CORROSIVE SUBLIMATE AS A GERMICIDE. BY CHARLES T. McCLINTOCK, A.M., OF ANN AWBOR, MICH. FROM THE MEDICAL NEWS, October i and 8, 1892. [ Reprinted from The Medical News, October 1 and 8,1892.] CORROSIVE SUBLIMATE AS A GERMICIDE.1 By CHARLES T. MCCLINTOCK, A.M., OF ANN ARBOR, MICH. When I began the experiments on which this paper is based, when in fact I had demonstrated the chief facts herein, I did not know that Koch's work on the germicidal value of sublimate had ever been seriously questioned. The papers by Geppert2 and Abbott3 are the only noteworthy ones bearing on this subject that I have been able to find. Geppert saw the danger of carrying over with the germs that had been in the disinfecting solution enough of the disinfectant to act as an antiseptic. One of his methods of experimenting was to have small sterilized dishes, in one of which was placed the germ and disinfectant, in a second a very weak solution of ammonium sulphide. After leaving the germ in dish No. i a given time, a portion was transferred to dish No. 2, and from this his tubes and plates were immediately made. Judging from my own experiments, he would have obtained dif- 1 A thesis submitted for the degree of Ph.D. in Michigan Uni- versity. 2 Geppert: " Zur Lehre von den Antisepticis," Berliner, klin. Wochenschr.. 1889. 3 Abbott: " Corrosive Sublimate as a Disinfectant against the Staphylococcus Pyogenes Aureus,'' Johns Hopkins Hospital Bul- letin, April, 1891. 2 ferent results had he left the germ for a longer time in the ammonium sulphide solution. True, he ac- complished what he was aiming at, viz., the pre- cipitation of the free sublimate, but it takes some little time for ammonium or hydrogen sulphide to dissolve or precipitate the mercury from the albumin- ate that is formed when sublimate comes in contact with albuminous bodies. This one can readily demonstrate by placing in a solution of ammonium sulphide a bit of cloth, or filter-paper, or still better egg-albumin, or a mass of anthrax-threads that have lain in sublimate for some time. Abbott also recognized the danger of carrying over enough of the sublimate to act as an antiseptic. He avoided this by high dilutions. Both of these observers, however, failed to take into account the formation by the mercury of a compound with the material of the germ itself, and while their results show that sublimate does not deserve the place as a germicide that Koch gave it, they still leave it with a germicidal power far beyond its due. Geppert found that oftentimes he could kill animals with a germ treated with sublimate, when he could get no growth from the germ on any of his media. He could offer no satisfactory explanation of these cases. The experiments herein recorded will, I think, ex- plain the cause of these discrepancies in his results. Geppert's experiments show that the blood has the power of freeing the germ from this compound of sublimate (the proof of the existence of this compound I shall present further on), and his numerous and trustworthy experiments along this line have made it unnecessary for me to go over the 3 ground again. From my experiments I should ex- pect to find a marked difference in this power between the blood, the subcutaneous tissues, and serous cavities, and, indeed, Geppert and Gruber1 found this difference. By introducing into the blood spore-threads that had been in the sublimate, Koch would have obtained results far different to those he obtained by introducing the spores beneath the skin of animals. The amount of sublimate carried along in combination with the thread and the con- tained germs would be enough to form an antiseptic pool for some distance around the thread, and while, nominally, the germ had been in contact with the germicide only a few minutes, in reality, before the slowly moving subcutaneous lymph could dissolve the compound of mercury, the germs would have been under its influence for hours or even days. Abbott also would have found his results different had he used liquid instead of solid media. Bouillon, gelatin, agar, etc., have the power of slowly dis- solving this organic compound of mercury, but, transfer a germ encapsulated by this mercurial com- pound to gelatin, and while the adjacent gelatin will slowly dissolve or decompose the compound, the result will be, if ever complete, that the germ is left lying in a pocket or pool of sublimate solution, which, if in sufficient quantity, may completely hinder its growth, although the germ is in nowise incapable of growing. Athough recognizing its 1 Gruber: " Ueber die Methoden der Priifung von Desinfek- tions-mitteln," Centralbl. f. Bacteriologie und Parasitenkunde, Bd. xi, Nos. 3 and 4. 4 advantages, I was loath to use bouillon in my ex- periments, because the argument of contamination is so applicable, and from the further fact that one surviving organism would give the same result as if hundreds had survived. The solutions of sublimate used in these experi- ments were made with chemical accuracy and were frequently renewed. They were kept in a dark closet in well closed, glass-stoppered bottles. At no time were the solutions more than two weeks old. The salt used in most of the experiments was one made by Merck, but, in order to be safe in my conclusions, another preparation was obtained from a different source, and solutions made from this. Afterward a portion of the salt was recrystallized several times, and this in turn used. In these experi- ments I have for the most part employed very resistant germs, as the bacillus subtilis and the staphylococcus pyogenes aureus. Both of these are germs that withstand great extremes of temperature, are very resistant to desiccation and possibly to chemical agents. The staphylococcus aureus was used because it is the germ against which our efforts at destruc- tion are most often aimed. The bacillus subtilis was chosen as being a very resistant spore-former. In contrast with these, I have made a few experi- ments on germs that do not have any great power of resistance, such as the germ of swine-plague, Eberth's germ of typhoid fever, which it will be remembered, is speedily destroyed at a temperature of 6o° C., and one of Dr. Vaughan's list of typhoid germs, the bacillus venenosus. 