egg is firmly coagulated. Filter through cotton. Tube in 10-cc portions and sterilize in Arnold sterilizer for 20 minutes on 3 successive days. After each heating, cool rapidly to prevent lowering of melting point. 173. Nutrient broth (for water analysis). Beef extract 3 gm Peptone (Bacto) 5 gm Distilled water 1,000 cc 167 TM 8-327 173-177 MEDICAL DEPARTMENT Prepare as for meat extract broth in basic media but adjust pH between 6.4 and 7.0. 174. Nutrient agar (for water analysis). Meat extract broth (as above) 1, 000 cc Agar 15 gm Add agar to broth and prepare as for meat extract agar in basic media but adjust pH between 6.4 and 7.0. 175. Lactose broth (for water analysis).—Prepare in same manner as for meat extract broth but with addition of 0.5 percent lactose and 1 cc of 1.6 percent alcoholic solution of bromcresol purple per liter. Adjust to pH 6.4 to 7.0, preferably pH 6.9. 176. Levine’s eosin methylene blue agar (for water analy- sis). Peptone 10 gm Dipotassium phosphate (K2HP04) 2 gm Agar 15 gm Distilled water 1, 000 cc Add ingredients, dissolve by boiling, and make up volume lost. No adjustment of reaction is necessary. Place 100-cc amounts in flasks and autoclave 15 minutes at 15 pounds. Just prior to use, melt above and to each 100 cc add: Lactose, 20 percent, sterile solution 5 cc Eosin, yellowish, 2 percent aqueous solution 2 cc Methylene blue, 0.5 percent aqueous solution 2 cc Mix thoroughly and pour plates. Allow to harden and incubate for sterility. Note.—It is permissible to add all of the ingredients to the stock agar at the time of preparation. Place in flasks and sterilize; plates may be prepared from this stock directly. 177. Brilliant green lactose bile broth (for water analysis). Peptone (Bacto) 10 gm Lactose 10 gm Distilled water 500 cc Dissolve and add— Fresh ox bile 200 cc or 20 gm dehydrated ox bile, dissolved in 200 cc distilled water. Then add--- Distilled water q. s. ad 975 cc 158 TM 8-227 '177—179 METHODS FOR LABORATORY TECHNICIANS Adjust pH to 7.4 and add— Brilliant green, 0.1 percent aqueous solution 13. 3 cc Distilled water, q. s. ad 1,000 cc Filter through cotton and autoclave for 15 minutes at 15 pounds. Final pH should be not less than pH 7.1 and not more than 7.4. 178. Tryptone-glucose-extract-milk agar (for milk analy- sis). Agar 15 gm Beef extract 3 gm Tryptone 5 gm Glucose 1 gm Distilled water 1,000 cc Dissolve by boiling over a free dame, make up volume lost, and adjust reaction to pH 7.0. Add— Skim milk 10 cc Dispense measured amounts (100 or 200 cc) in flasks, or place 10 to 12 cc in test tubes. Autoclave 15 minutes at 15 pounds. 179. McLeod’s gonococcus medium.—a. Stock medium. Peptone. - 10 gm Sodium phosphate, dibasic (Na2HP04) 2 gm Distilled water 1,000 cc Dissolve by heating to 60° C. Then add— Ground lean meat 1 lb Heat to 60° C. for 45 minutes, then steam in autoclave for 30 minutes. Filter, adjust to pH 7.4, and add— Agar : 12 gm Dissolve with scant boiling, bottle in 100-cc amounts, autoclave 15 minutes at 15 pounds, and store. b. Gompleted medium. Stock medium 100 cc Blood, human, citrated 10 cc Add blood to melted stock medium while still very hot. Mix thor- oughly and pour plates. Use same day as prepared. Note.—Concentrated amino-acids and peptone inhibit growth of gonococcus; the heating of blood checks this inhibition, hence the use of “chocolate” or heated blood agar. The inhibiting effect of peptone on delicately growing micro-organisms is due to some oxidized substance produced in heat steriliza- tion of the medium, hence the McLeod method reduces these oxidized bodies by having the peptone in contact with the meat during extraction. 159 TM 8-227 180-183 MEDICAL DEPARTMENT 180. Semisolid media.—a. General.—General media containing 0.1 to 0.3 percent agar are known as semisolid. Many different types of semisolid media may be prepared, differing in agar content, in the basic liquid medium used (infusion broth, peptone broth, etc.) and in special ingredients that may be added (serum, ascitic fluid, carbo- hydrates, indicators, etc.). These media are used especially in the study of anaerobic bacteria and in the cultivation and fermentation studies of gonococci and meningococci. b. Bade formula. Infusion broth (beef, veal, etc.) or other type liquid media 1,000 cc Agar 1 gm Add agar to broth and dissolve by boiling. Adjust reaction to pH 7.6. Tube in 10-cc quantities and serilize in autoclave at 15 pounds for 15 minutes. Before use, melt agar and drive off dissolved oxygen by boiling for 10 minutes; cool to 45° to 50° C. and inoculate. 181. Calcium carbonate broth (for pneumococci). Meat infusion broth 1.000 cc Glucose 10 gm Dissolve by heating. Adjust pH to 7.6. Place clean marble chips in bottom of test tube and add broth. Sterilize for 15 minutes by the fractional method in Arnold sterilizer. 182. Potato-glycerin-blood agar (for H. pertussis). Sliced potato . 100 gm Glycerin, 4 percent aqueous 200 cc Mix and steam in autoclave. Remove 50 cc of the resulting glyc- erin extract of potato. Glycerin extract 50 cc Sodium chloride 0.6 percent 150 cc Agar 5 gm Add ingredients, melt in autoclave, and place 2 to 3 cc in test tube and sterilize. Sterile defibrinated rabbit blood or human blood 2 to 3 cc Add in equal volume to sterile tubes of above media, mix, and slant. pH is approximately 6 and no adjustment is necessary. 183. Robertson’s medium (for anaerobes). Beef heart, fresh, with all fat, fascia, and blood vessels removed, ground 500 gra Peptone 10 gm Distilled water 1,000 cc 160 TM 8-227 183-186 METHODS FOR LABORATORY TECHNICIANS Mix ingredients and bring to a boil. Adjust pH to 8.0 and allow to simmer 1 y2 hours; readjust pH. Separate broth from meat and place in flasks. Autoclave 15 minutes at 15 pounds. Place meat on clean filter paper and dry in oven at 56° C. for 48 hours. Place the desired quantity of dried heart in a test tube and add 10 cc of above broth. Autoclave, cool broth, and titrate. Adjust reaction for acid drift and adjust so that media will have a final pH of 7.4 to 7.6. Resterilize. 184. Dieudonne’s alkaline blood agar (for Vibrio comma). Blood, beef, defibrinated 150 cc Potassium hydroxide (normal) 150 cc Mix and steam in Arnold sterilizer 80 minutes. Then melt— Nutrient agar (pH 6.8) 700 cc To 7 parts of agar add 3 parts of above alkali-blood mixture. Pour plates and allow to harden uncovered but protected with paper. Place strips of filter paper between dish and cover to absorb moisture and ammonia. Incubate 15 hours at 37° C. before using. 185. Petroff’s medium (for M. tuberculosis).—This medium is composed of meat juice, eggs, and a minute amount of gentian violet or brilliant green. a. In a cool place infuse 500 gm of beef or veal in 500 cc of a 15 percent solution of glycerol in water; after 24 hours place in sterile press and collect the extract in a sterile container. h. Immerse washed eggs in 70 percent alcohol for 10 minutes. Pick out with sterile tongs, flame, and break in a sterile container. Add 1 part meat juice to 2 parts eggs by volume (1 egg equals approximately 25 cc). o. Add 1 percent alcoholic solution of gentian violet or brilliant green to make a final proportion of 1 to 10,000. Thoroughly mix ingredients, tube, slant, inspissate, and sterilize in Arnold sterilizer by discontinuous method. 186. Cystine blood agar (for P. tularensis). Beef or veal infusion 1, 000 cc Agar 15 gm Proteose-peptone (Difco) 10 gm Sodium chloride 5 gm Dissolve by heating; adjust reaction to pH 7.8; autoclave 15 min- utes at 15 pounds, and filter. Place in flasks and resterilize. Final 406303°-—41 11 161 TM 8-237 186-191 MEDICAL DEPARTMENT pH of medium should be between 7.3 and 7.4. Before use, add to the above— Cystine (preferable) or cystine hydrochloride 1 gm Glucose 10 gm Dissolve by heating in Arnold sterilizer and then sterilize for 30 minutes. Cool to 50° C. and add— Kabbit or horse blood or serum, sterile 50 cc Tube aseptically, slant, and incubate for sterility. 187. Hiss’ serum-water medium (for fermentation tests). Clear, sterile serum 1 part Distilled water 3 parts Mix and heat in Arnold sterilizer for 15 minutes. Add 1 per- cent of the desired carbohydrates, dissolved in a little hot water. To each 1,000 cc of medium, add 1 cc (or more, if required) of a 1,6 percent alcoholic solution of bromcresol purple. Tube and sterilize for 20 minutes in Arnold sterilizer on 3 successive days. Incubate for sterility. 188. Potato medium.—Select large white potatoes, peel, and scrub thoroughly under running water. Cut cylinders from the po- tato with cork borer. Cut these obliquely into wedge-shaped pieces and place in running water overnight to reduce acidity. Place in tubes and add 2 cc of distilled water; autoclave for 15 minutes at 15 pounds. 189. Glycerol agar. Meat infusion agar 1,000 cc Glycerol (pure) 30 cc Mix by heating and adjust pH to 7.2. Tube, autoclave for 15 minutes at 15 pounds, and slant. 190. Loeffler’s medium (for C, diphtheriae).—Collect beef blood in sterile containers. Allow to clot without removing. Loosen clot with sterile rod and store in refrigerator. Pipette off clear serum. To 3 parts serum add 1 part meat infusion broth, pH 7.6, containing 1 percent glucose. Mix by stirring, tube, and inspissate on a slant, gently raising temperature to about 85° C. Hold at this temperature until the serum is firmly coagulated. Sterilize by frac- tional method (for 20 minutes on 3 successive days) in the Arnold sterilizer. After sterilization, paraffinize cotton plugs and incubate tubes for sterility. 191. Tellurite medium (for C. diphtheriae).—Melt meat ex- tract agar or 0.2 percent dextrose agar, 10 cc in tube or larger meas- ured quantity in flask, and cool to 50° C. For each 10 cc of medium, 162 METHODS FOR LABORATORY TECHNICIANS TM 8-227 191-195 add 1 cc of citrated or defibrinated rabbit blood and 1 cc of a sterile 2 percent solution of potassium tellurite. Mix and pour into Petri dishes. Note.—An excellent tellurite medium may also be prepared by adding 5 cc of Bacto-tellurite blood solution to 100 cc of Bacto-dextrose proteose No. 3 agar, heating to 80° C., cooling to 50° C., and pouring plates. 192. Sabouraud’s media (for fungi).—a. Conservation. Peptone 30 gm Agar 20 gm Tap water 1,000 cc Dissolve, tube, and autoclave 30 minutes at 8 pounds. b. Differential. Maltose, crude , 40 gm Peptone 10 gm Agar 20 gm Tap water 1, 000 cc Mix all ingredients except maltose, bring to a boil, and then add maltose; filter if necessary. Autoclave 30 minutes at 8 pounds. No adjustment of reaction is necessary. 193. Corn meal agar. Water, distilled 1, 500 cc Corn meal 62. 5 gm Heat at 60° C. for 1 hour. Filter through paper and adjust volume to 1,500 cc. Add— Agar 19 gm Heat in Arnold sterilizer for 1*4 hours. Filter through cotton, tube, and sterilize in autoclave at 15 pounds for 15 minutes. Adjust- ment of reaction is not necessary. 194. Chocolate blood agar.—Add 5 percent of sterile defibrinated blood to melted meat infusion agar at 50° to 55° C., mix carefully to avoid bubbles, and slowly raise the temperature to 75° C. Pour into plates or tube and slant. Incubate to determine sterility. 195. Enriched agar media.—a. Blood agar.—Add 5 to 10 per- cent of sterile defibrinated or whole blood (human, rabbit, or horse) to infusion agar (preferred) or extract agar that has been melted and cooled to 45° C. Pour plates or prepare slants and incubate for Sterility. b. Serum agar.—To 1,000 cc of melted infusion agar at 45° to 50° C., add 100 cc of sterile normal horse serum. Place in sterile tubes and slant, or pour into plates. Incubate to determine sterility. Sterile ascitic or hydrocele fluid may be used instead of the serum. 163 TM 8-227 196-198 MEDICAL DEPARTMENT 196. Glucose agar.—To 1,000 cc of melted extract agar or infu- sion agar, add 10 gm of glucose and heat slowly until dissolved. Ti- trate and adjust the reaction to original pH of the agar. Sterilize by the fractional method. 197. Glucose brain broth (for streptococci and anaerobes). Fresh calf brain ,5 to 10 gm 1 percent glucose veal infusion broth pH 7.8 35 cc a. Prepare flask of veal infusion broth (par. 170), adjust reaction to pH 7.8, and add glucose (1 gm per 100 cc of medium). h. Wash several pieces of calf brain, 1 cc in size, in running water and place into the bottom of a large tube (200 by 25 mm) ; add the infusion broth. Autoclave at 15 pounds for 20 minutes and cool. c. Remove 10 cc of the supernatant fluid and check the reaction; if the reaction is pH 7.4 to 7.6, it is satisfactory; but if there has been a greater acid drift, adjust to pH 7.6. Estimate from the titration of the 10-cc portion the amount of NaOH required to adjust reaction for bulk of the broth. d. Then fill the desired number of tubes with similar quantities of the brain tissue and broth (pH adjusted, if necessary). Sterilize in the autoclave at 15 pounds for 20 minutes. Incubate at 37° C. to determine sterility. 198. Liver infusion agar (for Brucella).—a. Basic formula. Agar 20 gm Distilled water 500 cc Beef liver infusion 500 cc Peptone (Difoo) 5 gm Sodium chloride 5 gm Place all ingredients in a suitable container, cover, and place in flowing steam for 1 hour. Remove and cool to 60° C. Adjust the pH at this time to 7.0. Place in flowing steam again for y2 hour. Decant and place in sterile flasks or tubes and sterilize 30 minutes at 15 pounds. The pH will drop to 6.6 during this sterilization and no adjustment is necessary as the organisms grow best at this pH. Two separate media are prepared, using one of the. following dyes: (1) Basic fuchsin 1/100,000 (1.0 cc of 0.1 percent aqueous solution to 100 co of medium). (2) Thionin 1/200,000 (1.0 cc of 0.1 percent aqueous solution to 200 cc of medium). b. Preparation of heef liver infusion for above.—Fresh beef liver, free from fat, is ground. To 500 gm of ground liver add 500 cc of tap water and place in flowing steam for 20 minutes. Remove lid and stir with glass rod in order to mix thoroughly. Continue heating 164 METHODS FOR LABORATORY TECHNICIANS TM 8-227 198-205 for 1 y2 hours. Remove and strain through wire screen or four layers of gauze. Autoclave 15 minutes at 15 pounds. 199. Liver infusion broth (for culturing blood for Bru- cella).—Same as liver infusion agar (par. 198), except omit agar and do not add dyes, 200. Russell’s double sugar. Meat extract agar, melted 1,000 cc Lactose (sterile 25 percent solution) 40 cc Glucose (sterile 25 percent solution) 4 cc Mix melted agar and two sugars and adjust pH to 7.2. Add 50 cc of 0.02 percent aqueous phenol red. Filter if necessary, tube, and sterilize in autoclave 25 minutes at 8 pounds. 201. Eosin methylene blue agar (for typhoid-dysentery isolation).—Prepare as for Levine’s eosin methylene blue agar in water analysis (par. 176) but reduce dye content one-half by using 1 cc of 2 percent eosin and 1 cc of 0.5 percent methylene blue per 100 cc of medium. 202. Bismuth sulfite agar (Wilson and Blair) (for typhoid group).—Use Bacto or other dehydrated product. Follow directions on bottle for preparation and sterilization. 203. Desoxycholate agar and desoxycholate-citrate agar (Liefson) (for typhoid-dysentery group).—To prepare these media, use a dehydrated product (such as those prepared by the Baltimore Biological Laboratory, Baltimore, Md.) and follow directions on bottles for preparation, sterilization, and use. 204. Selinite-F enrichment medium (for typhoid group iso- lation from feces and urine). Sodium acid selinite (anhydrous) 4 gm Peptone 5 gm Sodiimi phosphate (anhydrous) 10 gm Distilled water 1, 000 cc Determine experimentally the portion of the monobasic and dibasic sodium phosphate which together with the peptone and sodium acid selinite will give a pH of 7.0. Dissolve the weighed ingredients in the distilled water by gentle heat, tube in 10-cc amounts, and sterilize in Arnold sterilizer for not over 30 minutes. 205. Bile medium (for typhoid group). Ox bile (or equal volume of 10 percent solution of Bacto-oxgall) 900 cc Glycerol 100 cc Peptone 20 gm 165 TM 8-227 205-208 MEDICAL DEPARTMENT Dissolve over water bath, tube or place in flasks, and autoclave 15 minutes at 15 pounds. 206. Simmons’ citrate agar. Magnesium sulfate 0. 20 gm Sodium chloride 5. 0 gm Ammonium acid phosphate (NH4H2P04) 1.0 gm Sodium citrate (5^H20) 2. 77 gm Distilled water 1,000 cc Agar 20 gm Dissolve chemicals in the distilled water; add agar and heat to dissolve. Adjust reaction to pH 7.2 and add— Bromthymol blue, 1.5 percent alcoholic solution 10 cc Filter through cotton, tube, and autoclave 15 minutes at 15 pounds. Slant with deep butt. Note.—Check reaction of medium with known cultures of E. coli, A. aero- genes, S. schottmuelleri, and E. typhosa before using routinely. 207. Jordan’s tartrate agar. Agar 20 gm Peptone (Difco) 10 gm Sodium potassium tartrate 10 gm Sodium chloride 5 gm Distilled water 1, 000 cc Dissolve ingredients by heating. Adjust pH to 7.4 and add— Phenol red, 0.2 percent alcoholic solution 12 cc Tube in 10-cc quantities and autoclave 15 minutes at 15 pounds. Note.—Cheek reaction of medium with known cultures of $. typhimurium, 8. enteritidis, 8. paratyphi, and 8. schottmuelleri before using routinely. 208. Lead acetate agar (for H2S production). Bacto-tryptone 20 gm Agar 15 gm Distilled water 1,000 cc Prepare basic medium, adjust to pH 6.8 to 7.0, and sterilize in auto- clave. Prepare for use by adding to each 100 cc of melted medium 0.4 cc of a sterile 25 percent solution of dextrose and 10 cc of a sterile 0.5 percent solution of basic lead acetate. Mix and dispense in tubes aseptically, with slant and deep butt. Incubate for sterility. Check reaction of medium with known strains of S. 'paratyphi and 8. schottmuelleri. 166 METHODS FOR LABORATORY TECHNICIANS TM 8-227 209-214 209. Tryptone broth, (for indol test). Distilled water 1, 000 cc Tryptone (Bacto) 10 gm Dissolve by heating and stirring, place 5-cc amounts in test tubes, and autoclave 15 minutes at 15 pounds. 210. Clark and Dubs’ medium (for Voges-Proskauer and methyl red tests). Proteose-peptone (Difco) 5 gm Dextrose 5 gm Dipotassium phosphate (K2HP04) 5 gm Distilled water 1,000 cc Dissolve by heating, filter, and make up volume lost. * Tube in 10- cc quantities and sterilize by fractional method. 211. Dunham’s peptone solution (for “cholera red” reaction and as base for carbohydrate media). Proteose-peptone (Difco) 10 gm Sodium chloride 5 gm Distilled water 1, 000 cc Dissolve by heating, adjust pH to 7.6, filter, tube in 10-cc amounts, and autoclave 15 minutes at 15 pounds. 212. Nitrate broth (for nitrate reduction test). Peptone (Bacto) 10 gm Potassium nitrate (KNG3) nitrite-free 1 gm Water, distilled, ammonia-free 1,000 cc Dissolve by heating, adjust reaction to pH 7.4 to 7.6, filter, tube in 10-cc amounts, and sterilize in autoclave at 15 pounds for 15 minutes. 213. Carbohydrate broth (for fermentation tests).—To 1,000 cc meat extract broth (par. 173), add 1 percent of the desired fer- mentable substance, dissolved in a little hot water. Then add I cc of 1.6 percent alcoholic solution of bromcresol purple per liter. Place 6-cc portions in fermentation tubes and autoclave 10 minutes at 7 pounds or by fractional method in Arnold sterilizer. 214. Bromcresol purple milk.—Remove the cream from sweet milk or purchase fresh, skimmed milk. Heat the comparatively fat- free milk in the Arnold sterilizer for 20 minutes. Siphon off the central four-fifths for use (discard the cream layer on top and the bottom portion containing sediment). To each 1,000 cc of fat-free milk, add 1 cc (or more, if required to give distinct blue color) of a 1.6 percent alcoholic solution of bromcresol purple. Tube in 10-cc amounts and sterilize in Arnold sterilizer by the fractional method. 167 TM 8-227 214—S18 MEDICAL DEPARTMENT Other indicators may be substituted for the bromcresol purple, such as azolitmin (10 cc of a neutralized 5 percent aqueous solution) to make litmus milk. Note.—For the many media not described in this section refer to “A Compila- tion of Culture Media” by Levine and' Schoenleiu, published by Williams and Wilkins Company, Baltimore, Md., or to any standard textbook on bacteriology. Media in a dehydrated powder form are obtainable and may be substituted for most of the media described in this section. They are accurately prepared by reliable commercial manufacturers, supplied in powder form, and include a wide range of complex media. Being dry, they may be preserved without deterioration in stoppered bottles and kept at room temperature. Section VII CARE OF LABORATORY ANIMALS Types of animals • 215 Reception quarantine 216 Housing 217 Rabbits 218 Guinea pigs 219 Mice 220 Albino rats__ 221 Monkeys 222 Paragraph 215. Types of animals.—Five types of laboratory animals are the rabbit, guinea pig, mouse, albino rat, and monkey. 216. Reception quarantine.—All animals received from an out- side source should be isolated for 10 days to 3 weeks in previously disinfected quarters, and found to be free from disease before mixing with regular stock. 217. Housing*.—Animal quarters should be kept clean, dry, and completely free from vermin. The optimum temperature for most animals is 65° to 70° F. with adequate ventilation. The standard large (10%-inch) and small (8-inch) animal jars are suitable for mice and rats; the large jar also can be used for a small guinea pig. The standard galvanized-iron animal cage (14 by 14 by 16 inches) will hold one rabbit or several guinea pigs. For use in breeding rabbits or guinea pigs, larger cages or pens, preferably with out- side runways, should be built. The bottom of the jar or tray in cage should contain an absorbent bed material, such as wood shavings: hay or straw may be used in large breeding cages. Clean quarters and renew bedding twice per week. 218. Rabbits.—a. The diet recommended consists of commercial “Rabbit Pellets” supplemented once or twice per week with feeding of greenstuff, such as carrots, lettuce, or celery tops. A diet con- 168 TM 8-227 218-220 METHODS FOR LABORATORY TECHNICIANS sisting of equal parts of oats, wheat, and barley, plus 10 percent of legume, soybean, or linseed meal is suitable. Alfalfa or timothy hay will serve both for food and bedding. Always keep plenty of water and a small piece of rock salt in the cage. b. Diseases.—(1) “Coccidiosis,” an intense and fatal enteritis, is the most serious disease. Observe new rabbits for this several days before adding to stock. (2) “Ear mange” is caused by a mite; can be cured by local application of a parasiticide. (3) “Snuffles” is a cold-like disease caused by a filterable virus. Isolate infected rabbits until 3 weeks after recovery. c. Breeding.—Keep one male (buck) for each 8 to 10 females (does). Females are ready for mating at age of 10 months and may be bred every 3 months thereafter (4 litters per year). Keep record of date bred; gestation period 31 days; 2 or 3 days before expected arrival of litter place small breeding box and ample supply of bedding in cage. Wean young after 8 weeks and separate sexes. 219. Guinea pig's.—a. Feeding.—Same as for rabbits, except they must have supplementary feeding of greenstuffs at least twice per week to supply vitamin C. b. Diseases.—(1) Salmonella infections, chiefly Salmonella typhi- murium and iS. enteritidis, are most dangerous of common diseases. Best method of control: Kill all potentially infected animals, sterilize room and cages, and obtain new stock. (2) Vitamin C deficiency is caused by lack of sufficient green- stuffs in diet. Characterized by coarse hair and mangy appearance. It is transmissible to young through mother. Treatment: improved diet. (3) Balantidium coli type of enteritis. c. Breeding.—Use colony breeding with four or five females in cage with one male; duration of pregnancy, 63 days. Wean young and separate sexes when 4 or 5 weeks old. 220. Mice.—Several different strains are used, such as, white mice, Swiss mice (also white), and C 57 strain (black). a. Feeding.—Commercial dog- or fox-chow checkers furnish an ample, balanced diet for growth and breeding; occasionally add piece of carrot or other greenstuff. Must have supply of fresh clean water in cage at all times. Mice will do well on simpler diets, such as the mixed grain diet listed above for rabbits, or dry bread with water or skimmed milk, with addition of cod liver oil once per week. 169 TM 8-227 220-222 MEDICAL DEPARTMENT b. Diseases.—Salmonella infections (mouse typhoid), caused by same organisms as for guinea pigs, are common and very dangerous. To control: Destroy all infected stock, sterilize room and cages, and obtain fresh stock. c. Breeding.—Colony breeding, with four or five females to one male; gestation period, 21 days; when well advanced pregnancy is observed, place female in individual jar. After 21 days, isolate young and return mother to breeding jar. Feed young same as adults, but addition of evaporated milk to diet hastens growth. 221. Albino rats.—a. Feeding.—Same as for mice. b. Diseases.■—If cages are kept clean and ample diet provided, rats are very resistant to disease. c. Breeding.—Young females are readj for breeding when 4 months old. Use colony method of breeding with four females and one male in cage; duration of pregnancy, 22 days; not necessary to remove pregnant female from breeding cage. Wean young and separate sexes when 21 days old. 222. Monkeys.—a. Feeding.—Monkeys will do very well on dog- chow checkers plus canned tomatoes, with occasional feeding of fruits and nuts (oranges, apples, bananas, peanuts, sunflower seeds, etc.). b. Diseases.— (1) Pneumonia, usually fatal. (2) Miliary tuberculosis. c. Breeding.—Breeding in captivity in small laboratories is not practical. 170 METHODS FOR LABORATORY TECHNICIANS TM 8-227 223-224 Chapter 8 METHODS OF STUDYING BACTERIA Section I. General 223 II. Direct methods 224—228 III. Staining methods 229-230 IV. Culture methods 231—243 V. Serological methods 244-247 ET. Animal experimentation 248—257 Paragraphs Section I GENERAL Paragraph General 181 223. General.—Micro-organisms may be studied and their charac- teristics determined by— a. Direct microscopical examination. h. Stained film examination. c. Cultivation on artificial culture media. d. Serological reactions—determination of certain immune sub- stances, by agglutination, complement fixation, animal protection, and other tests. e. Pathogenicity—the disease-producing capacity, if any, on man, animals, or plants. Section II DIRECT METHODS Hanging drop 224 Cover glass 225 Darkfield 226 Brownian movement 227 Active motility 228 Paragraph 224. Hanging1 drop.—With a small applicator ring the concavity of a concave slide with vaseline. Place a loopful or small drop of bacterial suspension on the center of a clean cover glass. Invert the slide and place its concavity over the cover glass so that the drop of fluid lies in the center. Turn over the slide and its attached cover glass. Press the cover slip firmly in place to prevent evaporation. Place the slide on the microscope stage and, with the low-power 171 TM 8-227 §24-328 MEDICAL DEPARTMENT lens, focus on the edge of the drop. Without changing the focus change to the high-dry objective, locate the drop, and focus on the bacteria. The oil immersion objective is used for still higher mag- nification. This permits the observer to see the micro-organism in the fresh state, to note some of its characteristics, particularly its motility if present, its size, shape, sometimes spores, capsules, and even to estimate its purity and observe its growth. It cannot be studied as well in this fresh state as when stained. 225. Cover glass.—Place a small loopful of the bacterial suspen- sion on a clean slide; over it place a cover glass and gently press the latter until it no longer floats. Examine as above. This method is simpler than the hanging-drop method and for most purposes equally satisfactory. It has the advantage of permitting observation with darkfield as well as by ordinary illumination. 226. Darkfield.—The illumination principle of this method is comparable to that which causes dust particles to be illuminated in a ray of sunlight. A cover glass preparation (ringed with vaseline) Thin cover glass Vaseline Hanging drop Concave slide Figueb 13.—Hanging drop. of material under examination is prepared, using thin slide and cover glass, and is placed on a specially prepared microscope; a darkfield condenser replaces the ordinary condenser, a “funnel stop” is placed in the oil immersion objective, specially intense light Ts used, and immersion oil is placed between the slide and condenser as well as on top of cover glass. All highly refractile objects, including bacteria, will be seen as bright objects against a black background. This is particularly used in the study of spirochetes in order to observe their peculiar motility. 227. Brownian movement.—All micro-organisms, living or dead, and also all other microscopic objects, observed by above methods, will have a dancing, trembling motion in the field, which will be magnified just as much as is the organism itself; this is “Brownian movement.” It is entirely a passive movement, just as is the bobbing up and down of a bubble on the surface of a pool. 228. Active motility.—This is a movement actively produced by the organism itself, causing it to change its position, often very rapidly, even to the extent of having individuals dart across the 172 TM 8-227 228-231 METHODS FOR LABORATORY TECHNICIANS field, as if they were in a hurry to get away. Not all micro-organ- isms possess this quality of active motility, a feature of value in identification. Section III STAINING METHODS Paragraph Simple stains 229 Special stains 230 229. Simple stains.—These are merely weak solutions of aniline dyes, applied to the slide for a brief period, then washed off. Those most commonly used for the staining of bacteria are methylene blue, fuchsin (red), crystal violet, safranin (pink), and bismarck brown. 230. Special stains.—More than a simple stain is required to make apparent the flagella, capsules, spores, and other special characteristics of a micro-organism. Differential stains utilize more than one dye. sometimes aided by a decolorizing agent and a “mordant” (any agent such as heat or phenol that will fix the dye more firmly); these include Gram’s stain, acidfast stains (for tubercle and lepra bacilli), Neisser’s stain (for diphtheria bacilli), and Wright’s stain (for protozoa). Section IY CULTUEE METHODS Paragraph Cultural characteristics. 231 Cultural requirements 232 Culture media 233 Culture planting 234 Transfer from test tube to test tube 235 Pour plate transfer 236 Plate method 237 Colony picking . 238 Incubation 230 Atmospheric conditions.. _ 240 Deep tube-tissue anaerobic method 243 Anaerobic jar method 242 Partial oxygen tension method 243 231. Cultural characteristics.—Under favorable conditions of food, moisture, and temperature, an organism multiplies to become, after many generations, a colony of many like organisms, visible to the naked eye. In fluid media these organisms scatter throughout the liquid, giving it a cloudy appearance, sometimes developing a color, odor, or appearance peculiar to that species. In solid media, such as agar, dispersion of the new growth is impossible; the organisms TM 8-227 231-235 MEDICAL DEPARTMENT pile up into a mass (a colony) the size, shape, and color of which may characterize the species. The differences in growth on different media, the temperature at which it grows best, and the requirements as to the presence or absence of oxygen are also important charac- teristics of each kind of micro-organism, 232. Cultural requirements.—Food materials and conditions of life and growth must be suitable before an organism will grow. Disease-producing bacteria will utilize as food material substances resembling the fluids and tissues of the body; beef broth (infusion or extract) is a basic constituent, sodium chloride an essential salt, and agar a hardening substance, of general culture media. The re- action of the medium, or its degree of acidity or alkalinity must be suitably adjusted; most bacteria prefer a neutral or slightly alkaline medium, others prefer one that is acid. Some bacterial species are more fastidious—grow only in media containing some special enrich- ment substance. There are some bacteria for which the particular artificial media for cultivation have not yet been found. 233. Culture media.—This may be liquid or solid, the latter made so by the addition of gelatin or agar. As the melting point of gela- tin is 28° C., it can be used only for cultivations at or below room temperature. The melting point of agar is about 99° C. and its solid!- fying point about 39° C., so agar may be used for those organisms which grow at body temperature (37° C.) or below. Certain sub- stances may be added to media, not for nutrition but to gain some specific reaction of the organism. The final step in the preparation of culture media consists of killing all the living organisms in it, that is, sterilizing it so that any growth present following subsequent inoculation may be assured to be from the organism present in in- oculum. A medium so prepared and sterilized will retain its service- ability until it desiccates, this tendency being reduced by storage in the ice box. 234. Culture planting.—Inoculation or transfer is usually ac- complished with a wire needle (platinum) supported in a holder, the wire end being either straight or bent in a loop. This wire must be sterilized before and after use by heating to redness through an open flame. Occasionally a sterile cotton swab or pipette may be used for transfer of inoculum. Every effort must be used to make the transfer without risk of contamination. 235. Transfer from test tube to test tube.—The best technic is to hold both tubes in one hand while making the transfer so that inoculating needle may pass from tube to tube without risk of dust contamination. The steps of the inoculation are as follows: 174 METHODS FOR LABORATORY TECHNICIANS TM 8-227 235-237 a. Sterilize needle (deposit upright in tumbler while preparing tubes). b. Place both tubes in left hand and remove cotton plugs (plugs to he kept sterile, held between fingers of right hand). c. Pass mouth of both test tubes through flame. d. Enter needle into tube No. 1 to obtain inoculum. e. Enter needle into tube No. 2 to plant bacteria. (1) Liquid media merely receives a touch of needle' loop. (2) “Stab” agar receives a stab of straight needle. (3) “Slant” agar receives surface stroke or broad brush of needle. /. Sterilize needle. g. Pass mouth of each tube through flame. h. Replug tubes. i. Label tubes with identification and date. 236. Pour plate transfer.—a. Agar in tube is melted and cooled to about 42° C. in water bath. b. Inoculation is made as in paragraph 235e(l). ose heart and lungs. Study for presence of pathological lesions and prepare smears and cultures. k. Place organs or tissues to be given histopathological study in a bottle of 10 percent,formalin or Zenker’s fluid. l. Remove animal from the board, wrap it firmly in paper, and dispose of it as circumstances permit, preferably by immediate incineration. TM 8-227 258-259 MEDICAL DEPARTMENT Chapter 9 PATHOGENIC BACTERIA Section I. Classification of bacteria ; 258-203 II. Bacteria of medical importance 264-318 III. Stock culture maintenance 319^321 IY. Preparation of autogenous vaccines 322-326 V. Production of diagnostic antigens and antisera 327-328 VI. Handling of bacteriological specimens 329-347 Paragraphs Section I CLASSIFICATION OF BACTERIA Paragraph General 258 Cocci > 259 Curved forms 1 260 Nonsporulating bacilli ; 261 Sporulating bacilli 262 Bacilli having branching characteristics , 263 258. General.—a. Bergey’s “Manual of Determinative Bacteri- ology” provides classification system and criteria for bacteriology. The genera and species names used in the following paragraphs are those found in the fifth edition (1939) of that manual. The outline classification, herein given, omits many groups, including only those species mentioned elsewhere in this text. For brevity and simplicity, no effort is made to keep each genus in its proper tribe or family as shown by Bergey. Bacteria in this key are classified, first, on basis of morphology of organism, second, on Gram-staining qualities, and third, on basis of other special characteristics. h. The full name of a bacterium consists of the name of the genus with initial letter capitalized, followed by the name of the species in small letters, with the entire name underscored when written, italicized when printed. The name of the genus may be abbreviated. 259. Cocci.—Cells spherical or somewhat elliptical; aerobic. a. Gram 'positive.—(1) Streptococcus forms.—Cells in short or long chains, never in packets. Genus Streptococcus. (a) Pyogenic group; generally (beta) hemolytic; four species and several serological types. Most important species, S. pyogenes. (h) Viridans group; not beta hemolytic .but may show varying de- grees of greening (alpha) of blood. Most important species, S. salivarius. (> X Dulcitol Lactose Saccharose Inositol ! • Indol , HjS pro- duction C i trate utilization | d-tartrate utilization Motility Salmonella cholerae- suis _ _ _ - - AG AG AG AG (AG) y + + + Salmonella pullorum AG AG — (AG) — — — — — + — — — Salmonella paratyphi _ AG AG AG — (AG) — — — — ( + ) — — + Salmonella enteritidis_ AG AG AG AG AG — — — — + + + + Salmonella schott- muelleri AG AG AG (AG) (AG) (AG) 4- + + Salmonella lyphimur- ium AG AG AG AG AG (AG) + + + + Eberthella typhosa . .. A A A (A) (A) — — — — + — + + Shigella dysenior iae A — — Shigella paradysen- teriae - A A (A) (A) + — Shigella alkalescens__. A A A A A — (A) — + — — — Shigella sonnei - A A A — — A A — — — — — — Shigella madam,pensis_ A A A A — A A — + — — --- “ Note.—Parentheses in symbols denote variable or delayed reaction. 189 TM 8-227 262-363 MEDICAL DEPARTMENT 262. Spomlating bacilli.—Rods producing endospores, usually Gram-positive. Often decompose protein media actively, a. Grow aerobically.—Mostly saprophytes. Genus Bacillus. (1) Pathogenic forms.—Nonmotile rods with square-cut to concave ends, occurring in long chains; central spores. Bacillus anthracis. (2) Nbnpathogenic forms.—Usually motile, having central or excentric spores. B. subtilis group (145 species). b. Grow only anaerobically.—-Often parasitic. Genus Clostridium. (1) Nonmotile rods.—Rods not swollen at speculation; spores central or excentric. Cl. perfringens. (2) Motile.—Rods swollen at speculation. (a) Spores terminal or subterminal. Spherical or nearly so. Cl. tetani. (b) Spores oval, central, or excentric. 1. Pathogenic to man—due to preformed toxin. (a) Cl. parabotulinum. (b) Cl. botulinum. £. Pathogenic to man—associated with gas gangrene. (a) Cl. novyi. (b) Cl. septicum. (c) Cl. bifermentans. (d) Cl. histolyticum. (e) Cl. fallax. 3. Not pathogenic to man; many species. Examples: (a) Cl. sporogenes. (b) Cl. tertium. 263. Bacilli having branching characteristics.—Show paral- lelism, slight branching, curving forms, V-shapes, clubbing at ends, and segmental staining. Gram-positive. a. Not acidfast.—Colonies more flat and moist like other bacteria; rods frequently club-shaped. Genus Corynebacterium. (1) True diphtheria organism. Slender rods, curved or straight, of variable lengths; granular or segmented; generally club-shaped. Met achromatic granules large except in gravis type. Moderate growth on ordinary media. G. diphtheriae. (2) The diphtheroid group of bacteria; 20 species. (a) Short, thick, straight rods; stain uniformly; luxuriant growth on ordinary media. G. pseudodiphthericum. (b) Medium-sized rods showing solid and barred forms; meta- chroma tic granules small; scanty and slow growth on ordinary media. C. xerose. 