COLON-AEROGENES GROUP OF BACTERIA 257 



The collecting outfit consisted of a large test tube and a strip 

 of tin one end of which was hammered into a spoon shape. The 

 tin was about an inch longer than the tube, in which it was 

 kept until the time of sampling, both being protected by a cotton 

 plug. This outfit was sterilized in the hot air sterilizer. In 

 taking a sample of soil the dry surface layers were usually removed 

 by a sterile knife and 10 to 15 grams of the soil collected in the 

 tube by means of the tin spatula. The samples were taken to 

 the laboratory immediately. 



Four different amounts of each sample were employed in the 

 isolation process, namely 0.01, 0.1, 0.5 and 1.0 gram portions. 

 At first these amounts were weighed out separately in watch 

 glasses, but subsequently they were approximated by comparison 

 with portions of known weight which were used as standards. 

 The different portions of soil were vigorously shaken with definite 

 amounts of water in dilution bottles. The shaking was continued 

 until a uniform emulsion was obtained. After standing long 

 enough for the heavy particles to settle out the supernatant 

 fluid was drawn off and plated on plain agar. The direct plating 

 method for isolation was employed throughout the investigation, 

 in preference to the liquid sugar medium enrichment method. 

 Although the latter method has often been employed, it was 

 feared that its use would disturb the original numerical relation- 

 ships of the bacterial types. 



Litmus lactose agar was at first used in the plating, but was 

 soon found to be very unsatisfactory. Very few red colonies 

 were observed on the plates, and at times not a single red colony 

 was discernable in as many as 20 to 30 plates after forty-eight 

 hours of incubation. This was in harmony with Burton and 

 Rettger's observations on the cloacae-aerogenes type of bacteria, 

 and those of Ayers who showed that alkali-forming bacteria in 

 milk would rarely be noticed on litmus lactose agar. 



The plain agar plates were incubated at 30°C. for forty-eight 

 hours. At the end of this period all of the coli-like colonies, 

 noted on examination with the low power objective, were sub- 

 cultured in lactose fermentation tubes. These tubes were incu- 

 bated at 30°C. from two to five days, at the end of which time 