5 While it will be noticed that the highly resistant germs withstand the influence of the sublimate for many hours, it is also to be noticed that even those with but feeble powers of resistance, as the bacillus of Eberth and the bacillus venenosus, withstand its action for a comparatively long time. As the anthrax-spore has been generally used as the test-organism in disinfection-experiments I began some work with it. The culture in the laboratory having been grown on artificial media for at least five years, had completely lost the power of spore formation. Through the kind- ness of Dr. Abbott, of the Johns Hopkins Univer- sity, I obtained a spore-forming culture of an- thrax. In growing this I found that but few spores were formed, and I then attempted to restore its powers by growing it in the orthodox manner; and after passing it through animals, growing it in various kinds of dilute bouillon, and on potatoes for many generations, I at last obtained potato-cul- tures with a fairly abundant spore-formation. On testing these for the thermal death-point I was sur- prised to find that they succumbed to a temperature of 920 C. maintained for one minute. On this account I abandoned the bacillus of anthrax in my further experiments. The variation in the power of resistance among anthrax-spores has long been known, but I suspect that it has not received the attention that it deserves from many experimenters* with germicides. The fact that spores are formed, or that the culture is virulent for susceptible animals, does not mean that the experimenter has a resistant organism, as it 6 is well known that even the sporeless anthrax- bacillus readily kills susceptible animals. In my earlier experiments I used hydrogen sul- phide to remove the sublimate, but I soon noticed variations in my results that I could not account for, these being more marked when I used i : 100 and saturated solutions of the sublimate. On re- flection I was convinced that these variations might be due to the hydrochloric acid formed by the action of the hydrogen sulphide on the sublimate. A calculation of the amount of the acid formed showed that this inference was correct. After this I used sodium carbonate, sodium hydrate, or barium carbonate to neutralize the acid formed. In most of the experiments barium carbonate was used, it being insoluble in water and in general better than the alkaline salts, as the mercury is imperfectly pre- cipitated except in neutral solutions. If the germ is to be used for inoculating animals the barium salt should not be used, as the barium chloride formed is itself highly poisonous. Noticing that the neutral salts, as sodium chloride, prevented the coagulation of albumins by sublimate, and that these, when so coagulated, were dissolved on the addition of the salt, I used sodium chloride in most of my experiments, in addition to the barium carbonate, in removing the sublimate. In the first series of experiments recorded, the apparatus used (Fig. i) was as follows: Into a bottle with a capacity of about thirty c.c. was fitted a cotton-wool plug; passing through this plug and reaching to the bottom of the bottle was a glass tube, also plugged for several inches of its length 7 with cotton-wool. In the bottle was placed a small quantity of barium carbonate, in the earlier experi- ments without, in the later with, sodium chloride. The apparatus was sterilized in the steam sterilizer. Fig. i. Fig. 2. In the bottle was then placed a measured quantity of the sublimate solution and afterward a measured quantity of the germ-containing liquid. After a given time, the glass tube was connected with a Kipp's generator, and through it hydrogen sulphide 8 passed. Then, with the platinum loop, three drops were removed from the bottle and placed in gelatin, which was then plated. For convenience in handling and examining without contamination, Petri dishes were used ex- clusively. As the colonies were at times very slow in developing, the Petri plates were kept in moist chambers to prevent drying. In most of my experiments no attempt was made to deter- mine how many of the germs were killed and how many survived. Such experiments have some scientific value, but are likely to be misleading. The fact that a control-plate shows one thousand colonies, while that exposed to the action of a disinfectant shows but fifty, gives but little insight into the ques- tion of disinfection. The few experiments that I have made in that direction seem to show that the majority of germs in any given culture are easily killed, and in looking over the experiments of others I find that this is confirmed. Experiments. 1891. Nov. 12. One c.c. of bouillon-culture of the staphylo- coccus aureus, twenty-four hours old; twenty c.c. of sublimate, 1 :200; time, thirty min- utes ; two Petri plates made. Nov. 16. Many colonies. Nov. 14. One c.c. of the same culture as the preced- ing ; twenty c.c. sublimate, 1 : 200; time, sixty minutes. Nov. 22. Many colonies. Nov. 15. One c.c. of bouillon-culture of the bacillus subtilis, eleven days old; twenty c.c. of a saturated sublimate solution ; time, thirty minutes. 