COLON BACILLUS TEST 99 



contamination in large amount, and the lactose bile medium 

 gives conclusive results regarding this. 



The presumptive colon bacillus test is to be supplemented 

 further as follows : Plate suitable dilutions of the substances to be 

 tested for sewage contamination (o.ooi cc., o.oi cc., o.io cc., 

 i. oo cc.) into lactose litmus agar Petri dishes, making two sets. 

 Incubate one set of these plate cultures at 20 C. and the other at 

 37 C. and note the following: 



1. The relative number of colonies which develop at the two temperatures. 



2. The number of acid-forming colonies. 



The time of incubation at the lower temperature (20 C.) 

 should be 3 days, although fairly conclusive results may be 

 noted at the end of the second day. The standard time of incuba- 

 tion at the higher temperature (37 C.) is 48 hr., although certain 

 results may be noted at the end of 24 and 36 hr. If the propor- 

 tion of high temperature colonies is high, it is indicative of the 

 presence of numerous bacteria derived from the intestinal tract. 

 If the high temperature colonies approximate (numerically) the 

 low temperature colonies, sewage contamination may be suspected. 

 If in addition many of the high temperature colonies show pink 

 or vermilion (on lactose-litmus agar), the sewage contamination 

 is practically proven. Both the colon bacilli and the sewage 

 streptococci show pink colonies on this medium, the latter being 

 the brighter, more vermilion in coloration. This coloration is 

 due to the formation of acid by the organisms named which reacts 

 with the litmus. Examine the pink colonies under the micro- 

 scope in order to determine which are the colon bacilli and which 

 the streptococci. As a rule, high temperature colonies should not 

 exceed i : 100 as compared with the low temperature colonies. It 

 must be kept in mind that the pink colonies may turn blue within 

 24 hr. due to the liberation of ammonia and amines. Red 

 colonies indicate lactose fermentation with formation of acid, but 

 since bacteria other than the colon bacillus form acid (notably 



