460 HILDA HEMPL HELLER 



present to grow and form a colony. Otherwise colonies may be 

 fished through agar that contains living invisible organisms of 

 other species, and the most deceptive sort of contamination will 

 take place. The medium should be clear and transparent. Our 

 standard agar medium for routine work is made of beef liver. 

 The usual proportion of one part of meat to two of water gave too 

 active a growth and too much gas. The medium is made as 

 follows : 



One part of ground beef liver and four parts of distilled water 

 are infused over night, boiled, and strained. To the broth add 

 1.5 per cent peptone, 0.5 per cent salt, and for ordinary purposes 

 make up with 2 per cent agar pH 7.2 (faintly alkaline to litmus). 



When unusually active gas-producers are present, high dilu- 

 tions and short (twelve hours) incubation periods are resorted to. 

 Such methods always suffice when rapidly growing species are 

 the ones to be isolated. But when slowly growing species are 

 sought in the presence of actively growing ones, other methods 

 are available. To absorb hydrogen, 1 per cent potassium nitrate 

 may be added to the agar (Veillon and Maze) . To prevent the 

 colonies of the rapidly growing types from outrunning the others, 

 use 3 per cent agar or old agar that has partially dried out, or pay 

 particular attention to enrichment of the desired species in the 

 inoculum and employ abundant dilution tubes. Do not depend 

 upon any colony method for the isolation of badly contaminated 

 slowly growing tissue invaders, but resort directly to guinea-pig 

 inoculation. For slowly growing non-pathogenic organisms mixed 

 with rankly growing gas-producers, try a sugar-free agar (von 

 Hibler,1908). 



There is an essential point in the employment of deep colony 

 tubes which must be observed. Otherwise the method is of no 

 more use than any other. Actively growing anaerobes fre- 

 quently leave their colonies and grow in the agar as though it were 

 a broth. 



This happens more readily with some types of organisms 

 than with others. B. Welchii is the chief offender and should be 

 avoided by heating whenever possible. A tube in which this phe- 

 nomenon has occurred is readily identified by holding it to the 



