[HARRISON à vanderi.eck] -ESCULIX bile salt ME1)L4 151 



A. To this medium varying amounts of iron citrate scales were 

 added as follows: 0.4% iron citrate, acidity + 2.6 neutralised to + 0.6. 



B. 0.3% iron citrate, acidity + 1.8, neutralised to + 0.6. 



C. 0.2%, iron citrate, acidity + 1.2, neutralised to + 0.6. 



D. 0.1% iron citrate, acidity + 0.65, neutralised to + 0.6. 



E. 0.02% iron citrate acidity + 0.12. made acid to + 0.6. 



Three plates were made from eacii medium (A.B.C.D. & E.) with 

 different amounts of />'. coll. 



1 containing 25 colonies, 2 containing 11 colonies, 3 containing 

 2 colonies. 



The plates were poured in the following way: 



One half cubic centimetre of tiie bacterial suspension was 

 placed in the Petri dish and well mixed with 10 c.c. of the medium, 

 after the plates had hardened 10 c.c. of the medium was poured on 

 top; this prevented the forming of surface colonies and reduced the 

 result of evaporation in the incubation. The appearance of the 

 plates befoi-e the growth of colonies was as follows: 



A. B. C. pale brown tone, colour increasing with the amount 

 of iron citrate. D. E. very clear, slight yellow colour. After 24 

 hours incubation at 37°C. the plates were examined. A. B. C. D. 

 were very satisfactory in number of colonies and appearance of 

 black fields, but E. was a complete failure. Comparing A. B. C. D. 

 with one another, D. attracted attention by the clear contrast between 

 the medium and the black colonies, and by the large size of the colo- 

 nies. They were at least twice as large as the colonies on the other 

 plates and kept increasing in size during the second day, until their 

 diameter was more than 3 m.m. 



It is not necessary to discuss why the method of neutralisation 

 recommended by the American Public Health Association is impos- 

 sible with a'sculin media. Complete neutraUsation precipitates all 

 the iron, and it is only a defect in the filtration if iron is retained in the 

 form of colloidal iron hydrate. This iron hydrate is dissolved again 

 when hydrochloric acid is added. Neutralisation after the addition 

 of the iron citrate may be a correction, but never part of the mani- 

 pulation. 



The previous directions for the making of icsculin bile salt agar 

 only hold good for the analyses of those substances which are added 

 in small quantities. Should large quantities of the material for 

 analysis be added the results are apt to suffer. For example, if 

 we place 5 c.c. of water in a Petri dish and add two tubes of sesculin 

 bile salt agar, each containing 8 c.c. of the medium, we have a dilu- 

 tion of 20 per cent. 



