)52 ROYAL SOCIETY OF CANADA 



Media stored in plugged test tubes lose considerable water by 

 evaporation, hence it can be used with advantage for such tests 

 as the one mentioned, but in some cases, it may be necessary to change 

 the medium for certain specific uses. 



Three points have to be observed in the preparation of bile salt 

 aescuUn agar media, and the neglect of any of these makes the media 

 useless. 



A. The minimum amount of sesculin in pure crystal form 

 (Merck) must be 0.1%. Less sesculin endangers the reliability 

 of the test, more produces no noticeable effect. ^Esculin is an ex- 

 pensive product, so that it is not advisable to use more than is abso- 

 lutely necessary. 



B. The amount of iron citrate (scales, Merck) must be at least 

 0.1%, as already explained. 



We shall mention here, however, one case in which more iron 

 citrate 0.3% was used without subsequent neutralisation. We 

 were testing spring-water for />'. coll and could not find any in .30 c.c. 

 water. For greater accuracy we filled a half gallon bottle with the 

 spring water, added peptone, œsculin and bile salt and incubated 

 at 37°C. At the end of 24 hours contents of the bottle were black, 

 and some of the water was plated with sesculin agar, but the lique- 

 fying alkaU bacteria were so numerous in the water after incubation 

 that they made the plates alkaline, and we had diffuse brown plates, 

 where we expected to find black colonies. As the colon bacillus 

 was in such small numbers compared with the alkaline forms we tried 

 to improve conditions by increasing the acidity of the plates Two 

 ways were possible: (a) by the addition of acid; (b) by the addition 

 of iron citrate. Both methods were tried. The mark was made 

 -f- 2.0 and 5 c.c. of the blackened spring-water was added to the 

 plates and then incubated at 37°C. After 7 days' incubation three 

 black colonies appeared on the (b) plates, which were identified 

 as B. coli. The (a) plates never showed any bacterial development. 

 This is an example of a case in which the amount of iron citrate 

 could be increased. Such acid plates are not transparent, as the 

 bile salt becomes cloudy as soon as the acidity is over 4- I.I. 



C. The acidity of the media should be + 0.6. A greater 

 acidity interferes with bacterial growth, while a smaller acidity 

 favours the development of too many bacteria, which crowd the 

 plates too much, besides the defect of the iron concentration already 

 mentioned. 



The experimental data upon which the recipe for sesculin bile 

 salt agar already given was based were as follows: — 