190 TM 8-327 263 METHODS FOR LABORATORY TECHNICIANS Species Dextrose ! Maltose Dextrin Glycerol t Galactose Saccharose Litmus milk Production of an exotoxin Production of hemolysis C. diphtheriae, type I . _ + + + + + C. diphtheriae, type II . . + — — — — — — + + C. diphtheriae, types III, IV, V__ + + + + + — — + + C. pseudodiphthericum . .. C. xerose + + — — + + — — — C. segmentosum _ _ _ + + + ± acid — — b. Acidfast.—Colonies more or less wrinkled and dry, more like molds. Slender rods, seldom filaments, which are stained with diffi- culty, but when once stained are acidfast. Cells sometimes show swollen, clavate or cuneate forms and sometimes even branched forms. Genus Mycobacterium. (1) Saprophytes, or parasites on cold-blooded animals; grow rap- idly on most media at room temperature (8 species). Examples: {a) M. lacticola. {b) M. phlei. (2) Parasites on warm-blooded animals; grow slowly on all media. («) Pathogenic for man. 1. M. tuberculosis variety hominis. 2. M. tuberculosis variety bovis. (b) Not pathogenic for man. M. avium. (3) Pathogenic for man. Will not grow on usual culture media. M. leprae. Section II BACTERIA OF MEDICAL IMPORTANCE Paragraph General r 264 Genus Staphylococcus 265 Genus Streptococcus 266 Diplococcus pneumoniae (pneumococcus) 267 Neisseria 268 Neisseria gonorrhoeae (gonococciis) 269 Neisseria intraeellularis (meningococcus) 270 Neisseria catarrhalis 271 Chromogenic bacteria 272 Pseudomonas aeruginosa (Bacillus pyocyaneus—bacillus of blue pus) 273 Pasteurella (hemorrhagic septicemia group) 274 Pasteurella pestis 275 191 TM 8—337 263-264 MEDICAL DEPARTMENT Diagnosis of plague in rodents . 276 Pasteurella tularensis 277 Malleomyces mallei (glanders bacillus) 278 Genus Brucella 279 Br. melitensis, Br. abortus, and Br. suis 280 Genus Hemophilus 281 Hemophilus influenzae 282 Hemophilus pertussis 283 Hemophilus duplex (Morax-Axenfeld bacillus) 284 Hemophilus ducreyi 285 Gram-negatice, aerobic, nonspore-forming enteric bacilli (family enterobac- Coli-aerogenes group (tribe Eschericheae) 287 Escherichia coli 288 Aerohacter aerogenes 289 Klebsiella pneumoniae 290 Genus Proteus 291 Typhoid-dysentery and paratyphoid-enteritis groups (tribe Salmonelleae) 292 Genus Salmonella 293 Genus Eberthella 294 Eberthella typhosa— 295 Laboratory examination of specimens for typhoid 296 Genus Shigella 297 Dysentery group 298 Laboratory examination of specimens for dysentery 299 Vibrio comma x 300 Family Bacillacae (spore-forming bacilli) 301 Genus bacillus 302 Bacillus subtilis (hay bacillus) 303 Bacillus anthracis 304 CL parabotulinum and Cl. botulinum 305 Corynebacterium diphtheriae (diphtheria bacillus): 306 Mycobacterium tuberculosis (tubercle bacillus) 307 Mycobacterium leprae (leprosy bacillus) 308 Actinomyces _* 309 Actinomyces bovis * 310 Actinomyces hominis '— 311 Actinomyces madurae 312 Examination of clinical materials for Actinomyces 313 Spirochetes 314 Borrelia recurrentis (relapsing fever spirochete) 315 Fusospirochetal disease (Vincent’s angina) 316 Treponema pallidum 317 Leptospira icterohaemorrhagiae (Weil’s disease—infectious jaundice) 318 Paragraph 264. General.—A brief description of the principal pathogenic bacteria and of certain nonpathogenic species commonly encountered in the examination of pathological materials is presented in this sec tion. Herein will be shown their important generic and specific char 192 METHODS FOR LABORATORY TECHNICIANS TM 8-227 264-266 acteristics and the chief laboratory procedures required for their identification, or for the identification of the infections in man caused by them. Organisms possessing similar morphological and Gram- staining properties or, in case of the enteric group, bacteria frequently found associated in pathological specimens, are presented together without regard to the sequence of presentation in the Bergey classifica- tion, with fhe exception that genera belonging to the same family are discussed together. 265. Genus Staphylococcus.—a. Habitat.—Common, potential or actual parasites, occurring on normal skin and body orifices, and in feces, therefore in dust, soils, and as culture contaminants; fre- quently the cause of suppurative lesions in man. h. Characteristics.—Moderate size cocci, in pairs or grape-like clusters; Gram-positive; grow freely, aerobically, on common culture media, giving in 24 hours at 37° C. medium size, low, convex, smooth, glistening colonies with an even edge; color of colony variable with species; some strains produce hemqlysis on blood agar. c. Staphylococcus aureus.—Golden-yellow colony; usually hemo- lytic; frequently found in boils, carbuncles, and other skin lesions; sometimes in blood cultures in the event of septicemia. d. Staphylococcus albus.—Porcelain-white colony; feebly patho- genic. e. Staphylococcus citrous.—Lemon-yellow colony. A nonpatho- gen ic saprophyte. f. Identi-fication.— (1) Microscopy.—Gram + staphylococci on di- rect or culture-stained spread. (2) Culture.—Blood agar plate, 24 hours at 37° C. gives colony features of staphylococcus, species determined by color of colony. Mote also presence or absence of hemolysis. 266. Genus Streptococcus.—a. Habitat.—Common pathogenic forms; also frequently on skin and body orifices without invasive tendency. Some species are the specific cause of infectious diseases. A number of saprophytic species are commonly present in dairy products and elsewhere. h. Characteristics.—Gram-positive cocci of medium size, in pairs or short chains, never in packets; grow best on blood or serum agar, aerobically, at 37° C., the 24-hour colony being small, circular, slightly raised, and surrounded at times by zone of hemolysis. Killed at 55° C., in 30 minutes. (1) Hemolytic group (beta type) have clear zoiie of hemolysis around colony on blood agar. (2) Viridans group (alpha type) have greenish zone around colony on blood agar. 406303° —41 13 193 TM 8-227 266-367 MEDICAL DEPARTMENT (3) Nonhemolytic group (gamma type) have no area of hemolysis or green zone around colony. c. Streptococcus pyogenes.—Colonies have beta zone of hemolysis 2 to 8 mm wide. Grow in long chains. Found in man in acute in- flammations, including septicemia, cellulitis, wound infections, middle ear or sinus disease or elsewhere. Tend to be more severe and generalized than Staph, aurens infections. The cause of scarlet fever; transmitted by nose, throat, and skin contaminations from cases or carriers, and of erysipelas. d. Streptococcus salivarius (S. viridavs. S. mitior).—This species is a parasite of the normal nose and throat, also encountered in dental abscesses, in endocarditis, and in some blood cultures. Grow in short chains. This colony readilv recognized on a blood agar plate by its greenish zone of hemolysis. ( Usually not pathogenic for small animals. Distinguished from Diplococcus pneumoniae by inability to ferment inulin and by not being bile soluble. ) e. Streptococcus lactis.—Is nonpathogenic. Occurs in milk and milk products and in mouth and intestinal tract of man. Colonies on blood plates produce no hemolysis or only trace of green. /. Streptococcus faecalis.—Is feebly pathogenic, found in feces of man and other animals. Sometimes found in inflammatory exudates and subacute endocarditis. No hemolysis on blood agar. g. Identification.—(1) Microscopy.—Gram stain of direct or cul- ture spreads will show Gram + cocci, singly, in pairs or in chains of varying length. The chain form is best seen in spreads made from liquid culture, or in liquid body fluids. (2) Culture.—Blood agar plates at 37° C. for 24 hours will give the small colony type and form of hemolysis classifying roughly the species. For routine clinical work the examination is usually limited to the study of colonies on blood agar and the results reported as the case may be: (a) Streptococcus, hemolytic. (b) Streptococcus, nonhemolytic. (c) Streptococcus viridans. 267. Diplococcus pneumoniae (pneumococcus).—a. Charae- teHstics.—Large lancet-shaped cocci, usually occurring in pairs; some- times found singly or in short chains. When in pairs, the adjacent ends of the cocci are usually bluntly rounded, and the opposite ends acutely pointed. In films from sputum, blood, and cultures on serum containing media, a definite capsule can be seen. Gram-positive stains well with aniline stains and special capsule stains. Poor growth on plain agar; grows best on blood or serum agar with pH 194 METHODS FOR LABORATORY TECHNICIANS TM 8-237 267 7.6 to 7.8. Colonies on blood agar plate, surface flat and smooth with edge sharply raised from the medium, surrounded by a narrow zone of alpha-hemolysis (green discoloration); some stains (types III and VIII) give characteristic mucoid colonies. Killed in 20 minutes or less at 55° C. Bile soluble and ferments inulin. Thirty- one distinct serological types have been identified; called D. 'pneu- moniae type I, II, etc., to XXXIII; however, types 26 and 30 apparently are identical with types 6 and 15, respectively. b. Habitat.—The principal cause of lobar pneumonia (over 90 percent) ; also may cause bronchitis, bronchopneumonia, conjunctivitis, otitis media, brain abscess, meningitis, endocarditis, and arthritis. Frequently present in normal mouths. Highly pathogenic for mice and slightly less so for rabbits. c. Identification.— (1) Direct microscopy.—Make spreads of speci- men on slide, fix, and stain by Gram’s method and/or Hiss’ capsule stain. Examine for diplococci showing typical morphology; if pres- ent confirm by procedures below. (2) Typing by Neufeld reaction.—This is the rapid method of choice for identification of type on materials direct from the patient, giving the type within 30 minutes. It is less applicable to typing of cultures or to detection of type in patients who have received one of the sulfanilamide compounds. Only after pneumococci have been shown by stained spread to be present in appreciable numbers, is this typing effort to be attempted. {a) Collection of specimen.—Small sample of sputum, carefully coughed up by the patient from deeper air passages, as free as pos- sible of saliva, is collected in a Petri dish or wide-mouthed bottle and should be typed within 1 hour of collection. Older specimens may be examined only if they have been kept on ice. Preferably, the sample is to be collected before beginning treatment with sulfa- pyridine (which interferes with this test). Samples with few pneu- mococci, or which otherwise give poor results by this test may be inoculated intraperitoneally into a mouse, and the mouse’s peritoneal washings 6 to 18 hours later used for this or other typing effort. Speci- mens of spinal fluid and cultures in blood or serum broth also may be typed directly. (b) Materials. 1. Platinum loop, 1-mm, for transferring sputum. &. Platinum loop, 4-mm, for serum and dye, 3. Loeffler’s alkaline methylene blue (not required if dye is already in type serum). ■4. Glass slides and cover glasses. 195 TM 8—237 267 MEDICAL DEPARTMENT 5. Typing sera (rabbit) : types I to XXXIII monovalent serum and group mixtures; mixtures A (types 1, 2, and 7) ; B (3, 4, 5, 6, 8); C (9, 12, 14, 15, 17); D (10, 11, 13, 20, 22, 24); E (16, 18, 19, 21, 28), and F (23, 25, 27, 29, 31, 32). These sera may be in capillary tubes, each with enough for one test, or in small bulk bottles. (o) Technic of test. 1. Divide three clean slides into halves by wax pencil and label halves “A, B, C, D, E, and F.” 2. Place tiny fleck of sputum in center of an area, with small loop. 3. Add typing serum, about five times as much as specimen used. J±. Add large loopful of methylene blue. Mix thoroughly and apply cover slip. 6. Let stand for 5 minutes (prepare other slides while waiting). 6. Examine under oil immersion objective for dark-blue diplococci surrounded by unstained area with definite outline. Only a small indistinct capsule can be seen around the pneumococci mixed with heterologous antisera. Large distinct halo surrounds the pneumococci which have been mixed with their type antisera. If none of the mixtures are found positive at first examination, yet pneumococci have been shown to be present by stained spread, reexamine these slides from time to time over a period of 30 minutes. 7. Positive mixtures having been determined, repeat the test procedure with each serum contained in that mixture, until the positive tvpe or types have been determined. (3) Culture.—Streak specimen (sputum, pus. peritoneal washings, or mouse’s heart blood) on blood plate and incubate 24 hours at 37° C.; then select well-isolated, typical greenish colonies and examine on stained slide for morphology. Transfer suspect colonies to calcium carbonate broth, incubate for 24 hours, and examine by confirmatory tests. V (4) Animal inoculation, (applicable when other tests are incom- plete).—(a) Select small piwe of tenacious sputum, wash through three changes of sterile saline, and emulsify in mortar with sterile saline. (b) Inject mouse intraperitoneally with 1 cc of suspect emulsion 196 METHODS FOR LABORATORY TECHNICIANS TM 8-337 267-26S or culture. The pneumococci grow rapidly in the peritoneum while most other bacteria die off. (c) When mouse appears sick, or after 6 to 8 hours, withdraw a drop of peritoneal exudate by a capillary pipette or hypodermic needle punctured through the abdominal wall. Examine micro- scopically, (d) If diplococci are numerous, kill the mouse and use peritoneal washings for typing and culturing; also make culture from heart’s blood. (e) If diplococci are not numerous on preliminary trial, retest in 24 hours or when mouse becomes definitely sick. (5) Confirmatory tests.—When indefinite results are obtained by the above tests, the identity of a pure culture of the suspect organism can be confirmed by the following examinations: (a) Bile solvability test.—Add 0.2 cc of sterile bile to 0.8 cc of culture; prepare a control by adding 0.2 cc of sterile saline to 0,8 cc ® Type I pneumococcus in sputum mixed with type II antiserum; no swelling of capsule. @ Type I pneumococcus in sputum mixed with type I antiserum ; swelling of capsule (Neufeld reaction). Figure 16.—O. pneumoniae (showing Neufeld reaction). of culture; incubate 2 hours and note clearing of the turbidity due to solubility in bile of the pneumococcus. Streptococci and other organisms are not bile soluble. (b) Inulin fomentation.—Inoculate a tube of inulin-serum water and incubate 37° C. for several days. This medium is usually fermented and coagulated by pneumococci but not by streptococci. (c) Agglutination tests.—Macroscopic tests with suspensions of the organisms and antipneumococcus sera. 268. Neisseria.—a. Characteristics.—Gram-negative cocci of vari- able growth vigor and pathogenicity. All members give a positive oxydase reaction. h. Habitat.—•N. gonorrhoeae (gonococcus) is the cause of gonor- rhoea; N. intraeellularis (meningococcus) is the cause of a specific meningitis. Both organisms may be readily demonstrated in the exudates from involved tissues. N. catarrJwMs and several other species, which are found in the nose and throat of normal individuals. 197 TM 8-327 268-269 MEDICAL DEPARTMENT are sometimes associated with certain epidemics of respiratory or eye infections. 269. Neisseria gonorrhoeae (gonococcus).—A strict parasite of man. Found in discharges from the genito-urinary system in acute or chronic gonorrhoea, and in the pus from gonorrhoeal con- junctivitis; rarely in the blood stream. a. Characteristics.—Oval or spherical cocci of moderate size, fre- quently arranged in pairs with adjacent sides flattened or slightly concave, resembling a pair of kidney beans side by side. In exudates, the cocci are fairly regular in size and shape and are usually inside the pus cells; in cultures, the cocci show variations in size. They are noncapsulated and Gram-negative. The cocci will not grow on plain agar, enrichment of media is needed; grow on moist chocolate agar at 37° C. in 24 hours to small, round, convex, grayish-white colonies. Growth is aerobic, favored by atmosphere of 10 percent C02. Highly susceptible to inimical agencies; when dried the cocci die in 2 hours; moist heat at 55° C. kills in 5 minutes; quickly killed by 1:4,000 silver nitrate; cultures kept at room temperature die in a few days, but at 37° C. they may survive several weeks. b. Identification.—Microscopic examination only is generally done; cultural confirmation done only under special conditions. (1) Microscopy.—Make direct spreads of the infected urethral, cer- vical, or conjunctival discharges on glass slides, fix with heat, and stain by Gram’s method. Examine the stained preparation for Gram- negative, coffee-bean shaped, intracellular or extracellular diplococci having the typical morphology of gonococci. Report whether diplo- cocci are intra- or extra-cellular, or both. Also report any other bac- terial forms present, noting for each whether Gram-negative or Gram-positive and whether coccus or bacillus; also the relative numbers and kinds of tissue cells present. (2) Ordinary culture methods.—These methods, especially in chronic urethral or cervical infections, will reveal only the secondary organisms which may occur, A special culture program is needed for growing N. gonorrhoeas. (3) Special culture program.—The cultural demonstration of the gonococcus is superior to direct spread examinations in cases of chronic gonorrhoea in both sexes and in all cases in the female, especially when material for examination is taken from the cervix. (a) Cultivation of gonococcus.—The cultivation of gonococcus, mixed with freer-growing micro-organisms, requires observance of the following special procedures: 1. Take specimens of representative material and apply directly to plate media. 198 METHODS FOR LABORATORY TECHNICIANS TM 8-227 269 2. Use a medium such as moist chocolate agar, which will readily grow the gonococcus in mixed culture. 3. Grow in 10 percent C02. If.. Identify the gonococcus-meningococcus group by colony form and oxydase reaction. S. Confirm the identification by carbohydrate fermentation tests. (6) Specimen taking and transmission (optional methods listed in order of preference). 1. Platinum loop is touched to drop of pus, to urethra, or to cleansed cervical os, and is immediately stroked broadly over a warm-culture plate at the bedside or clinic chair. 2. Sterile swab is similarly contaminated with the suspected material at the bedside or clinic chair and immediately placed in a tube containing 1 cc of nutrient broth for prompt transmission to laboratory and inoculation of warm-culture plate (broad spread of 0.1 cc of this broth). Figure 17.—Gonorrhoeal pus. (Film from urethra showing intracellular and extracellular N. gonorrhoeae.) 3. For delayed inoculation (up to 8 hours), the swab-broth tube No. 2 is stored in icebox until culture-plate inoculation is made. (o) Culture media. 1. Chocolate agar, soft, moist, and warm. 2. The media of McLeod is elsewhere described. 3. Difco “Proteose No. 3 Agar'’ and “Bacto Hemoglobin” may be combined. (d) Incubation.—At 37° C. in 10 percent C02 in closed jar, 24 to 48 hours. (e) Examination of culture.—Observation made of two features: 1. Colony form; convex, slightly opaque colonies, 1 to 3 mm in diameter, with undulated margins. Their slight opacity and characteristic undulated margins serve to differentiate them from colonies of streptococci and diphtheroids. 199 TM 8-227 269-270 MEDICAL DEPARTMENT 2. Oxydase reaction; Flood a segment of the plate with 1 cc of 1 percent aqueous solution of dimethyl paraphenylene diamine hydrochloride. (The McLeod program similarly uses 1 percent tetramethjd paraphenylene diamine hydro- chloride, giving the colonies a bright purple color, is more expensive, but has the advantages of a more rapid reaction and not killing the cocci in 30 minutes, as does the dimethyl.) Gonococcus colonies develop a pink color which on further oxidation becomes maroon and finally black. Streptoccus and diphtheroid colonies fail to undergo this color change. Caution is indicated not to be mislead by a mere darkening of the surrounding media. Spreads made and stained from the oxydase-positive colonies must verify the tinctorial and morphological properties of the micro-organisms, as this stain is not entirely specific for the Neisseria group. Medium-sized, convex and translucent colonies which give the oxydase reaction may be accepted as gonococci if they consist of Gram-negative diplococci; in cases of doubt, that is, if appearance of colonies is not entirely characteristic or when the complete identification is of special importance, subcultures are made and the fermentation reactions and ability to grow on ordinary agar are determined. (The dye does not interfere with the staining properties of the gonococcus, though it does interfere with its cultivation, if it has proceeded beyond the pink stage.) 270. Neisseria intracellularis (meningococcus).—a. Charac- teristics.—Similar to the gonococcus, but found in different locations and possessed with different invasiveness; distinguishable by sero- logical tests. Divided into five types by serological behavior, types 1 and II and less commonly types III, IV, and V; types I and III, and II and IV, respectively, are very closely related. Responsible for en- demic and epidemic cerebrospinal meningitis in man; may be found in and isolated from infected spinal fluid, blood, or nasopharyngeal secretions of patients suffering with cerebrospinal meningitis and from the nasopharyngeal secretions of carriers. Highly susceptible to inimical agents; cocci die in less than 3 hours when dried and kept at room temperature; killed by moist heat at 55° C. in less than 5 minutes; cultures die in a few days when kept at room temperature. h. Identification.— (1) Macroscopic.—Macroscopic appearance of the spinal fluid is to be noted and reported. Normal fluid is water- 200 METHODS FOR LABORATORY TECHNICIANS TM 8-337 270 clear and colorless. Meningitis fluid is more or less turbid. Color, tur- bidity, blood, and clot are to be noted. Blood, if fresh, may have come from the spinal puncture and make examination of the fluid difficult. (2) Microscopy.—An immediate presumptive diagnosis of menin- gococcic meningitis may be made by direct study of cerebrospinal fluid. {a) Stained films of suspected spinal fluid.—Centrifuge the fluid, prepare spreads of the sediment on glass slides, fix, and stain by Gram’s method. Examine for typical Gram-negative, coffee-bean shaped, in- tracellular diplococci. If present, they should be considered as meningococci and tentatively reported as such, to be confirmed by cul- ture and agglutination tests. The presence of other organisms and the relative number and kind of tissue cells are also reported. (6) Ceil counts of spinal fluid.—Make total and differential counts, comparable to the counting of blood cells. The relative number of polymorphonuclear and mononuclear leucocytes are to be noted; the former are usually enormously increased in cerebrospinal meningitis. (3) Culture of sediment of spinal fluid.—Plant several loopfuls of sediment on warm blood agar plate. Inoculate tube of warm serum dextrose broth with 1 cc. Incubate cultures at 37° C. for 18 to 24 hours and observe for the typical Gram-negative, coffee-bean shaped diplococci. Cultures are generally pure; if mixed, pure growth is to be obtained by subcultures on solid media (as for the gonococcus). Pure cultures are used for fermentation tests to rule out N. gonort‘hoeae, and for tube-agglutination tests. (4) Culture of blood.—This is not a routine procedure. The meningococcus may be recovered from the blood by routine methods, in anomalous infections with septicemia, with or without meningitis. (5) Culture of nasopharynx.—This is done for the detection of car- riers only. The nasopharynx of convalescents and of potential car- riers are touched with a sterile applicator or inoculating needle, and this inoculum is spread diffusely onto warm blood agar or chocolate agar plates. After incubation at 37° C., suspect colonies are fished to warm serum dextrose broth for confirmation of identity. (6) Agglutination tests of pare cultures.—A presumptive slide ag- glutination may hasten the procedure and cast out atypical organisms. A macroscopic tube-agglutination test with polyvalent meningococcic antiserum is used for final proof of identity. Occasionally type deter- mination will be indicated. Most of the saprophytic NeisseHa are salt or serum sensitive; to rule out nonspecific clumping, it is necessary, in all agglutination tests for meningococci, to run controls using normal horse serum (diluted 1:10) and saline. 201 TM 8-237 270-271 MEDICAL DEPARTMENT (a) Presumptive test.—Place a drop each of polyvalent antimen- ingococcic serum (1:10), normal horse serum (1:10), and sterile saline on separate areas of a slide; emulsify bacteria (portion of sus- pected colony) in each drop; observe for clumping of organisms. (b) Macroscopic test tube agglutination test.—Add 0.6-cc amounts of each serum, diluted to one half of titer shown on vial, into labeled tubes. Use separate tube for polyvalent and for each type antimenin- gococci serum (usually I and II only) to be tested. Another tube re- ceives 0.6 cc of normal horse serum (diluted 1:10) and last tube receives 0.6 cc of saline. To each tube add 0.5 cc of suspension of cocci being tested and incubate overnight at 45® to 55° C, or for 2 hours at 37° C. and overnight in icebox. (c) Result.—If the organism is a meningococcus, it should aggluti- nate in tube containing polyvalent and homologous type serum and not in other tubes. If clumping occurs in either control tube, the test is unsatisfactory. (7) Fermentation reactions.—With material from a pure culture, inoculate tubes of serum water media containing four pivotal sugars (see table XI) and incubate at 37° C. 271. Neisseria catarrhalis.—a. Characteristics.—Gram-negative diplococci. In sputum, the organisms are shaped like coffee beans and may be both intra- and extracellular; in cultures, they are generally larger and are found in pairs and tetrads. Grow freely, forming large colonies in 21 hours. They are normally found in the nose and throat ; have meager pathogenicity; and may be found, incidentally, in in- flammatory secretions especially of respiratory area. A number of closely related Neisseria, also found in respiratory area, are included on differential chart (table XI). b. Identification.—(1) Microscopy.—Make Gram-stained spreads of the infected secretions and examine for Gram-negative cocci. These organisms are larger than meningococci, may not be arranged in pairs, and may be intracellular. (2) Culture.—(a) Inoculate plain agar and incubate at 22° C.; N. catarrhalis will grow, gonococcus and meningococcus will not. (b) Pure culture is inoculated into sugar series in serum water media. (See table XI for results.) 202 TM 8-227 271-273 METHODS FOR LABORATORY TECHNICIANS Species Dextrose | Maltose j Levulose Sucrose Agar growth t O So Q Agg. with menin- gococci serum Special colony feature N. gonorrhoeae A Small, round, convex. N. intracellularis _ _ A A — — i — + Small, round, bluish gray. N. catarrhalis_ _ — — — — + + — Large, grayish white. N. sicca A A A A + + — Large, wrinkled, impossible to emulsify. N. perjlava_ _ A A A A + + — Greenish yellow, adherent to me- dium. N. jlava A A A — — — — Yellow. N. subflava A A — — ± ± Greenish yellow, adherent to me- dium. N. flavescens __ _ — — — — ? ? — Golden yellow. Table XI.—Differentiation of various species of Neisseria A indicates formation of acid. 272. Chromogenic bacteria.—a. A large number of bacteria, when cultivated upon suitable media, give rise to characteristic colors which aid in their identification. For each species the color is usually constant and dependent, to some extent, on the conditions of cultivation. Examples: (1) Staphylococcus aureus and citrous. (2) Serratia marcescens (Bacillus prodigiosus.) (3) Pseudomonas aeruginosa (Bacillus pyocyaneus) and other bac- teria producing water-soluble bluish-green or yellowish-green pigment, frequently found in soil, water, and feces. (4) Sulfur bacteria, red, purple, etc., found in decomposing organic matter. (5) Yellow to orange pigment-producing bacilli (genus Flavo- hacterium) found in water. h. Most of the chromogenic bacteria found in the dust, soil, water, cesspools, and other sites of decomposing organic matter are non- pathogenic. Many of them, being in dust, may contaminate labora- tory cultures. Most of the Gram-negative chromogenic bacilli, Flavobacteria and related chromogens, are incapable of producing disease in man or animals. The one species here described has feeble pathogenicity and is frequently associated with infected wounds. 273. Pseudomonas aeruginosa (Bacillus pyocyaneus—Ba- cillus of blue pus).—a. Characteristics.—Small, slender, motile 203 TM 8-227 273-275 MEDICAL DEPARTMENT rods; Gram-negative, non-acidfast, noncapsulated; aerobic and faculta- tive anaerobic (pigment formed only in presence of oxygen) ; grows readily on simple media at 37° and also at 25° C. Colony on agar; large, spreading colony, grayish with dark center and translucent edge, irregular, fluorescent yellowish-green color, surrounding media taking a greenish color. b. Habitat.—Widely distributed in nature, on skin and in upper respiratory tract. It is often a secondarj' invader of inflammatory areas in the wake of another incitant; occasionally is the primary cause. It may be found in the stools of children suffering from diarrhoea, in purulent discharges, and in wound infections. c. Identification.—(1) Culture is made on plain agar, incubated at 30° C. for several days. (2) Observe colony and depth of media for color change. (3) Confirm microscopic morphology of the resultant cultured growth, 274. Pasteurella (hemorrhagic septicemia group).—Small, Gram-negative rods showing bipolar staining. Litmus milk not coagulated. Aerobe, facultative anaerobe; nonmotile or motile; fre- quently pathogenic, producing characteristic hemorrhagic infections in man and animals. Includes— a. P. pestis, causing plague in man and rodents. b. P. tularensis. causing tularemia in man and rodents. c. P. avicida d. P. muricida e. P. cuniculicida associated with fowl cholera or hemorrhagic [septicemia of birds or lower animals. 275. Pasteurella pestis.—a. Habitat.—A parasite of rats and other rodents causes plague in man. Transmitted by the bite of infected rat flea, or by contact or contamination with rodent, human case, or carrier. b. Characteristics.—Short, thick bacillus; pleomorphic, especially in 3 percent salt agar; bipolar staining; grows readily on agar at 37° C. with raised, translucent, grayish-yellow, glistening, viscid growth. May live for months in bodies of dead animals. Agglu- tinated by plague antiserum. Infections by inoculation for small ani- mals; subcutaneous injection into guinea pigs provokes local edema followed by inflammatory swelling of regional lymph nodes, and a generalized infection to death in 2 to 5 days. Post mortem appear- ance: glands enlarged, surrounded by hemorrhagic exudate; small grayish, necrotic areas in liver and spleen; bacilli found in local lesions, bubo, internal organs, especially spleen, and blood. c. Collection of specimens.—(1) Pus or gland fluid from bubos, 204 METHODS FOR LABORATORY TECHNICIANS TM 8-237* •275* aspirated by syringe or collected after incision. (May be forwarded to distant laboratories on agar slants.) (2) Portions of affected tissues, removed at operation, to be for- warded in sterile bottles. (3) Blood specimens, taken during period of septicemia. (4) Autopsy materials, preferably bubo, lung, liver, and spleen. (5) Sputum, in cases of pneumonic plague. (6) Rodent: the whole rodent, shipped in fruit preserving jar, sealed. d. Microscopic examination.—(1) Stain films from suspect mate- rials by Gram’s method, and with methylene blue or dilute carbol- fuchsin (for bipolar staining). (2) The presence of typical Gram-negative, short, ovoid, polar- staining bacilli, including many degenerated and poorly stained forms, is suggestive but not conclusive evidence of P. pestis infection. e. Culture.—(1) Inoculate surface of blood agar, glycerol agar, and 3 percent NaCl agar plates. (2) Plant blood specimen into nutrient broth and incubate before plating. (3) Incubate cultures at 30° to 35° C. for 36 to 48 hours. (4) Observe growth and transfer to agar, broth, litmus milk, gelatin, tryptone broth, lead acetate medium, and sorbitol broth for further study. (See par. 2616.) /. Agglutination.—Macroscopic method is preferred, to avoid the spontaneous clumping confusing the microscopic test. (1) Make suspension of young agar culture in normal saline, using only the fine supernatant emulsion remaining after period of settling. (2) High-titer agglutinating serum (horse) is generally used. (3) Test is of greatest value in identifying suspect cultures, positive titer being interpreted in comparison with the titer of same serum tested with a known plague antigen. (4) Test is of little value as applied to patient’s serum, for agglu- tinins do not appear in patients suffering from plague until about ninth day. (5) Salt solution controls are necessary in all tests, to detect auto- agglutination. g. Animal inoculation. Caution: Animals should be freed of all ecto-parasites, prior to use, by dipping in an antiseptic solution. Then place in glass jars covered with fine mesh gauze to prevent access or escape of any para- sites. When handling animals, living or dead, protect the hands and arms by wearing rubber gloves and long-sleeved gown. 205 TM 8-227 875—277 MEDICAL DEPARTMENT (1) Inoculate guinea pigs or mice subcutaneously with small amount of the original specimen, or with a loopful of suspected cul- ture. Putrefied materials may be applied to the freshly shaven ab- domen of a guinea pig (plague bacilli penetrate the abraded skin, contaminants do not). (2) If P. pestis is present, the animals will develop characteristic lesions and die in 2 to 5 days with characteristic post mortem ap- pearance. Cultures of P. pestis may be isolated from the lesions. 276. Diagnosis of plague in rodents.—a. Post mortem appear- ance will usually evidence the natural infection in rodents. (1) Bubo, with hemorrhagic spots and areas of gray necrosis. (2) Subcutaneous and general congestion. (3) Granular liver, with punctate hemorrhage and gray-yellow spots. (4) Congested spleen. (5) Pleural effusion. b. Bacilli may be found in bubo, liver, spleen, and blood, and iso- lated from thence for study in pure culture by methods used for clinical materials. c. Shipment to a distant laboratory for examination: The entire carcass is placed, without any preservative, in a tightly sealed con- tainer which is packed in a second container to avoid breakage and escape of contents. The package must be shipped by express; fed- eral laws prohibit the shipment of plague-infected materials by mail. Decomposition may be avoided by surrounding the inner container with ice or “dry ice.” Label package “Perishable—for bacteriologi- cal examination—please expedite.” 277. Pasteurella tularensis.—a. Characteristics.—Small, Gram- negative, nonmotile rods; pleomorphic, bacillary and coccoid forms; stained best with carbol-fuchsin and crystal violet, show bipolar staining; fail to grow on ordinary media; aerobic; require specially enriched media for growth. An organism which grows on plain agar or in broth is not P. tularensis. Growth on serum-glucose- cystine agar, 2 to 5 days at 37° C.; minute, grayish-white colonies. Fairly susceptible to inimical agencies; killed by moist heat at 56° C. in 10 minutes. Agglutination tests of great value in diagnosis of disease by serum study, or in identification of cultures; agglu- tinins may persist for 20 years after recovery and a positive serum agglutination does not necessarily mean active infection. P. tula- rensis antiserum also agglutinates Bmcella antigens to about one- fourth of its titer. P. tularensis is the cause of “tularemia,” a plague- like infection of rodents, especially rabbits, and occasionally in man. 206 TM: 8-227 277 METHODS FOR LABORATORY TECHNICIANS Generally transmitted from rodents to man by infected, blood-sucking insects such as flies, ticks, lice, fleas, and bedbugs, or by direct handling of infected rabbits or squirrels. Accidental laboratory infections occur, due to its ability to invade unbroken skin. b. Microscopic examination.—Of value— (1) To study morphology of organisms. (2) To rule out M. tuberculosis by observing acidfast stain of spreads made from pathological materials. c. Culture.—(1) Piece of infected tissue, pus, fluid, or blood is planted on slants of glucose-cystine agar or blood cystine agar. Incu- bate at 37° C. for 3 to 5 days. (2) Blood agar plates also are planted to detect other organisms. (3) Observe cystine slants for characteristic colonies. If negative, continue observation for 21 days; if growth occurs, identify organism by stained spread, pure culture transplants, and macroscopic ag- glutination tests with high-titer immune serum. (4) Cultures made from blood and lesions of man are usually unsatisfactory. Cultures should be made from heart’s blood, spleen, lymph nodes, and liver of guinea pigs following inoculation with material from patient. d. Animal inoculation.— (1) Inoculate guinea pigs, rabbits, or mice with suspected materials from glands, ulcers, or blood— (a) Subcutaneously; and (b) Rubbed on the recently shaven, abraded abdomen, if other bacteria are present. (2) Result: death in 5 to 10 days (generally) with characteristic lesions: (a) At site of inoculation, hemorrhagic edema, no pus. (b) Bubos, cervical, axillary, or inguinal. (c) Glands enlarged and filled with dry, yellow, caseous material. (d) Spleen enlarged, dark. (e) Liver contains discrete, white, caseous granules. (/) Organisms can be seen in spreads and be cultured from spleen, liver, bubo, and blood. e. Agglutination reaction.—Macroscopic tube method preferred. (1) Set up agglutination tests of patient’s serum against P. tu- larensis and Brucella (abortus or melitensis) antigens. Incubate in water bath at 45° to 55° C. for 12 to 18 hours. (2) Agglutination of P. tularensis by serum in dilutions of 1 to 80 or higher is considered diagnostic of tularemia, provided there is no cross agglutination with Brucella. Agglutinins appear in the patient’s blood after the first week of the disease and usually increase rapidly. 207 TM 8-327 277-279 MEDICAL DKPAHTMENT (3) Identity of a suspect culture may be established by a similar test, using a suspension of the organisms and serial dilutions of a P. tularensis antiserum of known titer. The resultant agglutination to be significant, must be present in dilutions approaching the known titer of the serum. 278- Malleomyces mallei (glanders bacillus).—a. Character- istics.—Slender, nonmotile, noncapsulated rods, with rounded ends. Gram-negative; bipolar staining common. Litmus milk slowly co- agulated and sometimes digested. Grows readily into round, moist, grayish-white, glistening, convex colonies on agar at 37° C. in 24 hours. Ferments no carbohydrates. h. Habitat.—The cause of glanders in horses, asses, and occasion- ally man. c. Identification.