CULTURAL TECHNIQUE 21 



difficult to obtain. The extent of the zone of influence of a 

 colony depends upon the organism and the medium. Inhibi- 

 tion by overcrowding is negligible when the number of col- 

 onies ranges from 100 to 200 on a 90-mm. plate. 



If larger numbers are present, this inhibitory action becomes 

 pronounced and a reduction in total content is to be observed. 

 This error, as well as tedious enumeration of very small col- 

 onies, makes it preferable to have properly prepared plate cul- 

 tures. In plates prepared from pure cultures of bacteria, i.e. 

 organisms all of one kind, this inhibiting tendency is much 

 less important. 



Dilution. The number of bacteria present in any given 

 material, as, for instance, milk, is of course unknown. Hence, 

 in preparing plate cultures, in order to have the proper num- 

 ber of colonies it is necessary to vary considerably the quan- 

 tities of milk used in the plates. The milk should be diluted 

 with sterile water or salt solution (water blanks). The extent 

 to which the dilution must be carried cannot be determined 

 with exactness, but with experience the analyst can judge 

 from the age of the milk, the conditions under which it was 

 produced, the temperature at which it has been kept, and, by 

 determination of the acidity, the dilutions which are most 

 likely to give satisfactory results. 



In the case of perfectly fresh milk, plates should be made 

 with 1/10, 1/100, and 1/1000 cc.; ordinary market milk 

 1/100, 1/1000, 1/10,000 cc.; milk having an acidity over 

 0.2 per cent, 1/1000, 1/10,000, and 1/100,000 cc. In the 

 case of sour milk and cream it will be necessary to use still 

 greater dilutions. , 



The various dilutions are obtained by the use of 9-cc. and 

 99-cc. water blanks in test tubes and 150-cc. Erlenmeyer 

 flasks. To obtain 0.1 cc. of milk, 1 cc. is placed in a 9-cc. 



