Appendix. 213 



shall be ventilated so as to insure a reasonably good cir- 

 culation of air in order to prevent the accumulation in 

 the incubator of gases which might be prejudicial to the 

 development of the bacteria. 



No definite period of incubation can be prescribed 

 which will be suitable for all the work of species deter- 

 mination, but in reporting results the period used shall 

 always be stated and form a part of the report. General 

 statements as to the necessary periods will be found in 

 connection with the principal tests. 



PRELIMINARY CULTIVATION. 



It is impossible to control completely the original 

 vitality of bacteria when ready for cultivation, because 

 in most cases the conditions for their optimum growth 

 are not known. Experience, however, has shown that, 

 when bacteria are submitted to a period of preliminary 

 cultivation or rejuvenation in nutrient broth and trans- 

 fers of young cultures made from one tube to another at 

 frequent intervals, the result is to put the bacteria into a 

 condition where subsequent cultures give greater uni- 

 formity in their characteristics than where this procedure 

 is not followed. The following shall be considered as 

 the standard procedure for this preliminary cultivation, 

 and all bacteria shall be so treated before proceeding to 

 the detailed tests. 



Procedure. — Make a transfer from an agar culture of 

 the bacterium to be tested into a tube of nutrient broth, 