9 Nov. 20. Few colonies. Nov. 15. Two c.c. of subtilis culture; twenty c.c. of satu- rated sublimate solution ; time, two hours. Nov. 25. No colonies. (Very acid after H,S, i. e., not enough BaCO3.) Nov. 18. One c.c. of a culture of aureus; three c.c. of water ; ten c.c. of sublimate, 1 : 200; time, sixty minutes. Nov. 23. Many colonies. Nov. 18. One c.c. of a culture of aureus ; twenty c.c. of a saturated sublimate solution; time, forty minutes. Nov. 26. No colonies. Nov. 26. One c.c. of a culture of aureus ; twenty c.c. of sublimate, 1 : 1000 ; time, fifty minutes. Nov. 30. Many colonies. Nov. 30. One c.c. of a culture of subtilis ; twenty c.c. of sublimate, 1 : 200 ; time, sixty minutes. Dec. 5. Two Petri plates were made ; one had three colonies, the other had eight. Nov. 30. One c.c. of subtilis culture ; twenty c.c. of sublimate, 1 : 1000; time, twenty-three and one-half hours. Dec. 5. Many colonies developed. Nov. 28. One c.c. of aureus culture; twenty c.c. of sublimate, 1 : 200 ; time, seventy minutes. Dec. 5. Many colonies developed. Nov. 28. One c.c. of aureus culture; twenty c.c. of sub- limate, 1 : 1000; time, eighteen and one- half hours. Dec. 5. No colonies developed. Dec. 10. Two colonies developed. Nov. 25. One c.c. of aureus culture ; twenty c.c. ofjsub- limate, 1: 200; time, sixty minutes. Dec. 3. Hundreds of colonies developed. Dec. 1. One c.c. of aureus culture; twenty c.c. of sub- limate, 1 : too; time, sixty minutes. Dec. 7. Few colonies developed. 10 Dec. 5. One c.c. of subtilis culture; twenty c.c. of a saturated sublimate solution ; time, seventy minutes. Two Petri plates were made. Dec. 11. One dish had four, the other had ten colonies. (In this experiment sodium carbonate was used instead of barium carbonate.) Dec. 5. A control-experiment was made to see if con- tamination was possible; no germ was employed, but all of the details of the other experiments were carried out. Dec. 11. No colonies developed. Dec. 8. One c.c. of swine-plague culture; twenty c.c. of sublimate, 1 : 1000; time, sixty minutes. Dec. 12. Hundreds of colonies developed. Dec. 8. One c.c. of swine-plague culture; twenty c.c. of sublimate, 1 : 200; time, sixty minutes. Dec. 12. Many colonies developed. Dec. 12. One c.c. of aureus culture ; twenty c.c. of sub- limate, 1:1000; time, eighteen and one- half hours. Two plates were made. Dec. 14. On one four, on the other six colonies ap- peared. Dec. 13. One c.c. of subtilis culture, filtered; twenty c.c. of a saturated sublimate solution ; time, eighty-five minutes. Two Petri plates of agar were made and kept in an incubator. Dec. 17. Many colonies developed. Dec. 13. Control. No germ was employed, but all else was carried out as in the other experiments. There was no development. 1892. Jan. 13. A fresh culture of staphylococcus aureus was obtained, and this was used in subsequent experiments. One c.c. of aureus culture; twenty c.c. of sublimate, 1 : 1000; time, sixty minutes. Jan. 17. Innumerable colonies had formed. In order to show that it was not a mere question of free sublimate preventing the germ from growing, there were taken from the flask used in the last experiment, after 11 the germ had remained in the sublimate sixty minutes and before the addition of H2S, etc., three loopfuls, which were placed in a tube of bouillon. From this bouillon- tube one loopful was removed to a tube of gelatin, and in turn one from this to a second gelatin tube; both tubes were plated. Jan. 27. No development had taken place in either plate. From this and other experiments I became con- vinced that the treatment with H2S and sodium chloride did more than remove the free sublimate in the solution-that is to say, it seemed to me that here were proofs that the sublimate formed a chem- ical combination with the investing membrane or coat of the germ, and that this was not removed by the process of dilution or by washing. Schaefer1 and Braatz2 have pointed out that this is true for silk threads and catgut. To test this conclusion I made the following ex- periments. Some egg-albumin was placed in sub- limate, i : 1000, for thirty minutes ; this was then placed on a filter and repeatedly washed with dis- tilled water; the washing being continued long after the filtrate failed to give a precipitate with ammonium sulphide. To show that the free sub- limate was removed, twenty drops were run into each of two bouillon-tubes containing about eight c.c. each. One of these tubes was inoculated with the bacillus subtilis, the other with the staphylo- coccus aureus; both gave luxuriant growths. Then 1 SchXefer : Berliner klin. Wochenschr., 1890. 2 Braatz : Centralbl. f. Bacteriol. und Parasitenkunde, Bd. viii, No. 1. a small quantity of the washed albumin, estimated at one-fifth of a c.c., was placed in a bouillon-tube and this was inoculated with the bacillus subtilis ; this tube also gave an abundant growth. In still another tube was placed a quantity of the albumin, estimated at one c.c. This tube was placed in the incubator for sixteen hours and then inoculated with the staphylococcus aureus. No development fol- lowed. A mass of anthrax-threads, grown in bouillon, was placed in i : 100 sublimate for thirty minutes; it was then placed on a filter and repeatedly washed. To show that free sublimate was re- moved, five c.c. of the filtrate were added to eight c.c. of bouillon and the tube inoculated with the staphylococcus aureus; it gave an abundant growth. A small quantity of the washed threads was placed in a bouillon-tube and a larger quantity in a second tube. Both tubes were inoculated with the staphy- lococcus aureus; the one containing the small quantity grew; the other remained sterile. The remaining portion of the threads was placed in a beaker with distilled water and ammonium sulphide added; the threads became intensely black from the precipitation of the contained mercury. Several days later the experiments were repeated with the egg-albumin and anthrax-threads, with similar re- sults. Still other experiments were made with filter- paper, cotton cloth, and silk threads, all giving the same results. The experiments show that the sublimate jinites with the egg-albumin, with the cellulose of the cotton cloth and filter-paper, with the silk, and 12 13 with some portion of the germ, as in anthrax, and that this is a chemical union, but that such fluids as bouillon can set free the contained sub- limate, and this, if in sufficient quantity, will pre- vent any growth in the bouillon. In order to test this further, egg-albumin was treated with sublimate, and, having been freed from the free sublimate by continued washing, it