—(1) Male guinea pig may be given intraperi- toneal injection of a small amount of infected material from a lesion, or a suspension of a culture. Result: Orchitis starts in 2 to 3 days; later there is tumefaction and pus formation (the “Straus reaction”). Post mortem findings in addition to the testicular lesions; subcutaneous abscesses and small miliary grayish-white nodules in liver, spleen, pancreas, and lungs; bacilli may be recovered from these lesions. (2) Complement fixation test of blood serum, using special glanders antigen. (3) Culture of aseptically removed specimens of pus from suspicious lesions, lymph gland, or other material onto 3 percent glycerol agar of pH 6.6 to 7.0 and potato medium given prolonged incubation at 37° C. Result; After several days or a week, round, whitish or yel- lowish colonies, characteristic on potato media as yellowish, semi- transparent honey-like growth becoming brownish or amber colored and tenacious, the medium becoming green or greenish brown. (4) Histopathological slide of suspect tissue lesion to show typical glanders tubercle. 279. Genus Brucella.—a. Description.—Minute rods with many coccoid cells; 0.5 by 0.5 to 2.0 microns; Gram-negative; do not show bipolar staining; all species pathogenic to man are nonmotile; do not liquefy gelatin; and fail to ferment any carbohydrates. h. Habitat.—Strict parasites, invading animal tissue, producing infection of the genital tract, the mammary gland or the lymphatic tis- sues, and the intestinal tract. Br. melitensis, Br. abortus, and Br. suis primarily infect goats, cows, and hogs, respectively, causing abortion and systemic infection; infectious to other domesticated animals; may infect man causing undulant fever (brucellosis). 208 METHODS FOR LABORATORY TECHNICIANS TM 8-227 280 280. Br. melitensls, Br. abortus, and Br. suis.—a. Descrip- tion.—Gram-negative, nonmotile coccobacilli as for genus. Br. mel- itensis and Br. suis grow aerobically; Br. abortus requires 10 percent C02 for initial isolation and early culture transplants. Growth on all media is slow; grows best on liver infusion agar with pH 6.6; 48- hour colonies on plate are small, circular, convex, amorphous, smooth, glistening, and entire. Agar cultures turn media brownish after 7 days. The three species are very closely related; may be separated with difficulty on basis of— (1) C02 requirement for growth. (2) Growth on media containing certain dyes. (3) H2S production. (4) Agglutinin absorption tests. b. Habitat.—Found in blood, urine, feces, exudates, and occasion- ally sputum and nasal drainage of human cases; also in milk, cheese, and other dairy products from unpasteurized milk from infected animals. c. Laboratory exam,motion of clinical material.— (1) Mi&roscopio.— Indistinguishable morphologically. However, Gram-stained smears from pathological lesions should be examined for the small Gram- negative rods described above. (2) Cultural.—While the organisms may be found in the blood early in the disease and during the febrile periods and in urine and milk specimens at irregular intervals, the percentage of positive cul- tures, even from proven cases is low. (a) Obtain specimen consisting of 10 to 12 cc of blood or 50 cc of urine or milk. Other body foci such as contents of ovarian cyst, synovial fluid, or excised glands may also be subjected to cultural study. (b) Inoculate two flasks containing 100 cc of veal infusion broth pH 6.6 with 6 cc of blood, several loopfuls of sediment from cath- eterized urine specimen, or several loopfuls of sediment and of cream layer from milk. Also streak specimen on two infusion agar plates. (c) Incubate one set of media in incubator at 37° C. for growth of Br. melitensis and Br. suis/ place other set of media in jar containing 10 percent C02 and incubate at 37° C. for Br. abortus. (d) Examine plates and Gram-stained films from broth after 24 to 48 hours and at frequent intervals thereafter for growth. Streak new plates from broth at least once per week, even if no evidence of growth is discernible. Observe cultures for at least 4 weeks before reporting as negative. 406303°—41 14 209 TM 8-227 280-281 MEDICAL DEPARTMENT (e) Identify any positive culture as belonging to this group by agglutination with antisera prepared against either Br. abortus, Br. melitensis, or Br. suis. Note.—Although not usually required, the species of young cultures can be determined by agglutinin absorption tests, by tests for H2S production and by ability of the organism to grow on media containing certain dyes (basic fuchsin and thionin). TableXII.—Differential characters of the three related species of genus Brucella Species 10 percent COs re quired for primary isolation HjS forma- Growth on media con- taining tion (days) Thionin Basic fuch- sin Br. melitensis 0 ± 1 2 + + + 0 + + + + + + 0 Br abortus + + 0 Br. suis 4 + + + (3) Animal inoculations.—Br. melitensis and Br. suis, and less con- stantly Br. abortus, may be isolated from infected material by sub- cutaneous inoculatipn into guinea pigs (preferably males). After 4 weeks, kill the animal; examine Gram-stained smears from the lymph glands, spleen, and liver; and make cultures from the liver, spleen, blood, and lymph nodes. This test is seldom used because of the great danger of laboratory infection. (4) Serological.— (a) Identification of pure cultures.—There is complete cross agglutination to titer between an antigen prepared with either species and antisera prepared against any other species. However, Br. abortus and Br. suis can be differentiated from Br. melitensis, but not from each other, by agglutinin absorption tests. (5) Serum from a patient taken after the fifth day of disease will usually contain agglutinins. Set up macroscopic agglutination tests in dilution of 1/20 to 1/640 or higher against a Brucella antigen (abortus, melitensis, or suis) and against Pasteurella tularensis anti- gen, Agglutination of Bimcella antigen in dilution of 1/100 or higher is considered to be significant. Cross agglutination in serum from patients with brucellosis or tularemia is frequently present, but is less marked with the heterologous antigen. Agglutinins may per- sist for years after recovery. This is the most valuable test for diagnosing Brucella infections and is the only one routinely used. 281. Genus Hemophilus.—Minute rods, sometimes almost coc- coid, sometimes threadlike and pleomorphic; Gram-negative; not 210 METHODS FOR LABORATORY TECHNICIANS TM 8-227 281-283 acidfast; nonmotile, nonsporing, nonencapsulated. Strict parasites; do not grow on common media but require for their cultivation acces- sory substances present in the blood and fresh vegetable tissue. H. influenzae, H. ducreyi, and H. 'pertussis are the three most important species. 282. Hemophilus influenzae.—a. Characteristics.—Very small, short rod; stains faintly, best by dilute carbol-fuchsin or Giemsa stain. Grows bests on media containing hemoglobin; subcultures on plain or serum agar fail to grow. Chocolate agar plate colony, 24 hours at 37° C.: small, pinpoint, transparent, smooth, raised. Tend- ency to grow best near colony of other aerobic organism, i. e., “satel- lite” colonies. Not subject to agglutination test, and does not have conspicuous biochemical activities. h. Habitat.—Commonly found in cultures of upper respiratory tract, their significance there questionable; probably not as name implies, related to the disease influenza. Occasionally found in pathological spinal fluids. “Koch-Weeks” bacillus, formerly called H. conjwnc- tivitidis, found in eye fluids in acute infectious conjunctivitis (“pink eye”) is now classified as H. influenzae. c. Identification.— (1) Koch-Weeks hacillus.—(a) Make slide spread from conjunctiva and stain by Gram’s method and with dilute carbol- fuchsin. (6) Observe for minute Gram-negative bacilli; often intracellular. (c) Culture is not informative except to reveal other organisms. (2) Spinal fluid, respiratory tract, and other suspect materials.— {a) Make culture on chocolate agar and incubate at 37° C. for 2 days. (h) Suspect colonies are identified by colony appearance, micro- scopic morphology of organisms, and failure of subcultures to grow on plain agar. Colony may be confused with a streptococcus colony. (c) Specific identification considers source of the specimen, hemo- lytic properties, and requirements of accessory growth factors. 283. Hemophilus pertussis.—a. Characteristics.—Like II. in- fluenzae, except bacilli, are more uniform in size, with less pleomor- phism, ferment no carbohydrates, and do not require accessory factors for growth, but cannot be distinguished on morphology alone. Col- ony on potato-glycerin-blood medium (pH 5.0) at 37° C. barely visible in 24 hours; plainly visible after 48 to 72 hours as small, grayish, raised, pearl-like growth. After several generations growth is freer, grayish, and glistening, becoming in a few days heavy, almost like the growth of typhoid bacilli; then transplants will grow on plain agar. 1). Habitat.—Constantly present in the respiratory secretions of whooping cough. 211 TM 8-237 283-285 MEDICAL DEPARTMENT c. Identification.—Cough plate method for isolating 77. 'pertussis is preferable to sputum cultures: Open Petri dish, containing potato- glycerin-blood medium, is held in front of the mouth during a cough paroxysm. The organisms, sprayed on the plate with droplets of secretion appear in colonies after 37° C. for 48 hours. Colonies are larger, more opaque and whiter than those of II. influenzae. 284. Hemophilus duplex (Morax-Axenfeld bacillus).—a. Characteristics.—Short, stumpy, moderate-size bacillus, often in diplo- form and chains. Cultivated only on media containing blood, serum, or ascitic fluid. On Loeffler’s blood slant colonies appear after 24 to 36 hours at 37° C. as small indentations which indicate a liquefac- tion of the medium. h. Habitat.—Found in eye and subacute infectious conjunctivitis. Not pathogenic for animals. c. Identification.—(1) Prepare slide spreads from conjunctival sac and stain with dilute carbol-fuchsin or Gram stain. (2) Short, stumpy bacilli in direct spreads are presumptively Morax-Axenfeld bacilli. (3) Culture on Loeffler’s blood slant or other special media may confirm. 285. Hemophilus ducreyi.—a. Characteristics.—Very small, ovoid rod, nOnrAptile, tendency to be in short chains and parallel rows; Gram-negative; tendency to be more deeply stained at the poles. In pus, the bacilli are often found within leucocytes. Difficult to cultivate; coagulated blood which has been kept for several days in sterile tubes (fresh blood will not do unless heated to 55° C. for 15 minutes) has been found to be a favorable medium. h. Habitat.—The cause of chancroid, the soft chancre, and found in the pus of ulcerating chancroidal ulcers, mixed with secondary infect ion, and in purer state in the chancroidal bubo. Not inoculable to lower animals, c. Identification.—Examination of spreads or cultures for II. ducreyi is seldom practiced because of the technical difficulties of identification, and the fact that chancroid lesions are usually dis- tinguishable as such without laboratory confirmation. (1) Direct diagnostic cultivation from chancroidal, lesion.—(a) Media: 1 cc of sterile rabbit blood (freshly drawn) is placed in each of several small tubes, allowed to clot, then heated to 55° C. for 15 minutes, and kept in ice box until used. (b) Thoroughly cleanse lesion with sterile water or salt solution. (c) Scrape material from bottom of ulcer or from beneath its edges, with a stiff platinum loop and plant in a tube of clotted blood by passing the wire around the clot. 212 METHODS FOR LABORATORY TECHNICIANS TM 8-227 285-287 (d) After 37° C. for 24 hours, the serum around the clot is stirred with the platinum loop and a spread is made and examined by Gram method. (e) Characteristic chains of Gram-negative bacilli, sometimes in pure, sometimes in mixed culture, will sufficiently identify the organism. (/) Transfer onto soft, moist blood agar of pH 7.2 may give in 48 hours pinhead size, transparent, gray colonies with a firm, finely granular consistency. (2) Culture from unruptured bubo.—Pus is withdrawn by aspira- tion with a sterile hypodermic syringe and needle. Cultured as in (1) above. 286. Gram-negative, aerobic, nonspore-forming enteric bacilli (family Enterobacteriaceae).—Gram-negative rods, widely distributed in nature. Grow aerobically. Many species are para- sitic for man, several of which cause typical disease; other species are saprophytes, or parasites on plants and animals. Grow well on ordinary culture media. All species attack certain carbohydrates forming acid, or acid and visible gas. May be motile or nomnotile. Nonspore forming. Has been divided into five tribes, only three of which (Eschericheae, Proteae and SalmoneUeae) contain species of interest in medical bacteriology. All of these bacteria are morpho- logically similar. They have many other characteristics in common, and serological as well as cultural methods may be required to defi- nitely identify a member of the group. 287. Coli-aerogenes group (tribe Eschericheae).—Motile or nomnotile rods, commonly occurring in the intestinal canal of normal animals, in the respiratory tract of man, or widely distributed in nature. All ferment dextrose and lactose with the formation of acid and visible gas. Do not liquefy gelatin except slowly by one species {Aerohacter cloacae). Separated into three genera on basis of results of methyl red test, Voges-Proskauer test, and ability to utilize citric acid as sole source of carbon. Table XIII.—Coli-aerogenes differentiation Genus and species Methyl red test Voges- Pros- kauer test Indol test Citrate utili- zation Gelatin lique- faction HiS formed Escherichia coli _ + — + — — — E. freundii__ .. _ + — ( + ) + — + Aerobacter aerogenes — + (-) + — (-) A. cloacae __ . — + — + + (-) Klebsiella'pneumoniae . _ (+) (-) — ( + ) — — Note.—Some species give variable results; (+) or (—) indicates usual reaction. 213 TM 8-237 288-290 MEDICAL DEPARTMENT 288. Escherichia coli.—a. Characteristics.—Coccoid to long rods, occurring singly, in pairs, and in long chains. Gram-negative. Motile or nonmotile. Not usually capsulated. Ferments many carbohy- drates, including dextrose and lactose, with formation of acid and gas. The large number of species formerly identified on basis of mo- tility and carbohydrate fermentation are now included within this species as varieties. b. Habitat.—Occurs in normal intestinal tract of animals; fre- quently found in soil and water as a result of fecal contamination. Sometimes acquires pathogenic power and may cause local or general infections; frequently causes infections of the genito-urinary tract; and invades the circulation in agonal stages of diseases. c. Identification.—(1) For isolating E. coli from water and sewage see section III, chapter 10. (2) For E. coli in feces, urine, etc., follow the procedure outlined under examination of feces for E. typhosa (par. 295) and identify according to the reactions in table XIII above. 289. Aerobacter aerogenes.—a. Characteristics.—Short rods with rounded ends, usually shorter and plumper than E. coli. They are aerobic, Gram-negative, nonspore-forming and frequently capsulated. Ferment many carbohydrates, including dextrose, lactose, and glycerol with formation of acid and gas. Do not liquefy gelatin. Colonies on solid media are large and very viscid. b. Habitat.—Widely distributed in nature; normally found on grains and plants; sometimes found in the intestinal canal of man and animals. It has been reported as the cause of cystitis. c. Identification.— (1) See section III, chapter 10, for method of isolating A. aero genes from water. (2) Isolate organism from feces, food, and soil by plating on eosin methylene blue agar or other media as described under E. typhosa and identify by characteristic biochemical reactions shown in table XIII. 290. Klebsiella pneumoniae.—a. Characteristics.—Short, plump, nonmotile, Gram-negative rods; aerobic, growing well on ordinary media; produces a large, mucoid colony on solid media. It has a large capsule which can be demonstrated readily in spreads from sputum, animal exudates, and other pathological material. Ferments dextrose, levulose, galactose, saccharose, and usually lactose with pro- duction of acid and gas. b. Habitat.—Common commensal in respiratory tract; occasionally found in soil, dust, and water. Associated with pneumonia and other inflammations of the respiratory tract. Occasionally found in various suppurative lesions of the body and may give rise to septicemia. 2H METHODS FOR LABORATORY TECHNICIANS TM 8-227 290-292 c. Identification.—(1) Examine stained spreads from pus, sputum, or fluid from lesions for Gram-negative encapsulated bacilli. (2) Inoculate eosin methylene blue agar plates or other media. Examine for mucoid colonies consisting of bacilli with typical mor- phology. Identify suspected colonies through cultural and biochem- ical tests. (3) Blood culture may be made by usual methods. Identify any suspect colonies as above. 291. Genus Proteus.—a. Characteristics.—(The only genus in tribe Proteae). Consists of highly pleomorphic, Gram-negative rods; filamentous and curved rods and involution forms are common. Generally actively motile. Characteristically produce ameboid col- onies on moist media and decompose proteins; gelatin is rapidly liquefied by most species. Ferment dextrose and generally sucrose, but not lactose, with formation of acid and small amount of gas. Usually Voges-Proskauer test is negative. Genus consists of eight species; type species is Proteus vulgaris. h. Habitat.—Putrefying animal and vegetable materials; found in feces, soil, and gunshot wounds. Certain Proteus strains, identified as X19, X2, and X Kingsbury, originally isolated from typhus fever cases, are used as antigens in the Weil-Felix test (see sec. V, ch. 10). One species, P. morgani, has been reported as the cause of mild enteritis. c. Identification.— (1) Most laboratories roughly identify any Gram-negative, motile bacillus that produces an ameboid colony on moist agar at 37° C. as belonging to the Proteus group and do not classify further. (2) However, one species of the genus, Proteus morgani, produces the typical ameboid colony only when grown on 1 percent agar at 21° to 28° C. Isolate pure cultures of this organism as described under E. typhosa (par. 295) and identify on basis of fermentation of dextrose and other hexoses only, with formation of acid and slight amount of gas. 292. Typhoid-dysentery and paratyphoid-enteritis groups (tribe Salmonelleae).—Motile or nonmotile, Gram-negative rods; grow aerobically; nonspore-forming; Voges-Proskauer test negative; gelatin not liquefied; and no spreading growth. Attack many carbo- hydrates with formation of acid or acid and gas. Certain species of genus Shigella and genus Eherthella attack lactose with gas forma- tion only. Tribe consists of three genera; genus Salmonella organ- isms ferment dextrose with the formation of acid and usually gas; genus Eherthella and genus Shigella organisms ferment dextrose with 215 TM 8-327 292-293 MEDICAL DEPARTMENT formation of acid, but no gas, Eherthella being motile and Shigella non motile. 293. Genus Salmonella.—a. Characteristics.—Usually motile, but nonmotile forms occur. Attack numerous carbohydrates with the formation of acid, and usually gas; lactose, saccharose, and salicin are never attacked. Do not form indol or liquefy gelatin. Differ from coli-aerogenes group in failing to ferment lactose; and from typhoid-dysentery group in forming gas from dextrose. Can be separated into 37 species, several of which are pathogenic for man, causing a typhoid-like fever, food poisoning, or an acute gastro- enteritis. All species pathogenic for man .are motile. h. Important species.—(1) S. paratyphi, the cause of paratyphoid. A fever in man. Characteristic reactions: never ferments xylose, rarely able to produce H2S, and fails to utilize citrate and d-tartrate. (2) S. schottnrnelleri, the cause of paratyphoid B fever in man. Characteristic reactions: ferments xylose and usually attacks inositol; HoS formed; citrate + and tartrate usually —. (3) S. typhimurium, (formerly S. aertrycke) a natural pathogen of rodents, especially mice, and many other animals; causes food poisoning in man. Characteristic reactions: very difficult to distin- guish from S. schottmuelleri, by means of either biochemical or sero- logical reactions, most reliable tests for separating them being— {a) S. typhimimum is usually tartrate +. (b) Agglutination reactions with “H” antigens in the specific phase. (4) S. enteritidis and its varieties are widely distributed among animals; sometimes the cause of food poisoning in man. Character- istic reactions: ferments xylose, but never attacks inositol; H2S + , citrate -f, and tartrate +. (5) S. hirschfeldii, the cause of a typhoid-like fever in man, some- times referred to as paratyphoid C bacillus. Found principally in Europe. Characteristic reactions: biochemical reactions similar to those of S. enteritidis; serologically, closely related to S. choleraesuis. (6) S. choleraesuis, two varieties, causing American and European hog cholera, respectively; occasionally infect man. Characteristic reaction: fails to ferment arabinose, a carbohydrate attacked by other Salmonella. c. Identification.—(1) Isolate paratyphoid fever group from feces, urine, or blood as described under E. fyphosa (par. 295). (2) Food poisoning group. Isolate pure cultures from feces or food (see sec. I, ch. 10). (3) Identify pure cultures by means of carbohydrate ferment a- 216 METHODS FOR LABORATORY TECHNICIANS TM 8-227 293-296 tions and other biochemical tests (sec. 1) and by agglutination reactions. (4) The Salmonella group, including E. typhosa, is very complex, serologically. Each species possesses from one to three distinct anti- genic components in the body of the bacillus (ktO” antigens') and other distinct components in the flagella (“H'’ antigens), the latter occurring in many species in two alternate phases, the specific phase and the group phase, each possessing different antigens. The same antigenic components may be found in several different species in various combinations. However, most strains of the pathogenic species listed above can be definitely classified on basis of— (a) Source of specimen. (b) Biochemical reactions. (c) Series of agglutination tests. 294. Genus Eberthella.—The organisms of this genus are defined as Gram-negative, motile rods, generally occurring in the intestinal canal of man, usually in different forms of enteric inflammation. Attack dextrose and several other carbohydrates with the formation of acid, but no gas; certain nonpathogenic species may attack lac- trose, saccharose, and/or salicin with formation of acid, but no gas. E. typhosa is the only species regularly pathogenic for man. 295. Eberthella typhosa.—a. Characteristics.—Actively motile, Gram-negative rods, possessing the general features of the tribe and genus. Never attack lactose, saccharose, or salicin. The normal smooth, motile form has one somatic and one flagellar antigen, thus producing both “H” and “O" agglutinins; nonmotile variants are rare. The somatic antigens are related to those of Salmonella en- teritidis and a number of other species of Salmonella. Colonies on plain agar, after 24 hours’ incubation at 37° C., are smooth, round, domed, grayish in color, transparent to opaque, with entire edge; after cultivation on artificial media, rough-type variants may develop. See paragraph 261&(2)() Second doAj. 1. Observe lactose fermentation tubes; record presence and percent, or absence of gas formation. ii. If gas has been formed in any tubes, inoculate the following media: (a) Streak on E. M. B. plate; plant from the tube inocu- lated with smallest amoimt of original water sam- ple and showing gas formation, 273 406303°—41 is TM 8-227 869 MEDICAL DEPARTMENT .00 Brilliant green lactose bile tubes; plant from at least three (preferably all) tubes showing gas formation, including tubes inoculated with smallest portions of original water sample. 3. Place original lactose tubes and transplants in incubator at 37° C. for another 24 hours. (c) Third day. 1. Make and record 48-hour readings on original lactose tubes; if no gas has been formed in any tube, make negative re- port; if gas has been formed in tubes inoculated with a smaller portion of water sample than at 24 hours, inoculate E. M. B. plate and B. G. L, B. and proceed as indicated for procedure (h), second day. 2. ()bserve 24-hour E. M. B. plate for typical coli or aerogenes type colonies; if present, select one or more (one of each type present) well-isolated colonies and transfer to small lactose fermentation tube and plain agar slant. The pres- ence of typical colonies within 48 hours is recorded in column D, W. D., M. D. Form 95, as positive. If no typi- cal colonies are found on plate and gas has been formed in corresponding B. G. L. B. tube, a new E. M. B. plate should be streaked from that tube. Reincubate any nega- tive plates. 3. Observe B. G. L. B. tubes for gas formation; gas in any amount is recorded in column C, W. D., M. D. Form No. 95, as positive. Reincubate any negative tubes. Jf. Place newly inoculated media and other media as indicated into incubator at 37°C. for 24 hours. (d) Fourth day. 1. Observe any 48-hour cultures, E. M. B. plates for typical colonies or B. G. L. B. tubes for gas formation, and record results. If positive, proceed as indicated for procedures 2 and 3, third day; if no typical colonies are present on plate and no gas has formed in any tube of liquid con- firmatory media, report as negative. 2. Observe secondary lactose tubes for gas formation and re- cord results. Reincubate. 3. If gas has been formed in lactose tube and not otherwise, make Gram-stained film from corresponding plain agar slant culture, examine for Gram-negative, nonspore- forming bacilli, and record in column F as “C.-A.” 274 METHODS FOR LABORATORY TECHNICIANS TM 8-227 369-370 (e) Fifth day. 1. Make 48-hour reading of secondary lactose tube and record. #. If gas formation has occurred in lactose tube that was nega- tive at end of 24 hours, make Gram-stained film and examine as above. 3. Prepare report on W. D., M. D. Form No. 95, using a stand- ard remark wherever applicable. Note.—In most infected water samples, gas formation will occur within 24 hours in all cases and the test can be completed as shown in above outline within 4 days of receipt of specimen. Sometimes, as indicated in procedure I for third and fourth days, lactose fermentation is delayed; in this case pro- ceed as if reaction had occurred within 24 hours, but note that 5 or more days will be required to complete test. 370. Standard remarks for use in reporting results of bac- teriological examinations. No. 1. Condition: No gas in any lactose tube. Colony count under 200 per cc. Remark: Potable bacteriologically. No evidence of fecal con- tamination. No. 2. Condition: No gas in any lactose tubes. Colony count over 200 per cc. Remark: Potability questionable. Colony count is high (“very high” if over 1,000). No. 3. Condition: Gas due to coli-aerogenes group in one or two large lactose tubes. Colony count low. Remark: Potability questionable. Coli-aerogenes group or- ganisms present in one (or two) 10-cc samples. No. 4. Condition : Gas due to coli-aerogenes group in one or two large lactose tubes. Colony count over 200 per cc. Remark: Not potable bacteriologically. Coli-aerogenes group organisms present in one (or two) 10-cc samples. Colony count high. No. 5. Condition: Gas in one or more original lactose tubes. Failure to demonstrate presence of coli-aerogenes group (E. M. B. plate and B. G. L. B. tubes negative, no gas in secondary lactose tubes, or gas due to spore-forming bacillus). Colony count under 200 per cc. Remark: Potable bacteriologically. Gas formation not due to coli-aerogenes group. No. 6. Condition: Same as No. 5, except colony count is over 200 per cc. Remark: Not potable bacteriologically. Gas formation not due to coli-aerogenes group. Colony count is too high. 275 TM 8-227 370-373 MEDICAL DEPARTMENT No. 7. Condition: Gas formation due to coli-aerogenes in three or more large lactose tubes or in a small lactose tube. Colony count high or low. Remark: Not potable bacteriologically. Tests reveal evidence of fecal contamination. No. 8. Special remarks—to be added after use of some other remark. a. Specimen days in transit to laboratory. b. Evidence of defective packing or collection. 371. Differentiation of members of the coli (genus Esche- richia)-aerogenes (genus Aerobacter) group.—A satisfactory identification of a coli-aerogenes group organism as Escherichia coli, E. freundi, or Aerobacter aerogenes can be based upon four tests (indol, methyl red (M. R.), Yoges-Proskauer (V. P.), and sodium citrate utilization). These tests are not routinely run but are sometimes re- quested as part of a sanitary survey of a watershed. E. coli (indol-f ; M. R.+ ; Y. P. — ; and citrate—) is considered to be of fecal origin; E. freundi (indol 2: ; M. R. + ; Y. P. —, and citrate +) and A. aero- genes (indol— ; M. R.— ; V. P. + , and citrate+ ) of nonfecal origin. 372. Bacteriological examination of swimming pool water.— This is carried out by the technic described for drinking water. The standard of purity recommended is identical with the standard for a potable drinking water. Since most swimming pools contain residual chlorine, sufficient to kill bacteria in sample between time of collecting and testing, it is required that such samples be collected into sterile water-sample bottles containing approximately 0.02 to 0.05 gin of sodium thiosulfate. Section IV BACTERIOLOGICAL EXAMINATION OF DAIRY PRODUCTS Bacteriological examination of milk 373 Bacteriological examination of cream 374 Determination of number of bacteria in plain ice cream 375 Paragraph 373. Bacteriological examination of milk.—a. References.— (1) Standard Methods for the Examination of Dairy Products, sev- enth edition, 1939, published by American Public Health Association. (2) Standard Milk Ordinance and Code of the United States Pub- lic Health Service. (3) Sections XII and XIII, AH 10-310. b. Definitions.— (1) Paw milk is untreated (except for refrigera- tion) milk. (2) Pasteurized milk is milk that has been treated with limited 276 METHODS FOB LABORATORY TECHNICIANS TM 8-227 373 heat by one of several methods, in order to kill most pathogenic bacteria. (3) Certified milk is an especially pure raw or pasteurized milk, generally for infant feeding, produced under the supervision of a medical milk commission of the county or state Medical Society, based on requirements of the American Association of Medical Milk Com- missions. (a) Certified raw milk: colony count should not exceed 10,000 per cc. (b) Certified pasteurized milk: colony count of not over 10,000 per cc before, and of not more than 500 per cc after pasteurization. (4) Standard Milk Ordinance and Code classifies and defines milk as— (a) Grades A, B, C, and D raw. (b) Grades A, B, and C pasteurized. (5) Grade A pasteurized milk is the grade usually sold for drink- ing purposes; must have colony count of not over 30,000 per cc, and must be prepared from grade A (50,000 per cc), or grade B (200,000 per cc) raw milk in plants meeting strict sanitary requirements. (6) Other grades of milk are based on definite sanitary require- ments for the production, distribution, and bacterial content. The allowable colony counts for raw milks are greater than for the cor- responding grade of pasteurized milk; also, the sanitary requirements are progressively less rigid and the allowable colony counts greater for grades B, C, and D milk, respectively. c. Collection of samples.— (1) Sample selected should be repre- sentative of the lot to be tested, free of extrinsic contamination and so preserved by the use of sterile or contamination-free containers, and iced from the time of collection to time of laboratory test, to prevent bacterial growth in transit. (2) Bottle should be picked at random from distribution channel, kept on ice, and set up in laboratory within 4 hours, if possible. (a) Protect cap and lip of bottle from contamination in transmis- sion by a tight-fitting, waterproof covering. (b) Pack in ice in upright position, keeping its temperature under 45° F. until examined. (c) If sample is to be sent to a distant laboratory by mail for a direct microscopic count, add 2 drops of formalin for each 10 cc of milk, fill a sterile 120-cc glass-stoppered bottle up to the stopper, label “Formalinized,” and prepare for mail shipment in a double mailing case. (3) Bulk milk may be sampled at the plant or in distribution by collection with sterile equipment and handled as in (2) above. 277 TM 8-337 373 MEDICAL DEPARTMENT (4) A plate count at a local laboratory is preferable to a direct count at a distant laboratory. (5) All samples should be properly identified with the essential information as to name of dairy, time of collection, source and grade of milk, preservative used (if any), and the address of the person to whom the report is to be submitted. The laboratory includes these data on its report and adds the time of the start of examination and results of test. d. Standard tests.—The following methods are applicable to sam- ples of milk received under differing conditions and according to the laboratory facilities locally available: (1) Agar plate method.—This consists in counting the number of visible colonies of bacteria in a culture made of a measured amount of milk in standard nutrient agar after 48 hours’ incubation at 87° C. This gives an estimate of the number of living bacteria present in the milk and is the test routinely used in Army laboratories. (2) Direct microscopic method.—Consists of an examination of stained films of milk and cream dried on glass slides. It is used in the central Army laboratories in the making of estimates of the number of individual bacteria (living or dead) in specimens of formalinized milk from outlying stations where laboratory facilities for agar plate method are not locally available. See “Standard Methods for the Examination of Dairy Products” for technic. (3) Methylene blue reduction method.—This method, usually known as the “reductase test,” is based on the fact that color imparted to milk by a small amount of methylene blue will disappear more or less quickly from incubated milk as a result of the consumption of the dissolved oxygen by growing bacteria. The results of this test compare favorably with those obtained by other methods. Due to the small amount of equipment, space, and experience required, it is applicable to small laboratories and in isolated posts. (4) Sediment test.—This depends upon appearance of standard filter disks after passage of 1 pint of milk. It is used as an index to the cleanness of milk. It has little utility in Army laboratories. (5) Tests for specific types or groups of bacteria.—Such tests, as for coli-aerogenes group, hemolytic streptococci, tubercle bacilli, and Brucella are described in “Standard Methods for the Examination of Dairy Products.” These tests are similar to the routine laboratory examinations -for those micro-organisms and are seldom done routinely. e. Standard plate count.— (1) First day.— {a) Agitate milk sample thoroughly, preferably by pouring back and forth from one sterile container to another. 278 METHODS FOR LABORATORY TECHNICIANS TM 8-227 373 (h) Using sterile dilution bottles containing exactly 9.0 cc or 99 cc of distilled water, prepare a series of dilutions depending upon expected colony count (based on grade of milk and results of previous examinations) ; the plate to be counted should have between 30 and 300 colonies; routinely, dilutions of 1/100, 1/1,000 and 1/10,000 are prepared. (c) Mix each dilution as prepared, by shaking rapidly up and down 25 times in arc of 1 foot. Transfer 1.0 cc of each dilution to properly labeled Petri dish. (d) Add tube of standard nutrient agar (tryptone-glucose-extract- milk agar), previously melted and cooled to 42° C., to each Petri dish and mix with sample by rotating and tilting the dish carefully. Allow to cool. (e) Incubate at 37° C. for 48 hours (plus or minus 3 hours). (2) Third day.— (a) Select the plate containing between 30 and 300 colonies and count all colonies, including those of pinpoint size. The use of a Quebec colony counter is recommended. If only a fraction of the plate is counted, determine total plate count by multi- plying the average number of colonies per square centimeter by a variable factor depending upon the average inside diameter of the Petri dishes being used (90 mm, multiply by 63.5; 91 mm by 65: 92 mm by 66.5). (5) Multiply the number of colonies found by the dilution factor to find colony count per cubic centimeter of sample. (c) Report; “Standard plate count. per cubic centimeter.” /. Methylene blue reduction method.—This test is most applicable to raw milk; asceptically drawn normal milk from healthy udders seldom, if ever, reduces methylene blue in less than 10 hours. (1) Methylene blue reagent.—Use only certified methylene blue thiocyanate tablets. Prepare fresh reagent weekly by dissolving one tablet of dye in exactly 200 cc (at room temperature) of sterile or freshly boiled distilled water. (2) Technic of test.— (a) Mix sample thoroughly and transfer 10 cc to a 12 to 15 by 150 mm test tube, fitted with a rubber stopper, (b) Add 1.0 cc of the methylene blue reagent. (c) Stopper the tubes immediately, label indelibly, and place in water bath at 37° C. Invert the tube once at end of 5 minutes, after which avoid agitation that might disturb the cream layer. (d) Observe tubes at frequent intervals (15 to 30 minutes) and record the end point (disappearance of the blue color from at least four-fifths of the contents of the tube). (3) Interpretation of results.—(a) Class 1.—Excellent, not decol- orized in 8 hours. 279 TM 8-237 373-375 MEDICAL DEPARTMENT (6) Glass 2.—Good, decolorized in less than 8 hours but not less than 6 hours. {c) Class 3.—Fair, decolorized in less than 6 hours but not less than 2 hours. (d) Class d.—Poor, decolorized in less than 2 hours. g. Tests for 'presence of call-aero genes group.—This tests consists of inoculating five tubes each of various portions (10 cc, 1.0 cc, y10 cc, Y\oo cc, etc.) of the milk to be tested into brilliant green lactose bile, or formate ricinoleate broth. If gas is formed, continue as with water for definite identification. 374. Bacteriological examination of cream.—Estimations of the bacteriological content of cream samples are made by using same methods as those for milk with the following exceptions: a. Measuring sample.—Mix sample; weigh 1.0 gm asceptically into a sterile butter boat or directly into a dilution bottle. h. Dilutions used.—The allowable bacterial content of cream (50,000 to 100,000 colonies per cc) is greater than for milk. Carry dilutions one or two steps farther when making agar plate count. 375. Determination of number of bacteria in plain ice cream.—a. General.—(1) In the bacteriological examination of ice cream and of ice cream mix before it is frozen, follow the same general methods as for milk: (a) Use the agar plate method. (h) Make direct microscopic count. (c) Sometimes test for coliform group. (2) Collect samples of at least 50-cc amounts in unopened cartons or in sterile, wide-mouthed, 125-cc bottles fitted with ground glass stoppers or metal caps. In sampling bulk ice cream, remove top inch of cream with sterile spoon, discard this, and use a second sterile spoon to collect sample. Collect representative samples of ice cream mix at periodic intervals. Send to laboratory immediately for examination; if laboratory is at some distance, keep sample properly refrigerated by packing in dry ice or in water containing cracked ice. b. Standard plate method.— (1) Preparation of sample.—Melt the frozen ice cream by placing the container in a water bath at 45° C. for a period not to exceed 15 minutes. (2) Methods of making dilutions.