61 



ceivable, of course, that such a test may be due to death of the colon 

 types. A transfer from this unconfirmed presumptive test to another tube 

 of lactose broth, which on incubation shows gas production, would be 

 excellent evidence that the gas forming organism in question was an an- 

 aerobe and that the negative confirmatory test was not due to death or over- 

 growth of the gas producer. 



The media for confirmatory tests which have found most favor among 

 American bacteriologists are litmus lactose agar, and Endo agar; and 

 more recently the simplified Eosin-Methylene-Blue agar was suggested by 

 the author. The litmus lactose agar has been discussed in detail in con- 

 nection with the isolation of the colon group. It will merely be added 

 here that Meyer suggests that by the addition of 3 percent agar, giving a 

 stiffer and dryer medium, more favorable results are obtained than with the 

 ordinary medium. 



The Endo Agar. This is perhaps the most extensively employed 

 confirmatory medium at the present time. It is the medium of choice of 

 the Public Health Service and is recommended in the Standard Methods 

 (1917 and 1920). The colon colonies produce a distinct red coloration, 

 often with a metallic sheen, and are quite easy to detect. Unfortunately 

 there has been, in the past, considerable lack of uniformity in the prepara- 

 tion of Endo. The method of adjustment of the reaction is very crude 

 and there was lack of agreement as to the quantity of indicator to be em- 

 ployed. Thus Kendall, and Prescott and Winslow recommended 0.1 c. c. 

 of a saturated basic fuchsin and 1 c. c. of a 10 percent sodium sulphite 

 per 100 c. c. of agar. The Army Laboratory Manual, employed exten- 

 sively during the war, recommends 0.18 c. c. of fuchsin and 2.5 c. c. 

 of a 10 percent sodium sulphite. In the Hygienic Laboratory Endo medium 

 the indicator consists of 0.5 c. c. of basic fuchsin and the equivalent of 2.5 

 c. c. of a 10 percent sodium sulphite solution. Some investigators, Rob- 

 inson and Rettger, have recommended decolorization with sodium acid 

 sulphite in place of sodium sulphite. 



The appearance of the colon colonies will depend to a considerable 

 extent on the concentration of the indicator, being very intense red and 

 showing the characteristic metallic sheen only with the higher concentra- 

 tions. The description of a typical colony of Pact, coli on one of these 

 mediums may therefore not tally at all with its appearance on supposedly 

 the same medium prepared in some other laboratory. It thus becomes 

 essential to detail, in each instance, the method of preparation of the 

 medium employed. We may well exclaim with Morse and Wolman, "What 

 shall the standard test for Bact. coli include if a mere difference in the 

 proportion of fuchsin and sulphite in two Endo media results in the one 

 showing typical colonies in 75 percent of the tubes, in which Bact. coli is 

 present, while the other shows the same colonies in only 14 percent?" 

 The apparent difference is due to a confusion of the term "typical" Bact. 

 coli colony. What is considered typical for one medium with one con- 

 centration of dye will of course not be typical for another. 



