II44-12 MANUAL OF METHODS FOR PURE CULTURE STUDY 



content) in 95% ethyl alcohol. Add 1 ml. of this fuchsin solution to 

 100 ml. of the agar (melted) also 0.125 g. anhydrous sodium sulfite 

 dissolved in about 5 ml. distilled water and pour plates immediately. 

 The medium should be light pink while hot and almost colorless after 

 cooling; as varying results may be obtained with different batches of 

 fuchsin, it is sometimes necessary to use a weaker stock solution of 

 that dye (e. g., 2% or occasionally only 1%). 



Brilliant-green-bile medium. Dissolve 20 g. dried oxgall and 10 g. 

 peptone in 1000 ml. boiling water; cook in a double boiler or steam 

 for an hour. Add 10 g. lactose, and filter through cotton or cotton 

 flannel. Adjust reaction to between pH 7.1 and 7.3. Add 0.013 g. of 

 brilliant green (85-90% dye content). This concentration of bile and 

 dye is adjusted to permit the growth of bacteria of the colon-aero- 

 genes group, but to restrain or prevent the growth of Gram-positive 

 organisms, which often confuse diagnostic routine. When used in 

 water analysis, and more than 1 ml. of water is added to each tube, the 

 medium should be made of suflSciently greater concentration so that 

 the final dilution will be the same as that above indicated. 



Levine's eosin-methylene-hlue agar. Dissolve by boiling: 



Distaied water 1000 ml. K^HPO^ 2 g. 



Peptone 10 g. Agar 15 g. 



Before sterilizing add to 100 ml. of the above: 2 ml. sterile 2.0% 

 aqueous solution eosin Y (dye content about 85%), and 2 ml. sterile 

 0.325% aqueous methylene blue (dye content about 85%). Just 

 before use add aseptically 5 ml. sterile 20% lactose solution. Re- 

 action not adjusted. Do not filter. 



Buffered peptone solution for methyl red and Voges-Proskauer 

 tests. Dissolve 7 g. peptone (Witte or Difco Proteose Peptone), 

 5 g. glucose and 5 g. K2HPO4 in 1000 ml. distilled water. Adjust 

 reaction to pH 6.9-7.0, and sterilize in the autoclave. 



Blood broth. Add 5% of rabbit, sheep, or horse blood, drawn 

 aseptically and defibrinated, to beef extract broth or meat infusion 

 broth. 



Blood agar. Prepare beef extract or meat infusion agar containing 

 2% (instead of 1.2%) agar. Melt 100 ml. of this, cool to 45°C,and 

 add 5 ml. of rabbit, sheep, or horse blood, drawn aseptically and 

 defibrinated. The medium should be poured into plates or slanted in 

 tubes very soon after adding the blood. 



Bismuth-sulphite agar (Wilson and Blair, 1926; formula from Diagnostic Procedures 

 and Reagents, A. P. H. A., 1941, p. 25). To 1 litre nutrient agar (2% agar, 0.5% beef 