— (a) Volumetric <—To reduce the percentage of error when using materials of high viscosity (melted ice cream, ice cream mix, condensed milk, etc.) it is necessary to use large amounts in making the first dilution. To make 1:10 dilu- tion, use 11 cc of sample to 99 cc of sterile water in dilution bottle. Most applicable to plant control work. 280 METHODS FOR LABORATORY TECHNICIANS TM 8—227 375-376 (h) Gravimetric.—This method is more accurate and should be used for most Army laboratory examinations. 1. Bring the sample to a suitable degree of consistency (about 10° C.) by allowing to stand at room temperature or by heating in water bath at 43° to 46° C.; using either method the total time required must not exceed 15 minutes. £. Obtain a butter boat or similar piece of apparatus, steril- ized in cotton-plugged test tube and a standard dilution bottle containing 99 cc of sterile distilled water and having opening of such size as readily to take the butter boat. 3. Remove the cotton plug from the test tube and slide the the butter boat forward until it projects about % inch beyond the end of test tube. Do not allow boat to touch any contaminated object. Weigh test tube and butter boat to the second decimal place. 4• Pipette 1 gram, or slightly more, of the sample into the butter boat and again weigh to the second decimal place, 6. Allow’ the butter boat and contents to slide into the dilu- tion bottle. (3) Technic of test.—Make further dilutions, if required, and con- tinue as for examination of milk. Section V RICKETTSIAE General 376 Habitat - - 377 Important species 378 Collection and transmission of specimens for examination 379 Weil-Felix reaction 3S0 Reaction in guinea pig * 381 Relationship between typhus species 382 Cultural examination 383 Microscopical examination 384 Paragraph 376. General.—a. Description.—Small pleomorphic diplobacil- lary micro-organisms; generally appear as diplobacilli in pairs but may occur singly. Their size depends upon various factors, the chief one being the source from which the infectious material is obtained. Rick- ettsiae are rarely found in the animal body except in the tunica vaginalis of infected guinea pigs which show scrotal swelling. In cultures, the various types of Rickettsiae vary greatly in size from 281 TM 8-327 376-378 MEDICAL DEPARTMENT barely visible forms up to long bacillary of filamentous forms. In tissues they stain purple with the Giemsa stain, but in exudate smears (tunica vaginalis) and in tissue cultures they stain best with the Machiavello stain, appearing as red bodies. Rickettsiae occupy an intracytoplasmic position in European and murine typhus, while in Rocky Mountain spotted fever they often appear in the nucleus. This is an important morphologic distinction between these two types. Rickettsiae grow only in the presence of living susceptible cells and in this respect resemble viruses. b. Vaccines.—Effective formalized vaccines have been prepared from tissue cultures, from infected animal tissues, and from infected insect vectors. 377. Habitat.—Rickettsiae are parasitic on insects (arthropods), animals, and man. The Rickettsiae pathogenic for man cause five clearly defined groups of diseases, many of which are primarily har- bored by rodents or other animals, being transmitted to man by infected lice, ticks, fleas, or mites. The blood, and hence all the organs of infected man or animal, is infectious, but Rickettsiae are found with extreme difficulty in preparations made from these tissues. The common locality in which to find them is in the tunica vaginalis of infected guinea pigs when scrotal swelling exists. In typhus, espe- cially the murine type, the mesothelial cells lining the tunica vaginalis are packed with them. In spotted fever the mesothelial cells in the tunica vaginalis show a sparse infection. The distinctive lesion in this disease is the presence of organisms in the smooth muscle cells as well as the endothelial cells of the arterioles and venules. a. Typhus fever group.— (1) European or epidemic typhus (R. prowazeki). (2) Murine or endemic typhus {R. mooseri). h. Rocky Mountain spotted fever {R. rickettsia) and related dis- eases.—(1) Sao Paulo exanthematic typhus. (2) Fieure Boutonneuse. (3) South African tick fever. c. Trench fever {R. quintana). d. UQ” fever (R. burneti). e. Tsutsugamushi fever group {R. nipponica).—Tsutsugamushi fever in Japan. 378. Important species.—a. R. prowazeki causes European or epidemic typhus fever, transmitted from man to man by the body louse (Pediculus humanus var. corporis). The micro-organisms oc- cur in the cells lining the alimentary tract of the infected louse and 282 METHODS FOR LABORATORY TECHNICIANS TM 8-227 378-379 so the disease is transmitted to man via the louse excreta. In from 5 to 7 days after the louse has fed on the blood of an infected man, the excreta of the louse becomes infective and may remain so for 5 days; the louse then dies and therefore is not a reservoir of the disease between epidemics. In man the most characteristic lesion resulting from this infection is in the vascular system, mainly in the skin and brain. This is a filth or war disease which was re- sponsible for about five million deaths in the period of the World War. “Tabardillo” or Mexican typhus belongs to this group. b. R. mooseri is the cause of endemic or murine typhus. It is normally a pathogen of rats and other rodents and is transmitted from rat to rat and from rat to man by rat fleas (X. cheopis and G. fasciatus). During epidemic periods it may be also transmitted from man to man by the body louse. In fleas the Rickettsiae may exist without causing their deaths. c. R. rickettsia is the agent of Rocky Mountain spotted fever. This disease in the western part of the United States is transmitted by the tick (Dermacentor andersoni), while in the eastern part of the United States, it is transmitted by a different tick (D. variables). There is a great variation in the severity of different strains of this disease. The Bitter-root Valley strain is very pathogenic for man and guinea pigs; outside of this strain the disease is equally severe in the East as in the West. The Rickettsiae proliferate in the tick without harm to it and are transmitted hereditarily to its descendants. In the tick, intranuclear forms are frequently found. Infected ticks may transmit the disease to man, monkeys, guinea pigs, rabbits, dogs, ground squir- rels, and other rodents. d. R. nipponioa is transmitted by the bite of larval form only of a mite (genus Trombicula). e. This disease has occurred in Australia and a few accidental cases have occurred in the United States as laboratory infections. The organism has been isolated from infected ticks in Montana. f. R. quintana causes trench fever. This is transmitted by the louse. Rickettsiae are found in the lumen of the bowel 5 days after the louse has fed on the blood of a patient; its excreta becomes infective and may remain so for at least 4 months. Infection occurs as the result of rubbing the excreta into the skin. This was a common disease in the trench life of the World War. 379. Collection and transmission of specimens for examina- tion.—a. Clear, sterile serum of patient for Weil-Felix agglutination test. 283 TM 8-227 379-381 MEDICAL DEPARTMENT b. Sterile defibrinated or citrated blood for animal inoculation. This material should be inoculated intraperitoneally into guinea pigs as soon as possible. c. Autopsy (man) : Fresh portions of sterile brain or spleen for animal inoculation; tissue fixed in formalin or Zenkers for histopath- ological examination. d. From guinea pigs; Slide preparations from scrapings of tunica vaginalis. European typhus is transmitted from guinea pig to guinea pig by intraperitoneal inoculation of an emulsion of infected brain. Murine typhus is transmitted to guinea pigs by an intraperitoneal inoculation of ground-up tunica vaginalis. Rocky Mountain spotted fever is transmitted from guinea pig to guinea pig by an intraperi- toneal inoculation of heart blood. 380. Weil-Felix reaction.—a. General.—(1) This is a macroscopic tube agglutination test used in diagnosing Rickettsial diseases. The antigen employed is a Proteus X strain, originally isolated from the urine of typhus patients. This organism bears no relationship with the Rickettsiae which cause the disease (though a common polysac- charide between Proteus X and Rickettsiae is said to exist). The test is done 2 hours at 56° C. and overnight in ice box. Only the non- motile or “O” variant, living or heat killed, is used. While the OX18 strain is used for typhus (both types) and Rocky Mountain spotted fever, the Kingsbury strain or OXK is used for tsutsugamushi fever. Agglutinins for Proteus X appear about the fourth day of the disease and gradually increase in titer from then on, to disappear soon after convalescence. The increase in agglutination titer is of significance. (2) The OXX9 agglutination test alone cannot be used in distin- guishing the two types of typhus fever from Rocky Mountain spotted fever. A positive agglutination titer of 1; 100 is significant and an increase in titer during the course of the disease is of first importance. In typhus a titer of 1:1,000 is frequent and even 1; 100,000 may rarely be attained. In Rocky Mountain spotted fever a titer of 1:10,000 has been found. h. Results. European or murine typhus OX19 +. Rocky Mountain spotted fever OXX9 +. Tsutsugamushi fever OXK + . 381. Reaction in guinea pig.—a. Transmission.—Transmission of an unknown infection to the male guinea pig is the most important single process for establishing the diagnosis, for it may induce fever or scrotal reactions or enable one to apply cross-immunity tests with 2S4 METHODS FOR LABORATORY TECHNICIANS TM 8-327 381-3852 known strains of typhus and spotted fever after the guinea pig has reacted. Human blood is commonly infective for guinea pigs only during the first 4 to 6 days of fever. The best method for establishing a strain is to inject large amounts (6 cc) of blood into guinea pigs by the intraperitoneal route. In transferring epidemic typhus from one animal to another, the guinea pig is sacrificed on the third day of fever and a brain suspension is inoculated intraperitoneally. For murine typhus, the ground-up tunica vaginalis is used. In Rocky Mountain spotted fever 1 cc of guinea pig blood is usually sufficient, but in some milder strains 4 cc may be necessary. The diagnosis of the disease in the guinea pig is based on a febrile reaction, presence and type or absence of scrotal lesions, presence of Rickettsiae in tunica vaginalis, the transfer of the disease from animal to animal, the de- velopment of a specific immunity, the presence of brain lesions, and the sterility of the blood and brain on ordinary culture media. b. Temperature.—The temperature of the guinea pig may be re- garded as normal up to 103.8 or 104; above this, abnormal. In the original guinea pig inoculation the temperature may declare itself at any time between 4 and 24 days. Once the disease has “taken,” the course in subsequent transfers remains more or less constant, provided the transfers are made at the same time in the same manner. In murine typhus the incubation period is from 3 to 4 days with slight fever and scrotal swelling. In epidemic typhus the period of incubation is from 8 to 10 days with fever lasting about 6 days. In Rocky Mountain spotted fever the period of incubation is about 6 days and fever is of about 5 days’ duration. c. Scrotal reaction.—This reaction is more frequently found in large male guinea pigs rather than in small ones. The typical reaction is characterized by swelling and redness. The testes are firm and cannot be pushed back into the peritoneal cavity. Scrapings of the tunica vaginalis from guinea pigs infected with murine typhus show many cells filled with Rickettsiae. These infected cells are called “Mooser bodies.” While scrotal swelling may occur in epidemic typhus, it is not the rule. Here only a few Rickettsiae are found. Scrotal swelling only occurs with a virulent strain of Rocky Mountain spotted fever. Here fewer Rickettsiae are found and intranuclear forms may be present. With the milder strains no scrotal swelling is present. 382. Relationship between typhus species.—a. Similarities.—• (1) Guinea pigs and monkeys recovered from one type of typhus are immune to the other. (2) Weil-Felix reaction with OX19 is present in both. 285 TM 8-227 382-384 MEDICAL DEPARTMENT (3) Brain lesions in experimental animals are indistinguishable. (4) Clinical course in man is somewhat similar in onset, febrile reaction, rash, and duration (though different in severity and sea- sonal incidence). b. Differences.—(1) Murine typhus produces a more rapid rise of temperature in guinea pig and a characteristic scrotal swelling. Scrapings from the tunica vaginalis show large, swollen endothelial cells, filled with Rickettsiae (Mooser bodies). European strain does not give obvious scrotal swelling in guinea pigs. (2) Murine typhus causes a febrile disease in the rat, with Rickett- siae in the scrotal sac; European strain causes inapparent infection in this animal. (3) Murine Rickettsiae injected into X-rayed rats will result in heavy diffuse infection; European Rickettsiae will not. (4) European typhus is more severe in man and occurs in winter; murine typhus tends to occur in summer. 383. Cultural examination.—This procedure is not routinely employed. The Rickettsiae will not grow on ordinary culture media. The various types of Rickettsiae have been grown by the following methods: a. By classical tissue culture methods. b. Inoculation into the yolk sac of fertile chick eggs made on the sixth day of incubation (Cox). c. Serum Tyrode agar medium on which susceptible tissue cells are placed on the surface of the medium (Zinsser, Plotz, and Enders). d. In insect (arthropod) louse vector (Weigl). 384. Microscopical examination.—Not applicable to the direct diagnosis but only to tissue, animal exudate, and culture materials. a. Giemsa stain.—Tissue specimens, fixed for y2 hour in alcohol, Zenker’s solution, or Regaud’s solution, are sectioned and stained for 10 to 24 hours with diluted (1: 50) Giemsa solution. Result: Rickett- siae appear as purple bodies. b. MachiaveUo staining method.—Tissue culture or exudate smears are stained by the following procedure: Stain with basic fuchsin (0.25 percent in distilled water) for 4 minutes; wash in water, then very rapidly with 0.5 percent citric acid solution, then wash rapidly with tap water; stain with methylene blue (1 percent aqueous solution) for 10 seconds; wash and dry. Result: Rickettsiae stain red, cells stain blue. 286 TM 8-227 384 METHODS FOR LABORATORY TECHNICIANS Table XV.—Interpretation of laboratory tests for rickettsial diseases Weil-Felix reaction Results'of guinea pig inoculation Disease OX18 OXK Scrotal swellings Brain lesions Immunity European typhus_ + + + — Rare; few Rickettsiae in tunica vaginalis exudate, only in- tracytoplasmic localization. + + Immune to itself and murine typhus. Murine typhus. _ + + + Usual; many Rick- ettsiae in tunica vaginalis (Mooser bodies). Only in- tracy toplasmic localization. + + Immune to itself and Pluropean typhus. Rocky Mountain. + + + Only with virulent strains (Bitter-root Valley strain). Few Rickettsiae in tunica vaginalis. May find intra- nuclear forms. + + Immune to itself but not to Eu- ropean or mu- rine typhus. “Q” fever. _ None _ _ Immune to itself only. Trench fever ? ? None. ... Immune to itself only. Tsutsugamushi fever. + + + None _ __ Immune to itself only. Section VI FILTERABLE VIRUSES Habitat 385 Characteristics 386 Classification 387 Collection of specimens 388 Preparation of virus suspensions 389 Filtration 390 Animal inoculations 391 Inclusion bodies 392 Serological tests 393 Cultivation of viruses 394 Babies 395 Paragraph 287 TM 8-227 385-387 MEDICAL DEPARTMENT 385. Habitat.—These are strict parasites, responsible for various pathological conditions in man, animals, birds, fish, insects, plants, and bacteria. They are the causative agents of many communica- ble diseases in man. These diseases are transmitted by contact with a diseased individual or carrier, by infected insects, or by the bite of an infected animal (e. g., rabies by bite of dog). The viruses live and multiply within the living cells of the host and usually infect a particular type of cell or cells from a limited group of tissues. 386. Characteristics.—Uncertainty exists as to the exact nature of viruses; whether living matter, as usually considered, or inanimate substance. The following characteristics are common to most viruses and are used to describe and identify them: «. Size is from 8 to 275 millimicrons (ni/x,). Larger ones can be stained and seen with the ordinary microscope (elementary bodies of vaccinia and psittacosis). By use of ultraviolet light, photographs have been made of the smaller ones. b. They are usually filterable. A large amount of virus is usually lost during filtration. c. The}7 cannot be cultivated in the absence of living, susceptible cells. d. Characteristic “inclusion bodies” develop in the cells of the host, infected with many virus diseases. e. They are resistant to many bactericidal agents. /. Many viruses have ability to produce protective immunity with specific viricidal and other antibodies. g. Ability to reproduce rapidly in the tissues of a susceptible host. k. Ability to adapt themselves to different hosts by repeated passage. 387. Classification.—Due to small size and parasitism for living cells it is impossible to classify viruses by their morphological or cultural characteristics. Viruses may be grouped by the character- istic affinity they have for certain tissues: a. Those having an affinity for skin are variola, vaccinia, animal pox diseases, herpes, varicella, lymphogranuloma inguinale, foot and mouth disease. 1). Those having an affinity for the nervous system are encephalitis lethargica, St. Louis encephalitis, encephalitis of horses, cattle, and sheep, poliomyelitis, lymphocytic chorio meningitis, and rabies. c. Those characterized by catarrhal or generalized infection are measles, mumps, psittacosis, yellow fever, dengue fever, distemper, swine fever or hog cholera, rinderpest, infectious anaemia of horses, fowl plague. d. Those characterized by tumor formation are fowl leukemia, Rous sarcoma of chickens, warts, infectious myxomatosis of rabbits. 288 METHODS FOR LABORATORY TECHNICIANS TM 8-227 388-390 388. Collection of specimens.—While some viruses are re- sistant, it should be borne in mind that many are fragile. Rabies and poliomyelitis virus may be conserved in 50 percent buffered glycerin, but other viruses should be kept in an ice box containing dry ice, or they may be frozen and dried. The frozen viruses in a liquid state are thawed at 37° rather than at room temperature. a. Infected tissue specimens collected for laboratory examinations, vary according to the disease suspected. Specimens may include— (1) Fresh tissue, or tissue fixed in 10 percent formalin, for micro- scopical examination for inclusion bodies. (2) Pieces of tissue are conserved in 50 percent buffered glycerin, made as follows: Stock solution A (Na2HP04) +28.4 grams per 1,000. Stock solution B (citric acid)+21 grams per 1,000. Take of solution A, 93.65 cc. Take of solution B, 6.35 cc. This makes 100 cc of buffer pPI 7.6. Then add 100 cc of glycerin. Divide in specimen jars and sterilize in autoclave. b. Liquid material (blood, urine, spinal fluid) is conserved by plac- ing material in pyrex Wasserman tubes, stoppering tightly with a cork stopper and covering cork with adhesive tape. This tube is then plunged in dry ice. Liquid virus material may also be kept by freezing and drying. c. Clear, sterile serum from patient or recovered animal may be tested for specific antibodies. 389. Preparation of virus suspensions.—Preparation of virus suspensions for animal inoculation or for titration: Tissues are ground up in a mortar with sand or alundum. Tyrode solution, Lockes solution, or broth may be used as a diluting fluid. A 10 percent sus- pension is often used but this depends upon the virus used. Centrifu- galize at 1,200 rpm to bring down larger particles, and inject super- natent liquid. If tissue has been left in glycerin, wash several times in diluting fluid before grinding. 390. Filtration.—Virus suspensions may be filtered to remove bac- teria or to determine the filterability of the virus. Of the many types of filters available, the Handler and Berkefeld diatomaceous earth filters are the most suitable for these purposes. These candles come in varying sizes and varying grades of porosity, “V” or coarse, “N” or medium, and “W” or fine for Berkefeld candles and numbers indicating pounds test for Handler’s. Even the largest viruses and many bacteria will pass through the “V” filter, most viruses through an “N,” and only the smallest viruses through the “W” candle. Asbestos 406303°—41 19 289 TM 8-237 390-391 MEDICAL DEPARTMENT filters of the Seitz type also are excellent; the “E.K.” filter pad corre- sponds closely in porosity to Mandler “W,” is used once, and then discarded. (Since these disks are often alkaline, they should be washed in distilled water and dried before use.) a. Filtering.— (1) Set up filter in filtering flask and sterilize in autoclave. (2) Attach side arm of flask to suction pump in series with manom- eter and trap. (3) Place virus suspension in glass mantle surrounding candle and draw into filter flask using minimum amount of vacuum. h. deeming of filters.—(1) If infectious material has been filtered, sterilize by covering filter as set up with 5 percent phenol or cresol, and allow to stand for a minimum of 1 hour. Rinse off with tap water. (2) There are several methods for cleaning filters, some of which are destructive, others tedious and time-consuming. One of the best em- ploys 1 percent KMn04 (potassium permanganate) which is drawn through the filter as set up (50-75 cc for a 1- by %-inch candle). Subsequently, a 5 percent solution of sodium bisulfite (NaHS03) is drawn through until all color has disappeared from the candle. Dis- tilled water is then drawn through until test of filtrate with BaCl2 (barium chloride) shows no precipitate. Filter should then be dried in incubator overnight before using. (3) Filters should be handled by the metal parts and should be carefully protected from contact with grease or oil since such sub- stances change the porosity and allow the passage through the filter of objects otherwise retained. (4) Alternative method: The simplest and easiest method involves the use of Berkchlor and is recommended when this product is avail- able. A 0.5 percent solution of Berkchlor is passed through the filter (50-100 cc for a 1- by %-inch candle). This is followed by distilled water until test with AgNOs (silver nitrate) shows no trace of chlorine (a white precipitate). In some instances candles may be boiled 15 to 20 minutes in Berkchlor, followed by distilled water as above. Filters should be subjected to brushing only when no other means for freeing them from clogging is successful. 391. Animal inoculations.—Test for the presence of specific virus by inoculating bacteria-free virus-containing material into sus- ceptible animals. If the type virus present is not known, inoculate different animal species with varying dosages and by several different routes. Due to the selectivity of many viruses for certain tissues in specific hosts, it is often necessary to pass a virus in series through several animals before it becomes sufficiently adapted to produce 290 METHODS FOR LABORATORY TECHNICIANS TM 8-227 391-392 recognizable symptoms. The virus specimens most frequently inocu- lated into animals, animal used, and type reaction to be noted are: a. Insect-home diseases (yellow fever and dengue fever).—Blood during first 3 days of fever or, in case of yellow fever, suspensions of liver and spleen may be injected subcutaneously or intraperitoneally into monkey; results in mild infection in monkey after 4 to 10 days. h. Neurotropic diseases.—Suspensions of brain or cord from the central nervous system are used: (1) Poliomyelitis (infantile paralysis).—Intracerebral or intra- nasal inoculation of suspension of spinal cord into monkey results in typical disease. (2) Encephalitis (lethargica and St. Louis types of sleeping sick- ness).—Intracerebral inoculation into monkeys and rabbits or mice results in encephalitis in 5 to 14 days. (3) Rabies.—Intracerebral inoculation of brain suspension or saliva from infected animal into rabbit, mouse, or guinea pig results in rabies within 21 days. c. Dermotropic diseases.—(1) Smallpox.—Inoculation of pus from pox lesion into monkey by scarification of skin produces local lesion. (2) Fever blisters (Herpes febrilis).—Inoculation of vesicular fluid on scarified cornea of rabbit causes a severe keratitis, or subdural inoculation of rabbit produces encephalitis, fatal in 4 to 6 days. d. Respiratory diseases.—(1) Influenza.—Filtrate of nasal secretions or scrapings, inoculated intranasally into ferret may result in pneu- monia. After ferret ‘“take,” anesthetized mice may be used. (2) Psittacosis (parrot fever).—Blood or filtered sputum may be injected into peritoneum of mouse, guinea pig, or parrot, resulting in typical disease after 7 to 10 days with focal necrosis in liver, the cyto- plasm of cells containing tiny coccoid inclusion bodies. e. Lymphogranuloma inguinale.—Pus from bubo injected intra- cerebrally into mouse or guinea pig results in encephalitis in 2 to 8 days. Note.—Bacteria-free pus from bubo, diluted 7 to 10 times with saline, or saline suspension of mouse brain after several serial passages, when killed by heat at 60° C. for 90 minutes on first day and for 60 minutes on second day, may be used as a Frei antigen after testing on immunes and non-immunes for potency and specificity. Recently elementary bodies from yolk-sac cultures have been used. 392. Inclusion bodies.—Small structures, the exact nature of which has not been determined, found within the cytoplasm or nucleus of infected cells during many virus diseases, are known as “inclusion bodies.” Smears or tissue sections may be stained with Giemsa’s, 291 TM, 8-227 392-395 MEDICAL DEPARTMENT Mann’s or Seller’s stain and examined microscopically. The demon- stration of inclusion bodies is of great diagnostic value based upon their selective affinity for certain cells, their position within the cell, and their acidophilic or basophilic staining qualities. For example, the diagnosis of rabies is based upon the demonstration of inclusion bodies (see par. 395). 393. Serological tests.—Most virus diseases produce a strong and lasting immunity in their host. The antibodies present in the blood serum are type specific and may be demonstrated by agglutination, precipitin, complement fixation, and neutralization tests. 394. Cultivation of viruses,—They will not grow on ordinary culture media. Any method which permits cells to remain alive or multiply will support virus growth provided living susceptible cells are present. Viruses may be grown “in vitro” in a modified Mait- land’s medium consisting of Tyrode’s solution, with or without addi- tion of fresh serum, and minced susceptible tissue; in proliferating cells in a plasma clot; or on serum-Tyrode-agar medium where cells are placed on the surface of the semisolid medium; or grown “in vivo” on the chorio-allantoic membrane or other tissues of a developing chick embryo. 395. Rabies.—Eabies is the only virus disease usually diagnosed upon the basis of laboratory findings, that is, the demonstration of inclusion bodies, the so-called “Negri bodies,” in smears or sections from the central nervous system of the infected animal. a. Characteristics.—Eabies is an infectious disease, affecting the central nervous system. It is most common in dogs but occurs in man and other animals. Transmission is by bite of infected animal (dog), whereby virus containing saliva is inoculated. The incubation period in dogs is from 2 to 8 weeks, sometimes longer; in man, usually about 40 to 60 days. The mortality rate of rabid animals is 100 percent, death occurring within 5 days. h. Collection and transmission of specimens for examination.—If a living animal is received, do not kill, but place in quarantine for 2 weeks or until death. If a dog is killed during early stages of disease, “Negri bodies” may not be found. The entire body of a suspected rabid dog that has died or been killed should be sent to the laboratory, if nearby; or send head and several inches of neck packed in ice. If the specimen is to be sent through the mail to a distant laboratory, remove brain and divide longitudinally into two equal parts; place one-half of brain in 10 percent formalin for sectioning; place other half in 50 percent buffered glycerol for use in making touch prepara- tions and for animal inoculations. For local examination, use fresh METHODS FOR LABORATORY TECHNICIANS TM 8-337 395 brain, selecting portions of the hippocampus major (Ammon’s horn), fissure of Rolando, cerebral cortex, or cerebellum. c. Examination for Negri bodies.—(1) Make smear of gray matter of brain on slide, or fix small piece of brain (Ammon’s horn), with cut surface up, on end of cork stopper and make touch preparations by gently touching three or four times with clean slide. (2) Fix for 2 minutes with methyl alcohol. (3) Seller’s stain.—(a) Reagents. 1. Methylene blue 15 gm Methyl alcohol 100 cc 2. Basic fuchsin 32 gm Methyl alcohol 100 cc Just before use, mix three parts of solution 1 with one part of solu- tion 2 and five parts of methyl alcohol. The chromatin should stain blue and cytoplasm red. If a clear-cut differentiation of cytoplasm and chromatin is not obtained, additional amounts of either solution 1 or 2 is added until the desired effect is obtained. The stock solutions should be stored in the refrigerator, (h) Technic for staining.—“Touch” preparations are flooded with the mixed stain for approximately 10 seconds, washed in water, dried and examined. The methyl alcohol serves as a fixative and no addi- tional fixation is necessary. (4) Mann’s stain.—(a) Reagents.—Keep as stock solutions— 1. Methyl blue (China blue), 1 gm, dissolved in 100 cc of distilled water. 2. Eosin, W. S., 1 gm, dissolved in 100 cc of distilled water. Prepare fresh stain for use daily. Add 3.5 cc of methyl blue solu- tion to 10 cc of distilled water, and then add approximately 3.5 cc of eosin solution gradually until correct amount is present (drop of stain on filter paper gives blue center surrounded by pink ring). Filter and use. (b) Technic for staining. 1. Flood slide with stain for 5 minutes. 2. Rinse with distilled water. 3. Allow to air-dry or dehydrate and mount in balsam. h. Examine microscopically for deep-pink stained round or oval Negri bodies from 10 to 20 microns in diameter. They are usually found within cytoplasm of blue-stained nerve cells, but may be extracellular. Red blood cells are larger and take an orange-red color. 5. Report—“Microscopic examination demonstrated (failed to demonstrate) Negri bodies.” 293 TM 8-327 395-397 MEDICAL DEPARTMENT (5) Sections of brain fixed in formalin or Zenker’s fluid may be made, carried through xylol and alcohols to water, stained with Mann’s stain, dehydrated, mounted in balsam, and examined as above. d. Animal inoculations.—If touch preparations are negative and persons or valuable animals have been bitten, an animal should be inoculated and the final diagnosis of rabies based upon the demon- stration or failure to demonstrate Negri bodies. (1) Rabbit.—Make subdural inoculation of 0.15 cc of 10 percent suspension in H20 of portion of hippocampus. Incubation time is IT days or more. (2) Young white /Swiss mice.—They are the preferred animal for use. Make intracerebral inoculation of 0.03 cc of 10 percent suspen- sion as above. Incubation time is 9 to 11 days. (3) Guinea gigs.—They are not very satisfactory; do not use if mice or rabbits are available. Section VII FUNGI General __ 396 Habitat 397 Descriptive terms 398 Materials for examination 399 Methods of examination 400 Paragraph 396. General.—The fungi are complex plant organisms, devoid of chlorophyll. The single-cell types, as the common budding yeast {Saccharomyces cerevisiae), grow and multiply much as do bacteria, except as to their method of multiplication (by budding, not by fission). Each individual cell combines the functions of nutrition and reproduction. Other fungi, the molds, are made up of many cells, usually cylindrical (hyphae), joined into filaments (mycelia) from which spores (small round cells) develop, the structure built up by the filaments and spores being characteristic for each species. 397. Habitat.—Fungi occur widespread in nature and to a less extent in disease. Saprophytic fungi obtain their food from dead plants or decaying materials. Parasitic fungi obtain their food from living animal or plant life. A few species of fungi are pathogenic and capable of producing minor or major skin infections (dermato- mycosis), hair infections (trichophytosis), bronchial infections (bronchomycosis). and certain specific generalized infections (blasto- mycosis, actinomycosis, and others). Some fungi have commercial 294 METHODS FOR LABORATORY TECHNICIANS TM 8-227 397-398 / Ch/amudospore Septot e Hcjpha Condi a. Cwvd,ophorm Fuseaui Spirals Pectinate body ■ Ascus Spores Blostospore •5pons Arthroepore Sprout my Spores Ascospore SporoCricHum Vesic/e Storiymo. ■ Stenjma Mucor Aspergillus Aspergillus Peniclllium Figure 19.—Characteristic morphology of typical fungi importance, such as those that give flavor to cheese and cause bread to rise (yeast). Many fungi attain laboratory attention because of their ubiquitousness in dust and their contamination of laboratory media. 398. Descriptive terms.—Fungi occur in many forms, often vari- able within species under different conditions of growth. Those that are pathogenic tend to grow differently in the tissues than on culture media. Their classification is too complex to be given here. A few) terms used in describing and classifying fungi are given below. 295 TM 8-227 398 ME DICAL DEPARTMENT a. Budding fungus.—Yeast-like fungus that grows by budding. In the tissues and in culture they appear as round or oval budding cells and may, but generally do not, develop rudimentary mycelia. b. Filamentous fungi.—Mold-like fungi that develop long filamen- tous threads with or without apparent spore formation. c. Hypha.—The single thread-like portion. d. Mycelium.—A group or matted mass of branching hyphae. e. Septa.—Divisions of hypha formed by transverse partitions. f. Spores.—Cells developed for the propagation or preservation of the species. g. Thallus.—The actively growing, vegetative organism as distin- guished from spores. h. Ascospores.—Group of spores, four or eight, enclosed in a sac or ascus. i. Endospore.—A spore formed within an outer envelope. j. Conidiophore.—Hypha bearing a spore or group of spores. k. Blastospore.—A spore formed by budding. l. Arthrospore.—A spore formed of segments of a hypha and re- leased by disarticulation. m. C Flamy do spore.—A large spore with tough and frequently double-contoured wall, undergoing encystment. n. Sterigma.—A short stalk bearing chains of conidia (as in As- pergillus). o. Sporangium.—A sac containing an indefinite number of spores at the end of a hypha (as in Mucor). p. Fuseaux.—Fusiform septate spores, produced by certain fungi (Trichophyton). q. Spirals.—Terminal coils seen in some species. r. Pectinate bodies.—Comb-like structures formed by some fungi. s. Cryptococous.—A genus of budding fungi devoid of ascospores and mycelia (e. g., Cryptococcus gilchristi, the causative organism of one form of blastomycosis). t. Saccha.romyces.—A genus of budding fungi having ascospores but no mycelia (e. g., Saccharomyces cerevisiae, brewers yeast, oval or spherical cells, cause alcoholic fermentation). u. Monilia.—A genus of budding fungi having no ascospores, mycelia of rudimentary type, and capable of fermenting certain sugars with the production of acid and gas (e. g., Monilia, psilosis, formerly thought to cause sprue). v. Endomyces.—A genus of budding fungi, having ascospores and segmented mycelia (e. g., Endomyces albicans, found in thrush). w. Madurella.—A genus of filamentous fungi characterized by sep- 296 TM 8-327 398 METHODS FOR LABORATORY TECHNICIANS tate, branching hyphae, and chlamydospores. They are contained in black granules of infected tissue (e. g., “Madura foot”; Madurella mycetomi in “Madura foot”). x. Nocardia {Actinomyces).—A genus of filamentous fungi charac- terized very fine, nonsegmented mycelial filaments and no spores (e. g., Nocardia bo vis, the “ray fungus” of “lumpy jaw” of cattle, actinomycosis of man). y. Sporotrichum.—A genus of filamentous fungi which appear in the tissues as oval spores and develop in cultures as mycelium with characteristic grouped spores or conidi a (e. g., Sporotrichum schenki, the causative agent of sporotrichosis, appearing in fresh spreads of the pus or tissue as oval or cigar-shaped cells, and when examined by hanging-drop culture appear as fine interlacing septate hyphae with oval or pear-shaped spores attached to the hyphae). z. Aspergillus.—A genus of filamentous fungi characterized by its spore organ. They are common and troublesome laboratory contami- nants, appearing on culture plates as cottony masses dotted with minute colored spots, becoming in older cultures profusely black, yellow, or green, according to the species. Microscopically, the col- ored spots are seen to be the spore organs, the spores (conidia) borne on aerial hyphae which terminate in a large rounded head with rows of spores projecting in all directions. The main cottony mass is a network of septated mycelial filaments (e.g., Aspergillus niger, the black mold). aa. Penidlliwm.—This genus differs in its spore organ in that the fertile hyphae show numerous branches, rather than a rounded head, bearing rows of spores, a structure somewhat resembling a broom. The color of the colony varies with the species—green, yellow, etc. A common variety is Penicillium break aide and its strains, it causes spoilage of cheese and other dairy products. ab. Phycornycetes.—A group of genera having, in addition to the mycelium, spores contained in a spherical, case-like structure (a spor- angium) at the end of a hypha. Species of this group frequently contaminate laboratory media and food products, and occur in soil, dust, and water (e. g., Mucor rnucedo, the blue-black mold). ac. PleomorpMsm.—This term refers to the great variation in characteristics of morphology and culture which many fungi undergo under different conditions of life. The ringworm group are particu- larly likely to undergo these degenerative changes, and once a culture has so changed, it cannot easily be restored to its original condition. Prolonged growth on suger-containing media leads to this permanent change, hence the use of “conservation agar” for stock-culture maintenance. 297 TM 8-227 398-400 MEDICAL DEPARTMENT ad. Ringworm group of fungi.—These filamentous fungi produce superficial skin infections, generally growing as leathery masses of closely interwoven hypae, growing slowly with development of bumps and ridges, and covered by a powdery or velvety “duvet”, of aerial hypliae. The next four genera belong in this group. The so- called athlete’s foot may be caused by the same group of fungi. ae. Micro sporum.—The small-spored ringworm fungus. In the dis- eased epidermis they appear as a fine mycelium, 1 to 5 microns in diameter, composed of rectangular elements; they penetrate into the hairs and grow up and down in the hair. When the infected hairs are examined, they are found to be encased with an irregular mosaic of small round spores about 2 microns in diameter (e. g., Micro- sporum audouini, causing the common ringworm of the scalp). af. Trichophyton.