284 BACTERIOLOGY. 



squeezed out, and the organisms present can thus be spread 

 throughout the gelatin. From this tube, which is No. 1, 

 transfers are made in the usual manner to tube No. 2, and 

 from this to tube No. 3. Gelatin plates (Petri dishes) are 

 are' then made in the usual manner and set aside for two 

 or three days to develop. 



The form of the colonies should then be carefully 

 studied, inasmuch as this is very characteristic. If possi- 

 ble, impression preparations of the surface colonies should 

 be made and stained with methylene blue. 



Since the colony is a pure culture of the anthrax bacil- 

 lus, transplantations to tubes in turn will yield pure cul- 

 tures. A stab culture in gelatin and a streak culture on in- 

 clined agar, and on .two potatoes in tubes, should be made. 

 The inoculation of the inclined media is made by simply 

 drawing the end of the platinum wire over the middle of 

 the surface of the agar or potato. The agar tube and 

 one potato tube are placed in the incubator at 37 to 39, for 

 one or two days, then removed and examined for threads 

 and spores. The other potato tube is allowed to develop 

 at a temperature of 15-18. Under these conditions spores 

 are not produced, but instead, marked involution forms will 

 be found. 



A tube of peptonless agar should be inoculated at the 

 same time as the above and allowed to develop in the incu- 

 bator. This growth, unlike that on the ordinary agar, will 

 be extremely rich in spores. The material obtained in this 

 way- will be utilized, subsequently, for the staining of 

 spores. The ordinary agar does not yield a good supply 

 of spores, inasmuch as the anthrax bacillus employed has 

 been cultivated artificially for many years, and, as a result, 

 shows an asporogenic tendency. If ^-1 drop of calcium 

 hydrate is added to the tube of ordinary agar, and this 

 then sterilized, it will yield an abundance of spores. The 

 absence of pepton and the presence of calcium favor spore 

 formation. 



