BACTERIOLOGICAL GRADE AGAR, GELATIN, AND PEPTONES 71 



"Loss on drying. Weigh accurately about 1 Gm., and dry at 100'^ to constant 

 weight: it loses not more than 7% of its weight. 



"Residue on ignition. Weight accurately about 500 mg., and heat slowly until 

 thoroughly charred. Cool, add 1 ml. of sulfuric acid, and ignite to constant weight: 

 the weight of the residue corresponds to not more than 15%. 



"Nitrite. To 5 ml. of a solution of the digest (1 in 50) add 0.5 ml. of sulfanilic- 

 a-naphthylamine T.S., mix, and allow to stand for 15 minutes: no pink or red color 

 develops. 



"Bacteriological test. The digest meets the following tests for bacteria-nutrient 

 properties. Prepare media of the following compositions: 

 " (a) 2 % of peptone, in water; 

 " (b) 0.1 % of peptone, in water; 

 " (c) 1 % of peptone, 0.5 % of dextrose, in water; 

 " (d) 1 % of peptone, in water; 

 " (e) 2 % of peptone, 1.5 % of agar, in water. 



''Adjust all media to a pH of 7.2 to 7.4. 



"Freedom from fermentable carbohydrate. To medium (a) add sufficient phenol- 

 sulfonphthalein T.S. to give a readable color, tube in Durham fermentation tubes, and 

 autoclave. Inoculate with a loop of 24-hour culture of Escherichia coli: no acid, or 

 only a trace in the inner tube, and no gas are produced during incubation for 48 hours. 



"Production of indole. Inoculate 5 ml. of medium (h) with Escherichia coli, incu- 

 bate for 24 hours, and test by addition of about 0.5 ml. of dimethylaminobenzaldehj-de 

 T.S.: it shows a distinct pink or red color which is soluble in chloroform. 



"Production of acetylmethylcarbinol. Inoculate 5 ml. of medium (c) with Aero- 

 bacter aerogenes, and incubate for 24 hours. Test by adding to the culture an equal 

 volume of sodium hydroxide solution (1 in 10), shake, and allow to stand at room tem- 

 perature for several hours: appearance of a pink color indicates the presence of 

 acetylmethylcarbinol. 



"Production of hydrogen sulfide. Inoculate 5 ml. of medium (d) with Salmonella 

 typhosa. 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. After 

 incubation for 24 hours, the lower tip of the lead acetate test paper shows little if any 

 darkening. After 48 hours, it shows an appreciable amount cf brownish blackening 

 (lead sulfide) . 



"Growth-supporting properties. In the foregoing tests the media support good 

 growth of Escherichia coli, Aerohacter aerogenes, and Salmonella typhosa. Medium (e) 

 stab-inoculated with a stock culture of Brucella abortus shows good growth in the line 

 of the stab after incubation for 48 hours. Slants of medium (e), inoculated with 

 Escherichia coli, Aerobacter aerogenes. Salmonella typhosa, Pseudomonas aeruginosa, 

 Staphylococcus aureus, and Staphylococcus albus, show characteristic growth after 

 incubation for 24 hours. Medium (e), to which about 5% of rabbit blood has been 

 added and which has been inoculated and poured into Petri dishes, show^s character- 

 istic alpha or beta zones about colonies of pneumococci and beta hemolytic streptococci 

 (serological groups A and B), recognizable within 24 hours and fully developed after 

 48 hours' incubation. Medium (e), to which about 10 % of blood has been added and 

 which then has been heated to 80 to 90° until the blood has turned chocolate-brown, 

 permits the growth of gonococcus colonies within 48 hours when incubated in an 

 atmosphere containing about 10 % of carbon dioxide." 



