58 



Clemesha, in 1912, found that in peptone bile salt, neutral red broth 

 containing 1 percent lactose, pure cultures of Bact. coli and Bact. aerogenes 

 begin to fall off in numbers after 24 hours. Burling and Levine observed 

 that the concentration of carbohydrates influences markedly the viability 

 of the colon group in culture media. With 0.3 percent glucose or lactose, 

 the number of viable organisms reached a maximum of 100 to 1,000 mil- 

 lion in about 10 hours and remained constant for about 7 days. Increasing 

 the amount of carbohydrates to about 0.5 percent resulted in death of 

 over 99 percent of the organisms in a week, whereas with a concentration of 

 1 percent of the carbohydrates a count of 100 million, after 24 hours, was 

 reduced to less than 10,000 (a reduction of 99.9 percent in 72 hours.) 



In preparing carbohydrate media the author has used the following 

 method with excellent results: 



The broth containing 0.5 percent of the carbohydrate is distributed in 

 sterile Durham fermentation tubes, using ordinary precautions but not 

 necessarily strict asepsis, and autoclaved at 10 pounds for 10 to 15 minutes 

 in a pressure steam sterilizer. Immediately after removing from the auto- 

 clave, the tubes are cooled by placing in running tap water after which 

 they are incubated at 37 C. for 24 to 48 hours to eliminate unsterile tubes. 

 Several tubes are inoculated with Bact. enteritidis or Bact. paratyphosum 

 and incubated for 24 hours to detect hydrolysis as shown by gas formation. 



Wagner and Monfort have recently reported that if gentian violet 

 (1-20,000 to 1-100,000) be added to lactose broth, the medium may be 

 prepared by a single heating in the Arnold or simply pasteurization. If 

 further experience proves gentian violet non-inhibitory to the growth of 

 Bact. coli., such a medium would be practically ideal, for not only is the 

 danger of inversion of the lactose eliminated, but growth of the spore 

 forming lactose fermenters is also reported to be checked. 



In recording the presumptive test with lactose broth (this applies to 

 lactose bile also), the production of 10 percent or more gas is regarded 

 as a positive presumptive test. If the amount of gas is less than 

 10 percent the test is regarded as questionable. If there is no gas it is of 

 course negative. Experience indicates that where the quantity of gas is 

 more than 70 percent the fermentation is very likely due to anaerobic spore 

 formers rather than to the colon group. 



Reliability and Value of the Lactose Broth and Lactose Bile 

 Presumptive Tests. In studies on the Ohio and Mississippi Rivers, 

 Fuller noticed that the results with these presumptive tests were far be- 

 yond what could be explained by ordinary pollution and much higher 

 than results obtained by a complete isolation test. Similarly in studies 

 on the Potomac River, Cummings found these tests were reliable when pol- 

 lution was excessive, but as purification progresses, the correlation be- 

 tween the presumptive and confirmatory tests become less marked. Using 

 Endo agar for confirmation 92 percent of positive presumptive tests were 

 confirmed in regions close to the source of pollution but in the vicinity of 

 oyster beds, which were further removed from pollution, only 47.5 per- 

 cent of the positive presumptive tests were found to contain members of 



