54 STUDY AND IDENTIFICATION OF BACTERIA 



Liquefaction of gelatin is a very important means of differentiating. When room- 

 temperature incubator is not at hand (20 to 22C.), it is better to put the inocu- 

 lated gelatin tube in the body-temperature incubator, and from day to day test the 

 power of solidifying with ice- water. If the organism digests the gelatin (a liquefier), 

 the medium will remain fluid when placed in ice- water; if the organism is a non- 

 liquefier, the medium in the tube becomes solid. Of course we lose the information 

 to be obtained from the shape of the area of liquefaction. 



For routine work the only sugar media used are glucose and lactose 

 bouillon. These are of the utmost importance in differentiating organ- 

 isms of the typhoid and colon group. Following Ford, these intestinal 

 bacteria have primarily been separated by their action on litmus milk 

 whether turning it pink or only slightly changing or not changing at 

 all the original color. 



Colony Isolation. Examine the colonies on Petri plate at first with the unaided 

 eye, then with a hand magnifying glass or low-power objective, using reflected and 

 transmitted light alternately. Having determined the presence of two or more dif- 

 ferent kinds of colonies, make a ring with wax pencil around one or more of each 

 kind of colony, numbering them. The slides or culture tubes used in determining 

 the species of organism present in the plate should bear the same number as that 

 of the colony from which the material was taken. A convenient procedure is to 

 put a loopful of water on a clean coverglass and emulsify material from a colony 

 in it. Then invert over a concave slide without vaselining the circumference of 

 the concavity. After examining for motility, smear out and dry the bacterial 

 preparation. Then fix in the flame and stain with aniline gentian violet for two 

 to five minutes. Wash and mount the preparation in water. Afterward pass 

 through the usual Gram technic. 



After this inoculate the various culture media from similar colonies. One may 

 inoculate a tube of bouillon from a single colony and later on inoculate the other culture 

 tubes. 



In testing for gas production it is better to use the Durham fermenta- 

 tion tube as small amounts of gas may not be easily detected with deep 

 stab cultures into glucose or lactose agar. 



If a Durham or Smith tube, or a slant of Russell's double sugar medium be not at 

 hand the production of gas may be determined by observing bubble formation on the 

 surface of the sugar bouillon culture. As none of the pathogenic cocci produce gas, 

 fermentation tubes are unnecessary where cocci are to be studied. The litmus milk 

 tube gives data as to acid production. 



An important point is to wait at least forty-eight hours (in the case 

 of M . melitensis, four to seven days) before reporting on the cultural 

 findings on the agar, blood agar or blood-serum slant or plate upon 

 which the material is smeared (pus, exudate, blood, etc.). 



Indol production is of but slight aid in differentiating organisms. 



