Cultivation 735 



generated. If filaments and filamentous masses are found to be 

 present in the granule, then the disintegrated products of the granule 

 are to be transferred by means of the platinum loop to melted i per 

 cent, dextrose agar-agar contained in test-tubes filled to a depth of 

 7 or 8 centimeters which have been cooled to about 4oC. 

 The material is to be thoroughly distributed throughout the melted 

 agar-agar by means of the loop, and the tube then placed in the in- 

 cubator. Several tubes should be prepared. At the same time a 

 number of granules, after washing in sterile water or bouillon, should 

 be placed on the sides of sterile test-tubes plugged with cotton and kept 

 at room temperature in the dark. The sugar-agar tubes inoculated 

 as above described should be examined from day to day for the 

 presence of the characteristic colonies in the depths of the agar-agar. 

 If very many colonies of contaminating bacteria have developed in 

 the tubes, it will probably be very difficult or impossible to isolate 

 the specific micro-organism. If there are few or no contaminating 

 colonies, then the colonies of the specific organism should be ex- 

 pected to develop in the course of two or three days to a week. 

 If a good number of living filaments of the micro-organism have 

 been distributed throughout the agar, the specific colonies that 

 develop will be very numerous in the depths of the agar, especially 

 throughout a shallow zone situated about 5 to 12 mm. below 

 the surface of the agar-agar. When the presence of the char- 

 acteristic colonies has been determined, slices or pieces of the agar 

 containing colonies are to be cut out of the tube by means of a stiff 

 platinum wire with a flattened and bent extremity. A piece of the 

 agar-agar is to be placed upon a clean slide and covered with a clean 

 cover-glass. It is to be examined under a low power of the micro- 

 scope, and an isolated colony selected for transplantation. By 

 obvious manipulations, under continuous control of microscopic 

 observation, the selected colony, together with a small amount of 

 the surrounding agar-agar is to be cut out, care being taken to 

 be sure that no other colony is present in the small piece of agar- 

 agar containing the colony. The small piece of agar-agar thus cut 

 out should not have a greatest dimension of more than 2 mm. The 

 piece of agar-agar is then transferred from the slide by means of a 

 platinum loop to a tube of sterile bouillon where it is thoroughly 

 shaken up to free it from any adherent bacteria. If there be any 

 reason to believe that the small piece of agar has been very much 

 contaminated with bacteria, it should be washed in a second tube of 

 bouillon, then the piece of agar-agar is to be transferred by means of 

 the platinum loop to a tube of melted sugar-agar cooled to 4oC. 

 It should be immersed deeply in the agar and the tube placed in the 

 incubator. If the colony thus transferred to the agar-agar is capable 

 of growth, in the course of some days it will have formed a good- 

 sized colony from which transplants in various culture-media may 

 be made." 



