86 PHARMACEUTICAL BACTERIOLOGY 



loop and return to its proper place). Rotate tube (replugged with the 

 cotton and held vertically) rapidly between the hands for twenty seconds, 

 to mix contents. By means of the platinum loop take two loopfuls (one 

 loopful may serve) from tube No. i (which you have just inoculated and 

 rotated) and pass them into tube No. 2. Plug both tubes, set aside tube 

 No. i, and rapidly rotate tube No. 2. Take two loopfuls from tube No. 2 

 and transfer to tube No. 3, and proceed as before. Now pour contents 

 of tube No. i into a sterile Petri dish, also numbered i ; contents of tube 2 

 into Petri dish 2; and tube 3 into Petri dish 3. Wait until the media in 

 the Petri dishes are solidified, and then set aside at the room temperature 

 to await developments. In the course of two or three days it will perhaps 

 be found that very many minute specks are visible in dish No. i, some one 

 hundred or more may appear in dish No. 2, and perhaps not more than ten 

 or twenty in dish No. 3. Observe carefully the several growths in dishes 

 2 and 3. Each visible growth indicates the development from a single 

 microbe. Are the several growths all alike, or do they differ? Differences 

 in color- and in outline of growths indicate different species of bacteria. 

 The several different kinds of bacteria may now be transferred to test- 

 tubes by means of the straight platinum needle or the loop, and the 

 observations may thus be extended. Transfers can be made to different 

 kinds of media, as agar, gelatin, agar-gelatin, beef broth, milk, prepared 

 potato, etc. 



C. Making Bacterial Counts. In order to determine the number of bac- 

 teria in any given substance, the same procedure as was just described is fol- 



PIG. 36. Petri dish. These dishes are among the essentials in the bacteriological 



laboratory. ( Williams.) 



lowed, with the difference that a definite amount of the thoroughly mixed 

 contaminated substance is added to a definite amount of culture medium in 

 the test-tubes in which the dilution mixtures are made. For example, we 

 will suppose that it is desired to determine the number of bacteria (per cc.) 

 in milk: Thoroughly mix the sample of milk by shaking it in the container. 

 Take o.i, 0.2, 0.5, or i cc. of the milk (by means of a sterilized graduated 

 pipette) and add it to 9 cc. of the liquefied culture medium in tube No. i ; 

 i cc. of tube No. i to tube No. 2, also with 9 cc. of medium; i cc. of tube 

 No. 2 to tube No. 3 (with 9 cc. of medium), following the other directions 

 as already given. Plate out as already explained, and watch developments. 



