70 MANUAL OF MICROBIOLOGICAL METHODS 



" (c) 0.1 % of peptone and 0.5 % of dextrose in water 

 '* (d) 1 % of peptone in water 



''Adjust all media to a final pH of 7.2 to 7.4. Place 5 ml. of (a) in Durham fermen- 

 tation tubes, and 5 ml. each of (6), (c), and (d) in ordinary test tubes. Autoclave the 

 media at 121° for 15 minutes. After autoclaving, and after standing for 24 hours, all 

 media are clear. 



''Presence of fermentable carbohydrate. Inoculate medium (a) with Escherichia 

 coli and with Streptococcus liquefaciens : acid is produced by E. coli but not by S. 

 liquefaciens during incubation for 24 hours. 



''Production of indole. Inoculate medium (6) with Escherichia coli and with Aero- 

 bacter aerogenes, and incubate for 24 hours. Test by adding about 0.5 ml. of p-di~ 

 methylaminobenzaldehyde T.S.: the appearance of a pink or red color (soluble in 

 chloroform) indicates the production of indole by E. coli. The A. aerogenes culture 

 gives a negative test. 



"Production of acetylmethylcarbinol. Inoculate medium (c) with Escherichia coli 

 and with Aerohacier aerogenes, and incubate for 24 hours. Test by adding to the 

 culture an equal volume of sodium hydroxide solution (1 in 10), shaking well, and 

 allowing to stand at room temperature for several hours: the appearance of a pink 

 color indicates the production of acetylmethylcarbinol by A. aerogenes. The E. coli 

 culture gives a negative test. 



"Production of hydrogen sulfide. Inoculate medium {d) with Salmonella iyphosa. 

 Hold a strip or loop of lead acetate test paper between the cotton plug and the mouth 

 of the test tube so that it hangs about 5 cm. above the medium. Then incubate for 

 24 hours: the lower part of the lead acetate test paper shows an appreciable amount of 

 brownish blackening (lead sidjide). 



PANCREATIC DIGEST OF CASEIN (A BACTERIOLOGICAL PEPTONE) 



"A grayish yellow powder, having a characteristic, but not putrescent, odor. 

 Freely soluble in water; insoluble in alcohol and in ether. The casein used in prepara- 

 tion of this digest meets the following specifications: 



Residue on ignition not more than 2.5 % 



Loss on drying not more than 8 % 



Free acid (as lactic acid) not more than 0.25% 



Fat not more than 0.5 % 



Reducing sugars not more than a trace 



Fineness All passes through a 20 mesh sieve 



"Degree of digestion. Dissolve 1 Gm. in 10 ml. of water. 



" (o) Overlay 1 ml. of the digest solution with 0.5 ml. of a solution of 1 ml. of glacial 

 acetic acid in 10 ml. of diluted alcohol: no ring or precipitate forms at the junction of 

 the two liquids, and when shaken no turbidity results (indicating the absence of 

 undigested casein). 



" (6) Mix 1 ml. of the digest solution with 4 ml. of a saturated solution of zinc 

 sulfate: a moderate amount of precipitate is formed (indicating the presence of 

 proteoses). Filter, and retain the filtrate for the next test. 



" (c) To 1 ml. of the filtrate from (6) add 3 ml. of water, and follow with 1 drop of 

 bromine T.S.: a violet-red color is produced, indicating the presence of tryptophane. 



"Nitrogen content. Determine the nitrogen content of the digest, previously dried 

 at 105° to constant weight, by the Kjeldahl method (see page 909) : not less than 10 % 

 of nitrogen (N) is found. 