was washed with 0.6 per cent, solution of sodium chloride. One, five, and ten drops of this filtrate respectively were added to tubes of bouillon and then inoculated with the staphylococcus aureus. The one with ten drops gave no growth, although inoculated a second time. Experiments with anthrax-threads gave the same results, i. e., after treatment with sublimate, then washing, then being placed in one hundred and fifty c.c. of a 0.6 per cent, solution of sodium chloride, then sixteen hours in the incubator; five c.c. of this liquid placed in a tube of bouillon prevented the growth of the bacillus subtilis. Again, a silk thread was placed in sublimate, 1 : 1000, for an hour ; it was then throughly washed and cut into two pieces, one of which was placed in distilled water, the other in five c.c. of blood; both were placed in the incubator for one hour. Both were washed and then placed in a solution of ammonium sulphide. The portion that had been in water be- came intensely black, while that from the blood was not discolored. In order to see if a 0.6 per cent, solution of sodium chloride could act as does blood in remov- ing the sublimate from germs, one c.c. was removed from the flask that in the experiment of January 13th 14 had for sixty minutes contained the germ and sub- limate, i : 1000, before adding hydrogen sulphide, etc., and placed in one hundred and eighty c.c. of sterilized 0.6 per cent, solution of sodium chloride; after standing for one hour plates were made. January 27th, no development had taken place. 1892. Jan. 19. One c.c. aureus ; twenty c.c. of sublimate, 1 : 100; time, thirty minutes. Jan. 23. Innumerable colonies had developed. From the flask used in this experiment before the addition of hydrogen sulphide, etc., one-tenth c.c. was removed and placed in one hundred c.c. of sterilized salt solu- tion ; from this one plate was made after twenty minutes and one after sixteen hours. Jan. 26. There was no development. Feb. 24. One c.c. of aureus culture; fifty c.c. of sub- limate, 1 : 1000; time, sixty minutes. Feb. 27. Many colonies had formed. Before adding hydrogen sulphide, etc., dilutions were made from the foregoing, so that the plates contained less than 1: 50,000,000 sublimate. Mar. 4. Four colonies had formed. Feb. 24. One c.c. of aureus culture; fifty c.c. of sub- limate, 1 : 100 ; time, sixty minutes. Feb. 27. One hundred and eighty colonies had formed. After the plate was made, the apparatus containing the germ, sodium carbonate, hydrogen sulphide, etc., was placed in the incubator for twelve hours and then a second plate made. Feb. 27. Thousands of colonies had formed. As it is improbable that the germ would multiply, at least to any considerable extent, in this combina- 15 tion of salts, both on account of lack of food- material and for the further reason that the hydrogen sulphide, and possibly the salts, would have a re- tarding influence on its multiplication, the conclu- sion seems warranted that the difference in the number of colonies on plates No. i and No. 2 was due to the fact that the incubator temperature and the prolonged action of the hydrogen sulphide and the salts had liberated many more germs from the sublimate than in the case of the first plate. From the second flask of Feb. 24, before the addition of hydrogen sulphide, etc., dilution-tubes were made, the plates con- taining less than 1 : 10,000,000 sublimate. Mar. 1. No development had taken place. Control-plates were made at the same time as the other plates of Feb. 24, i. e., one c.c. of aureus cultures; fifty c.c. of water. Feb. 27. Innumerable colonies had formed. In the succeeding experiments a modification of the apparatus previously in use was made. The germ was placed in a sterilized Erlenmeyer flask plugged with cotton-wool, and into this was placed the required amount of sublimate solution. From time to time one c.c. was removed with a sterilized pipette from this flask and placed in a test-tube con- taining a small quantity of barium carbonate and sodium chloride. This test-tube (Fig. 2) contained a plug of cotton, through which passed a small tube also plugged. This small tube passed to the bottom of the test-tube. The test-tube and contents were, of course, thoroughly sterilized in the steam steril- izer before being used. After adding the one c.c. 16 of germ and sublimate, a stream of hydrogen sul- phide was passed through the small tube ; then three drops of the liquid were removed with the platinum loop and placed in gelatin, which was then plated. The cultures of staphylococcus aureus used from this time on were treated as recommended by Geppert,1 i. e., they were grown in bouillon, in a flask containing sand, the continued shaking of which tended to hinder the formation of clumps. The culture was afterward filtered not less than twice through several inches of glass-wool contained in an Allihin tube. By this means all of the larger clumps were removed, but the microscope showed that there still remained small aggregations of the germ,'although invisible to the eye. Feb. 24. One c.c. of aureus culture; fifty c.c. of sub- limate, 1 : 1000; time, sixty minutes. Mar. 1. Nine colonies had formed. (Sodium hydrate was employed instead of barium carbonate.) Before the addition of sodium hydrate or hydrogen sulphide a portion was taken from the foregoing and diluted until the plate contained less than 1 : 50,000,000 sublimate. Mar. 1. Three colonies had formed. Feb. 24. One c.c. of aureus culture; fifty c.c. of sub- limate, 1:100; time, sixty minutes. Feb. 27. One hundred and eighty colonies had formed. Before adding H2S, etc., one c.c. was taken from the foregoing and diluted until the plate contained less than 1 : 10,000,000 sublimate. Mar. 1. No development had taken place. 1 Geppert: " Zur Lehre von den Antisepticis," Berliner klin. Wochenschr., 1889. 