[background noise] -Since clinicaland epidemiological importance is attachedto biochemical types of C. Diphtheriae, it is often desirableto determine these various types. Through observing the colony formationon chocolate Telluride plates, pellicle formation on broth, fermentation of carbohydrates , and production of soluble hemolysinsin broth. [silence] In order that resultsmay be comparable and reliable, the methods of determiningthese biochemical type should be uniform with all workers. [silence] Three carbohydrates are usedin the typing of C. Diphtheriaee. Glycogen 5%, starch 2%,and dextrose 10% each sterilized in distilled water. [silence] Three-tenths of a milliliterof each of the sterile dextrose, starch, and glycogen solutions is added,aseptically, to each of several tubes containing three millilitersof Bromcresol purple broth. -Each tube isthen marked to indicate content. [silence] When tubes containingeach of the three carbohydrates have been prepared,they're arranged in rows for inoculation. [silence] The carbohydrate broth tubesare incubated overnight to test sterility. [silence] After incubation,the carbohydrate broths are examined for sterility. One tube of plain brothis then placed in the rack with each set of three Bromcresolpurple carbohydrate tubes in regular rows, one for each culture. [silence] With a capillary pipette,a broth culture of C. Diphtheriae is taken from one of several tubes and used to inoculatea tube of plain broth and a set of three carbohydrates. [silence] Two drops per tube, dextrose, [silence] starch, [silence] and glycogen. A McLeod chocolate Telluride plateis then inoculated with one drop from the same pipettebefore it is discarded. McLeod's Telluride Agaris used only for typing. [silence] The plate is streaked with a bent needle. [?] -It is numbered. [silence] All tubes are then markedwith the culture number. With a clean pipette,another culture of C. Diphtheriae is taken and used to inoculate the next set of plain broth tubes,three carbohydrates, and a plate. [silence] After all sets of tubes in the rackare inoculated and marked, they're incubated. After 48 hours, the broth culturesand chocolate plates are brought from the incubator. The colonies on the chocolate platesare examined for type and size. This is a gravis-type colony. Irregular margins, rough surface,concentric markings, and low conical form. [?] -This is a mitis-type colony. Regular margins, smooth, glisteningsurface, and smaller size. [silence] A similar type called intermediushas characteristics similar to mitis but is still smaller. This is a minimus type colony,a distinct type, but often confused with intermedius. The colonies are extremely minute. The next step in typingC. Diphtheriae is the hemolysin test for which the 48-hour plain broth culturesare used. Sterile tubes are placed in an empty rack. -With a sterile pipette,one-half milliliter of plain broth culture is drawn from one of the tubes, [silence] transferred to a sterile tube, [silence] and labeled. [?] -With a sterile pipette,the next plain broth culture is drawn and transferredto another sterile tube until each broth culturehas been used. [silence] Using a pipette, a 2% suspensionof washed human blood cells is taken from a flask. It is essential to use human cells. Others give aberrant results. One-half milliliterof the erythrocyte suspension is then added to each tube of culture. -The tubes are shaken. [silence] They're placed in a water bathat 37 degrees centigrade. [silence] After one hour,they're refrigerated overnight. [silence] The next morning,the hemolysin tests are read. [silence] The tube on the left shows no hemolysis. One on right shows hemolysis. [silence] Hemolysin production isa characteristic of the mitis type. [silence] The data is recorded for later reference. The gravis, intermedius, and minimus typesdo not produce hemolysis in this test. At this time or, even earlier, it is wellto examine the fermentation tests, as some cultures,after an initial acidity, revert to an alkaline reaction. Pellicle should also be looked for,as they are erratic in time and place of appearance. Incubation of the cultures is continued. After one week's incubation,the final readings are made on the fermentation tests. Pellicle formation, the whitish gumon the surface of the culture is a characteristic of the gravis type, so, too, is the fermentation of starchand glycogen. These tubes show no pellicle formation. The starch and glycogen are not fermented. These properties are characteristicof all types, except gravis. Failure to ferment dextroseis distinctive of the minimus type. After all datahave been recorded for the test, the types of C-Diphtheriaeare determined .