APPENDIX 169 



alkaline (using as indicator litmus paper), filter, boil for half an 

 hour. Add 260 c.c. litmus solution (Kubel and Tiemann), and 

 boil for ten minutes ; add 30 grammes of chemically pure milk- 

 sugar, and boil for fifteen minutes. Add the hot litmus milk- 

 sugar solution to the liquid agar solution (cooled to 60 C.) ; 

 shake well, and make faintly alkaline ; then add 4 c.c. of a 

 hot sterile solution of 10 per cent, water- free soda and 20 c.c. of a 

 freshly prepared solution of 0*1 gramme crystal violet (B. Hb'chsf) 

 in 100 c.c. warm sterile distilled water. The result is a meat- 

 water peptone nutrose agar containing 13 per cent, litmus and 

 O'Oi per 1000 crystal violet. The medium can be kept in tubes 

 or in small flasks containing enough for three or four plates. It 

 is sufficient to sterilize once in current steam for thirty minutes. 



After the plates are inoculated they should be thoroughly 

 dried uncovered, either in the laboratory or preferably in the 

 incubator. They are then covered, inverted, and incubated. 



This medium is chiefly used in the isolation of B. typhosus 

 and in particular to differentiate this bacillus from B. coli and 

 other organisms. After sixteen to twenty-four hours at 37 C. 

 the colonies can be distinguished from one another. The B. coli 

 colonies are red, not transparent, and have a diameter of 2 to 6 

 millimetres, but considerable variation in size and degree of 

 colour are met with. The B. typhosus colonies are blue, with a 

 violet tinge ; they are transparent and resemble dewdrops, and 

 have a diameter of I to 3 millimetres, seldom larger. 



Drigalski-Conradi medium is rather a trouble to prepare, and 

 is not always satisfactory in use. 



Dieudonnes Alkaline Blood agar. Prepared as follows : 



Equal parts of normal caustic potash solution and defibrinated 

 ox blood are mixed and sterilized in the autoclave (Solution A). 

 Nutrient agar of ordinary composition but exactly neutral to 

 litmus is prepared (Solution B). Seven parts of B are mixed 

 with three parts of A and poured into Petri-dishes. 



When the mixture of blood and alkali is heated a part of the 

 latter is absorbed, but the final agar still preserves a very strong 

 alkalinity corresponding to about 0*6 per cent, of potassium 

 hydrate. The free alkali and the alkaline combinations formed 