17 Control from the foregoing culture of aureus, one c.c. in fifty c.c. of water. Feb. 27. Innumerable colonies had formed. In the following experiments a culture of staphy- lococcus aureus, twenty days old, was used. It was grown with sand and filtered as usual. Mar. 1. One c c. of aureus culture ; fifty c. c. of sub- limate, 1:100. Time thirty minutes. Mar. 4. Innumerable colonies had formed. Dilution plates made as usual. Mar. 4. No development had taken place. Time sixty minutes. Mar. 4. Numerous colonies had formed. Dilution plates after sixty minutes. Mar. 4. No development had taken place. Time ninety minutes. Mar. 4. Many colonies had formed. Dilution plates after ninety minutes. Mar. 4. No development had taken place. Time two hours. Mar. 4. Innumerable colonies had formed. Dilution plates after two hours. Mar. 4. No development had taken place. One c.c. of aureus culture; fifty c.c. of sublimate, 1:1000; time, thirty min- utes. Innumerable colonies formed. Dilution plates (March 4). No develop- ment took place. Mar. 1. One c.c. of aureus culture; fifty c.c. of sub- limate, 1 : 1000; time, sixty minutes. Mar. 4. Innumerable colonies had formed. Dilution plates (March 4). No develop- ment took place. Mar. 1. One c.c. of aureus culture; fifty c.c. of sub- limate, 1: 1000 ; time, ninety minutes. Mar. 4. Many colonies had formed. Dilution plates (March 4). No develop- ment took place. 18 Mar. i. One c.c. of aureus culture; fifty c.c. of sub- limate, i : 1000; time, two hours. Mar. 5. Many colonies had developed. Dilution plates (March 5). No develop- ment took place. In making the dilutions, one loop of the germ and sublimate was placed in a tube of bouillon con- taining eight c.c.; from this, one loop was removed and placed in a tube of gelatin. If the loop held one one-thousandth of a cubic centimeter, which was approximately its capacity, the sublimate in the plates from the 1 : 100 would be 1 : 6,400,000,000; in those made from the 1 : 1000, the plates would contain 1 : 64,000,000,000. Mar. 9. One c.c. of aureus culture; fifty c.c. of sub- limate, 1 : 1000; time, thirteen hours. Mar. 13. Thirteen colonies had formed. It occurred to me that if sodium chloride pre- vented the coagulation of albumin by sublimate, a solution of the double chloride of mercury and sodium or a mixture of the two salts should prove a better germicide than sublimate alone. In order to test this, several experiments were made, but the results failed to confirm my hypothesis. Mar. 14. One c.c. of aureus culture; forty c.c. of sub- limate, 1 : 100; twenty c.c. of a 10 per cent, solution of sodium chloride. Mar. 16. After thirty minutes, innumerable colonies had formed; after forty-five minutes, in- numerable colonies had formed ; after sixty minutes, innumerable colonies had formed. At the same time and from the same cul- ture of aureus, one c.c. was added to sixty c.c. of sublimate, 1 : 100. 19 Mar. 16. After thirty minutes, innumerable colonies had formed; after forty-five minutes, in- numerable colonies had formed ; after sixty minutes, innumerable colonies had formed; after twelve and one half hours, ninety- eight colonies had formed; after fifteen hours, there was no development. In the case of the fifteen-hour plate, ammonium sul- phide alone was used, instead of the usual method. In order to test the effect of having more albumin- ous material present, a companion set of experiments to the foregoing was made with the same culture of the staphylococcus aureus, but grown in gelatin. Both cultures were grown in the incubator for five days. The gelatin was, of course, liquefied by the germ. Mar. 14. One c.c. of aureus culture (in gelatin); forty c.c. of sublimate, 1 : 100; twenty c.c. of a 10 per cent, solution of sodium chloride. After twelve and one-half hours (March 19), very many colonies had developed; after fifteen hours (March 19), two hundred colonies had formed ; after thirty-six hours, (March 19), no development had taken place. Mar. 18. One c.c. of bouillon-culture of the bacillus subtilis ; fifty c.c. of sublimate, 1 : 1000. After four and one-half hours (March 22), very many colonies had developed; after eighteen hours (March 22), forty col- onies had formed; after twenty-four hours (March 22), forty-two colonies had formed; after forty-two hours (March 22), no de- velopment had taken place. A second set of experiments comparing the cul- ture grown in the ordinary way, i. e., in bouillon with sand and then filtered, with that grown in 20 gelatin was made. Both cultures were eight days old. Mar. 18. One c.c. of aureus culture (in gelatin); fifty c.c. of a saturated solution of sublimate. After sixty minutes (March 23), no de- velopment had taken place; (March 27), three colonies had formed ; after four hours (March 27), no development had taken place; after eleven hours (March 27), no development had taken place. One c.c. of a bouillon culture of the staphy- lococcus aureus; fifty c.c. of a saturated solution of sublimate. After sixty minutes (March 25), no de- velopment had taken place; (March 27), four colonies had formed ; after four hours (March 25), no development had taken place; after eleven hours (March 25), no development had taken place. One c.c. of aureus culture (in gelatin); fifty c.c. of sublimate, 1 : 100. After sixty minutes (March 23), no de- velopment had taken place; (March 24), very many colonies had formed ; after four hours (March 23), no development had taken place; (March 24), many colonies had formed ; after eleven hours (March 23), no development had taken place ; (March 26) fourteen colonies had formed ; (March 27) twenty-one colonies had formed. One c.c. of bouillon culture of the staphylococ- cus aureus ; fifty c.c. of sublimate, 1 : 100. After sixty minutes (March 23), innumer- able colonies had formed ; after four hours (March 23), many colonies had formed; (March 24), innumerable colonies had formed; after eleven hours (March 23), no development had taken place; (March 27), no development had taken place. One c.c. of aureus culture (in gelatin) ; fifty c.c. of sublimate, 1 : 1000. 