—The large-spored ringworm fungus. The my- celium consists of chains of oval or rectangular spore-like bodies 5 to 8 microns in diameter, in regular alinement. Various species com- monly produce ringworm of the scalp, beard, skin, and nails (e. g., Trichophyton tonsurans, which is found only within the medulla of the hair). ag. Epidermophyton.—A genus of skin-invading fungi, appearing as long,, interlacing filaments, never invading the hair as do trichophy- tons. May be readily recognized by direct miscroscopic examination of skin scraping (e. g., Epidermophyton cruris, causing the common “Dhobie itch” or ringworm of the groin and other areas). ah. Achorion.—A genus of filamentous fungi of which a species causes favus of scalp (Achorion schoenleini). 399. Materials for examination.—a. Hairs, skin scales, or scrap- ings from lesions. h. Tissue masses or scrapings from internal lesions. c. Secretions or excretions from infected areas. d. Incidental cultures (contaminants, etc.). 400. Methods of examination.—a. Choice of method.—Method varies with expectancy of findings. Skin scrapings and hair may yield informative data by immediate direct methods only and be less informative on culture or animal inoculation, being difficult to grow, to infect, or to identify exactly. Some species give the desired in- formation only on cultivation or animal inoculation, especially in the case of tissue invaders. A few pathogens may be detected by histo- pathological examination. h. Microscopic examination.—(1) Collection of specimen (prefer- ably in laboratory by a medical officer).—After cleansing the affected part with alcohol, such materials as hairs, nails, scales, or bits of 298 METHODS FOR LABORATORY TECHNICIANS TM 8-227 400 tissue may be scraped into a sterile Petri dish. Moist specimens should be prepared and examined without delay. Biopsy specimens should be divided, one-half for direct examination, the other half for fixation and histological examination. All specimens should be ob- tained from an active, infected area and not from dried or inactive lesions. Collection of sputum for this examination requires especial care to avoid mouth contaminants by previously rinsing mouth with sterile saline solution, the expectoration then to be placed in a sterile Petri dish and examined within a few hours. (2) Fresh -preparation for direct study.—Outline a vaseline circle on a slide, place the material under examination within this circle, add a few drops of 10 percent sodium hydroxide, cover with a cover glass, and examine after a period of digestion—a few minutes to 12 hours. Fungi resist the digestive action of the hydroxide and retain their form, whereas tissue elements disappear. Avoid mistaking arti- facts, resembEng yeast-like organisms and hyphae. (3) Stained spreads.—Moist materials may be spread on a slide as for bacteriological study, for bacterial stains and for a polychrome (Wright or Giemsa) stain. The former stains will reveal bacterial content, the latter will assist in the study of any fungi present but will also bring out cellular detail and may lead to the discovery of a protozoan infection as leishmaniasis. c. Cultivation.—(1) Seventy percent alcohol may be used to cleanse scales or hair of contaminating bacteria by allowing the specimens to soak therein for about 1 hour prior to placing them on culture media (control culture to be made of the alcohol to detect con- taminating spores in it). Materials, with or without above prepara- tion, should be placed upon media having a pH of 5 to 6, unfavorable for bacterial growth. Inoculate several Sabouraud’s maltose agar tubes, planting the inoculum into a slightly broken surface of the slant. Incubate at room temperature (22° C.) and at 37° C. Some pathogenic fungi grow better at 37° C. Many fungi show cultural differences when grown at 22° and at 37° C. Retain the cultures for at least 4 weeks before considering them negative. (2) Observe the cultures daily, but do not open the tubes unless definite growth is observed, then make a subculture as soon as the tube is opened. Subcultures are made on Sabouraud’s maltose agar or on his conservation agar; the former is best for primary isolation, the latter for storage and the study of characteristics. In studying the yeast-like organisms, corn meal agar plates are useful for isola- tion, purification, and low-power study under the microscope. These are best inoculated by so streaking the plates that the wire produces a slit in the medium. 299 TM 8-227 400 MEDICAL DEPARTMENT (3) Cultures may be studied by observing colony characteristics by naked eye, under low-power magnification, and by slide prepara- tions, fresh or stained, for microscopic examination. Instead of removing, as for bacterial spread examination, a surface loopful of the colony, it is best to remove, with a stiff wire, a fragment of the culture supported intact in a fragment of the culture media; this is placed in a drop of lactophenol (equal parts of lactic acid and phenol) or of water, on a slide, and covered with a cover glass. In studying these slides the microscope light is modified to provide a subdued light (by lowering of substage and control of diaphragm). Study hyphae, branching, budding, sporulation, septation, etc., for descriptive report of the cultured fungus. (4) Hanging-drop cultures in maltose broth, afford another method of study. d. Animal inoculation.—Most of the fungi are not pathogenic for laboratory animals. A few species have animal pathogenicity and animals may therefore be used in determining the characteristics of only those few species. The form of the fungi seen in tissues tends to be quite different from the form seen in cultures. Some of the yeast-like fungi, producing as a group, blastomycosis, are pathogenic for animals; diagnostic information is, in these, obtainable by the subcutaneous, intramuscular, intraperitoneal, or intravenous inocula- tion of the suspected material, or preferably, a pure culture of the isolated fungus. The mouse, rat, guinea pig, or rabbit may be so used, and observed for a prolonged period for local or general evidences of infection. TM 8-237 401 METHODS FOE LABORATORY TECHNICIANS Chapter 11 SPECIAL SEROLOGICAL METHODS Section I. Human blood groups 401-405 H. Sero-diagnosis of syphilis 406-414 Paragraphs Section I HUMAN BLOOD GROUPS Groups and their application 401 Determination of blood groups 402 Cross-typing 403 Universal donor 404 Preparation of typing serum 405 Paragraph 401. Groups and their application.—a. According to the classi- fication of Landsteiner, recommended by the National Research Council, human blood is divided into four main groups or types, i. e., “0,” “A,” “B,” and “AB.” This grouping depends upon the presence or absence in the blood cells of the two agglutinogens A and B. Both are present in the AB type, while neither is present in the 0 type which should be referred to as “zero.” b. The confusion between the older classifications by Jansky and by Moss is eliminated by this newer grouping. The following table will show the relationship between the systems: Occurrence Official Jansky Moss (percent) 0 I IY 43 A II II 40 B in III 7 AB IV I 10 c. The practical application of blood grouping is in its relation to transfusion. These transfusions are given to replace blood losses, to combat various sorts of shock and in the treatment of certain infections. The division into these groups is made necessary because the blood serum of certain of these groups will produce dangerous or even fatal reactions in the blood of certain other groups. d. If the blood cells of the donor are agglutinated by the serum of the recipient this danger is greatest. Such a donor must not be used. If, however, the cells of the recipient are agglutinated slightly by the serum of the donor, there is less danger. This is because the TM 8-237 401-403 MEDICAL DEPARTMENT donor’s blood is injected into the recipient’s circulation so slowly that its serum is generally diluted beyond its agglutinating strength. Such a donor can be used only in the gravest emergency. e. The donor must belong to the same group as the recipient, except those in group 0. This group is referred to in more detail in paragraph 404. 402. Determination of blood groups.—a. The only reagents re- quired for the test are sera from blood groups A and B. The only apparatus required comprises— (1) Microscopic slides. (2) Hypodermic syringes fitted with 24-gage needles. (3) Wood applicators. h. The test is made as follows: (1) Divide a slide down the middle with a wax pencil. (2) Mark the left side A and the right B. (3) Withdraw enough A serum for the test into one syringe and enough B serum into another by perforating the rubber stoppers of the ampoules containing the grouping sera. (4) Place 1 drop of A serum in the center of the A side of the slide and a drop of B serum in the center of the B side. One drop of each is sufficient for the test. (5) Puncture the finger or the ear lobe of the person to be tested and transfer a minute drop of blood by means of a clean applicator to the drop of A serum, mixing to a smooth suspension of the cells. With a fresh applicator transfer a like drop to the B serum and mix thoroughly. Never use the same applicator for both sera. (6) Allow to stand for 5 minutes, occasionally rolling or tilting the slide to insure thorough mixing. Any agglutination of the red cells must take place within this time limit. If it is difficult to distinguish between true agglutination and rouleau formation, stir again with an applicator, as rouleaux will be broken up to a smooth suspension thereby, but true agglutination will be unaffected. (7) The reading of the reaction is best expressed by the following illustration. (8) Occasionally there will be a doubt as to the agglutination in weakly reacting blood. It will be wise then to use a suspension of the cells in saline. One drop of the blood to be typed in 0.5 cc of Saline tested in the same manner as whole blood, will almost always give a reaction which can be read clearly, even if it is necessary to use the low-power magnification of the microscope to determine the presence or absence of agglutination. 403. Cross-typing.—Occasionally the bloods of donor and re- cipient, even though clearly of the same group, will not match, that 302 METHODS FOR LABORATORY TECHNICIANS TM 8-227 403 O" 'A’ "B Cfrovvjp fVB. Figure 20.—Blood grouping. is, there will be some agglutination of the donor’s cells by the patient’s serum or of the patient’s cells by the donor’s serum. While this may not be fatal, it is potentially a grave danger. It is to be avoided by cross-typing. This is done in the following manner: a. Take 1 or 2 cc of prospective donor’s blood and the same amount of the recipient’s blood in separate Wright’s capillary ampoules or in small Kahn tubes. h. Mark the appropriate tubes clearly as DS (donor’s serum) and RS (recipient’s serum). c. Set aside until the serum separates. If necessary, centrifuge to hasten separation. d. Transfer a drop of freshly drawn blood to 1 cc of salt solution from both donor and recipient and mark the appropriate tubes DC (donor’s cells) and RC (recipient’s cells). Mix to an even suspension. e. Divide a clean slide as for the standard typing test and mark the left side and the right side /. Place 1 drop of donor’s serum on the left side and 1 drop of recipient’s serum on the right, using fresh capillary pipettes for each transfer. g. Mix 1 drop of recipient’s cells with the donor’s serum and 1 drop of donor’s cells with recipient’s serum, using fresh capillary pipettes for each transfer. h. The remainder of the test is done in the same manner as for the standard typing. Any agglutination evident within 5 minutes 303 TM 8-227 403-405 MEDICAL DEPARTMENT should disqualify the donor and another should be tried until one is found giving no trace of agglutination. This is especially true when there is any grade of reaction between donor’s cells and recipient’s serum. 404. Universal donor.—Any donor belonging to group 0 is con- sidered to be a universal donor, all other things being equal. This is on the assumption that his serum contains no iso-agglutinins for other groups. This may be true but should not be relied upon with- out cross-typing except in dire emergency. There are by no means infrequent instances of 0 donors whose ceils are moderately aggluti- nated by the particular recipient’s cells or vice versa. Unpleasant or dangerous consequences attend their employment. An excellent rule to follow in selecting donors is: Whenever possible use one from the recipient’s group and always cross-type. 405. Preparation of typing serum.—a. General.—Occasionally an emergency may arise when typing serum is badly needed and none is at hand. Strong B serum is difficult to obtain because of the scarcity of B donors in general. It is wise, therefore, always to have an up-to-date list of donors among the local detachment with a note as to the strength of the reaction in each case. Enough serum can be obtained from 30 to 50 cc of blood to last over almost any emergency and is easy to prepare. Reasonable care as to maintaining sterility while taking the blood and preserving the serum will keep the serum at full strength for many weeks. Choose only donors whose reaction is strong, rapid, and clear-cut. It is wise to take the A and the B blood at different times to avoid all possibility of mixing or mistaking them. b. Collecting blood.—It has been found best to collect the blood in sterile culture tubes and not more than 15 cc to a tube for ease in handling and to secure the maximum yield of serum. The blood will be collected according to the directions given in paragraph 40a. c. Separating serum.—(1) Remove plugs and flame the mouths of the tubes lightly to burn off wisps of adhering cotton. Do not replug the tubes. (2) Centrifuge at 1,500 rpm for 5 minutes. (3) Decant or pipette off the clear serum into fresh, sterile tubes and recentrifuge to get rid of all stray red cells. (4) Collect in a sterile graduated cylinder to find the exact amount of the serum yield. d. Preserving type A serum.—(1) Have ready— {a) A 10 percent aqueous solution of sodium citrate. (b) A 1 percent aqueous solution of neutral acriflavin. (c) A 0.5 percent aqueous solution of basic fuchsin. 804 METHODS FOR LABORATORY TECHNICIANS TM 8-227 405-406 (2) To each 9 cc of clear A serum add 1 cc of the sodium citrate solution. (3) To each 10 cc of citrated A serum add 0.2 cc of the acriflavin solution and 0.2 cc of the basic fuchsin solution. (4) Mix thoroughly. Store in 2- or 4-cc sterile vials with sealed rubber stoppers. Keep in refrigerator when not in use. e. Preserving type B serum.—(1) Have ready— {a) A 10 percent sodium citrate solution as above. (b) A 1 percent aqueous solution of brilliant green. (2) To each 9 cc of clear B serum add 1 cc of the citrate solution, as above. (3) To each 10 cc of the citrated B serum add 0.2 cc of the brilliant green solution. (4) Mix and store as for the A serum. /. The addition of the citrate and stains to the sera serves both to preserve them from bacterial growth and to make them more easily distinguishable from each other. When the stains are not available, the A and B sera may be preserved by simple freezing, provided the laboratory has an electric refrigerator with a freezing compartment. The sera may then be frozen in sterile Kahn tubes each containing 1 cc of serum. The tubes should be plugged with cotton and the plugs sealed with paraffin immediately after freezing. They should be kep>t in the freezing compartment and thawed out as needed. Section II ParagTP’ih General 4u6 Standard Kahn test 407 Quantitative Kahn test 408 Spinal fluid Kahn test 409 Quantitative Kahn test for spinal fluid 410 Complement fixation test for syphilis (Craig-Wassermann method) 411 Two-tube Kolmer test (sheep system) 412 Spinal fluid complement fixation test 413 Colloidal gold test 414 SERO-DIAGNOSIS OF SYPHILIS 406. General.—a. Three standard tests are here outlined: Kahn precipitation. Craig-Wassermann, using the human hemolytic system. Two-tube Kolmer, using the sheep hemolytic system. h. The quantitative methods are confined to the Kahn test alone, since it is believed that more clear-cut reactions are achieved by this method. Technic for spinal fluid reactions are given for both Kahn and Kolmer tests. 406303°—41 20 805 IM 8-337 407 MEDICAL DEPARTMENT 407. Standard Kahn test.—a. Separate serum from clot and cen- trifuge until entirely free of cells. h. Inactivate serum in water bath (56°) for 30 minutes. c. Mix Kahn antigen by placing 1 cc of antigen in a mixing vial and the required amount of 0.85 percent salt solution (as indicated by titer on antigen bottle) in another vial, then pour salt into antigen and quickly pour from one vial to the other 12 times. Allow to stand for 10 minutes. d. The standard Kahn test is a three-tube test. The tubes are placed in the rack three deep. e. After the antigen has stood for 10 minutes it is shaken well to insure an even mixture and is then pipetted into the bottom of the tubes in the following varying amounts: Back tube, 0.0125 cc Middle tube, 0.025 cc Front tube, 0.05 cc A Kahn antigen pipette (total capacity 0.25 cc) is used to deliver the antigen. Caution: The antigen suspension is reliable for only one-half hour after dilution. If the number of tests is so great as to take more than a half hour to pipette, the first dilution should be dis- carded at the end of the half hour and a new dilution made with which to finish the tests. (A skilled technician can pipette antigen for 200 tests in 30 minutes.) /. The inactivated serum of the patient is then added to the tubes containing the antigen, 0.15 cc to each tube. g. Three controls are set up at the same time the test is run. The same amounts of antigen (e above) are used. (1) Control No. 1.—Add 0.15 cc of 0.85 percent salt solution to antigen in each of the three tubes. (2) Control No. 2.—Add 0.15 cc of known positive serum to antigen in each of the three tubes. (3) Control No. 3.—Add 0.15 cc of known negative serum to antigen in each of the three tubes. h. All tubes are shaken for 3 minutes. i. Salt solution is added to all the tubes in the following amounts: Back tube, 0.5 cc Middle tube, 0.5 cc Front tube, 1.0 cc j' All tubes are shaken enough to mix the salt solution and antigen- gerum mixture thoroughly. 306 TM 8-227 407-408 METHODS FOR LABORATORY TECHNICIANS k. Tests are then read. A negative reading shows homogeneous cloudiness with no visible floes. A four-plus reading shows complete flocculation in a water-clear fluid. One-, two-, and three-plus read- ings show degrees of flocculation and cloudiness of the fluid varying from the negative to the four-plus. 408. Quantitative Kahn test.—To be done only on sera that show a three- or a four-plus reaction with the standard Kahn test. a. Separate serum from clot and centrifuge until entirely free of cells. b. Inactivate serum in water bath (56° C.) for 30 minutes. c. Mix Kahn antigen by placing 1 cc of antigen in a mixing vial and the required amount of 0.85 percent salt solution (as indicated by titer on antigen bottle) in another vial, then pour salt into antigen and quickly pour from one vial to the other 12 times. Allow to stand for 10 minutes. d. While antigen is aging for 10 minutes dilutions are made of the serum as follows, using six tubes: Tube No. 1 0.6 cc salt solution + 0.4 cc serum. 0.5 cc salt solution+ 0.5 cc of mixture from tube 1. 0.5 cc salt solution-f 0.5 cc of mixture from tube 2, 0.5 cc salt solution-)-0.5 cc of mixture from tube 3. 0.5 cc salt solution+ 0.5 cc of mixture from tube 4. 0.5 cc salt solution + 0.5 cc of mixture from tube 5. 2 3 I 4 5 6 The dilutions will then be as follows: Tube No. Dilution 1 1:2.5. - 2 1:5. 3 1:10. 4 1:20. 5 1:40. 6 1:80. The dilutions may be carried higher if the serum potency demands it. One tube may be run with an undiluted specimen of serum if it is apparent that the unit reading should be less than 10. e. Six Kahn tubes are placed in the Kahn rack in a row, then 0.025 cc of antigen suspension is pipetted to the bottom of each of these tubes. 307 TM 8-237 408-410 MEDICAL DEPARTMENT /. Then 0.15 cc of each of the serum dilutions is added to the corresponding Kahn tube, beginning with tube No. 6. That is— 0.15 cc from serum dilution tube No. 6 into Kahn tube No. 6; 0.15 cc from serum dilution tube No. 5 into Kahn tube No. 5, etc. g. All tubes are shaken for 3 minutes. h. Add 0.5 cc of salt solution to each tube. i. Shake rack and read results. The highest dilution in which there is a + + + or a + + + + reaction shows the quantitative reading. It may be arbitrarily expressed by multiplying the dilu- tion factor of that tube by four and noting the result as units, for example, if the highest dilution showing a + + + reaction is tube No. 1. the reading in units will be 4+2.5 or 10 units. It may be re- ported by simply giving the series of readings of the six or more tubes in sequence, as + + + +, + + + +, + + +, +, —, —. 409. Spinal fluid Kahn test.—a. Centrifuge spinal fluid until free of cells. b. Pipette 1.5 cc of clear spinal fluid into centrifuge tube. c. Add 1.6 cc of saturated ammonium sulfate solution. d. Place in 56° water bath for 15 minutes. (Globulin can be read at this point.) e. Centrifuge at high speed for 10 minutes. The precipitated globulin will be packed in the bottom of the centrifuge tube. Pour off the supernatant fluid and drain by inverting the tube on a clean towel for several minutes. /. Add 0.15 cc of 0,85 percent salt solution and shake gently until the precipitate is dissolved. g. Mix antigen by placing 1 cc of antigen in a mixing vial and the required amount of 0.85 percent salt solution (as indicated by titer on antigen bottle) in another vial, then pour salt into antigen and quickly pour from one vial to the other 12 times. Allow to stand for 10 minutes. h. Pipette 0.01 cc of antigen suspension into the bottom of a Kahn tube. i. Pipette 0.15 cc of the globulin solution from the centrifuge tube into the Kahn tube which contains the 0.01 cc of antigen suspension. j. Shake for 3 minutes, add 0.5 cc of salt solution, and read results. 410. Quantitative Kahn test for spinal fluid.—a. Centrifuge spinal fluid until free of cells. b. Pipette 3 cc of clear spinal fluid into centrifuge tube. c. Add 3 cc of saturated ammonium sulfate solution. 308 METHODS FOE LABORATORY TECHNICIANS TM 8-2S7 410-411 d. Incubate, centrifuge, drain, and dilute antigen as in the stand- ard spinal fluid Kahn procedure. e. Add 0.3 cc of salt solution and dissolve precipitate. This so- lution may require centrifugation to free it from undissolved particles. /. While antigen is aging, dilutions are made of the globulin solution as follows, using five tubes: Tube No. 1 This is the 0.3 cc. of globulin solution in the centrifuge tube. 0.6 cc salt solution + 0.15 cc from tube No. 1. 0.4 cc salt solution +0.4 cc from tube No. 2. 0.1 cc salt solution +0.2 cc from tube No. 3. 0.2 cc salt solution +0.2 cc from tube No. 4. 2__ 3 4 6 g. This is a five-tube test. Place five Kahn tubes in the rack in a row. h. Pipette antigen that has stood for 10 minutes into Kahn tubes, 0.01 cc into the bottom of each tube. i. Pipette 0.15 cc of dilutions of globulin solution into tubes contain- ing the antigen beginning with the last tube. That is— 0.15 cc from dilution tube No. 5 into Kahn tube No. 5; 0.15 cc from dilution tube No. 4 into Kahn tube No. 4, etc. j. Shake 3 minutes, add 0.5 cc of salt solution and read results in sequence, for example, + + + +, + + + +, + +, -f, —. 411. Complement fixation test for syphilis (Craig-Wasser- mann method).—a. Glassware and apparatus. Pipettes: 0.2 cc graduated to 0.01 (for titrations); 1 cc grad- uated to 0.01 to tip; 10 cc graduated to 0.1 cc. Test tubes, 100 by 12 mm, heavy wall, without lip. Test-tube racks carrying 2 rows of 10 tubes each. Amboceptor cutter and set of 3 ambocepter paper markers (3, 4, and 5 mm). Centrifuge and graduated centrifuge tubes. Water baths, one at 37° C. and one at 56° C, Refrigerator at 6° to 8° C. All glassware should be chemically clean. h. Reagents.—(1) Patient's serum.—The serum should be separated frpm the clot and centrifuged until all cells are thrown to the bottom ao9 TM 8-337 411 MEDICAL DEPARTMENT of the tube, then it is poured or pipetted from the top and placed into a clean tube. The serum mast he entirely free of cells. Before the test is run, all sera are inactivated in 56° water bath for 30 minutes. (2) Salt solution.—An 0.85 percent salt solution made with distilled water is used. That is, 8.5 gm of chemically pure sodium chloride in 1,000 cc of distilled water. (3) Red-cell suspension.—A 5 percent suspension of human red cells is used. This is prepared by collecting blood (from a normal person belonging to group “0”) in a flask which contains 2 percent citrate in 0.85 percent salt solution, approximately 10 cc of blood to 30 cc of the citrate solution. The red cells are thoroughly washed by centrifuging 4 or 5 times, the supernatant fluid being removed each time and replaced with fresh salt solution. On last washing, centrifuge for 3 minutes at a speed of 1,200 rpm, then read volume of red cells by graduations on the centrifuge tube, remove supernatant fluid, and add salt solution to make a 5 percent suspension. A fresh suspension of red cells is made for each day the tests are run. The speed of the centrifuge and the time of centrifuging are both con- trolled so that uniform suspensions may be made from day to day. (4) Amboceptor paper.—Serum from rabbits which have been im- munized to human red cells (type 0) is soaked up on filter paper and dried in the air. It is kept for 3 months before use. (5) Complevient.—The serum used is that of two or more guinea pigs. Serum from a single pig must not be used because of the variabil- ity of the complement content in the serum of individual animals. To each 2 cc of fresh serum obtained the day before the test, add 3 cc of salt solution, making a 40-percent dilution. If lyophilized com- plement is used, regenerate the frozen and dried serum as directed on the ampoule and make the 40-percent dilution from this, as directed above. (6) Antigen.—An alcoholic extract of ether-extracted powdered beef heart, containing 0.4 percent cholesterol. c. Method employed.—The method employed is a two-tube qual- itative test. A cholesterolized alcoholic extract of powdered beef heart is used as antigen and the antihuman hemolytic system (Craig’s modification) is utilized. Assuming that all reagents are new and titrations are unknown, it is best to make a 40-percent dilution of the complement and use 0.1 cc as two units. This is merely an arbitrary dose to be used in the amboceptor titration and the complement must be titrated after the amboceptor unit is found. 310 TM 8-227 411 METHODS FOR LABORATORY TECH NICIANS d. Amboceptor titration.— (1) Amboceptor paper is cut in strips 3 mm wide, marked in varying lengths, and tested as follows: Tube No. Salt solution (cc) Complement (cc of 40 percent) 5 percent blood cell suspension Amboceptor paper (mm) l._ 0. 9 0. 1 0. 1 3 by 1 2 . 9 . 1 . 1 3 by 1H 3 . 9 . 1 . 1 3 by 2 4 . 9 . 1 . 1 3 by 2 y2 5 .9 . 1 . 1 3 by 3 6 . 9 . 1 . 1 3 by 3 ya 7 .9 . 1 . 1 3 by 4 8 . 9 . 1 . 1 3 by 5 9 . 9 . 1 . 1 None 10 .9 None . 1 3 by 10 (2) Incubate in water bath at 37° C. for 1 hour, shaking every 10 minutes, then read the titration. The first tube showing complete hemolysis contains one unit of amboceptor. Use two units, or twice that amount of paper, for the test. e. Complement titration.— (1) The complement to be used is the 40-percent guinea pig serum, prepared in the manner previously described. (2) The amboceptor unit is now known and two units are used in the complement titration as follows: Tube No. Complement (cc of 40 percent) Salt solution (ce) 5 percent red cell suspension Amboceptor units 1 0. 02 0. 9 0. 1 2 2 . 03 . 9 . i 2 3 . 04 . 9 . i 2 4 . 05 . 9 . i 2 5 . 06 . 9 . i 2 6 . 07 . 9 . i 2 7 . 08 . 9 . i 2 8 . 09 . 9 . i 2 9 . 10 . 9 . i 2 10 . 20 . 8 . i None (3) Incubate at 37° for 1 hour, shaking the tubes every 10 minutes to liberate the amboceptor from the paper and keep the red cells in suspension. Read the titration. 311 TM 8-227 411 MEDICAL DEPARTMENT (4) The first tube showing complete hemolysis is noted and the amount of complement in that tube is called one unit. Twice this amount, or two units should be used in the test. The control, tube No. 10, should show complete inhibition of hemolysis since it contains no amboceptor. /. Antigenic strength titration.— (1) A 1 to 10 dilution of the anti- gen is made by placing 1 cc of antigen in a small flask and adding 9 cc of 0.85 percent salt solution. The salt solution should be added drop by drop while the flask is being shaken constantly. The re- sultant mixture should present a markedly cloudy appearance. (2) The amboceptor unit and complement units are now known. The antigen titration is carried out as follows: Tube No. Antigen emulsion Salt solu- tion Positive human serum Comple- ment units 5 percent red cell suspension Ambo- ceptor units 1 0. 02 0. 9 0. 1 2 m O 0. 1 2 2 . 03 . 9 . 1 2 +3 . 1 2 3 . 04 . 9 . 1 2 s . 1 2 4 . 05 . 9 . 1 2 Q O . 1 2 5 . 06 . 9 . 1 2 CO u . 1 2 6 . 07 . 9 . 1 2 o t+H . 1 2 7 . 08 . 9 . 1 2 o I'- . 1 2 8 . 09 . 9 . 1 2 . 1 2 9- . 10 . 9 . 1 2 a . 1 2 10 none 1. 0 . 1 2 c3 . 1 2 11 _ none 1. 1 none 2 p . 1 2 12 none 1. 2 0. 1 none d h-i . 1 2 (3) The tubes are incubated for 30 minutes in the 37° water bath before addition of the hemolytic system (amboceptor and red cells) and for 1 hour after the addition of the hemolytic system with shaking of the tubes every 10 minutes. (4) It is noted that tube No. 10 has no antigen emulsion and should therefore show complete hemolysis. Tube No. 11 has neither antigen nor positive human serum and should also show complete hemolysis. Tube No. 12 has neither antigen nor complement and should show no hemolysis. If these control tubes do not comply with these require- ments, the titration should be rejected. (5) In the remaining tubes, the smallest amount of antigen which has completely fixed the complement in the presence of positive serum and therefore shows no hemolysis, is called the antigenic unit. The antigenic dose to be used in the test is twice the antigenic unit. 312 METHODS FOR LABORATORY TECHNICIANS TM 8-337 411 g. Procedure for the test.—(1) For each serum to be tested two tubes are necessary, a front and a rear tube. In addition, for each run of tests there must be a control set of two known positive sera and two negative sera. Three single tubes for control of the anti- complementary properties are added. (2) Each unknown serum and known positive and negative serum has a front and a rear tube: Front tube Rear tube 0.9 cc salt solution. . 0.1 cc serum (patient’s or control) 2 units antigen _ . _ ... 0.9 cc salt solution. 0.1 cc serum (patient’s or control). 2 units complement. 2 units amboceptor. 0.1 cc red cells. 2 units complement _ Incubate at 37° for 30 minutes. 2 units amboceptor _. . _ 0.1 cc red cells _ ... Incubate 1 hour at 37° C. with shaking every 10 minutes. Set in ice box for 1 hour. (3) The three control tubes for the anticomplementary properties of the antigen are set up as follows: Tube No. 1 Tube No. 2 Tube No. 3 0.8 cc salt solution . __ __ 0.7 cc salt solution... . 0.1 cc normal serum 8 units antigen _ 0.7 cc salt solution. 6 units antigen__ . 8 units antigen. 2 units complement .. 2 units complement 2 units complement. Incubate at 37° for 30 minutes 2 units amboceptor.. 2 units amboceptor 2 units amboceptor. 0.1 cc red cells 0.1 cc red 0.1 cc red cells. Incubate 1 hour at 87° C. with shaking every 10 minutes. Set in ice box for 1 hour. h. Results.—The positive control tubes should show complete inhi- bition of hemolysis in the front tube and complete hemolysis in the rear tube, while the negative controls should show complete hemolysis, both front and rear. In the tubes containing the unknown sera, if syphilitic, the front tube should show a complete inhibition of hemolysis or varying degrees of inhibition, according to the 313 TM 8-327 411-412 MEDICAL DEPARTMENT length of time after infection, the amount and character of treat- ment, and other factors influencing the strength of the reaction. If syphilis is not present, the front tube containing the patient’s serum should show complete hemolysis. All rear tubes should show complete hemolysis. The three antigen control tubes should show complete hemolysis. 412. Two-tube Kolmer test (sheep system).—a. Glassware and apparatus. Pipettes: 0.2 cc, graduated to 0.01 cc; 1,0 cc, graduated to 0.01 cc; 10.0 cc, graduated to 0.1 cc. Test tubes, 100 by 12 mm, heavy wall, without lip. Test tube racks, carrying 2 rows of 10 tubes each. Centrifuge and centrifuge tubes. Water baths: inactivating, set at 56° C.; incubating, set at 37° C. Refrigerator, running at 6° to 8° C. All glassware should be chemically clean and should be used dry or rinsed out with normal saline solution just before using. Never use any glassware containing the slightest degree of water. h. Reagents.—(1) Patient's serum (par. 40).—The serum must be inactivated at 56° C. in the water bath for 30 minutes just before using. If the serum to be tested has previously been inactivated, only a 10- minute inactivation is necessary just before running the test. (2) Salt solution.—This is an isotonic solution of sodium chloride. Add 0.85 gram of chemically pure sodium chloride (Merck’s blue label) to 100 cc of distilled water. (3) Sheep cell suspension (indicator antigen).—Collect the blood by bleeding the sheep from the external jugular vein into 1 to 3 percent sodium citrate solution. The blood may be received into a flask containing a handful of sterile glass beads and shaken well to defibrinate it. Either method prevents clotting. The former method is preferable. Filter a small amount of the blood through cotton into a graduated centrifuge tube, allowing twice as much blood as will be required for the test to be run. Add 2 or 3 volumes of salt solution. Centrifuge at tenth speed for 10 minutes. Repeat this washing five times. On the last washing, centrifuge at the tenth speed for exactly 15 minutes. Do not vary the time or speed, so as to insure the same percent suspension when the cells are finally diluted for use in the test. Read the volume of the cells in the centrifuge tube, carefully remove the supernatant fluid, and prepare a 2-percent suspension by washing the cells into a flask with 49 volumes of salt solution. Always shake well before using to 314 TM 8-227 412 METHODS FOR LABORATORY TECHNICIANS secure an even suspension, as the cells rapidly settle to the bottom of the flask on standing. (4) Gbmplement.—See paragraph 4115(5). (5) Amboceptor.—Glycerolized amboceptor may be obtained by requisition. (6) Antigen.—While the titration factors are given on all reagents issued by the Army Medical School, it is advisable to keep watch that they retain their potency. Once in 3 weeks is about the proper fre- quency to check up by titration. The technic is given below. c. Titration of amboceptor.—(1) Prepare a dilution of 1:100 am- boceptor as follow’s: Glycerolized amboceptor (50 percent) 2cc Salt solution 94 co Phenol (5 percent in salt solution) 4co This is to be kept in the refrigerator as a stock solution and is good for several weeks, (2) Dilute this stock solution for the titration as follow's: Stock amboceptor (1:100) 0. 5 cc Salt solution 4. 5 co This will be 1; 1,000 in strength. (3) In a series of 10 tubes, prepare higher dilutions as follows: Tube No. Amboceptor, 0.5 cc Plus cc saline 1 _ o o o T— r— None. 2 - 1:1, 000 0.5 (1:2,000). 1.0 (1:3,000). 1.5 (1:4,000). 2.0 (1:5,000). .5 (1:6,000). .5 (1:8,000). .5 (1:10,000). .5 (1:12,000). .5 (1:16,000). 3 1:1. 000 4 o o o *— r— 5 1:1, 000 6 1:8, 000 7 ... 1:4, 000 8 -. 1:6, 000 9 _ 1:6, 000 10 - 1:8, 000 Mix the contents of each tube thoroughly. (4) Prepare a 1:30 dilution of the regenerated complement (see par. 4115(5)) by diluting 0.2 cc of the regenerated complement with 5.8 cc of salt solution. (5) Prepare a 2-percent suspension of sheep cells in salt solution. 315 TM 8-227 412 MEDICAL DEPARTMENT (6) In a series of 10 tubes set up the amboceptor titration as shown in the following table: Tube No. Amboceptor, 0.5 cc Complement, 1:30 Saline Sheep cells, 2 percent 1 _ 1:1, 000 1:2, 000 1:3, 000 1:4, 000 1:6, 000 1:6, 000 1:8, 000 1:10, 000 1:12, 000 1:16, 000 0.3 cc to all tubes__ 1.7 cc to all tubes. _ 0.5 cc to all tubes. 2 3 4 5 6_ 7 8 9. 10.. _ Mix the contents of each tube thoroughly. (7) Incubate in the water bath at 38° C. for 1 hour. (8) Read the amboceptor unit. The unit is the highest dilution of amboceptor that gives complete hemolysis. (9) Two units of amboceptor are used in the complement and antigen titrations and in the final test. Example: if the unit equals 0.5 cc of the 1:6,000 dilution, then two units will equal 0.5 cc of the 1:3,000 dilution. Dilute just enough of the stock amboceptor for the titrations and the number of tests to be run. d. Titration of complements—(1) Prepare a 1:30 dilution of the complement (see c (4) above. (2) Dilute the antigen as indicated by the dilution factor on the antigen bottle, by placing the required amount of salt solution in a small flask and adding the antigen drop by drop, shaking the flask continually until the antigen has all been added. Prepare enough for the complement titration and for the final test. (3) In a series of 10 tubes, set up the titration as follows: Tube No. Comple- ment (1:30) Antigen dose Salt solu- tion Ambocep- tor 2 units Sheep cells, 2 percent 1 (cc) 0. 1 (cc) 0. 5 (cc) 1. 4 U (cc) 0. 5 (cc) 0. 5 2. . 15 . 5 I. 4 . 5 . 5 O <*-■ 3 ... . 2 1. 3 d . 5 . 5 d 4 . 25 . 5 1. 3 o Jh t- 2 . 5 . 6 o 6_ _. . 3 . 5 1. 2 CO o _ X . 5 , 5 CO 3 r< o 6 . 35 . 5 1. 2 a . 5 . 5 £ rC C3 H 7 . 4 . 5 1. 1 . 5 . 5 X! 8 . 45 . 5 1. 1 o . 5 . 5 s 9 . 5 . 5 1. 0 33 . 5 . 0 cj 10. ... None None 2. 5 None . 5 316 METHODS FOR LABORATORY TECHNICIANS TM 8-227 412 (4) The smallest amount of complement just giving sparkling hemolysis is the exact unit. The next higher tube is the full unit, which contains 0.05 cc more complement. In the antigen titration and in the final test, two full units are used and are so diluted as to be contained in 1.0 cc, as in the following example: Exact unit 0.3 cc Full unit .35 cc Dose (2 full units) .7 cc (5) To calculate the dilution to employ so that 1.0 cc will con- tain the dose of 2 full units, divide 30 by the dose (0.7). This equals 48, therefore 1.0 cc of a 1:43 dilution will contain the required 2 full units. e. Titration of antigen.— (1) Prepare a 1:80 dilution of antigen by adding 0.1 cc, drop by drop, with continual shaking, to 7.9 cc of salt solution in a large test tube or a small flask. (2) Higher dilutions are then prepared as follows: Quantity of dilution Salt solution added Resulting di- lution 4 cc of 1:80 _ - 4 cc 1:160. 1:320. 1:640. 1:1,280. 1:2,560. 4 cc of 1:160_ - 4 cc -_ __ _ 4 cc of 1:320- . - _ 4cc__ __ 4 cc of 1:640 __ _ 4 cc _ 4 cc of 1:1,280 ___ 4 cc (3) Arrange five rows of test tubes with six tubes in each row. (4) (a) In the first tube of each row place 0.5 cc dilution 1:80. (b) In the second tube of each row place 0.5 cc dilution 1:160. (c) In the third tube of each row place 0.5 cc dilution 1:320. (d) In the fourth tube of each row place 0.5 cc dilution 1: 640. (e) In the fifth tube of each row place 0.5 cc dilution 1:1,280. (/) In the sixth tube of each row place 0.5 cc dilution 1:2,560. (5) Heat 3 cc of moderately to strongly positive syphilitic serum in a water bath at 56° C. for 15 to 20 minutes and prepare five dilu- tions in large test tubes as follows: Tube No. Seram (ce) Saline (cc) Resulting dilu- tion Cc of serum in 0.5 cc of dilution 1 1. 