ISOLATION OF ANAEROBES 461 



light with a control. Such tubes are to be regarded as ''enrich- 

 ment cultures. " Thus their colonies may be of great use when 

 directly inoculated onto another agar series. They are of no use 

 when inoculated into a liquid medium. The close observation of 

 this phenomenon of ''permeating growth" cannot be too earnestly 

 insisted upon. 



The deep colonies of anaerobes are highly characteristic. 

 Surface colonies are quite characteristic but are obviously sub- 

 ject to many more outside influences than are deep ones. Often 

 colonies of different strains in the same species are different and 

 sometimes colonies of one type of anaerobe resemble those of an 

 entirely different type. But carefully made agar shakes often 

 give a beautiful picture of the flora of a wound or of a culture. 

 They are very easily observed with a hand lens and may be as 

 closely approached as may surface colonies. Aerobic growth is 

 easily distinguished from anaerobic growth. My routine method 

 of testing for impurity of culture has been to make three dilution 

 shakes on liver agar. The first and second tubes tell whether or 

 not the culture is pure. The third usually furnishes colonies 

 suitable for fishing. I was able to isolate, in two series of three 

 agar tubes each, a strain of oedematiens type that had been over- 

 grown 1 : 500 by a vibrion septique. 



Technique of sowing and fishing. Boil the tubes of agar for a 

 minute or two, remove them from the water, shake them, boil 

 them a little longer, shake them again to remove the air, then cool 

 them to 45°. Do not boil them for ten or fifteen minutes or the 

 cotton will become saturated with moisture. For ordinary pur- 

 poses use three tubes to each culture. For new and important 

 material of doubtful nature or for shyly growing organisms among 

 rankly growing ones, use more tubes. Inoculate tube 1 with 

 oneloopful of culture and roll it, tip it, and roll it four or five times. 

 Take a Pasteur pipette^ of large bore, flame it, draw up agar of tube 



1 It is to be noted that few laboratory workers today understand the making 

 of strong and serviceable Pasteur pipettes, and I hope to be pardoned for de- 

 scribing so simple an operation. Meeker burners are best for this purpose. Heat 

 the glass in the portion of the flame where the heat is nearly uniform for a con- 

 siderable distance. In a blowpipe or Bunsen flame this is above the cone; in 

 the flame of the Meeker burner it is half an inch above the base. Turn the glass 