21 After sixty minutes (March 23), innumer- able colonies had formed ; after four hours (March 25), innumerable colonies had formed; after eleven hours (March 25), no development had taken place; (March 26), one hundred and fifty colonies had formed. One c.c. of aureus culture (in bouillon) ; fifty c.c. sublimate, 1 : icoo. After sixty minutes (March 25), very many colonies had formed ; after four hours (March 25), no development had taken place; (March 26), there were too many colonies to count; after eleven hours (March 24), no development had taken place; (March 26), many colonies had formed. Klein, in 1884, in reviewing Koch's work, makes the statement that mercuric chloride is of no more germicidal value than vinegar. Actuated by curi- osity more than anything else, I made a few experi- ments on this point. The experiments made are possibly too few to draw any conclusions from, but they are given for what they are worth. Apr. 12. One c.c. of subtilis culture; fifty c.c. of sub- limate, 1 : 1000. After twenty-two hours (April 15), many colonies had formed ; after forty-one hours (April 15), seven colonies had formed. Apr. 12. One c.c. of subtilis culture; fifty c.c. of a saturated solution of sublimate. After seventy minutes (April 15), eight colonies had formed. One c.c. of subtilis culture; fifty c.c. of sub- limate, 1 :too. After twenty-two hours (April 18), no development had taken place ; after forty- one hours (April 18), no development had taken place. 22 One c.c. of subtilis culture; fifty c.c. of vinegar. After sixty minutes (April 15), very many colonies had formed ; after one and one-half hours (April 15), very many colonies had formed ; after twenty-two hours (April 18), many colonies had formed. One c.c. of aureus culture; fifty c.c. of sub- limate, 1 :1000. After twelve hours (April 15), forty-one colonies had formed; after twenty-three hours (April 16), no development had taken place. One c.c. of aureus culture; fifty c.c. of vin- egar. After twelve hours (April 16), no develop- had taken place; after twenty-three hours (April 16), no development had taken place. The vinegar used in the foregoing experiments was titrated with sodium hydrate, and gave a frac- tion over 7 per cent, of acetic acid. A different sample of vinegar was obtained and another set of experiments made. Apr. 30. One c.c. of aureus culture; fifty c.c. of sub- limate, 1 :1000. After thirteen hours (May 4), thirty-one colonies had formed; after sixteen hours (May 5), very many colonies had formed; after twenty-three hours (May 5), two col- onies had formed. One c.c. of aureus culture; fifty c.c. of vin- egar. After thirteen hours (May 5), six col- onies had formed; after sixteen hours (May 5), no development had taken place ; after twenty-three hours (May 5), no de- velopment had taken place. One c.c. of subtilis culture; fifty c.c. of sub- limate, 1 : 1000. 23 After twenty-three hours (May 5), no de- velopment took place; after thirty-nine hours (May 5), no development took place. One c.c. of subtilis culture; fifty c.c. of vin- egar. After twenty-three hours (May 5), fifty colonies had formed ; after thirty-nine hours (May 5), thirty-five colonies had formed. This sample of vinegar gave on titration 6.3 per cent, of acetic acid. Experiments with Eberth's Germ of Typhoid Fever. The culture from which this germ was grown was one brought by Dr. Vaughan from Koch's labora- tory some four years ago. It had, therefore, been grown on artificial media for at least four years, and we may reasonably suppose that its power of resist- ance to deleterious influence had been weakened by this long exposure to adverse conditions. Apr. 7. One c.c. of a culture of Eberth's germ ; fifty c.c. of sublimate, 1 : 1000. After six minutes (April 12), two hundred colonies had formed ; after ten minutes (April 12), eighty two colonies had formed ; after fifteen minutes (April 12), forty-one colonies had formed ; after thirty minutes (April 12), twenty-one had formed; after sixty minutes (April 12), no development had taken place; after three and one-half hours (April 12), no development had taken place. Control, using water instead of sublimate. Apr. 12. Innumerable colonies had formed. The culture used in the foregoing experiments was grown in alkaline bouillon in the incubator for thirty-six hours. 24 Apr. 19. One c.c. of a culture of the bacillus vene- nosus j1 fifty c.c. of sublimate, 1 : 1000. After thirty minutes (April 23), about four hundred colonies had formed. In another series of experiments with the typhoid germs a comparison was made between the germs grown in bouillon and those grown on agar. Unfor- tunately the bouillon-culture was three days old, while the agar-culture was but thirty-six hours old. In the case of the agar-culture a few c.c. of bouillon were poured into the agar-tube and the germ rubbed off the surface with a platinum needle and the whole filtered. Apr. 27. One c.c. of a culture of bacillus venenosus; fifty c.c. of sublimate, 1 : 1000. Bouillon-culture - after thirty minutes (April 30), about three hundred col- onies had formed. Agar-culture-after thirty minutes (April 30), about one hundred and fifty colonies had formed. Bouillon-culture-after forty-five minutes (April 30), about three hundred col- onies had formed. *Agar-culture-after forty-five minutes (April 30). Bouillon-culture - after sixty minutes (April 30), about two hundred and fifty colonies had formed. Agar-culture-after sixty minutes (April 30), no development had taken place. Agar-culture-after sixty minutes (May 3), five colonies had formed. *Bouillon-culture-after two hours. Agar-culture-after two hours (May 3), one colony had formed. 1 One of Dr. Vaughan's list of typhoid germs. 