0 4. 0 1:5 0. 1 2 . 5 4. 5 1:10 . 05 3 . 5 9. 5 1:20 . 025 4_ 2. 0 (1:20) 1. 0 (1:20) 2. 0 1:40 . 0125 5__ ___ ... . 4. 0 1:100 . 005 317 TM 8-327 412 MEDICAL DEPARTMENT (6) To each of the six tubes add dilutions as follows: 0.5 of 1: 5 to first row, 0,5 of 1:10 to second row. 0.5 of 1: 20 to third row. 0.5 of 1:40 to fourth row. 0.5 of 1:100 to fifth row. (7) Add 1.0 cc of complement dilution carrying two full units to all 30 tubes. (8) In a separate rack, set up a serum control carrying 0.5 cc of 1; 5 serum and 1,0 cc of complement (2 full units); also a hemolytic system control carrying 1.0 cc of salt solution and 1.0 cc of complement (2 full units). (9) Shake the tubes gently and place in the refrigerator at 6° to 8° C. for 15 to 18 hours, followed by water bath at 37° C. for 10 minutes. (10) Add 0.5 cc of amboceptor (2 units) and 0.5 cc of a 2 percent suspension of sheep cells to all 30 tubes and to the control tubes. (11) Mix thoroughly and place in water bath at 37° C. for 1 hour and make readings. The serum and hemolytic controls should show complete hemolysis. (12) Chart the results as per the following example observed with a strongly positive serum: Serum (in 0.5 Antigen dilutions (in 0.5-ec dose) cc) 1:80 1:160 1:320 1:640 1:1,280 _ 1:2,560 0.005__ + + _ _ 0.0125 — + + + + + + + + + + + 0.025 + + + + + 4- + + + + + + + + + + + + 0.05 + + + + + + + + + + + + + + + + + + + + + 0.1 + + + + + + + + + + + + + + + + + + + + + + + (13) The dose of antigen to employ in the final test is the largest amount giving a 4 plus reaction with the smallest amount of serum. If three dilutions of antigen give 4 plus reactions with the smallest amount of serum, the dose is midway between the highest and the lowest. /. Procedure for test.—(1) Having ascertained the exact amounts of the reagents to be used by the above methods, set up the two- tube Kolmer test on the various blood sera for diagnosis as indicated in the following table: 318 TM 8-327 412 METHODS FOR LABORATORY TECHNICIANS to »— 1 1 1 1 1 1 1 1 1 1 Tube No. p to to Patient’s serum (cc) 0. 5 : None (0.5 cc saline) Antigen (cc) Ten minutes at room tem- perature. o o Comple- ment (2 full units) (cc) Refrigerator at 6° to 8° C. 15 to 18 hours, then 10 to 15 minutes in water bath at 37° C. 0. 5 . 5 Ambocep- tor (2 units) (cc) 0. 5 . 5 Sheep cells (2 per- cent) (cc) Water bath at 37° C. for 1 hour, then read. (2) Tube No. 1 is the test tube; No. 2 tube is the serum control tube and should show complete hemolysis. If tube No. 2 shows any residual cells, the serum is anticomplementary and should be so reported. (3) An antigen, an amboceptor, and a sheep-cell control should be set up with each lot of sera tested, as follows: Oomple- Ambo- Sheep Tube Saline Antigen ment (2 ceptor cells (2 No. (cc) (cc) units) (cc) (2 units) (cc) percent) (cc) a o o 1-1 ® S - § 03 A U o -*4> c3 £ O CO 4-2 Antigen control should show 1 0. 5 0. 5 1. 0 O 00 .a 0. 5 0. 5 o3 A complete hem- 6 c3 £ 5 bC S mm Rubber Bo nd Cellophone Swot / in. Square Figtjkh 27.—NIH anal swab. (c) Mount the cellophane square in water on a microscopic slide and examine under a microscope. 450. Tapeworm segments.—It is necessary to differentiate seg- ments of beef or pork tapeworms passed in the feces. This is done as follows: a. Clean and relax the segments by shaking in physiological saline. b. Place the specimen between two glass slides and press it flat. c. Hold it up to a strong light (so that the light shines through it), and count the number of lateral branches of the uterus. (Since these lateral branches subdivide, they are to be counted where they arise from the main part of the uterus.) 451. Blood.—Thick films are very useful in demonstrating micro- filariae. For details of this technic see paragraph 437. 452. Tissue.—There are several specialized technics employed in the diagnosis of tissue parasites, most of which are not entirely satis- factory. Need for these technics is not very common, but when neces- sary, the technic is probably best performed by the medical officer in charge. Details of these technics are available in standard texts. 453. Urine.—a. In cases of heavy infection with the vesical blood fluke, eggs may be found in the urine, particularly in the last portion passed. The technic follows: 372 METHODS FOR LABORATORY TECHNICIANS TM 8-227 453-455 (1) Have the patient pass urine into a urinalysis glass. (This should especially include the last portion of urine voided.) (2) Let the material settle for 15 to 20 minutes. (3) Take up a small portion of the sediment in a pipette and place it on a microscopic slide. (4) Examine under the microscope. h. In cases of light infection this procedure may yield negative results. It is then necessary to centrifuge a representative portion of the urine for 1 to 2 minutes and examine some of the sediment under a microscope. 454. Sputum.—In many cases of suspected helminthic infection of the respiratory passage, examination of the sputum is necessary. The technic follows: a. Einse the mouth thoroughly with diluted hydrogen peroxide. h. Pass sputum into a jar, c. Transfer small bits of sputum, particularly blood-flecked por- tions, to a microscopic slide. d. Examine under a microscope. 455. Transmission of specimens.—a. From time to time it is necessary to send specimens to other laboratories, sometimes for iden- tification, sometimes for study purposes. In all such cases complete notes should accompany the material and should include such data as locality, host, date, collector’s name, number of specimens obtained, condition of the specimens, tissue, organ or medium from which recovered, and any other pertinent information. Material should be treated for shipment as indicated below. (1) Eggs.—Feces containing eggs may be diluted with water and agitated until an even mixture is obtained. To this mixture is added an equal volume of steaming (80° C.) 10 percent forjnalin. This will also fix any nematode larvae present. (2) Larvae and adults.—Larvae and adult helminths should first be shaken in physiological saline. This cleans and relaxes the speci- mens. They may now be fixed by adding an equal volume of steam- ing (80° C.) 5 percent formalin to the saline containing the worms. (3) Pathological tissues.—Pathological tissues may be fixed in 10 percent formalin, or if it is available, Zenker’s fluid. (4) Intermediate hosts.—Intermediate hosts may be fixed and pre- served in TO percent alcohol. h. Specimens should always be carefully packed to avoid breaking or spilling of the contents. Containers should be filled with the preserving liquid to avoid breaking of the specimens if the package is handled roughly. Jars and glass vials packed separately in a box with excelsior, shredded paper or cotton, and marked “Fragile” will usually survive. 373 TM 8-227 456 MEDICAL DEPARTMENT Chapter 14 ENTOMOLOGICAL METHODS Section I. General 456-457 II. Arthropods of medical importance 458-477 III. Handling and shipment of specimens 478-479 Paragraphs Section I GENERAL Paragraph 456. General.—a. Medical entomology is the study of insects and insect-like animals (arthropods) and their relation to human disease and discomfort. These arthropods may be associated with a number of diseases of importance to troops in garrison, camp, or campaign. The principal disease relationship of these forms is that of trans- mission: malaria and yellow fever by certain mosquitoes, plague by fleas, and typhus by lice. Also of importance are the arthropods that cause disease directly, they themselves serving as pathogenic or- ganisms. The itch mite of man, for example, invades the human skin and produces a severe irritation; this disease (scabies; the itch) caused thousands of men to be admitted to hospitals during the World War. Many arthropods, as nuisances, cause discomfort, to troops, such as the annoyance provoked by the common house flies and mosquitoes, especially when they occur in large numbers. b. Due to the widespread distribution of arthropods, and to their close association with troops at all stations, it is necessary that Army personnel be prepared to determine whether the species present in a locality are likely to be of medical importance. This may entail col- lection of representative forms, making a tentative identification, and if certain specimens are likely to be important, forwarding them to large central laboratories where positive identification may be ac- complished. The medical technician is not expected to know all of the various species by their scientific names, but if called upon to send in a representative sampling, for example, of mosquitoes in the vi- cinity, he should be able to send mosquitoes, and not a various as- semblage of small beetles, flies, midges, fleas, moths, etc. It is the purpose of this section to familiarize the medical technician with the 374 TM 8-227 456-458 METHODS FOR LABORATORY TECHNICIANS medical importance of various arthropods and to aid him in roughly identifying the forms. 457. Classification.—Classification of arthropods is based upon a system of organization. As an army is divided into corps, which are further divided into divisions, then brigades, regiments, battalions, companies, etc., down to the individual men, similarly animals are grouped into phyla, which are subdivided into classes, orders, families, genera, and species. The phylum Arthropoda may be divided into several classes of which only four will be here considered, namely, Insecta (insects), Arachnida (ticks, mites, spiders, scorpions, etc.), Myriapoda (centipedes and millipedes), and Crustacea (crayfish, shrimp, etc.). These classes can be further divided again and again until the individual species are reached; the yellow fever mosquito may be classified as follows: Phylum—Arthropoda. Order—Diptera. Genus—Aedes. Class—Insecta. Family—Culicidae. Species—Aegypti. The scientific name of an animal is a combination of the names of the genus and the species: the scientific name of the yellow fever mosquito is Aedes aegypti. Section II ARTHROPODS OF MEDICAL IMPORTANCE Procedure iu identification of specimens 458 Crustacea of medical importance j. 459 Myriapoda of medical importance 460 Millipedes 461 Centipedes 462 Arachnida of medical importance 463 Scorpions 464 Whip scorpions 465 Spiders 466 Mites 467 Ticks 468 lusecta of medical importance 469 Anoplura (lice) : 470 Diptera (flies, mosquitoes, etc.) - 471 Mosquitoes 472 Mosquito-like insects _ 473 Stout-bodied biting flies 474 Nonbiting flies (filth flies and myiasis-producing flies) 475 Heteroptera (bedbugs, kissing bugs, cicadas, etc.) 476 Siphonaptera (fleas) 477 Paragraph 458. Procedure in identification of specimens.—a. Since all members of any of the above classes possess certain common 376 TM 8-237 458-462 MEDICAL DEPARTMENT characteristics that differentiate them from the other classes of arthropods, use can be made of these characteristics in making “classi- fication keys,” These keys enable the worker to identify specimens. A working example of such a key follows: 1. If the specimen has three or four pairs of legs 2 If it has numerous pairs of legs 3 2. With three pairs of legs (fig. 34) Insecta (par. 469) With four pairs of legs (fig. 31) Arachnida (par. 463) 3. Lives in water (fig, 28) Ciaxstacea (par. 459) Lives on land (fig. 29) Myriapoda (par, 460) b. An examination of the above key will reveal that identification of specimens is not too difficult a matter. It is simply necessary to “run it through the key.” If by running the specimen through the key it should prove to be an arachnid, it is necessary only to turn to the section on Arachnida where a key to this class will be found. By subjecting the specimen to several such keys, identification can be made. c. The key is only an aid, not a final proof of identification. Since in the insect class alone there are thousands of species, it is obvious that the keys and outlines in this manual will not serve to identify all specimens. They will, however, enable the worker to identify many forms, even though the identification may not be entirely accurate in all cases. For more satisfactory identification this manual should be supplemented by standard texts, and for absolute identifica- tion specimens may be transmitted to entomological centers. 459. Crustacea of medical importance.—Crustaceans (crayfish, shrimp, etc.) are of little importance to the medical soldier, but are worthy of mention because a few species are associated with human disease. Several species of microscopic forms serve as intermediate hosts in the transmission of certain intestinal worms. A typical (but medically unimportant) crustacean is illustrated in figure 28. 460. Myriapoda of medical importance.—Myriapods include the centipedes (Ghilopoda) (fig. 29®) and the millipeds {Diplo- poda) (fig. 29@). They may be differentiated by the number of legs on each body segment, centipedes possessing one pair of legs per segment, whereas millipedes have two pairs on each segment. 461. Millipedes.—The millipedes may for practical purposes be omitted from the list of offenders. They have no fangs and are, therefore, harmless so far as venomous species are concerned. 462. Centipedes.—Certain centipedes, on the other hand, are very important. Although nearly all of the species possess fangs, they are for the most part unable to penetrate the human skin. While 376 METHODS FOR LABORATORY TECHNICIANS TM 8-227 462-463 Figure 28.—Crayfish (a crustacean). ® Centipede. ® Millipede. s Figure 29.—Myriapods. no deaths have been recorded from centipede bite, painful injury can be accomplished. Species of Scolopendra, Geophilus, and Lithohius are capable of injury. 463. Arachnida of medical importance.—The class Arachnida is very important from a medical standpoint, containing many species that serve as transmitters of disease, as well as species that cause disease directly. The following key will serve to separate the more important orders: 1. With abdomen divided into segments 2 Abdomen not divided into segments 4 2. Body divided by constriction into two main parts (fig. 31) Spiders (par. 466) Body not divided by such a constriction « 3 3TT TM 8-227 463-466 MEDICAL DEPARTMENT 3. Minute species (smaller than a pinhead in size) (fig. 32) Mites (par, 467) Medium-sized species (larger than a pinhead in size) (fig. 33) Ticks (par. 468) 4. With spine at tip of tail (fig. 30®) Scorpions (par. 464) Without spine at tip of tail (fig. 30®) —Whip scorpions (par. 465) 464. Scorpions.—Scorpions (fig. 30®) are offensive to man be- cause of their sting, which is accomplished by a spine at the tip of the tail (abdomen). Although many of the smaller species are harmless because they are not able to penetrate the human skin, some species, particularly of the genus Centruroides are very important. In the city of Durango in northern Mexico one species, G. suffusus, causes on the average of 50 deaths per year. The 20 or more common species ® Scorpion @ Whip scorpion. Figure 30.—Comparison of scorpion and whip scorpion. of the southern United States are generally capable of producing only a painful sting. Of interest to soldiers is the fact that some scorpions have a tendency to crawl into shoes during the night. 465. Whip scorpions.—Whip scorpions (fig. 30(D) are very fero- cious in appearance hut are entirely unimportant from a medical standpoint. Their only interest to soldiers is the tendency to confuse them with scorpions. 466. Spiders.—Although all spiders (fig. 31) produce venom, only a few possess fangs sufficiently powerful to penetrate the human skin. Of most importance to the soldier is the “black widow” spider, Latrodectm mactm£. This is a small black spider, which can be dis- tinguished by the reddish hour-glass marking on the underside of its abdomen. It may be found in grass, shrubs, old outhouses, and privies. Its bite produces severe symptoms and in some cases death. 378 TM 8-237 £66-467 METHODS FOR LABORATORY TECHNICIANS Figube 31.—Black widow spider (an arachnid). Tarantulas present a ferocious appearance, but compared to the black widow their bite is mild. Their hairy bodies serve as a con- venient resting place for many bacteria and secondary infection of the wound is common. 467. Mites.—All species of mites (fig. 32) are very small, many being barely visible to the naked eye. In general, only three forms are of medical importance. a. The common itch mite of man, Sarcoptes scahiei, tends to be found where many people may be forced to live together under un- hygienic conditions. In times of national emergency where many soldiers come in close contact with each other, and especially where bathing facilities are meager, cases of scabies may appear. When the mites attack, they usually invade the skin between the fingers, and spread to other parts of the body from these foci. The females bur- Figuke 32.—Itch mite of man. 379 TM 8-227 467-468 MEDICAL DEPARTMENT row into the skin and lay their eggs in the tunnels made during their migrations. The intense itching which this skin invasion produces results in scratching, with subsequent secondary infection. h. The genus Trombicvla contains two very important species. The most important is the Japanese chigger, T. okarrmshi, which transmits Japanese River Fever. This disease is very similar to Rocky Mountain Spotted Fever and is as fatal to man. These mites generally occur on small rodents, especially field mice, which serve as reservoir hosts of this disease. These mites are widely distributed throughout certain parts of Japan. c. The other species of medical interest is the American chigger or redbug. These tiny mites, hardly larger than a pinpoint, are larval forms of Trombicula irritans. Since these larvae are very numerous in the fields during the late spring and early summer, troops work- ing in high grass and weeds during warm weather are subjected to their attacks. The larvae, which have only three pairs of legs instead of four pairs, attach themselves to the exposed parts of the body where their bites produce an intense itching that may last several days. This species is widely distributed in the United States, and is a common cause of annoyance to soldiers. 468. Ticks.—a. The ticks are important chiefly as transmitters of harmful organisms and incidentally as direct cause of disease. They have a widespread occurrence, particularly in the tropics and sub- tropics. For purposes of general classification ticks can be divided into two groups: The soft-bodied ticks (family Argasidae) and the hard-bodied ticks {Ixodidae). (1) “Head” concealed beneath front margin of body, shield on upper surface of body absent (fig. 33(1)), soft-bodied ticks (&(1) below). (2) “Head” not concealed beneath front margin of body, shield on upper surface of body present (fig. 33(D), hard-bodied ticks (6(2) below). h. (1) Soft-bodied ticks.—The members of this group (Argasidae) (fig. 33®) are not fixed parasites as are the hard-bodied ticks {Ixo- didae). Their habits are similar to bedbugs, visiting the host for a blood meal, then returning to the cracks and crevices in which they live. Of the two genera that belong to this family, the genus Omi- thodorus contains nearly all the species of medical importance The members of this genus are very similar in appearance to those of the genus Argas, but may be differentiated since the edges of the body are rounded, whereas these edges are sharp-angled in Argas species. Eyes are frequently present in Ornithodorus and absent in Argas. 380 METHODS FOR LABORATORY TECHNICIANS TM 8-227 468 Soft-bodied ticks, may transmit relapsing fever from animals, hu- man cases, or carriers to other animals or to man. The tick acquires the spirochetes (Borrelia duttoni) when it takes a blood meal, har- bors them for prolonged periods, and later infects the person (or animal) when it takes another blood meal. Hereditary transmission from adult tick to its offspring, through the egg stage, may occur. Opossums and armadillos may serve as reservoirs from which the ticks acquire their infections. The more important species are O. mou- bata in Africa, O. talaje in South and Central x\.merica and Mexico, and O. turicata in the southern United States. (2) Hard-bodied ticks.—The members of this family (fig. 33®) differ in feeding habits from soft-bodied ticks in attaching themselves ® Soft-bodied tick. Figure 33.—Ticks. ® Hard-bodied tick. to their hosts and feeding for long periods of time. They are by far the more widely distributed group, being very well represented in tropical, subtropical, and temperate regions. Of the eight genera belonging to this family, the genus Der-macentor is by far the most important. Others of importance are Haemaphysalis and Ixodes. In general, ditferentiation of the hard-bodied ticks is rather difficult and for such identification the worker is referred to standard texts. Hard-bodied ticks are found associated with various human diseases, the most important of wdiich are Rocky Mountain Spotted Fever, tularaemia, and tick paralysis. Rocky Mountain Spotted Fever, an acute infectious disease of man having a high mortality, is caused by a rickettsial organism (Demiacentroxenus rickettsi). The tick of most importance in the transmission of this micro-organism to man is the Rocky Mountain wood tick, Dermacentor andersoni. The 381 TM 8-327 468-470 MEDICAL DEPARTMENT North American rabbit tick, Haemaphysalis leporis-palustris, is im- portant in transmitting the disease from rodent to rodent. Tularae- mia (rabbit fever), caused by Pasturella tularensis, may be trans- mitted to man by the bites of the wood tick D. andersoni. Again, the rabbit tick, H. leporis-palustris, is important in transmitting the disease among rodents (rabbits). c. In addition to transmitting disease, many ticks are able to inflict injury by the bites alone. Certain species, particularly the wood tick, D. andersoni, and various species of Ixodes are offensive in this re- spect. The bites of these species rarely cause a form of paralysis, and in a few cases, deaths have resulted. The exact mechanism by which the paralysis is effected is not known, but it is probable that the tick secretes some sort of neurotoxin into the wound. d. At times the technician may be presented with small forms that resemble ticks in appearance, but have only three pairs of legs. These forms may be the larval stages (called seed ticks), and should not be confused with insects. 469. Insecta of medical importance.—The class Insecta also contains many species of medical importance. In this group are in- cluded such offensive forms as morquitoes, lice, fleas, bedbugs, etc. The following outline will serve as a guide in classifying the more important orders. Those orders of medical importance are marked with an asterisk (*). For more complete information and keys the reader is referred to standard entomological texts. Insect order Common names of forms Figure *Anoplura Lice .. _ _ 35 Coleoptera Beetles, weevils _ _ _ _ _ _ _ 34® *Diptera Flies, mosquitoes, midges, etc 40 *Heteroptera Bedbugs, kissing bugs, cicadas, etc 43 and 44 Hymenoptera Ants, bees, wasps, etc 34® Lepidoptera Butterflies, moths, skippers 34® Orthoptera _ _. Roaches, crickets, grasshoppers 34® *Siphonaptera Fleas 45 Only the above insect groups indicated as of medical importance will be discussed below. The other groups have been included in order that the technician may observe examples of those groups and thus avoid confusion with important species. He should bear in mind, however, that this list is far from complete. 470. Anoplura (lice).—There are two species of lice that infest man, namely, the head louse and body louse {Pediculus hummius) 382 METHODS FOR LABORATORY TECHNICIANS TM 8-227 470 ® Cockroach (Orthoptera). ® Beetle {Coleoptera). ® Moth (Lepidoptera). ® Ant (Hymenoptera). ' ? Figure 34.—Insects. 383 TM 8-227 470 MEDICAL DEPARTMENT and the crab louse {Phthints pubis). The head louse and body louse are two varieties of the same species, and are called P. humanus var. capitis and P. humanus var. corporis, respectively. The head and body lice and the crab lice are easily differentiated by the length of the body in proportion to its width. Both varieties of P. humanus are about three times as long as they are broad, whereas crab lice are as broad as they are long (see fig. 35). a. The head louse lives among the hairs of the head of its host and attaches its eggs near the base of the hairs by means of a glue formed in a special gland. The hairs around the ears and back of the head are most frequently used as sites for depositing the eggs. b. The body louse usually infests the clothing along the seams, where it attaches its eggs to the fibers of the cloth. Woolen cloth - ® Body louse. ® Crab lOUge. Figure 35.—Lace (Anoplura). ing seems to be the material of choice of the body louse, because the eggs may be easily attached to the wool fibers. c. Man is affected by the head and body louse in two ways: by the direct, mechanical effect of the bites, and by their transmission of pathogenic organisms. The bites produce minute hemorrhagic spots which are accompanied by irritation, often with intense itching, leading to scratching and secondary infection. d. Among the infectious diseases transmitted by lice are typhus, trench fever, relapsing fever, and plague. European or epidemic typhus which resulted in many deaths among soldiers in the Euro- pean armies in the World War, is caused by a rickettsial organism, Rickettsia prowazeki, transmitted by the head and body lice (espe- cially the latter). Transmission may be accomplished in three ways: by deposition of the louse’s feces on the injured skin, by 384 METHODS FOR LABORATORY TECHNICIANS TM 8-227 470-471 crushing the insect against the skin, and by its bite. Trench fever is caused by Rickettsia quintana and transmitted by the bite of the body louse. e. A relatively mild type of relapsing fever caused by the spiro- chete, Borrelia recurrentis, is transmitted by lice when infective lice are crushed on the skin. The disease is not transmitted by the bite. /. It has been shown recently that lice on marmots in western Montana were naturally infected with the organism of plague, Pasturella pestis. g. The crab louse generally frequents the pubic hairs, but it has been found also on other hairy parts of the body, the legs, armpits, beard, eyebrows, and eyelashes. As in the case of the head louse, the crab louse lays its eggs attached to the hairs of the host. This insect provokes some local irritation, but has not yet been incriminated in the transmission of any disease. (T) Larva of house fly. (D Pupa of house fly. Figure 36.—Flies. 471. Diptera (flies, mosquitoes, etc.).—The Diptera is another arthropod order which contains species that may serve both to trans- mit disease and to cause it directly. The importance of some mos- quitoes and tsetse flies in malaria and sleeping sickness is common knowledge, but there are many other mosquitoes and biting flies that carry disease. There are many species living and breeding in close contact with Army personnel that may cause disease directly. It is well known that in their life cycles flies pass through egg, larval, pupal, and adult stages (fig. 36). The larvae (maggots) of some species may gain entrance to the human body and invade the tissue, such an invasion of tissue with fly larvae being spoken of as myiasis. Since the order Diptera contains so many species with variance of form and habit, it is impossible to present a simple key to all the major groups. The following key-outline will, however, serve as a convenient guide in separating the forms. It should be remem- 406303°—41 25 385 TM 8-227 471-472 MEDICAL DEPARTMENT bered that this outline is one of convenience, and for a more accu- rate classification standard texts should be consulted. Since the mosquitoes constitute the most important dipterous group, their classification is treated in more detail, and the technician should be able to make tentative examination of kinds of mosquitoes even though the other forms may be regarded more lightly. Separate any specimens collected as follows: 1. Fragile (mosquito-like) (fig. 40(D) 2 Stout-bodied (housefly and horsefly-like) (fig. 40@) 3 2. Scales on wings (fig. 38) Mosquitoes (par. 472) No scales on wings (fig. 40) Mosquito-like flies (par. 473) 3. Biting flies (fig. 410) Stout-bodied biting flies (par. 474) Nonbiting (fig. 41@) Filth and myiasis flies (par. 475) 472. Mosquitoes.—a. The family Culicidae to which the mosqui- toes belong may be divided into three subfamilies. Of these latter groups the mosquitoes are the only ones that suck blood and are of medical interest. They may be easily distinguished from other com- mon mosquito-like insects by the presence of scales on the veins and margins of the wings (fig. 38). h. Mosquitoes are classified into several genera, most of which will be omitted since they are unimportant as disease transmitters. The forms mentioned in this manual are the malaria mosquitoes (genus Anopheles), the yellow-fever mosquitoes (genus Aedes), and the com- mon house mosquitoes (genus Gulex, etc.). c. The adult anophelines (genus Anopheles) can generally be dif- ferentiated from the adult culicines (genera Aedes, Gulex, etc.) by the presence of spots on the wings (fig. 38), these spots being absent in the latter groups. Other characters that serve to differentiate anophelines and culicines are the position of the body when at rest (fig. 37) and the structure of the mouth parts, especially the palpi (fig. 37). Differences between eggs, larvae, and pupae of anophelines and culicines are illustrated in figure 37. d. To a certain extent members of the genus Aedes can be dif- ferentiated from the other culicines by the presence of white bands on the legs, this character being generally absent in the others. The character is, however, not a valid one, and is given here merely as a suggestion in tentative classification. For more accurate identifi- cation the worker is referred to standard texts. e. Malaria, a common, sometimes fatal, infection caused by various protozoan parasites (Plasmodium) and discussed elsewhere in this manual, is transmitted by mosquitoes of the genus Anopheles. Among the more important species are A. guadHmaculatus and 386 TM 8-227 472 METHODS FOR LABORATORY TECHNICIANS Male female male female 1, Anopheles: A, eggs; B, larva; C, pupa ; D, adult; E, wing of adult; F, mouthparts of adult male and female. 2, Aedes: A, eggs; B, larva ; O, pupa ; D, adult; E, wing of adult; F, mouthparts of adult male and female. S, Gulex: A, typical raft of eggs. Figure 37.—Mosquitoes. (Comparison of various stages of anopheline and culicine mosquitoes.) 387 TM 8-237 472 MEDICAL DEPARTMENT ® A. crucians. @ A. punctipennis. ® A. maculipennis. ® A. quadrimaculatus. ® A. pseudopunctipennis. ® A. altimanus. Fiqdh* 38.—Wings of Anopheles mosquitoes. 388 TM 8-227 472-473 METHODS FOR LABORATORY TECHNICIANS A. maculipennis in the United States, and A. albimanus and A. pseu- do punctipennis in Mexico and Central America. In the West Indies, Panama, and northern South America, A. alhimanus, A. pseudo- punctipennis, and A. punctimacula are the principal malaria-carrying species. Most important in the Philippines is A. mmimus. The wings of several common species are illustrated in figure 38. Larval characters are shown in figure 39. /. Yellow fever is a very fatal virus disease which at times may reach epidemic proportions. It is transmitted by Aedes aegypti. Although many other species of Aedes have been experimentally incriminated in the transmission of yellow fever, A. aegypti is the only one proven to transmit the disease naturally. g. Dengue (breakbone fever) is a virus disease transmitted by mosquitoes. Although not a very fatal disease, during epidemics it causes a great deal of disability. Among the more important trans- mitters of this disease are Aedes aegypti and A. albopictus. h. Filarial worm infections are also transmitted by mosquitoes. In these diseases the mosquitoes serve as intermediate hosts, and when feeding on a person the filarial larvae are transferred to the human host. Although the common house mosquito of the Southern states (Culex guinguefasciatus) is usually said to be the principal vector in filarial infections, many other species included in several genera have been incriminated. 473. Mosquito-like insects.—These are very small, hairy flies that may be confused with mosquitoes. They are differentiated from that group, however, by the absence of scales on the wings. a. The sand flies (family Psychodidae) include several species of the genus Phlebotomus, some of which transmit “Pappataci” fever, kala azar, tropical sore, and some other diseases. b. The gnats (family Simuliidae) include one group of medical interest, the genus Simulium. Certain species are instrumental in the transmission of onchocerciasis in Africa, Guatemala, and southern Mexico. c. The midges (family CMronomidae) are most liable to be con- fused with mosquitoes. The larvae of this family are the familiar “blood worms” found so often in stagnant water. The medically important genus is Culicoides, since many of the species are very annoying biters. Of even more importance is the fact that species of Culicoides transmit a filarial worm from man to man throughout tropical Africa. d. The crane flies (family Tipulidae) are of no medical importance but are mentioned in passing (fig. 40). These large, mosquito-like forms are commonly encountered in warm weather and are almost 389 TM 8-327 473-474 MEDICAL DEPARTMENT ma or breathing pore Palmate hairs Porsal view of an Anophelme Larva brush antenna mandible A. alb imanus tnaxilu A. punchpennis \Ventral and Porsai views of an Anopheline Larva A. pseudopuncdipennis Hypopygium of male Anopheles alhimanus and A. puncilpennis Note. —1. Dorsal view of an Anopheline larva. 2. Ventral view of the head of an Anopheline larva. S. Dorsal view of the head of an Anopheline larva, k. Hypopygium (male sex organ) of an adult A. albimanus. 5. Hypopygium of adult male A. punctipennis. 6. Palmate hairs (hair tufts) of various Anopheline larvae; a, c, e, and g are the hair tufts; b, d, f, and h are the individual hairs. Figure 39.—Structural characteristics of Anopheles mosquito larvae and hypopygium of adult males. always thought by the layman to be large mosquitoes. They may be differentiated from mosquitoes quite easily, since they have no scales on the wings. 474. Stout-bodied biting flies.—This group includes the horse- flies, deer flies, stableflies, and tsetse flies. The species of this group 390 METHODS FOR LABORATORY TECHNICIANS TM 8-227 474 are of interest due to disease transmission as well as annoyance from their bites. a. The horseflies {Tabarms) and deer flies {Ohrysops) (family Tabanidae) (fig. 40) are voracious blood-feeders. In addition to this annoying habit, which is limited to the females, species of Ohrysops serve as transmitters of a filarial worm infection and of tularaemia. Species of Tabanus have been recorded as transmitting anthrax to man and tularaemia to guinea pigs. b. The stablefly, Stomoxys calcitrans (family Muscidae), is a biting fly that resembles the common housefly very much in appearance. Occasionally before a rain it will be noticed that the houseflies are very annoying, and may even bite. When this occurs, it is not the housefly as is commonly believed, but the stablefly. They may be differentiated from houseflies by their biting mouth parts (fig. 41). It has been suggested, but not proven, that the stablefly is a vector of poliomyelitis, anthrax, and tetanus. Both males and females feed on human beings. c. The tsetse flies (family Muscidae) are of extreme importance in Africa, certain species of the genus Glossina being known to transmit ® Crane fly. ® Horsefly. Figure 40.-—Flies. 391 TM 8-227 474^-475 MEDICAL DEPARTMENT © Head of stablefly showing biting mouth parts. ® Head of housefly showing nonbiting mouth parts. Figure 41.—Fly mouth parts, Gambian and Rhodesian sleeping sickness. Both males and females take blood meals. The trypanosomes causing sleeping sickness may be transmitted directly through mere mechanical action, or after undergoing a developmental cycle in the tsetse fly. These flies are unique in depositing larvae rather than laying eggs. 475. Nonbiting' flies (filth flies and myiasis-producing' flies).—This group includes the houseflies, flesh flies, blue and green metallic-colored flies, and other species usually associated with decay- ing filth and garbage. These flies are of interest because they produce disease in two ways, by mechanical transmission of harmful organ- isms o'r by direct invasion of the human body. The mechanical transmission of disease is easily accomplished by the flies’ habits of frequenting filth, garbage, excreta, etc. The housefly is important in the direct transmission of many human diseases, particularly typhoid fever, and including dysentery, tuberculosis, cholera, and anthrax. In addition to transmitting disease by passing from in- fectious material to food, it lays eggs in the infective filth and the maggots and adults that follow are infected with the pathogenic organisms. The housefly {Musca domestica) is the most important member of this group. Others are the flesh fly (Sarcophaga), the nonbiting stablefly (Muscina stabulans), the lesser housefly (Fannia canicularis) and the blue and green metallic-colored flies {Cochliomyia, Lucilia, and Calliphora). In addition to transmitting human disease, some of the above, along with other species, serve to infect man directly. This is accomplished 392 METHODS FOR LABORATORY TECHNICIANS TM 8-227 475 by the gravid females depositing eggs or larvae on the skin or mucous membranes. The eggs hatch and the larvae penetrate the tissues, causing myiasis. In many species laying of eggs on the human host is purely ac- cidental, or the females may have been attracted to a sore or open wound, but in other forms definite attempts are made to deposit eggs on certain parts of the body that the larvae may hatch and invade the tissue. Another not uncommon method of infection is for a per- son to eat food upon which a fly has deposited eggs. The larvae hatch, and if present in considerable numbers, may produce severe irritation of the intestinal mucosa. These larvae may be found in feces sent to the laboratory for examination and should not be con- fused with parasitic worms. Some species lay eggs in the nostrils, and the maggots developing in the nasal passages have been known to cause extensive tissue damage and even death. Although in many cases the sites of invasion of these maggots serve as sites for the en- trance of harmful bacteria, some fly larvae are known to keep the Figure 42.—Stigmal plates of fly larvae. Note.—A, Blowfly (Calliphora) ; B, Green-bottle fly (Luoilia) ; G, Blue-bottle fly (Cynomyia); D, Screw-worm fly (Cochliomyia) ; E, Botfly (Oasterophilus) ; F, Warble fly (Dermatobia) ; G, Flesh fly (Sarcophaga) ; H, Black blowfly (Phormia) ; I, Biting stablefly (Stomoxys); J, Nonbiting stablefly (Muscina) ; K, Flesh fly (Wohlfahrtia) ; L, Housefly (Musca) ; M, Cattle botfly (Hypoderma) ; N, Sheep botfly (Oestris). 