ISOLATION OF ANAEEOBES 463 



pipette again, flaming it well, and use it to isolate two or three more 

 colonies. Other workers employ other methods, which are prob- 

 ably as good. Burri (1902) recommends the use of tubes open at 

 both ends with an autoclaved rubber stopper placed in the lower 

 end. Some use the loop only for purposes of dilution. Some heat 

 the end of the test tube and expel the agar column by force of the 

 steam thus generated. It is necessary to break the tube at the 

 bottom only when an aerobe is present. Burke used a dissecting 

 lens with stand for fishing colonies; Dr. Meyer finds a binocular a 

 great help in some cases. He sections the agar with a sterile blade 

 when researching for minute colonies that are rare. Some workers 

 prefer to attach a rubber tube or a teat to the pipette used in fish- 

 ing. It is theoretically wrong to fish the colonies from the top of 

 the column of agar without removing it from the tube, because the 

 capillary may pass ungerminated organisms, but such a method 

 might prove practical when used with discretion. Some workers 

 fish the colonies with a platinum needle, but this would hardly 

 prove as satisfactory as a pipette method. 



Methods of single-bacillus isolation. Isolation of a single bacil- 

 lus has been resorted to for the separation of anaerobes. Miss 

 Robertson found that the India-ink method of Burri (Besson 1913) 

 exposed the organisms too much and they failed to germinate. I 

 used the Barber method for some time for blackleg and vibrion- 

 septique organisms, and found that the exposure killed vegetative 

 forms and that spores were necessary to give a growth. I fished 

 from apparently pure cultures various numbers of organisms, from 

 one to ten, into meat tubes and used for a type strain the tube 

 that grew and had received the fewest bacilli. I found the method 

 wasteful of time, material, eyesight, and nervous energy, and have 

 abandoned it. My employment of the apparatus was, however, 

 far from being as skillful as that of Dr. Barber. I explained my 

 difficulties to Dr. Barber and he (1920) has made a careful statis- 

 tical study of the behavior of various anaerobes when isolated by 

 his technique. He was successful when inoculating various media 

 with different anaerobes in securing 62 growths from 400 single 

 bacilli, and 93 growths from 211 single spores. Vegetative rods 



