ISOLATION OF ANAEROBES 457 



it was the first method for the isolation of anaerobes that I learned 

 to use. It is a perfectly feasible method, but I find it to be less 

 satisfactory than others for various reasons. 



The difficulty of regulating the amount of moisture on the sur- 

 face of the plate or slant is the primary drawback to the use of 

 surface methods. Aerobic cultures differ fundamentally from 

 anaerobic ones in this respect. They are, so to speak, self-regula- 

 ting in their moisture content. WTien a plate is poured, the sur- 

 face of the agar is exceedingly moist, and the organisms planted 

 in it grow rapidly till their growth is inhibited by the drying at- 

 mosphere of the incubator. Moisture conditions are fairly uni- 

 form in ordinary bacteriological technique; colonies of most 

 species are discrete and characteristic. The colonies, when 

 few, are usually pure; the viable aerobes usually all form colonies, 

 and the method as a whole is easy and practical. But with an- 

 aerobes the moisture content of the medium and the moisture on 

 its surface become of great importance. I have known agar in 

 deep tubes of medium, which had given perfect results with black- 

 leg colonies, to refuse to give a growth of blackleg when it was 

 somewhat old and dried out, though the agar had nowhere, as yet, 

 separated from the side of the tube. The addition of sterile dis- 

 tilled water made the medium as fertile a soil as fresh agar. I 

 have encountered aerobes which grow to the surface of the agar of 

 a shake, but not in colonies upon its surface. How much more 

 would dryness affect the growth of the more delicate anaerobic 

 organisms on the surface of a plate! Even the hardy tetanus or- 

 ganisms, which grow well in dry deep agar, often refuse to grow on 

 its surface. In order to produce discrete anaerobic colonies plates 

 must be dried after pouring. They must be dried j ust long enough 

 and not too long. This period varies with the composition, age 

 and thickness of the agar, with the humidity of the atmosphere, 

 and with the moisture present in the anaerobic jar. It takes time 

 and patience to learn to adjust the period for drying the plates. 

 Then when the culture is sown and the plates are ready to incu- 

 bate, what have we for anaerobic methods? A variety of available 

 atmospheres for the growth of the organisms almost as great as is 

 the number of workers in the anaerobic field : Hydrogen, carbon- 