393 TM 8-237 475-476 MEDICAL DEPARTMENT wounds very clean. Maggots living in the wounds of soldiers in- jured in battle have been noted to remove the debris and bone frag- ments and thereby promote rapid healing, the basis of the maggot treatment of certain types of wounds. Among the more important myiasis-producing flies are the flesh flies {Sarcophaga and Wohlfahrtia), screw-worm fly {Cochliomyia), blue- bottle or blowfly (Galliphora), green-bottle fly (Lucilia), housefly {Mused), warble-fly {Dermatobia), botfly {Gasterophilus), lesser housefly (Fannia), cattle bot (Hypoderma) and sheep bot {Oestris). Identification of the adult flies is rather difficult and will not be attempted herein, but reference should be made to standard texts. Maggots (fig. 360) are likely to be recovered in stool examinations! and may be presented for identification from breeding areas around Army camps. These larvae, especially in the stage before pupation, can be identified by the pattern of the stigmal plates. These plates are a pair of tiny chitinous structures located at the hind end of the maggot. They may be removed from the maggot and by using the microscope compared with those of the species illustrated in figure 42. In this figure only the left stigmal plate is illustrated. For descriptive details consult standard texts. 476. Heteroptera (bedbugs, kissing bugs, cicadas, etc.).— Although the term “bug” is applied to all members of the class Insecta., strictly speaking only the members of the order Heteroptera are true bugs. The true bugs are very common, many living in water, while some abound on plants and feed on the juices. Some are blood suckers and are very troublesome or even dangerous to man. Figure 43.—Bedbug (Heteroptera). a. The common bedbug {Cimex lectularius) (fig. 43) is world-wide in distribution and is a temporary parasite of man, feeding on his blood and living and breeding in the cracks and crevices of beds and other furniture, and in the walls and floors of his home. In the absence 394 METHODS FOR LABORATORY TECHNICIANS TM 8-227 476-477 of the human host, bedbugs will feed on lower animals. Although the bedbug has been charged with the transmission of human disease, notably European relapsing fever, kala azar, tularaemia, and plague, there has been no definite proof, and its only interest to the medical soldier is the local irritation that some persons suffer from the bites. b. Certain assassin bugs (fig. 44) are of very definite medical in- terest in some localities. These bugs, called kissing bugs because they usually take their blood meals from the lips, are important in the trans- mission of Chagas’ disease. This disease, caused by a trypanosome Fioure 44.—Kissing bug (Heteroptera). {Trypanosoma cruzi), is chiefly a childhood disease, but it not infre- quently occurs in adults and is very fatal. It occurs in South America, especially Brazil. The South American kissing bug, Triatoma megista (synonyms Gonorhinus, Panstrongylus), is particularly important to preventive medicine since it serves as the principal vector of this disease. Closely related species occur in the southern United States, but no human cases of Chagas’ disease have been reported from this country. 477. Siphonaptera (fleas).—There are seven species of fleas of interest to the medical soldier: four because they are associated with human disease; three because they are commonly encountered and 395 TM 8-227 477 MEDICAL DEPARTMENT apt to be confused with those of medical importance. In this group also are to be found insects that both transmit disease and cause it directly. a. The human flea (Pulex irritans) (fig. 45®) is found wherever man lives, but is widely distributed throughout the Western States, especially California. Like the bedbug, this flea lives in the cracks and crevices of the home, in the floors, rugs, and bedding, emerging at night to attack the hosts. The human flea feeds readily on dogs, squirrels, and other animals as well as on man. h. The chigoe {Tunga penetrans) is the smallest flea known, and passes its life cycle as a fixed parasite of man and animals. The skin between the toes is most frequently attacked, with irritation and swelling. This results when the female fixes her mouth parts in the Figure 45.—Fleas (Siphonaptera). © Human flea (Pulex Irritans). © Dog flea (Ctenocephalus canis). ® Chicken flea (Echnidnophaga gallinacea). ® Temperature zone rat flea (Geratophyllus Jasciatus). © Tropical rat flea (Xenopsylla cheopis). ® Heads of human flea (left) and tropical rat flea (right) showing, arrangement of stout bristles in relation to eyes. (Note that in human flea a stout bristle is directly below the eye, whereas in the tropical rat flea it is in front of the eye.) 396 METHODS FOR LABORATORY TECHNICIANS TM 8-227 47.7 Figure 45.—Fleas (Siphonnptera■)—Continued. 397 TM 8-337 477 MEDICAL DEPARTMENT skin. The swelling encircles the entire insect except for a small open- ing at the hind end. It is through this opening that the chigoe is able to get air, and to lay her eggs which drop to the ground. Fol- lowing this egg laying, the flea shrivels up and dies. Secondary in- fections of the attacked sites is common. The chigoe is widely dis- tributed in tropical America and tropical Africa. It resembles the chicken flea very much in appearance (fig. 45(D), c. The tropical rat flea (Xenopsyllo cheopis) (fig. 45®) is the most important vector of disease, particularly of bubonic plague. This flea is widely distributed in tropical regions throughout the world, and as a rule is not found in colder climates. It is distributed in the western and southern parts of the United States. Although primarily a parasite of the rat, during an epidemic it transmits plague from rat to rat, rat to man, and man to man. d. The temperate zone rat flea {Geratophyllus fasciatus) (fig. 45®) is another species of medical importance. It, too, maintains plague among rats and transmits the disease to man. It is world-wide in distribution, but for the most part is confined to the temperate zones. e. The dog and cat fleas (Gtenocephalus canis and Gtenocephalus felis) (fig. 45®) are world-wide in distribution. The dog flea is widely distributed throughout the temperate climates of the United States and is the dominant ectoparasite of domestic pets, especially dogs and cats. The cat flea on the other hand, is more prevalent in warm climates, but may also be found in temperate regions. The cat flea has a wider range of hosts, but both species may infest man, rats, and other mammals. These two species may be found in enormous numbers in homes where cats and dogs are kept as pets and allowed to sleep in the house. /. The mouse flea (Leptosylla segnis) is a common ectoparasite of mice and rats in the Eastern Hemisphere but is also widely distributed in the Americas. This species is important in transmitting plague from rat to rat. g. The chicken flea {Echnidnophuga gallinazea) (fig. 45®) is of interest because it is commonly encountered and may be confused with more important species. It is very similar in appearance to the chigoe. h. The most important human disease transmitted by fleas is plague, that age-old destroyer of mankind, caused by a bacillus (Pasturella pestis). Of all the fleas known to transmit this disease to man the tropical rat flea is by far the most important. The temperate zone rat flea is able to transmit this disease but does not have much oppor- tunity to do so since plague is somewhat limited to tropical countries. The human flea is considered important in transmitting plague among 398 TM 8-227 477-479 METHODS FOR LABORATORY TECHNICIANS small animals and in times of epidemic may transmit the disease to man. Although other fleas may serve in this respect during epidemics, they are of very minor importance. i. Murine or endemic typhus, a mild infection with Rickettsia mooseri, is also transmitted by fleas. This infection, although not very common, is found along the South Atlantic and Gulf Coasts. Although this disease may be transmitted from man to man by human body lice, it is commonly transmitted from rat to man by the tropical rat flea and the temperate zone rat flea. j. Some fleas, particularly the dog fleas, cat fleas, and human fleas, serve as intermediate hosts in certain helminth infections. Section III HANDLING AND SHIPMENT OF SPECIMENS Paragraph Collection of specimens 478 Preparation, identification, and shipment 479 478. Collection of specimens.—a. Collect lice and ticks by pick- ing them with forceps from the person or animal infested. Before picking ticks, a few drops of chloroform or ether should be placed on the specimens. This kills the tick and permits easy withdrawal of the mouth parts from the skin. If this procedure is not followed, the heads frequently remain in the puncture sites resulting in secondary infection. b. Collect fleas from infested persons by picking. They may be collected from rats by killing the rat and combing out the fleas with a fine-toothed comb. c. Adult mosquitoes may be collected by trapping them in a wide- mouthed bottle while resting or feeding. “Killing bottles” used for this procedure may be made by moistening cotton with chloroform or ether and stuffing it in the bottom of the bottle, then covering cotton with a circle of blotting or filter paper. This killing bottle may also be used to kill other arthropods. d. Mosquito larvae may be collected by dipping from water in which they breed. e. Mites may be collected by scraping infected areas of skin. 479. Preparation, identification, and shipment.—a. Prepara- tion.— (1) Fleas, lice, and mites may be mounted in balsam on micro- scopic slides employing the usual technical methods of dehydrating and clearing. (2) Spiders and ticks may be studied unmounted. 399 TM 8-227 479 MEDICAL DEPARTMENT (3) Larger insects may be mounted by pinning through the thorax. (4) Arthropod larvae may be killed by dropping into very hot water. They may be preserved in 70 percent alcohol. b. Identification.—(1) Using this manual as a guide, make a tenta- tive identification of the specimen. (2) Check the identification with figures and descriptions in any standard texts available, especially those given in the appendix. (3) If the specimen appears to be of medical importance, or if it belongs to .a medically important group, forward it to the Army Medical Museum, Washington, D. C., for accurate identification. c. Shipment.—(1) Pack the specimen loosely in a small box between strips or sheets of lens paper or soft toilet tissue. (2) Do not pack the specimen in cotton, because this material clings to the specimen and makes manipulation difficult. (3) Send all data relating to specimen, including source, date, col- lector, conditions under which collected, and any other pertinent remarks. (4) Mark the package “Fragile.” 400 METHODS FOR LABORATORY TECHNICIANS TM 8-227 480-481 Chapter 15 PATHOLOGICAL METHODS Seotkust I, Gross pathology methods 480-486 II. Histologic technic 487-493 Paragraphs Section I GROSS PATHOLOGY METHODS The morgue 480 Restoration of body 481 Embalming 482 Fixation of tissue 483 Shipping wet-tissue specimens 484 Gross specimens I 485 Preparation 486 Paragraph 480. The morgue.—a. General.—The morgue should have light, ventilation, good artificial illumination, running water, and gas. A floor drain and an overhead water tap to which a short hose is attached are desirable, A cabinet should be provided for the instruments and these should be sterilized in a creosote solution and then thoroughly washed, completely dried, and carefully placed in the cabinet after each autopsy. If an autopsy table is not supplied one can be im- provised by covering a wooden table with galvanized iron or sheet lead, allowing a gentle slope to the foot of the table and a drain with a pipe fixture to open over the floor drain. The table should be thoroughly scrubbed with a creosote solution after each autopsy. Rubber gloves should be used by all engaged in performing autopsies or handling the organs. These should be washed while on the hand with soap and water, then reversed as they are removed and the insides similarly cleaned. Both surfaces should be thoroughly dried and covered with talcum. They need not be sterilized but should be thor- oughly cleaned after each use. h. Collection of specimen#.—Specimens removed during the autopsy for histologic examination are placed immediately in fixing solution. Gross specimens which are to be preserved should be kept moist and placed in a preserving solution as early as possible. 481. Restoration of body.—At the conclusion of the autopsy all excess fluid should be removed from the cavities, the rectal, vaginal, and urethral openings closed, the organs and sternum replaced, and 406303°—41 26 401 TM 8-327 481-483 MEDICAL DEPARTMENT the incision sewed, using the “baseball” stitch. Begin at the upper end of the incision, sew from within out, taking liberal bits of skin and muscle, keep the string taut, and use uniform stitches about 2 cm apart. The body is then thoroughly washed, taking care to remove all blood stains especially from the hair, face, and hands. If one has been careful throughout the necropsy to keep the body clean so that the blood has not dried on the skin, the cleansing is a simple matter. If the head has been opened, the base of the skull is filled with plaster of paris, the skull cap replaced and the scalp sutured in a similar manner to that just described for the body. The brain is not replaced in the skull but with the other organs in the body cavity. If the spinal canal has been opened it is stuffed with cotton or oakum over which the spines are replaced and the incision then sewed as above. 482. Embalming1.—a. Preparation of body.—The embalming of the head is readily done by the undertaker when the chest is open, but in his absence may be done very easily by anyone else. If shav- ing is necessary it must be done before the face is embalmed. The undertaker’s pressure bottle with several tubes armed with long metal cannulae, which are tied into the carotids and subclavian arteries, is most convenient. Pressure is obtained with a pump. If this is not available an alpha enema syringe will suffice. The nozzle is tied into the upper thoracic aorta. The open end of the aorta as well as any leaking arteries (internal mammaries) must be closed with clamps or tied. b. Technic.—Undertaker’s embalming fluid or a 10 percent solu- tion of formalin in water, to which a few drops of eosin solution are added to give it the faintest possible tinge of pink, may be used. As the fluid is pumped into the arteries and begins to drive blood before it out of the veins, the face and ears must be massaged and molded with a gauze sponge into a natural pose, with eyes and lips closed. The hands should also be massaged until white. When the tissue be- comes blanched and firm the process is complete. The same process is applied to the legs, the fluid being injected through the femoral arteries. Some formalin should be allowed to stand for a time in the body cavity. It is well to soak the organs in a 10 percent formalin solution for several hours before replacing them in the body, making incisions in the'solid organs and numerous punctures in the gastro- intestinal tract if they have not been opened. Undertaker’s harden- ing compound, oakum, or cotton should be spread over the organs after they have been replaced. 483. Fixation of tissue.—Pieces of tissue not more than 1 cm in thickness will be selected from representative parts of the various 402 TM 8-227 483-485 METHODS FOR LABORATORY TECHNICIANS tissues and fixed in approximately 20 times their volume of 10 per- cent formalin. The formalin solution will be changed the following day and again immediately before packing for mailing. Such tis- sue is designated “wet tissue.” 484. Shipping wet-tissue specimens.—For mailing small frag- ments of tissue the double mailing case (item No. 41270) is satis- factory. The wide-mouthed bottle (item No. 40590 taking a No. 20 cork, item No, 77700) will prove satisfactory as it will fit in the mailing case. For protection it should be carefully surrounded with absorbent cotton. The label should be marked “First class mail, rush, specimen for diagnosis.” Shipments exceeding 4 pounds in weight will be made by express after having obtained procurement authority from the Curator of the Army Medical Museum, Seventh Street and Inde- pendence Avenue, SW., Washington, D. C. 485. Gross specimens.—a. General.—If preservation of color is not a factor, as when an organ is to be sent to the Army Medical Museum for examination, fix in abundant 10 percent formalin taking care that all parts are in contact with the solution. Bulky specimens, as a liver, should be sliced or injected. After fixation, this may be shipped in a minimum amount of solution, using any watertight container available. h. Preservation of color.—(1) Procedure.—To preserve color in gross specimens, they should be quickly washed in water to remove excess blood and placed in Kaiserling’s solution No. 1. It is neces- sary to arrange the specimens in this solution as it is intended for them to appear when finally mounted, as they will become fixed as placed in the solution and it is difficult to alter them after this fixation. The length of time in the No. 1 solution varies from 1 to 7 days, depending on the size of the specimen. If the specimens are very large, it is advisable to inject fixative into the blood vessels, or to impregnate the tissues by means of a syringe and needle. The specimen should be supported by cotton or suspended by strings so that it will not be in contact with the con- tainer. Avoid direct light during all steps. (2) Fixative.—(a) Kaiserling'1 s solution No. 1. Formalin 400 cc Water 2,000 cc Potassium nitrate 30 gm Potassium acetate 60 gm (b) Kaiserling's solution No. 2. Alcohol 95 percent 403 TM 8-227 485-486 MEDICAL DEPARTMENT (c) Kaiserling"’ s solution No. 3. Potassium acetate 200 gm Glycerin 400 cc Sodium arsenate 100 gm Water 2,000 cc Note.—If sodium arsenate is not available some crystals of thymol, menthol, or sodium salicylate may be used instead, but the arsenate is preferable as a fungus deterrent. (3) Care after fixation.—After fixation, the specimen is drained and blotted, and then placed in 95 percent alcohol (Kaiserling’s solu- tion No. 2). When the maximum color has returned, which it will do in a few minutes or an hour or so, the specimen is to be removed from the alcohol and thoroughly washed and then preserved in Kaiserling’s solution No. 3, It is necessary to watch the develop- ment of the color, for after it has reached a certain point it will begin to fade and it is impossible to restore it again. Over-fixation in solution No. 1 is to be avoided; therefore, if the specimen is to be shipped some distance, it should be run through the alcohol and for- warded in solution No. 3. It is possible to develop some color in formalin-fixed tissue, but it is not satisfactory. 486. Preparation.—a. The preparation of museum specimens must be left largely to the ingenuity of the operator and only a few general principles can be given. The surface to be displayed should represent as large a section as possible of the whole organ and both the exterior and interior of the organ should be shown. In the case of solid organs such as the liver, a thick slice (5 cm) should be preserved, as it is impossible to fix a whole liver properly. The thickness of the slab should allow for the removal of a thin layer at a later date to freshen the surface. This is particularly true of the lung, in which case one-half or even the whole organ may be preserved. Nothing solid should be allowed to touch the surface of the fresh tissue until it is fixed and hardened. h. It is injurious to pack cotton firmly into a cavity, since after fixation the lining of the cavity will appear merely as a mold of the cotton. If a hollow organ must be held open it is best to distend it with fixing fluid for a day or two before cutting into it. If this is no longer possible, and it must be propped open with cotton, this should at least be inserted very loosely. c. The heart, after being opened, should be stretched on an im- provised frame in such a way as to display to advantage the chief lesion, or it may be clamped together and held in its original form by a few temporary stitches during fixation. 404 METHODS FOR LABORATORY TECHNICIANS TM 8-227 486-487 d. The stomach or portions of the intestine can be filled with Kaiser ling's fluid or 10 percent formalin and ligated at the ends until hardened, after which they can be bisected longitudinally. Other- wise, they may be opened, stretched on a board with thread so that the mucosa is exposed, and immersed in the fixing fluid. e. In the case of the kidney, one-half of the organ cleanly cut, forms a satisfactory specimen. Section II Paragraph Formulas 487 Frozen section method 488 Routine paraffin method 489 Rapid paraffin method (Mallory and Wright, method No. 3) 490 Special stains 491 Decalciflcation 492 Giemsa’s staining method 493 487. Formulas.—a. Mayer’s albumin. White of egg 50 cc Glycerol 50 cc Sodium salicylate 1 gm Shake well together and filter into clean bottle. b. Acid-alcohol.—One percent of hydrochloric acid in 70 percent ethyl alcohol. c. Kinyoun’s carbol-fuchsin. Basic fuchsin (rosaniline hydrochloride) 4 gm Phenol crystals 8 gm Alcohol, 95 percent 20 cc Water 100 cc d. Decalcifying fluid.—Ten percent nitric acid in 10 percent formol saline, or the following mixture; Formic acid 50 cc Formalin, 10 percent 50 cc See directions for use at end of this chapter. e. Eosin. Eosin Y (di-sodium tetrabromfluorescein) 0.5 gm Alcohol 25.0 co Distilled water 75.0 cc This solution will keep indefinitely. HISTOLOGIC TECHNIC 405 TM 8-337 487-488 MEDICAL DEPARTMENT /. Harris' hematoxylin. Hematoxylin 1 gm Alcohol 10 cc Dissolve dye in alcohol. Alum (ammonium or potassium) 20 gm Distilled water 200 cc The alum is dissolved in water with the aid of heat, and then the alcoholic solution of the dye added. The mixture is brought to a boil rapidly and then 0.5 gm of mercuric oxide (red oxide) added. The solution at once assumes a dark purple color and as soon as this occurs it is cooled by plunging the flask into cold water. For use, 4 percent glacial acetic acid is added to the mixture, as this increases the precision of nuclear staining. g. Oil red O (Sudan III or Sudan IV) fat stain. Oil red O 1 gm Acetone 50 cc Seventy percent alcohol 50 cc 488. Frozen section method.—It is possible with this method to prepare a slide for examination in a few minutes so that it is par- ticularly applicable to “operating-room diagnosis.” It is also the method of choice when it is desired to stain for fat. a. Materials. Automatic freezing microtome, and sharp knife. Tank of C02 and connection; the tank to be mounted inverted. A shallow dish filled with water. A mounted needle, glass rod drawm to dull point, or pair of fine, smooth-pointed forceps. Two pyrex test tubes. Bottle of 10 percent formalin. Bottle of 1 percent ammonia water. Bunsen burner or alcohol lamp. Glass slides and cover glasses. Seven small glass dishes. Harris’ hematoxylin. Eosin. Ninety-five percent alcohol. Absolute alcohol. Carbol-xylol. Canada balsam. Blotting paper. 406 TM 8-227 488-489 METHODS FOR LABORATORY TECHNICIANS h. Technic.—If the tissue has not already been fixed, a block about 0.5 cm thick is boiled for 1 minute in 10 percent formalin in a test tube and rinsed in tap water. It is placed on the freezing stage of the microtome with a few drops of water and frozen, pressing it gently with the finger during this process. Do not freeze too hard; cut at 12 to 16 microns, remove the sections from the knife with the finger, and float them in water. Select full sections and transfer in turn to the following, which are in small shallow glass dishes: Harris’ hematoxylin, 30 to 60 seconds. One percent ammonia water, until blue. Tap water, rinse. Eosin, 5 to 15 seconds. Ninety-five percent alcohol, rinse. Absolute alcohol, 5 seconds. Carbol-xylol, 5 seconds. Mount on slide in Canada balsam. 489. Routine paraffin method.—a. Materials.—The following are required in addition to those enumerated in paragraph 488; Oven with automatic control, temperature 56° C. Paraffin, refined, melting point 52° to 56° C. Containers for paraffin in oven: beakers, or casseroles and Stender dishes. Chloroform. Acid-alcohol. Xylol. Paper or metal forms for molding blocks. Two basins or photograph developing trays. Ice. Rotary microtome. Sharp knife. Hone and strop. Camel’s-hair brushes, one pointed, one 1 to 1 y2 inches wide. Twelve Coplin jars. b. Fixation.—Specimens of tissue which are to be examined microscopically should be fixed as quickly as possible after surgical removal or after death of the patient. TM 8-237 489 MEDICAL DEPARTMENT Fixative Formula Fixation time After treatment Formalin alcohol Neutral formalin _ 10 cc 95 percent alcohol . _ 90 cc 12 to 24 hours _ _ __ Wash in water. Transfer to 80 percent alcohol. Absolute alcohol Absolute alcohol (changed after 3 to 4 hours) 12 to 24 hours _ ___ 80 percent alcohol. 10 percent formalin. Neutral 40 percent formaldehyde__ 10 parts Water _ 90 parts Calcium carbonate (to neutralize). 24 hours Store in same fluid. Zenker acetic _ Potassium bichromate _ 2.5 gm Corrosive sublimate ... 5 to 8 gm Distilled water _ _ . 100 cc Glacial acetic acid (just before use)__ 5 cc 12 to 24 hours Wash 12 to 24 hours in running water. Transfer to 80 percent alcohol. Helly (Zenker-for- mol). Potassium bichromate . _ 2.5 gm Corrosive sublimate __ 5 to 8 gm Distilled water ______ 100 cc Formalin (just before use) _ 5 to 10 cc 12 to 24 hours Wash 12 to 24 hours in running water. Transfer to 80 percent alcohol. Schaudinu _ _ _ Mercuric chloride (saturated aqueous 2 parts solution). Absolute or 95 percent alcohol _ 1 part Glacial acetic (proportion of 5 percent added just before use). 48 hours. (Renew after 24 hours.) 70 percent alcohol. Transfer to 80 percent alcohol. Table XX.—Tissue fixatives 408 METHODS FOR LABORATORY TECHNICIANS TM 8-227 489 Carnoy__ _ _ __ Absolute alcohol __ . _ _ 60 cc Chloroform __ 30 cc Glacial acetic acid (add just before use) _ _ 10 cc 1)4 to 3 hours (not over 3 hours). Absolute alcohol, 12 to 18 hours. Chloroform, 2 changes, 1 hour each. Chloroform-paraffin, 4 hours. Formol bichloride.- Corrosive sublimate (saturated aqueous 45 cc solution, about 6.9 percent). 40 percent formalin _ 5cc At least 48 hours (change after 24 hours). 80 percent alcohol. Bouin Picric acid (saturated aqueous solution, 75 cc 1.22 percent). Formalin . _ _ __ _ . 25 cc Glacial acetic acid _ _ _ _ . 5 cc 18 to 24 hours Wash in 50 percent alcohol. 70 percent to remove picric acid. Transfer to 80 percent alcohol. 4:09 TM 8-227 489 MEDICAL DEPARTMENT A 10 percent solution of formalin is the most convenient and gen- erally practicable fixative. For finer cellular studies and some special stains, it is necessary to fix in one of the chromate solutions, of which Zenker’s is the most popular. If it is desired to stain for glycogen, aqueous fixatives must be avoided as glycogen is soluble in water, and absolute alcohol must be used. Blocks of tissue should be about 0.5 cm in thickness and they should be placed in an excess of the fixative agent to insure thorough im- pregnation. If formalin is used, the tissue may be kept in it indefinitely. If fixation is in Zenker’s, the blocks are to remain in it but 24 hours, then washed in running water for 24 hours and preserved in 80 percent alcohol. c. Embedding and cutting.—Blocks not more than 0.5 cm thick that have been fixed in 10 percent formalin or Zenker’s fluid are placed in the following: (1) Ninety-five percent alcohol, 2 to 4 hours. (2) Absolute alcohol, 2 to 4 hours. (3) Chloroform, 2 to 4 hours. (4) Chloroform saturated with paraffin, overnight in warm place at about 37° C. Note.—The above steps are carried out in wide-mouthed, tightly corked bottles. (5) Paraffin, 2 to 4 hours in oven. (6) Embed in paper or metal forms, with desired surface down, being sure to eliminate air bubbles. To prevent crystallization of paraffin, the mold should be immersed in ice water while paraffin is still melted. (7) Trim block so that opposite edges about tissue are parallel, leaving narrow margin of paraffin. (8) Mount on metal block holder by heating latter, pressing on block, and immersing all in ice water. (9) Cut sections as thin as possible, under 10 microns. Be sure knife is sharp, tightly clamped in microtome, that its edge inclines toward block just enough so that block misses back surface of knife, and that lower edge of block and knife edge are parallel. (10) Lay sections on surface of water sufficiently warm to insure complete spreading of section (40° to 50° C.). If sections are in ribbons, they may be separated by touching while in the water with the edge of a heated scalpel. (11) Float section onto slide that has been very lightly smeared with Mayer’s albumin. (12) Drain off water and place slide in oven for y2 hour to fix albumin. 410 METHODS FOR LABORATORY TECHNICIANS TM 8-227 489-491 d. Staining.—In the following steps, use a series of Coplin jars lined up in proper order. (1) Remove paraffin by immersing slide in xylol for several minutes, then in absolute alcohol 1 minute. (2) Ninety-five percent alcohol 1 minute. (If tissue has been fixed in Zenker’s, add sufficient iodine to the alcohol to give it a light port-wine color. This is to remove the precipitate of mercuric chloride, and requires 5 to 10 minutes. Rinse in clear 95 percent alcohol.) (3) Wash in tap water. (4) Harris’ hematoxylin, 10 minutes. (5) Differentiate in tap water until nuclei are blue. (6) Eosin, 2 minutes. (7) Ninety-five percent alcohol, to remove excess eosin. (8) Absolute alcohol, 1 minute. (9) Xylol, two changes, 2 minutes each. (10) Mount in Canada balsam. 490, Rapid paraffin method (Mallory and Wright, method No. 3) .—Tissues already fixed in formalin or fresh tissues boiled 2 to 3 minutes in 10 percent formalin may be used. Blocks should not be more than 5 mm thick. a. Acetone, two or three changes, 1 to 2 hours. h. Benzene, two changes, 30 minutes. c. Paraffin, two changes, 45 minutes each. d. Following the above method, proceed as in step (6) under routine paraffin method (par. 489c). 491. Special stains.—a. Stain for fat.—(1) Stain frozen sections for 1 minute in oil red 0 solution. (2) Wash in water. (3) Counterstain in Harris’ hematoxylin for 1 to 2 minutes. (4) Develop blue of hematoxylin in tap water. (5) Blot as dry as possible. (6) Clear in aniline. (7) Mount in glycerol. The fat is stained red. h. Stain for acid fast bacilli.—(1) Remove paraffin in xylol (2 changes) then place in absolute alcohol for 1 minute, 95 percent alcohol 1 minute, water 1 minute. (2) Stain 1 hour in oven at 56° C. in Kinyoun’s carbol-fuchsin. (3) Rinse in water. (4) Decolorize in acid alcohol until only thicker portions are pink (a minute or two). 411 TM 8-227 491 MEDICAL DEPARTMENT (5) Wash in water until a pale pink. (6) Stain in Loeffler’s methylene blue a few seconds. (7) Wash quickly. (8) Ninety-five percent alcohol-absolute alcohol-xylol, two changes. (9) Mount in balsam. c. MacC album's stain for bacteria.—(1) Paraffin sections affixed to the slide are passed through xylol and alcohols to water, and stained for 10 minutes to y2 hour in the Goodpasture mixture prepared as follows: Thirty percent alcohol 100. 00 cc Basic fuchsin 0. 69 grn Aniline 1. 00 cc Phenol crystals 1. 00 gm (2) They are then washed in water and differentiated in 40 percent formaldehyde. This requires only a few seconds, the bright red color being washed away and replaced by a clear rose. Sections are next washed in water and counterstained in a saturated aqueous solu- tion of picric acid. The section remains in this until it assumes a purplish color (3 to 5 minutes); then it is washed in water and differ- entiated in 95 percent alcohol; the red reappears and some of it is washed out as is some of the yellow of the picric acid. The sections are then washed in water and stained for 3 to 5 minutes in Sterling's gentian violet prepared as follows: Crystal violet 5 gm Alcohol 10 cc Aniline 2 cc Water 88 cc (3) Then washed in water and immersed in Gram’s iodine solution 1 minute. Iodine 1 gm Potassium iodide 2 gm Water 300 cc (4) The slides are finally blotted dry without washing, treated in equal parts of xylol and aniline until no more color comes away, passed through two changes of xylol, and mounted in balsam. Gram-negative organisms are stained red; Gram-positive, blue. d. Wilder's stain for reticulum.—(1) Remove paraffin with xylol, then through absolute and 95 percent alcohol to water. (2) Potassium permanganate, 0.25 percent, 1 minute. (May use 10 percent phosphomolybdic acid instead.) 412 METHODS FOR LABORATORY TECHNICIANS TM 8-327 491 (3) Rinse in distilled water. (4) Place in diluted hydrobromic acid, 1 minute. (Merck’s con- centrated 34 percent, 1 part; distilled water, 3 parts.) After phos- phomolybdic acid this step may be omitted. (5) Wash in tap water and then in distilled water. (6) Dip for 5 seconds or less in 1 percent uranium nitrate (sodium-free). (7) Wash 10 to 20 seconds in distilled water. (8) Place in silver diamino hydroxid (foot), 1 minute. (Prepara- tion : To 5 cc of 10.2 percent silver nitrate, add ammonium hydroxide drop by drop until the precipitate which forms is dissolved. Add 5 cc of 3.1 percent sodium hydroxide and just dissolve the resulting precipitate with a few drops of ammonium hydroxide. Make up to 50 cc with distilled water.) (9) Dip quickly in 95 percent alcohol. (10) Reduce in: distilled water, 50 cc; 40 percent neutral formalin (neutralized with magnesium carbonate), 0.5 cc; 1 percent uranium nitrate, 1.5 cc. (11) Wash in distilled water. (12) One minute in 1: 500 gold chloride solution (Merck’s reagent). (13) Rinse in distilled water. (14) One to two minutes in 5 percent sodium thiosulfate. (15) Wash in tap water. (10) Counterstain if desired with hematoxylin. (Overstain may be reduced with acid-alcohol and then neutralized with tap water; do not use ammonia water.) (17) Dehydrate and mount. e. Masson's stain {modified).—(1) Solutions,—(a) Picric acid solution. Alcohol absolute — 100 Picric acid to saturate (about 6 percent). (b) Weigerfs acid iron chloride hematoxylin. Hematoxylin 1.5 percent in 95 percent alcohol (aged) 1 part Liquor ferri 1 part Mix just before using. (c) Liquor ferri. Liq. ferri. sesquichlorate 4 cc Distilled water 95 cc Hydrochloric acid 1 cc 413 TM 8-227 491-492 MEDICAL DEPARTMENT id) Biebrich scarlet. Biebrich scarlet, 1 percent 9 cc Acid fuchsin, 1 percent 1 cc Glacial acetic acid 0.1 cc (e) Pkosphomolybdic-phosphotungstic acid mixture.—Equal parts of 5 percent solution of each acid. (/) Fiber stain. Fast green (FCF) 2.5 gm Acetic acid, 2,5 percent solution 100 cc To prepare: Boil 100 cc distilled water and remove from flame; add immediately 2.5 gm aniline blue or fast green (FCF). Boil a short time to dissolve dye. Add 2.5 cc glacial acetic acid. Cool and filter. (Aniline blue may be used instead of fast green in same quantity and prepared similarly.) (g) Salicylic acid balsam.—A few crystals of acid salicylic added to an ounce of balsam. (Or a few drops of salicylic acid-xylol may be placed on the section just before mounting in neutral balsam.) (2) Technic.— (a) Paraffin sections through xylol and alcohol to saturated alcoholic picric acid, 2 minutes. (b) Wash 3 minutes in running water. (c) Weigert’s acid iron hematoxylin, 6 minutes. (d) Rinse in water. (e) Biebrich scarlet, 2 minutes (4 minutes if aniline blue is used in step h below). (/) Rinse in distilled water. {g) Mordant 1 minute in phosphomolybdic-phosphotungstic acid mixture. (A) Stain 2 minutes in fiber stain (fast green or aniline blue). (i) Differentiate 1 minute in 1 percent aqueous acetic acid. (y) Rinse in water. (A) Dehydrate and mount (alcohols, acetone, xylene, (xylene, 2 changes), salicylic acid balsam). 492. Decalcification.—Tissues containing bone are fixed in 10 percent formalin, then placed in the decalcifying fluid until the lime salts are removed, changing fluid daily. This requires from 1 to 7 days and may be determined by piercing the block with a needle. Suspend the tissues in neutral 10 percent formalin over magnesium carbonate until blue litmus paper is not changed by touching the tissue to it. Then wash in running water for 24 hours, after which the blocks are ready for dehydration and embedding, beginning with >step (1) of routine paraffin method (par. 489c). 414 METHODS FOR LABORATORY TECHNICIANS TM 8-227 493 493. Giemsa’s staining method.—a. Reagents.—(1) Stock ace- tone.—To 500 cc of acetone add 0.1 cc of acetic acid, glacial. (2) Stock Giemsa’s stain.— (a) Dissolve 0.5 gm of Giemsa’s stain (certified powder) in 33 cc of glycerin at 55° to 60° C. for 11/2 to 2 hours. To this add 33 cc of methyl alcohol which has been heated to the same temperature. Caution: Heat alcohol in a screw-top bottle in a paraffin oven or in hot water. Keep away from flame. When the solution has cooled, filter and keep in a tightly stoppered bottle. (h) A satisfactory Giemsa’s stain may be also prepared in the same manner, using 0.6 gm of Azure II eosin and 0.16 gm of Azure II in 50 cc of glycerin and 50 cc of methyl alcohol. The Azures replace the certified powder. h. Technic.— (1) Bring sections (Zenker-, Schaudinn-, or formalin- fixed sections) to water in the usual way for each technic. (2) Rinse in distilled water. (3) Place in Giemsa’s stain 3 to 4 hours observing depth of stain at the end of each hour (should be somewhat overstained). (4) Remove from stain, drain, blot, and wave in air to dry slightly. (5) Differentiate in the stock acetone, under observation. (6) Drain slightly and place in xylol (2 changes); mount in cedar wood oil (immersion). 415 TM 8-327 MEDICAL DEPARTMENT Appendix LIST OF REFERENCES AH 40-205, Military Hygiene and Sanitation. AR 40-210, 215, 220, 225, 230, 235, 240, and 245. The Prevention of Communicable Diseases of Man. AR 40-305, Corps Area and Department Laboratories. AR 40-310, Collection and Preparation of Laboratory Specimens. AR 40-405, Army Medical Library. AR 40-410, Army Medical Museum. AR 40-1705, Medical Supplies. Medical Supply Catalog, IT. S. Army (for standard supply items). Standard Methods of Water Analysis, American Public Health Association. Standard Methods of Milk Analysis, American Public Health Asso- ciation. Diagnostic Procedures and Reagents, American Public Health Asso- ciation, 1941. Bergey: Manual of Determinative Bacteriology, Williams and Wilkins, 1939. Bodansky and Bodansky: Biochemistry of Disease, MacMillan, 1940. Bray: Synopsis of Clinical Laboratory Methods, Mosby, 1938. Cantarow and Trumper: Clinical Biochemistry, Saunders, 1939. Clark: The Determination of Hydrogen Ions, Williams and Wilkins, 1928. Comstock: Introduction to Entomology, Comstock Publishing Co., Ithaca, New York, 1933. Craig and Faust: Clinical Parasitology, Lea and Febiger, 1940. Dodge: Medical Mycology, C. V. Mosby Co. Dorland: Medical Dictionary, W. B. Saunders Co. Fales and Kenny: Inorganic Quantitative Analysis, Appleton-Cen- tury, 1939. Fox: Insects and Disease of Mam, P, Blakiston’s Son & Co., 1925. Gay: Agents of Disease and Host Resistance, Thomas, 1937. Gortner: Outlines of Biochemistry, Wiley and Sons, 1929. Gradwohl: Clinical Laboratory Methods and Diagnosis, Mosby, 1938. Hawk and Bergeim: Practical Physiological Chemistry, Blakiston, 1937. Herms: Medical Entomology, MacMillan, 1939. 