25 One c.c. of a culture of Eberth's germ ; fifty c.c. of sublimate, i : 1000. Bouillon-culture-after thirty minutes, (April 30), thirty-two colonies had formed. Agar-culture-after thirty minutes (April 30), one colony had formed. Bouillon-culture-after forty-five minutes (April 30), twenty-six colonies had formed. *Agar-culture-after forty-five minutes (April 30). Bouillon-culture - after sixty minutes (April 30), twelve colonies had formed. Agar-culture-after sixty minutes (May 5), no development had taken place. *Bouillon-culture-after two hours (May 5< Agar-culture-after two hours (May 3), no development had taken place. (Plates marked * were through an oversight destroyed.) Sublimate solution has not been extensively used for the disinfection of feces, writers on this subject generally stating that the large amounts of albumin- ous material present tend to delay its germicidal action. In order to see if it was merely a question as to the quantity of albumins present, the follow- ing experiments were undertaken. Apr. 25. A portion of a stool of feces, estimated at one hundred grams, from a healthy individual, was placed in a beaker and one liter of sublimate, 1 : 1000, added. Over the mouth of the beaker was tied a sheet of filter-paper moistened with sublimate. Through this paper was passed a sterilized glass tube plugged with cotton. After twenty-four hours this tube was connected with the hydrogen sulphide generator, and the gas passed for some time. Three plates were 26 then made, one from the liquid and two from the solid portion of the fecal mass. Apr. 28. Each of the plates contained hundreds of col- onies. No attempt was made to determine the species, but judging from the colonies alone, I would say that there were at least four species present. In another experiment made April 29, I proceeded as in the foregoing, with the exception that the portion of feces taken was estimated at not more than twenty-five grams, to which was added one liter of a saturated solu- tion of sublimate. After twenty-four hours the plates were made as in the previous experiment. The plate made from the liquid did not develop. The two plates from the solid matter each had many colonies. The amount of hydrochloric acid formed when hydro- gen sulphide was passed was doubtless sufficient to kill all germs, if any were living in the liquid. Note.-It will be observed that the experiments given in this paper are not always arranged in chronologic order. In several instances when an experiment bore on a particular point it was placed under that heading, although made at a different time. The almost universal practice of using sublimate, 1:1000, for disinfecting purposes is based on Koch's1 statement: " By a single application for but a few minutes, without any previous preparation of the objects to be disinfected, an absolute disinfection of even the most resistant organism is guaranteed." Gartner and Plagge2 claim that the staphylococ- cus pyogenes aureus is killed by sublimate, 1 : 1000, 1 Koch: Mittheilungen aus dem Kaiserlichen Gesundheits- amte, 1881. 2 Gartner and Plagge: Verhandlungen der Deutschen Gesell- schaft fur Chirurgie, vierzehnten Congress, 1885. 27 in eight seconds, the proportions of organisms and sublimate being the same as I have used. Tarnier1 and Vignal state that it is killed in two minutes. Abbott2 credits it with a much higher resistance. In contrast with these statements my experiments show that, exceptionally at least, the germs with- stand the action of sublimate as follows: Staphylococcus pyogenes aureus; sublimate, 1 : 1000; twenty-three hours. Staphylococcus pyogenes aureus; sublimate, 1 : 100; eleven hours. Staphylococcus pyogenes aureus; sublimate, satu- rated ; one hour. Bacillus subtilis; sublimate, I : 1000; forty-one hours. Bacillus subtilis; sublimate, saturated ; eighty-five minutes. Swine-plague ; sublimate, 1 : 200 ; one hour. Typhoid germs ; sublimate, 1 : iogo; one hour. Germs in feces; sublimate, 1: 1000; twenty-four hours. Germs in feces; sublimate, saturated, twenty-four hours. It may be urged against the experiments that years of use by surgeons and practitioners have demon- strated that sublimate is a germicide. In reply to this I may cite the fact that the same credence was given to Listerism and the same long experience was urged as confirmatory; and, again to the more recent practice of aseptic surgery (which was the real secret of success in using both carbolic acid and 1 Tarnier and Vignal: Arch, de M€d. experimentale et d'Anat- omic pathologique, 1890. 2 Abbott: " Corrosive Sublimate as a Disinfectant against the Staphylococcus Pyogenes Aureus," Johns Hopkins Hospital Bulletin, April, 1891. 28 sublimate), in which the results are better than with any of the antiseptics. Frommel,1 with an experience of some two hun- dred laparotomies, using at various times carbolic acid, sublimate, and asepsis, gives the preference to the last, and his results are practically in accord with all recent surgery. The resisting power of the tissues to the invasion of germs has not received due attention from writers on antiseptics. Whether the power resides in the leukocytes, blood-serum, or what not, does not matter. Laboratory experience has shown that in order to be sure of infection, with most germs at least, we must introduce relatively large quantities. The fact that a given operation was performed antiseptically and no infection followed, by no means warrants the conclusion that the antiseptic was the cause of the non-infection. It is to be noticed that in these experiments, the germs were placed under the most disadvantageous conditions, while for the disinfectant the conditions were the most favorable that could be devised- z. e., so far as possible the germs were isolated from each other and distributed throughout a large quan- tity of the disinfectant. In nature and at the operating-table these conditions do not obtain. Germs are generally found in masses, are contained in albuminous fluids, and hidden or surrounded by such matters as are but slowly permeable by fluids that do not coagulate them; whereas such a fluid as sublimate solution would, in some cases at least, be 1 Frommel, Review of, in Centralbl. f. Bakteriol. und Parasiten- kunde, 1891. 