416 METHODS FOR LABORATORY TECHNICIANS TM 8-227 Kelser: Manual of Veterinary Bacteriology, Williams and Wilkins, 1939. Kolmer and Boerner: Approved Laboratory Technic, Appleton- Century, 1938. Kolthoff, I. M. (translation by N. H. Furman) : Vblumetric Analysis, Yols. I and II, Wiley and Sons, 1928-1929. Kracke; Diseases of Blood and Atlas of Hematology, Lippincott, 1937. Lewis and Hopper: An Introduction to Medical Mycology, Year Book Publishers, 1940. Manson-Bahr: Tropical Diseases, Williams and Wilkins, 1940. Matthews: Physiological Chemistry, Wood, 1939. Mattice: Chemical Procedures for Clinical Laboratories, Lea and Febiger, 1936. Mallory: Pathological Technique, W. B. Saunders Co., 1938. Peters and Van Slyke: Quantitative Clinical Chemistry, Williams and Wilkins (revision to be published in summer of 1941). Rice: Textbook of Bacteriology, Saunders, 1936. Riley and Johannsen: Medical Entomology, McGraw-Hill, 1938. Simmons, Gentzkow, et al.: Laboratory Methods of the United States Army, Lea and Febiger, 1935. Sinclair: Microbiology and Pathology, Davis, 1940. Skinner et al.: Methods of Analysis, Association of Official Agricul- tural Chemists, Washington, D. C., 1940. Stitt, Clough, and Clough: Practical Bacteriology, Hematology, and Animal Parasitology, Blakiston, 1938. Sutton, F. (revision by A. D. Mitchell): Volumetric Analysis, Blakiston, 1935. Taber: Medical Dictionary, F. A. Davis Co., 1940. Thomson and Robertson: Protozoology, Wood, 1929. Thorne and Ward: Ephraim’s Inorganic Chemistry, Nordeman- Elsevier, 1940. Todd and Sanford: Clinical Diagnosis by Laboratory Methods, Saunders, 1939. Topley and Wilson: Principles of Bacteriology and Irmrmnity, Wil- liams and Wilkins, 1936. Treadwell, F. P. (translated and revised by Wm. T. Hall) : Analytical Chemistry, Yol, I, Qualitative, 1932, and Yol. II, Quantitative, 1930, Wiley and Sons. Zinsser and Bayne-Jones: Textbook of Bacteriology, Appleton- Century, 1939. 406303°—41 27 417 TM 8-227 MEDICAL DEPARTMENT Zinsser, Enders, and Fothergill; Immunity: Principles and Applica- tions in Medicine and Public Health, MacMillan. A System of Bacteriology, Medical Research Council, London, 1929- 1931. Handbook of Chemistry and Physics, editor in chief C. D. Hodgman, The Chemical Rubber Co., 1941. Pharmacopoeia of the United States, Mack Printing Co., 1936. Van Nostrand’s Chemical Annual, edited by J. C. Olsen, D. Van Nostrand Co., 8th Edition. Virus and Rickettsial Diseases, Harvard University Press, 1940. 418 TM 8-327 INDEX Paragraph Page Acanthocheilonema perstans 445 367 Aceto-acetic acid in urine, test 69 69 Acetone in urine, tests 68 68 Achromia 31 23 Acid; Hydrochloric, constant boiling 51 55 Hydrochloric, in gastric contents 91, 92 91. 92 Lactic, test for, in gastric contents 93 95 Normal solution 51 55 Picric, purification 104 107 Proof wood stain 12, 13 9 Salts in gastric contents 91 91 Uric, in blood 105 109 Acid-alcohol, histological 487 405 Acid digestion mixture 101 103 Acidfast stain 145,491 149,411 Acidimetry 50 53 Actinomyces 300-312 234 Actinomycosis, laboratory diagnosis 313 235 Adhesives 53 61 Aedes 472 386 Aeration apparatus for urea nitrogen 78 78 Aerobacter aerogenes 289 214 Aestivo-autumnal fever 433 353 Agar. (See Culture media.) Agglutination: In- Brucellosis 280 209 Cholera 300 223 Tularaemia 277 206 Typhoid fever 296 217 Materials used 244 179 Methods; Macroscopic 245 179 Microscopic 246 180 Rapid slide 247 181 Air, carbon monoxide 116 130 Albumen: Detection, in urine 65 65 Determination, in urine 77 76 In serum 114 121 In urine: Exton’s test 65 65 Heat and acetic acid test 65 65 Osgood-Haskins’ test 65 65 Robert’s test 65 65 Sedimentation method 77 76 419 TM 8-237 INDEX Albumen—Continued. In urine—Continued. Paragraph Page Tests for .1 65 65 Turbidimetric method 77 76 Standards (table VI) 77 76 Alcohol; Ethyl, in— Blood 117 132 Spinal fluid 117 132 Urine 117 132 Standards (table IX) — 117 132 Test meal 88 88 Alcoholic sodium hydroxide cleaning solution 43 39 Aldehyde test for Leishmania 427 342 Alkalimetry 50 53 Alkali reserve of blood plasma 115 123 Amboceptor— Antihuman, titration 411 309 Antisheep, titration 412 314 Ammonia— In urine 79 81 Production by bacteria, test , 156 153 Amoeba (table XVI; fig. 21) 416 325 Amoebiasis (table X) 416 325 Amoebic dysentery (table XVII) 416 325 Ampoules, preparation 18 11 Anaerobes, Robertson’s media 183 160 Anaerobic— Bacilli 305,354-361 227,258 Bacteria: Classification 262 190 Culture methods 241-243 176 Partial oxygen tension 243 178 Cocci. 362,363 263,264 Jar (fig. 14). Anal swab (fig. 27) 444, 449 364, 369 Ancylostoma braziliense 446 368 Ancylostoma duodenale 444 364 Animal parasites in gastric contents 86 87 Animals. (See Laboratory animals.) Anisocytosis... 31 23 Anopheles 472 386 Anoplura (fig. 35) 469, 470 382 Anstie’s reagent for ethyl alcohol 117 132 Anthrax, bacillus 304 226 Anticoagulants for blood 97 99 Antigen: Antibody reaction., 244 179 Bacterial, preparation 327 244 Craig-Wassermann, titration 411 309 Kolmer, titration 412 314 420 INDEX TM 8-227 Paragraph Page Antisera, production 328 246 Ants 469 382 Apparatus: Aeration, for urea nitrogen 78 78 Carbon dioxide, Van Slyke 115 123 Appearance of urine 60 64 Arachnida 463 377 Argas •_ 468 380 Arnold sterilizer 132 145 Arthropods 458-477 375 Collection 478 399 Shipment 479 399 Asbestos filters . 45 46 Ascaris lumhricoides 444 364 Assassin bugs (fig. 44) 476 394 Autoclave, use for sterilization 131 145 Autogenous vaccines, preparation 322-326 242 Autopsy: Collection of specimens: Bacteriological 344 251 Histological 480 401 Laboratory animals 257 184 Restoration of body 481 401 Bacillary dysentery and amoebic dysentery, differential diag- nosis (table XVII). Bacilli, classification of— Nonspore-forming 261 188 Spore-forming 262 190 Bacillus anthracis 304 226 Bacillus subtilis 303 225 Bacteria: Classification 258 186 Coli-aerogenes group 287 213 Colony picking from plates 238 176 Condition of growth 124 142 Cultural characteristics 231 173 Definition 119 139 Growth and reproduction : 123 142 Growth requirements 232 174 In gastric contents 86 87 Incubation of cultures 239 176 Morphology 120 140 Morphology of colonies 125 142 Motility 121) 228 141, 172 Paratyphoid-enteritis group 292 215 Staining > 122 141 Study of, methods: Cover glass ; 225 172 Darkfield j — _______ 226 172 Hanging drop 1 224 171 421 TM 8-227 INDEX Bacteria—Continued. Paragraph Page Transferring to tubed media 235 174 Typhoid-dysentery group 292 215 Bacterial— Antigens, preparation 327 244 Food poisoning 348-350 253 Spore, definition 120, 301 140, 225 Bacteriologi cal: Examination of— Cream 374 280 Ice cream 375 280 Milk 373 276 Water 366-372 270 Specimens, handling 329-347 246 Bacteriology of war wounds 351-365 255 Balances 42 35 Balantidium coli 416 325 Bancroft’s filaria 445 367 Bang’s microburette 44 40 Bedbugs (fig. 43) 476 394 Beef tapeworm 441, 444 361, 364 Bees 469 382 Beetles 469 382 Bence-Jones protein in urine 65 65 Benedict’s, tests for sugar 66, 80 67, 81 Benzidine test for occult blood 94 96 Bibliography App. 416 Bile: Collection of specimens 339 250 Medium 205 165 Pigments— In blood serum 113 119 In urine, tests 70 69 Bilirubin— In blood serum 113 119 In urine, tests 70 69 Van den Bergh test 113 119 Bismarck brown stain 141 147 Black widow spider (fig. 31) 466 378 Blastocystis hominis 425 341 Bleeding time 36 28 Blood 21-40 13 Agar 195 163 Alkali reserve of plasma 115 123 Animal 256 183 Anticoagulants 97 99 Bile pigments 113 119 Bilirubin 113 119 Calcium in serum 112 117 Carbon dioxide capacity of plasma 115 123 Carbon dioxide combining power of plasma 115 123 Carbon monoxide 116 130 422 INDEX TM 8—227 Blood—Continued. Paragraph Page Casts 76 72 Chlorides 108 113 Cholesterol 109 114 Collection of specimens 22, 40, 97, 335 13, 30, 99, 249 Creatinine 104 107 Culture- 40,296,335 30,217, 249 Ethyl alcohol 117 132 Fibrinogen in plasma 114 121 Films 436-438 357 Glucose 106 111 Glucose tolerance test 107 112 Groups (fig. 20) 401, 402 301, 302 Helminths found in 445, 451 367, 372 Icterus index of serum 113 119 In urine 72 71 Nonprotein nitrogen 101 103 Occult, tests for 94 95 Oxalated 97 99 Phosphatase in serum 111 116 Phosphorus, inorganic 110 115 Preparation of protein-free filtrate 100 101 Preservatives • 99 101 Proteins 114 121 Serum 98 100 Specimens: For analysis 97 99 Method of obtaining 97 99 Sugar 106 111 Sulfonamides 118 134 Table of normal values (table X). Urea— Clearance 103 105 Nitrogen 102 104 Uric acid 105 109 Van den Bergh reaction in serum 113 119 Blowfly 475 392 Body louse 470 382 Boeck-Drbohlav medium 422 337 Books, reference App. 416 Borrelia duttoni 468 380 Borrelia recurrentis i 315,470 235,382 Borrelia vincentii 316 236 Botfly 475 392 Botulinus toxin, identification 350 254 Brain broth, glucose 197 164 Breakbone fever 472 386 Bromcresol purple milk 214 167 Broth. (See Culture media.) Brown Jar for anaerobes 242 177 423 TM 8-237 INDEX Brownian movement 121, 227 141, 172 Brucella______ 279, 280 208, 209 Media for culturing 198, 199 164, 165 Brucellosis ... 280 209 Bubas 427 342 Buchner funnel 46 48 Buffer solution, bacteriological 151 151 Bugs (figs. 43 and 44) 469, 476 382, 394 Bulk staining for protozoa 421 335 Burettes, calibration 44 40 Butterflies . 469 382 Calcium in blood serum 112 117 Calibration of burettes and flasks 44 40 Calliphora _ _ _ x _ . 475 392 Capillary pipettes 17 11 Capsules, Hiss’ stain. 146 149 Carbohydrate broth 213 167 Carbol-fuchsin stain, dilute 140 147 Carbon dioxide— Apparatus, Van Slyke 115 123 Capacity of blood plasma 115 123 Combining power of blood plasma (table VIII) 116 123 Carbon monoxide-— In air and blood 116 130 Standards for * 116 130 Care of needles and syringes 40 30 Carrier 415 324 Casoni test 446 368 Casts in urine 76 72 Cat flea 477 395 Cells in gastric contents 86 87 Cements 53 61 Centipedes (fig. 29) 460,462 376 Centruorides suffusus 464 378 Ceratophyllus fasciatus 477 395 Cercaria 441 361 Cerebrospinal fluid: Collection of specimens - 337 249 Ethyl alcohol 118 134 Glucose 106 111 Cestodes _ 440 361 Chagas’ disease.... — 429, 476 347, 394 Chancroid, bacillus 285 212 Charcot-Leyden crystals 424 339 Chemical balance 42 35 Chicken flea 477 395 Chiggers 467 379 Chigoe :. ,. 477 395 Chilomastix mesmili 416 325 Chilopoda 460 376 Paragraph Page 424 TM 8-327 INDEX Paragraph Page Chinese liver fluke 444 364 Chironomidae 473 389 Chlorides in blood 108 113 Chocolate blood agar 194 163 Cholera, diagnosis 300 227 Cholera-red test for Vibrio comma 221, 300 170, 227 Cholesterol in blood 109 114 Chromogenic bacteria 272 203 Chrysops 474 390 Cimex lectularius (fig. 43) 476 394 Citrate utilization test 161 154 Clark and Lub’s medium 210 167 Classification keys for arthropods 458 375 Classification of— Arthropods 457 375 Bacteria 258 186 White blood cells (fig. 6) 32 25 Cleaning— Fermentation tubes 43 39 Fluid, for grease spots 55 62 Glassware 43, 128 39, 143 Solutions 43 39 Clear fluid test meal 88 88 Clonorchis sinensis 444 364 Clostridium: Bifermentans 360 262 Botulinum 305 227 In food poisoning 348 253 Novyi 358 262 Parabotulinum 305 227 In food poisoning 348 253 Perfringens 357 261 Proteolytic species 361 262 Septicum 359 262 Tetani 355 258 Clot retraction time 37 28 Clotting time 35 27 Coagulation time 35 27 Cocci: Anaerobic 362, 363 190, 264 Classification 259 186 Cochliomyia 475 392 Coleoptera (fig. 34) 469 382 Coli-aerogenes group 287 213 In milk 373 276 In water 369, 371 271, 276 Colloidal gold; Test, of spinal fluid 414 320 Solution, preparation 414 320 Colony characteristics (table XIV). 425 TM 8-337 INDEX Color— Paragraph Page Index 25 18 Of gastric contents 85 85 Of urine 59 64 Colorimeters 96 98 Colorimetry 95 98 Complement fixation— For amoebiasis.. . 424 339 Tests— Anti-human system 411 309 Anti-sheep system 412, 413 314, 320 Complement, titration, for— Craig-Wassermann 411 309 Kolmer__ 412 314 Concentration method, feces for protozoal cysts 418 331 Conorhinus • 476 394 Convoluted filaria 446 368 Corn meal agar 193 163 C orynebacterium— Classification 263 190 Diphtherias 306 228 Loeffler’s medium 190 162 Tellurite medium 191 162 Virulence tests 306 228 Neisser’s stain 144 149 Pseudodiphthericum 306 228 Xerose 306 228 Count: Platelet 34 25 Red cell (fig. 2) 24 14 Reticulocyte 33, 34 25 White blood cell (fig. 3) 26 18 Counting chamber (fig. 1) 24 14 Cover glass preparations 225 172 Crab louse 470 382 Craig modification of Wassermann test 411 309 Crane flies 473 389 Crayfish (fig. 28). Cream, bacteriological examination 374 280 Creatinine in blood 104 107 Creeping eruption 446 368 Crescents. (See Plasmodium falciparum.) Cresol solution 151 151 Cross-typing of human blood 403 302 Crustacea (fig. 28) 459 376 Crystals in— Gastric contents - . 86 87 Urine. 76 72 Ctenocephalus canis 477 395 Ctenocephalus felis 477 395 426 INDEX TM 8-227 Paragraph Page Culex 472 386 Culicidae 472 386 Culicoides 445, 473 367, 389 Culture- Media; Adjustment of reaction 164 155 Amoeba 422 337 Bile 205 165 Bismuth sulfite agar 202 165 Blood agar 195 163 Brilliant green lactose bile medium for water 177 158 Bromcresol purple milk 214 167 Calcium carbonate broth 181 160 Carbohydrate broth 213 167 Chocolate blood agar 194 163 Clarification of 165 156 Clark and Lub’s 210 167 Com meal agar 193 163 Cystine blood agar 186 161 Desoxycholate agar 203 165 Desoxycholate-citrate agar 203 165 Dieudonne’s alkaline blood agar 184 161 Dunham’s peptone solution 211 167 Eosin methylene blue agar 201 165 Extract: Agar 169 157 Broth 168 156 For— Intestinal bacilli 200-214 165 Milk 178 159 Water 173-177 157 Glucose— Agar 196 164 Brain broth 197 164 Glycerol agar 189 162 Hiss’ serum-water 187 162 Indicators for 152 152 Infusion— Agar.. 171 157 Broth 170 157 Jordan’s tartrate agar 207 166 Lactose broth for water 175 158 Lead acetate * 208 166 Levine’s eosin methylene blue agar for water 176 158 Liver infusion— Agar for Brucella 198 164 Broth for Brucella 199 165 Loeffler’s blood serum 190 162 McLeod’s for Neisseria gonorrhoeae 179 159 Nitrate broth — 212 167 427 TM 8-237 INDEX Culture—Continued. Media—Continued. Nutrient— Paragraph Page Agar for water 174 158 Broth for water 173 167 Gelatin 172. 157 Petroff’s (tubercle bacilli) 185 161 Potato 188 162 Potato-glycerin-blood agar 182 160 Robertson’s (anaerobes) 183 160 Russell’s double sugar 200 165 Sabouraud’s (fungi) 192 162 Selinite-F enrichment 204 165 Semisolid 180 160 Serum agar 195 163 Simmon’s citrate agar 206 166 Special sugar base 211 167 Tellurite 191 162 Tryptone broth 209 167 Tryptone-glucose-extract-milk agar 178 159 Use of 233 174 Methods for protozoa: Blood and tissue flagellates 427, 429 342, 347 Intestinal protozoa 422 337 Cutaneous leishmaniasis 427 342 Cyclops 446 368 Cylinders, graduated 44 40 Cylindroids 76 72 Cyst: Intestinal (table XVI). Iodine stain 418 331 Cysticercosis 444 364 Cysticercus 441 361 Darkfield examination— For Treponema pallidum 317 237 Technic 226 172 Decalcification of bone 492 414 Decalcifying fluid 487 405 Deer flies 474 390 Delhi boil 427 342 Dengue 472 386 Dermacentor andersoni 468 380 Dermacentroxenus rickettsi 1 468 380 Dermatohia J 475 392 Di-acetic acid in urine, test 69 69 Dichromate cleaning solution 43 39 Dientamoeba fragilis 416 425 Dieudonne’s cholera medium 184 161 Differential white blood cell count (fig. 4)— 30 23 Digestion mixture, sulfuric-phosphoric acid 101 103 INDEX TM B-227 Dilution table (table VTT). Paragraph Page For urea clearance 103 105 Diphtheria bacillus 306 228 Diphtheroid bacilli 306 228 Diphyllobothrium latum 444 364 Diplococcus pneumoniae (fig. XVI) 181, 267 160, 194 Diplopoda 460 376 Diptera 469, 471 382, 385 Disinfectant solution 151 151 Disinfection of bacteriological specimens 345 251 Dog flea - 477 395 Dracunculus medinensis 446 368 Dunham’s peptone solution 211 167 Dwarf tapeworm 444 364 Dyes, solubility 137 147 Dysentery, bacillary 298, 299 219, 222 Dysentery bacillus (tables XIV and XVII). Eberthella typhosa (table XIV) 295 217 Echinococcus granulosus 446 368 Echnidnophaga gaUinacea 477 395 Egg: Helminth (fig. 26). Medium 422 337 Ehrlich’s reagent, preparation of 71 70 Ehrlich’s test for urobilinogen 71 70 Embalming 482 402 Emptying time of stomach 85 85 Endamoeba— Coli (table XVI) 416 325 Gingivalis (fig. 21). Histolytica 416 325 Endolimax nana (table XVI). Enterobius vermicularis 444 364 Entomology 456-479 374 Eosin methylene blue agar for feces 201 165 Epithelial— Casts 76 72 Cells in urine 76 72 Erythrocytes 24, 31 14, 23 Count (fig. 2) 24 14 In urine 76 72 Escherichia coli . 288 214 Espundia 427 342 Ethyl alcohol in blood, urine, and spinal fluid 117 132 Ewald test meal 88 88 Exton’s reagent 65 Eye worm 445 367 Fannia 475 392 Fasciola hepatica 444 364 Fasciolopsis buski 441, 444 361, 364 429 TM 8-227 INDEX Fat stain 487 405 Fatty casts 76 72 Fecal examination 418 331 Feces: Collection of specimens .. 340 250 Examination for helminths.. 442, 449 362, 369 Helminths found 444 364 Occult blood 94 96 Fermentation— Tests, carbohydrate broth 213 167 Tubes, cleaning 43 39 Ferric chloride test for aceto-acetic acid 69 69 Fibrinogen in plasma 114 121 Filarial- Worm infections 472 386 Worms 445 367 Filariform larva 441 361 Filterable viruses. {See Viruses.) Filters: Asbestos 45 46 Bacterial 390 289 Fritted glass 45 46 Paper 45 46 Filth flies 475 392 Filtrate, protein-free blood 100 101 Filtration 45 46 Of virus suspensions 390 289 Finger puncture 22 13 Fixatives, tissue (table XX) 483 402 Flagellates 416, 426-429 325, 342 Blood and tissue 426-429 342 In gastric contents.. 86 87 Intestinal 416 325 Flasks, volumetric, calibration of 44 40 Fleas (fig. 45) 477 395 Flesh fly 475 392 Flies 469, 471 382, 385 Flukes 440,441 361 Fly— Larvae, stigmal plates (fig. 42). Mouth parts (fig. 41). Food— % Poisoning, bacterial ; 348-350 253 Remnants in gastric contents 86 87 Formaldehyde as preservative for urine. 58 63 Fractional gastric analysis ... 92 92 Francis' cystine blood agar 186 161 Fritted glass filters 45 48 Frozen section method for tissues 488 406 Paragraph Page 430 TM 8-227 INDEX Fungi (fig. 19) 396-400 294 Animal inoculation 400 298 Corn meal agar 193 163 Cultivation 400 298 Examination of clinical materials 400 298 Sabouraud’s media 192 163 Funnels . 46 48 Fusiform bacilli (fig. 18). Fusobacterium plauti-vincenti 316 236 Fusospirochaetal disease 316 236 Gametocytes. (See Plasmodium spp.). Gametogony (table XIX). (See Plasmodium spp.). Gas— Formation by bacteria 153 153 Gangrene, causative organisms 356 260 Gasterophilus 475 392 Gastric— Analysis: Fractional method 92 92 Indicators 91 91 Method of Rehfuss 92 92 Test meals 87, 88, 89 88, 89 Topfer method 91 91 Contents: Acid salts 91 91 Amount 85 85 Animal parasites and ova 86 87 Bacteria 86 87 Cells 86 87 Color 85 85 Combined HC1 91 91 Crystals 86 87 Flagellates 86 87 Food remnants , 86 87 Free HC1 91 91 Lactic acid 93 95 Layering 85 85 Macroscopic examination 85 85 Microscopic examination 86 87 Occult blood 94 96 Odor 85 85 Organic acids 91 91 Physical examination 85,86 85,87 Tissue fragments 86 87 Total acidity 91 91 Yeasts _ 86 87 Gelatin, nutrient 172 157 Geophilus 462 376 Gerhardt’s test for aceto-acetic acid 69 69 Paragraph Page 431 TM 8-227 INDEX Giardia lamblia 416 325 Giemsa’s stain 29 22 For tissue sections 493 415 Glanders 278 208 Glass— Cutting 15 10 Funnels 46 48 Handling 14 9 Tubing: Bending 16 10 Blowing bulbs 19 11 Glassware: Cleaning 128 143 Preparation for use 129 144 Trimming damaged pieces 20 11 Globulin in serum 114 121 Glossina (table XVIII) 474 390 Glucose: Agar 196 164 Benedict’s quantitative method for 80 81 Brain broth 197 164 In blood and spinal fluid 106 111 In urine 66, 80 67, 81 Tolerance test 107 112 Glycerol agar 189 162 Gnats 473 389 Gonococcus (fig. 17). Graduated cylinders 44 40 Gram’s method for staining 142 147 Granular casts 76 72 Granulocytes 31 23 Grease— Spots, removal 55 62 Stopcock 52 61 Groups, blood (fig. 20) 401, 402 301, 302 Guinea pig, inoculation, for Mycobacterium tuberculosis 307 231 Guinea pigs, care 219 169 Guinea worm 446 368 Haemaphysalis 468 • 380 Haemaphysalis leporis-palustris 468 380 Hammarsten’s test for bilirubin 70 69 Handling of arthropods 478 399 Hanging-drop preparations (fig. 13) 224 171 Harris’ hematoxylin 487 405 Hayem’s solution 24 14 Head louse 470 382 Helminth egg (ovum) (fig. 26). Helminths 440, 444 351, 364 Transmission 455 367 Hemoglobin 23 14 Paragraph Page 432 TM 8-227 INDEX Hemophilus 282-285 211 Heteroptera (figs. 43 and 44) 4fi9f 47fi 382, 394 Hiss’— Serum-water medium 187 162 Staining method for capsules 146 149 Histamine test meal 89 89 Histologic technic 487-493 405 Hookworm 441, 444 361, 364 Horseflies 474 390 Housefly 475 392 Howell-Jolly bodies 31 23 Howell’s method prothrombin time 38 28 Hulett and Bonner constant boiling acid 51 55 Human flea 477 395 Huppert-Nakayama test for bilirubin 70 69 Hyaline casts j 76 72 Hydatid disease 446 368 Hydrochloric acid: Combined, in gastric contents 91 91 Constant boiling 51 55 Free, in gastric contents 91 91 Normal solution 51 55 Hydrogen ion concentration of urine 81 83 Hydrogen sulfide production by bacteria: Culture medium for testing _ 208 166 Test 157 153 Hymenolepis nana 444 364 Hymenoptera (fig. 34) 469 382 Hypoderma 475 392 Ice cream, count of bacteria 375 280 Icterus index of blood serum 113 119 Ilosvay’s test for nitrate reduction 155 153 Indican in urine, tests 67 67 Indicator solutions, preparation 48 50 Indicators (tables III and IV) A 47, 91, 152 49, 91, 152 Tables 49 51 Indol— Production, test 154 153 Test, tryptone broth 209 167 Inoculation of media 234 237 174 Insecta 469 382 Insects 456,457 374,375 Intestinal bacilli, characteristics 261, 286 188, 213 lodamoeba hutchlii (table XVI). Collection of specimens 417 330 Iodine stains: Protozoan cyst 418 331 Removal 56 62 Iodine tenth normal, solution 51 55 Iron hematoxylin technic for protozoa 420 334 Paragraph Page 406303°—41 28 433 TM 8-227 INDEX Itch mite (fig, 32) 467 379 Ixodidae 468 380 Jansky, classification of blood groups 401 301 Japanese river fever 467 379 Jaundice, infectious 318 240 Jordan’s tartrate agar 207 166 Kahn test; Quantitative, for— Blood serum 408 307 Spinal fluid 410 308 Standard, for—- Blood serum • 407 306 Spinal fluid 409 408 Kaiserling’s solutions 485 403 Kala azar 427 342 Kenyoun’s carbol-fuchsin 487 405 Keys, classification of arthropods 458 375 Kissing bugs (fig. 44) 476 394 Klebsiella 'pneumoniae 290 214 Kolmer two-tube complement fixation tests 412, 413 314, 320 Labels, lacquer 54 62 Laboratory— Animals: Albino rats 221 170 Autopsy 267 184 Bleeding 255 183 Guinea pigs 219 169 Housing 217 168 Inoculation 248-254 150 Mice 220 169 Monkeys 222 170 Rabbits 218 168 Reports 6-8 5 Supplies 5 5 Lacquer, vinylite, for labels 54 62 Lactic acid in gastric contents 93 95 Landsteiner, classification of blood groups 401 301 Lange’s test for acetone 68 68 Latrodectus mactons (fig. 31) 466 378 Lead acetate medium 208 166 Leishmania (figs. 22 and 23) 426, 427 342 Lepidoptera (fig. 34) 469 382 Leprosy, bacillus 308 233 Leptospira icterohaemorrhagiae 318 240 Leptosylla segnis 477 395 Leucocyte count 26 18 Leucocytes in urine 76 72 Levy counting chamber (fig. 1) 24 14 Paragraph Page 434 INDEX TM 8-237 Paragraph Page Lice (fig. 35) 469, 470 382 Life cycle, malaria (fig. 25). Lithobius 462 376 Liver infusion media for Brucella 198, 199 164, 165 Liver puncture 427 342 Loa loa 445 367 Loeffler’s— Medium for C. diphtheriae 190 162 Methylene blue stain * 139 147 Lubricants, stopcock 52 61 Lucilia 475 392 Lymphocytes 31 23 MacLean’s test for lactic acid 93 95 McCallum’s stain for bacteria in tissue 491 411 McLeod’s medium for Neisseria gonorrhoeas 179 158 Machiavello’s stain for Rickettsiae 384 286 Macrocytosis 31 23 Macroscopic examination of gastric contents 85 85 Malaria (fig. 25; table XIX) 430-439, 472 349, 386 Maggot treatment of wounds 475 392 Maggots 471 385 Malleomyces mallei 278 208 Mann’s stain for Negri bodies 395 292 Manson’s blood fluke 444 364 Masson’s stain 491 411 Maurer’s dots 433 353 Mayer’s albumin fixative 487 405 Media. {See Culture media.) Medical entomology 456-479 374 Medical supplies-.: 3—5 4 Merozoites. (See Plasmodium spp.) Metachromatic granules 120 140 Methods of weighing 42 35 Methyl— Orange 48 50 Red 48 50 Test 159 153 Medium 210 167 Methylene blue reduction test, for milk 373 276 Mice, care 220 169 Microburettes : 44 40 Microcytosis 1 31 23 Microfilaria 445 367 Microscopes 9-11 7 Midges 473 389 Milk, bacteriological examination 178, 373 169, 276 Millipedes (fig. 29) 461 376 Miracidium 441 361 Mites (fig. 32) 467 379 Molar solution 50 53 435 TM 8-227 INDEX Monkeys, care 222 170 Monocytes — 31 23 Morgue 480 401 Morpholgy of— Bacteria (fig. 15). Fungi (fig. 19). Mosquitoes (figs. 37, 38, and 39) 472 386 Mosquito-like insects 473 389 Moss, classification of blood groups 401 301 Moths ----- 469 382 Motility; Active, of bacteria 121, 228 141, 172 Brownian movement 227 172 Mouse flea 477 395 Mouth parts of flies (fig. 41), Mucocutaneous leishmaniasis 427 342 Musca domestica 475 392 Muscidae 474 390 Muscina stabulans 475 392 Museum specimens, preparation 486 404 Mycobacterium: Classification 263 190 Leprae 308 233 Tuberculosis 185, 307 161, 231 Ziehl-Neelsen stain 145 149 Myiasis 471,475 385,392 Myiasis-producing flies 475 392 Myriapoda (fig. 29) 460 376 Nakayama’s reagent, preparation 70 69 Necator americanus 441, 444 361, 364 Needles and syringes, care 40 30 Neisseria 268-271 197 Neisser’s stain 144 149 Nemathelminthes 440 361 Nematodes 440 361 Nephelometric method of standardizing vaccines 325 243 Nessler’s solution, preparation 78 78 Neufeld’s typing of Diplococcus pneumoniae (fig. 16) 267 194 Newcastle’s bacillus 298 219 Nigrosine stain for spirochetes 148 150 NIH anal swab (fig. 27) 444, 449 364, 369 Nitrate— Broth 212 167 Reduction by bacteria 155, 212 153, 167 Nitrazine paper for urine pH 81 83 Nitric acid cleaning solution 43 39 Nitrogen: Non-protein, in blood 101 103 Standard solution 101 103 Urea, in blood 102 104 NNN medium 427 342 Paragraph Page 436 INDEX TM 8-227 Paragraph Page Non-biting flies 475 392 Non-protein nitrogen in blood 101 103 Normal— Solutions. 50 53 Values (blood) (table X). Novy, MacNeal, and Nicolle (NNN) medium for Leishmania_ 427 342 Obermayer’s reagent, preparation 67 67 Obermayer’s test for indican 67 67 Occult blood 94 96 Odor of gastric contents 85 85 Oil red O fat stain 487 405 Olef’s method 34 25 Onchocerca volvulus 446 368 Organic acids in gastric contents 91 91 Oriental— Blood fluke 444 364 Lung fluke 448 369 Sore 427 342 Ornithodorus sp 468 380 Orthoptera (fig. 34) 469 382 Osgood-Haskins test for Bence-Jones protein 65 65 Ova: Helminth (fig. 26). In gastric contents 1 86 87 Oxalic acid, normal solution 51 55 Oxidation 50 53 Oxygen tension, partial 243 178 Oxyuris vermicularis 444 364 Panstrongylus 476 394 Paper filters 45 46 Paradimethylaminobenzaldehyde reagent. 71 70 Paraffin sections of tissues, methods for preparing 489, 490 407, 411 Paragonimus westermani 448 369 Paratyphoid-enteritis group 292 215 Partial oxygen tension 243 148 Pasteurella 274 204 Pestis 275,477 204,395 Tularensis 186, 277, 468 161, 206, 380 Pathogenic helminths 444 364 Pathological methods 480-486 401 Pathology, gross specimens 485 403 Pediculus humanus 470 382 Percentage solutions, preparation 151 151 Persistent filaria 445 367 Petroff’s medium for Mycobacterium tuberculosis 185 161 pH, colorimetric determination 164 155 pH of urine. 81 83 Phenaphthazine paper for urine pH 81 83 Phenolphthalein 48 60 437 TM 8-227 INDEX Paragraph Page Phlebotomies 427, 473 342, 389 Phosphatase in blood serum 111 116 Phosphorus, inorganic, in blood serum 110 115 Phthirus pubis . 1 470 382 Picric acid, purification 104 107 Pin worm ; 444 364 Pipettes: Calibration 44 40 Capillary 17 11 Use 44 40 Plague __ 275, 276, 470, 477 204, 206. 382, 395 Plasma: Albumin and globulin 114 121 Alkali reserve of blood 115 123 Carbon dioxide capacity 115 123 Fibrinogen 114 121 Phosphorus in blood 110 115 Proteins 114 121 Plasmodium * 430 -439 349 Differential diagnosis (table XIX). Falciparum 433 353 Malariae 432 352 Ovale 434 354 Vivax •_ 431 349 Platelet count 34 25 Platyhelminthes 440 361 Pneumococcus. (See Diplococcus pneumoniae.) Poikilocytosis 31 23 Polar body stain, Neisser’s method 144 149 Pork tapeworm 444 364 Potassium— Dichromate, tenth normal solution __ 51 55 Oxalate solution 151 151 Permanganate, tenth normal solution 51 65 Potato-glycerin-blood agar 182 160 Potato medium 188 162 Polychromasia 31 23 Precipitation reactions . _ 50 53 Preservatives for— Blood 99 101 Urine 58 63 Proctoscopic examination 417 330 Protein-free blood filtrate 100 101 Proteins, in blood and serum_ 114 121 Proteus group ; 291 215 Prothrombin time 38 28 Protozoa _ -----— •___ 415-439 324 Blood and tissue 426-439 342 Intestinal 416-425 325 Pseudo-casts. - 76 72 438 TM 8-227 INDEX Paragraph Page Pseudomonas aeruginosa 273 203 Psychodidae 473 389 Pulex irritans 477 395 Pus casts 76 72 “Q” fever, Rickettsia burneti 378 282 Quartan malaria 432 352 Rabbit tick 468 380 Rabbits, care 218 168 Rabies ' 395 292 Animal inoculation with virus 395 292 Negri bodies 395 292 Van Gieson’s stain for Negri bodies 149 150 Rantzman’s test for acetone 68 68 Rats, albino, care 221 170 Reaction of urine 61 64 Red blood cells 24, 31 14, 23 Count 24 14 In urine 76 72 Redbugs 467 379 Red cell suspension, for— Craig- Wassermann r 411 309 Kolmer 412 314 Redia 441 361 Reductase, bacterial, test 158 153 Reduction 50 53 Reference books App. 416 Rehfuss’ method of gastric analysis 92 92 Relapsing fever 315, 468, 470 235, 380, 382 Reports, laboratory 6-8 5 Retention test meal 87 88 Reticulocytes 31-34 23 Rhabditoid larva 441 361 Rickettsia (table XV) 384, 468, 477 286, 380, 395 Burneti 378 282 Mooseri 378, 477 282, 395 Nipponica 378 282 Prowazeki 378, 470 282, 382 Quintana 378, 470 282, 382 Rickettsi 378, 468 282, 380 Rickettsial infections 377 282 Collection of specimens 379 283 Guinea pig inoculation 381 284 Weil-Felix reaction 380 284 Ringer's solution 422 337 Roaches 469 382 Roberts’ test for albumin in urine 65 65 Robertson’s medium for anaerobes 183 160 439 TM 8-337 INDEX Paragraph Page Rocky Mountain spotted fever, laboratory diagnosis. 378, 380- 384, 468 282, 284, 380 Rosenbach’s test for bile pigments 70 69 Ross’ test for acetone 68 68 Roundworms 440 361 Russell’s double sugar medium 200 165 Sabouraud’s media for fungi 192 163 Sahli’s method for free HC1 92 92 St. John’s wheat broth 422 337 Saline solution, physiological 151 151 Salmonella 261, 293 188, 216 Food poisoning 348 253 Samples of urine 57 63 Sand flies 473 389 Sarcophaga 475 392 Sarcoptes scabiei (fig. 32) 467 379 Scales, trip 42 35 Schaudinn’s solution 419 333 Schick test 1 306 228 Schilling differential count (fig. 5) 30, 32 23, 25 Schistosoma 444, 447 364, 369 Schizogony (table XIX). (See Plasmodium spp.) Schizonts. (See Plasmodium spp.) Schtiffner’s dots 431 349 Scolopendra 462 376 Scorpions (fig. 30) 464 378 Screw-worm fly 475 392 Seatworm — 444 364 Sediment in urine 76 72 Sedimentation rate 39 29 Seed ticks 468 380 Selinite-F enrichment medium 204 165 Seller’s stain 395 292 Serum: Agar 195 163 Albumin 114 121 Bile pigments in blood 113 119 Bilirubin in blood 113 119 Blood, separation 98 100 Calcium in blood 112 117 Globulin 114 121 Icterus index 113 119 Phorphorus in blood 110 115 Proteins 114 121 Table of normal values (table X). Sheep liver fluke 444 364 Shigella 298 219 Shipment of arthropods 479 399 Sholl tip for microburettes and pipettes 44 40 Sigmoidoscopic examination 417 330 440 INDEX TM 8-227 Paragraph Page Simmon’s citrate agar 206 166 Simuliidae 473 389 Simulium 446, 473 368, 389 Siphonaptera 469, 477 382, 395 Sleeping sickness, African 428 345 Smears; Blood, thick and thin methods, 436-438 357 Fecal - 418 331 Sodium— Carbonate: Normal solution, preparation 51 55 Solution 151 151 Chloride solution 151 151 Citrate solutions 151 151 Hydroxide, normal solution, preparation 51 55 Nitroprusside test for acetone 68 68 Thiosulfate, tenth normal solution 51 55 Solids, total, in urine 64 65 Solutions: Molar 60 53 Normal 60 53 Normal and tenth normal, preparation 51 55 Standard 61 55 Sparganosis 446 368 Sparganum 446 368 Specific gravity of urine 62 65 Specimens— Bacteriological: Autopsy material *_ 344 251 Bile 339 250 Blood 335 249 Blood serum 336 249 Cerebrospinal fluid 337 249 Data to accompany 346 252 Disinfection 345 251 Feces 340 250 From— Ear and mastoid 331 248 Eye 330 247 Nose, sinuses, and nasopharynx 332 248 Throat and tonsils 333 248 Urethra and prostate 342 251 Wounds 343 251 Handling 329-347 247 Pleural, pericardial, or other fluids from serous cavities ' 338 250 Sputum 334 248 Urine 341 250 Blood, drawing 97 99 Museum, preparation 486 404 Spermatozoa in urine 76- 72 TM 8-227 INDEX Spiders (fig. 31) 466 378 Spinal fluid: Colloidal gold test 414 320 Ethyl alcohol 117 132 Glucose 106 111 Kahn test 409 308 Kolmer test 413 320 Spirochetes 314 235 Stain for 148 150 Spleen puncture 427 342 Spore, bacterial, definition 120, 301 140, 225 Spore-forming bacilli 301 225 Spores, staining 147 150 Sporocyst 441 361 Sporogony (table XIX). (See Plasmodium spp.) Sporozoites. (See Plasmodium spp.) Sputum: Collection of specimens 334 248 Concentration in, of Mycobacterium tuberculosis 307 231 Examination for helminths 448, 454 369, 373 Helminths found 448 369 Stablefly ,v. 474 390 Stain: Acidproof, for wood 12, 13 9 Bismarck brown 141 147 Carbol-fuchsin, dilute 140 147 For— Negri bodies 395 292 Protozoa: Harris’ hematoxylin 421 336 Iron hematoxylin 420 334 Spirochetes 148 150 Spores 147 150 Giemsa’s 29 21 Loeffler’s methylene blue 139 147 Wright's 29 21 Staining: Methods; Gram’s 142 147 Neisser’s 144 149 Nigrosine 148 150 Ziehl-Neelsen 145 149 Of bacteria 122 141 Stains, stock solutions 137 147 Standard: Nitrogen solution 101 103 Solutions, preparation 51 65 Standards: Albumin (table VI). For determination—• Alcohol 117 132 Carbon monoxide 116 130 Paragraph Page 442 TM 8-227 INDEX Staphylococci 265 193 Staphylococcus in food poisoning 348 253 Starch indicator, preparation 51 55 Sterilization; By- Boiling ----- 133 146 Chemicals 134 146 Dry heat 130 145 Filtration i 135 146 In- Arnold sterilizer 132 145 Autoclave 131 145 Stigmal plates (fig. 42) 475 392 Stippling 31 23 Stock culture maintenance 319-321 240 Stomach— Contents. (See Gastric contents.) Emptying time 85 85 Stomoxys calcitrans 474 390 Stool: Examination for— Helminths 442, 449 362, 369 Protozoa 418 331 Helminths found 444 364 Stopcock, lubricants 52 61 Stout-bodied biting flies 474 390 Strauss’ test for lactic acid 93 95 Streptococci, identification 266 193 Strongyloides stercoralis 444 364 Substitution reactions — 50 53 Sugar: Benedict’s quantitative method 80 81 In- Blood 106 111 Spinal fluid 106 111 Urine: Benedict’s qualitative test 66 67 Determination 80 81 Sulfaguanidine in blood and urine 118 134 Sulfanilamide in blood and urine 118 134 Sulfapyridine in blood and urine 118 134 Sulfathiazole in blood and urine 118 134 Sulfonamides in blood and urine 118 134 Sulfuric acid, normal solution 51 55 Sulfuric-phosphoric acid digestion mixture 101 103 Supplies; Laboratory ; ! 5 5 Medical 3-5 4 Nonstandard items 4 4 Standard items 3 4 Swab, NIH anal (fig. 25) 444, 449 364, 369 Paragraph Page 443 TM 8-227 INDEX Paragraph Page Swimming pool water, bacteriological examination 372 276 Syphilis 317 237 Complement fixation tests 411-413 309 Precipitation tests 407-410 306 Tabanus 474 390 Taenia 440, 441, 444, 450 361, 364, 372 Tallquist method (hemoglobin) 23 14 Tarantula 467 379 Tartrate utilization test 162 154 Tellurite medium for Corynebacterium diphtherias 191 162 Temperate zone rat flea 477 395 Temperature corrections (table II). Tertian fever 431 349 Benign tertian fever 431 349 Malignant tertian fever 433 353 Test meals for gastric analysis 87-89 88 Tetanus bacillus 355 268 Threadworm 440 361 Ticks (fig. 33) 468 380 Tissue: Examination, for helminths 446, 452 368, 372 Fixation (table XX) 483 402 Fragments in gastric contents 86 87 Sections, frozen section method 488 406 Shipping 484 403 Toluene as preservative for urine 58 63 Topfer’s method for gastric analysis 91, 92 91, 92 Total— Acidity of gastric contents 91 91 Solids in urine 64 65 Trematodes 440 361 Trench fever 378, 470 282, 382 Treponema pallidum 317 237 Triatoma megista 476 394 Trichina worm 446 368 Trichinella spiralis 446 368 Trichinosis 446 368 Trichocephalus trichiurus 444 364 Trichomonas hominis 416 325 Trichuris trichiura 444 364 Trisodium phosphate detergent ’ 43 39 Trombicula 467 379 Trophozoite: Intestinal (table XVI). Malarial. (See Plasmodium spp.) Tropical rat flea 477 395 Trypanosoma (table XVIII) 426, 428, 429, 482 342, 345, 347, 402 Trypanosomiasis 364 264 Tryptone broth 209 167 444 INDEX TM 8-227 Paragraph Page Tsetse flies (table XVIII) 474 390 Tsuchiya’s reagent, preparation 77 76 Tsutsugamushi, Rickettsia nipponica 378 282 Tubing, glass, bending 16 10 Tularaemia 468 380 Agglutination test 277 206 Tunga penetrans 477 395 Typhoid-dysentery group (table XIV) 261, 292 188, 215 Typhus and fleas 477 395 Typhus fever: Endemic type (murine) 378,477 282,395 Epidemic type (European) 378, 470 282, 382 Laboratory diagnosis 380-384 284 Typing sera, preparation 405 304 Uffelman’s test for lactic acid 93 95 Urea— Clearance 103 105 Nitrogen 78, 102 78, 104 Urease paper, preparation 78 78 Uric acid in blood 105 109 Urine: Aceto-acetic acid 69 69 Acetone 68 68 Albumin: Qualitative 65 65 Quantitative 77 76 Ammonia 79 81 Appearance 60 64 Bence-Jones protein 65 65 Bile pigments 70 69 Bilirubin 70 69 Blood- 72 71 Collection of specimens 57, 341 63, 250 Color 59 64 Day and night samples 57 63 Di-acetic acid 69 69 Ethyl alcohol 117 132 Examination for helminths 447, 453 369, 372 Glucose: Qualitative * 66 67 Quantitative 80 81 Helminths found 447 369 Hydrogen ion concentration (pH) 81 83 Indican I 67 67 Occult blood 94 96 Preservatives 58 63 Quantity 63 65 Reaction 61 64 445 TM 8-227 INDEX Urine—Continued. Sediment: Paragraph Page Character 76 72 Microscopic examination 73 71 Organized 76 72 Unorganized 76 72 Sediments (figs. 8 and 9). Single samples 57 63 Specific gravity 62 65 Sugar: Qualitative 66 67 Quantitative 80 81 Sulfonamides 118 134 Total solids. __ ; 64 65 Twenty-four-hour samples 57 63 Urea nitrogen 78 78 Urobilinogen ; 71 70 Vaccines, autogenous 322-326 242 Vacuum wax 53 61 Van den Bergh test for bilirubin 113 119 Van Gieson’s stain 149 150 Van Slyke carbon dioxide apparatus 115 123 Venipuncture 40, 97 30, 99 Vesical blood fluke 447 369 Vibrio comma 184, 300 161, 223 Vibrion septique 359 262 Vincent’s angina (fig. 18) 316 236 Vinylite lacquer 54 62 Virulence tests for Corynebacterium diphtheriae 306 228 Virus diseases: Collection of specimens 388 289 Inclusion bodies 392 291 Serological tests 393 292 Viruses 385-395 288 Voges-Proskauer test 160, 210 154, 167 Volumetric— Glassware 44 40 Solutions 50 53 Water: Bacteriological examination 366-372 270 Culture media 173-177 157 Swimming pool, bacteriological examination 372 276 Warble fly ’ _ 475 392 War wounds, bacteriology 351-365 256 Wasps 469 382 Wax, vacuum 53 61 Waxy casts 76 72 Weighing, methods 42 35 446 TM 8-227 INDEX Weights— And volumes of water (table I). Paragraph Page Of water weighed in air 44 40 Weil-Felix reaction 380 284 Weil’s disease 318 240 Welch’s bacillus 357 261 Whip scorpions (fig. 30) 465 378 Whipworm 444 364 White blood— Cell (fig. 3) 26, 30, 31 18, 23 Count differential (fig. 5) i 30 23 Cells in urine 76 72 Whooping cough 283 211 Wilder’s stain for reticulum 491 411 Wood tick 468 380 Wounds: Collection of specimens 1 — 343 251 War, bacteriology 351-365 256 Wright’s— Method of standardizing vaccines 325 243 Stain 29 21 Wuchereria bancrofti 445 367 Xenopsylla cheopis 477 395 Yeasts in gastric contents 86 87 Yellow fever 391, 472 290, 386 Ziehl-Neelsen 145 149 Zinc sulfate centrifugal flotation technic 418, 449 331, 369 [A. G. 062.11 (5-23-41).] order of the Secretary of War: G. C. MARSHALL, Chief of Staff. Official ; E. S. ADAMS, Major General, The Adjutant General. Distribution : D 8 (10) ; B 8 (5); R 8 (SH 5); Bn 8 (8); IC 8 (2). (For explanation of symbols see FM 21-6.) 447 ». 0. COVERNMENT FRUITING OFFICE: l»4l For sale by the Superintendent of Documents, Washington, D. O.