29 hours, or possibly days, in reaching the germs con- tained in pus, blood-clots, feces, etc., and then would require further hours or days to destroy the germs after reaching them. Again, as pointed out by Gruber,1 solid media, as gelatin and agar, and room-temperatures are not favorable to the growth of bacteria that have been under the influence of disinfectants. Recognizing as we must, from daily experience in the laboratory, that our artificial media are at best but poor substitutes for the natural media on which germs grow, having in mind that the germs often fail to grow or grow but feebly on such media -with the foregoing facts in mind and yet seeing the experiments show that even under these adverse conditions the germs are still capable of growth after having been in sublimate for hours or even days, we are forced to the conclusion that the germicidal power of solutions of sublimate has been enormously overestimated. However, the proof that sublimate is not a good germicide is no proof that it may not be valuable as a disinfectant. As is well known, of all substances it has the greatest antiseptic power, and, if my inter- pretation of its action be correct, it follows that a germ treated with sublimate, and possibly with solutions more dilute than i : 1000, unless, per- chance it gets into the blood or is exposed to very exceptional conditions, is powerless to grow-that is to say, it is probable that a spore of subtilis or 1 Gruber: " Ueber die Methoden der Priifung von Disinfek- tions-mitteln,'' Centralbl. f. Bakteriologie und Parasitenkunde, Bd. xi, Nos. 3 and 4. 30 anthrax treated with sublimate, 1 : 1000, and then thrown on the soil or into water will not germinate, owing to the fact that the capsule of sublimate sur- rounding it is not removed. The following is a summary of the results of my experiments, and the conclusions that it seems to me they justify : 1. The high rank heretofore given corrosive sub- limate as a germicide is without warrant and was based on faulty experiments. These faults were in the main two: among the older experimenters, carrying over with the disinfected material enough of the sublimate to act as an antiseptic; in the later experiments, as those of Abbott,1 the failure to recognize the fact that the organic compound of the sublimate with the investment of the germ, especially with the use of solid media, is sufficient to act as an antiseptic; and from a failure to obtain a growth, the false conclusion is drawn that the germ is dead. 2 The very varying power of resistance in dif- ferent cultures, as pointed out by Esmarch,3 and insisted on by Gruber,3 is an all-important factor to be noted in determining the germicidal value of any agent. 3. That sublimate forms with cellulose, as cloth, 1 Abbott: " Corrosive Sublimate as a Disinfectant against the Staphylococcus Pyogenes Aureus," Johns Hopkins Hospital Bulletin, April, 1891. a Esmarch: " Milzbrandsporen als Test-Objekten bei Priifung von Disinfektionten," Zeit. f. Hyg., 1889. 3 Gruber: " Ueber die Methoden der Priifung von Desinfek- tions-mitteln,'' Centralbl. f. Bakteriologie und Parasitenkunde, Bd. xi, Nos. 3 and 4. filter-paper, etc., with silk, with albuminous bodies, with some part of bacteria, probably the envelop, a chemical compound that cannot be removed by any amount of washing with water. Thus sub- limate when acting on a germ, forms a capsule around it that protects the germ for a time from the further action of the sublimate, and, in turn forms an impenetrable barrier to the growth of the organism, unless removed. This barrier may be removed with salines, and is more rapidly removed in proportion to the renewal of the saline, condi- tions that are fulfilled in the circulating blood. The action of sublimate on bacteria is probably closely analogous to that of alcohol. All experience leads us to believe that absolute alcohol is immediately fatal to protoplasm, and the only way that we can ex- plain the fact that spores may survive in absolute alcohol for weeks, is on the assumption that the alcohol somehow changes the envelop of the spore, rendering it impermeable. 4. The presence of a gelatinous envelop in many, if not all, bacteria, at certain stages of their life- history has not received the attention it deserves. De Bary 1 insisted on this long ago. Now and then, in staining old cultures of bacilli especially, one may notice a halo or dim line surrounding the organism, generally at a distance about that of the diameter of the organism itself. I have observed this frequently, but cannot state just what the con- ditions are under which it occurs. Of course, this or something similar must be present in all germs forming zobglea, as well as in the capsule-forming 31 1 De Bary: 11 Vorlesungen fiber Bakterien," 1886. 32 germs. This envelop probably serves the organism in protecting it against temperature, dehydration, and chemical agents. {Note.-Since writing the foregoing I have re- read De Bary's statements as to the envelop of bac- teria. He points out that it is generally present, but far more prominent in bacteria grown on solid media, for the reason that being gelatinous, it is largely dissolved in fluids. If this be true, and if my explanation of the action of sublimate be true, it follows that had I used germs grown on solid media the results of the experiments would have been far more unfavorable for the sublimate, and, the foregoing suppositions holding, had the germs been air-dried their resistance to sublimate would have been yet more marked.) 5. That while sublimate has no great germicidal power, it does not follow that it is not a valuable disinfectant. Whether the germs contained in solutions treated with sublimate and disposed of as such material usually is, do or do not grow remains to be proved. The Medical News. Established in 1843. A WEEKL Y MEDICAL NEWSPAPER. Subscription, $4.00 per Annum. The American Journal OF THE Medical Sciences. Established in 1820 A MONTHL Y MEDICAL MAGAZINE. Subscription, $4.00 per Annum. COMMUTA TIOH RA TE, $7 jo PER ANNUM. LEA BROTHERS & CO. PHILADELPHIA.