PURE CULTURES 51 



solidification may set in and prevent the even distribu- 

 tion of the bacteria in the medium. These precautions 

 should be borne in mind when agar media are used, 

 especially where they are employed for quantitative 

 determination of the number of organisms in solutions. 



Preparation of Nutrient Agar Medium. 

 Ex. 20. Make a nutrient broth, using 



(1) Lemco's extract of meat, 5 grams. 

 Witte's peptone, 10 grams. 

 Water, 500 c.c. 



(2) Take 15 grams of agar-agar and scak it for twelve hours 

 in 500 c.c. of water. Mix (i) and (2) and boil or heat in the 

 steam sterilizer twenty to thirty minutes until the agar and other 

 materials are dissolved. Neutralize and bring the hot solution 

 to proper acidity, as in Ex. 17 (3 and 4). Cool to 4o-5o C., and 

 clarify with white of an egg, as in Ex. 17 (5). Then heat in 

 steam sterilizer for i J hours, and filter when hot in a steamer or 

 through Chardin filter paper in a hot water funnel, first wetting 

 the filter paper with boiling water.- If not clear and free from 

 opaque spots when cool, reheat again, clarify, and pass through 

 a Chardin filter paper. Run 8 to 10 c.c. into test-tubes, and 

 sterilize as in Ex. 1 7 (6). Agar media filter very slowly. Where 

 an autoclave is available, place the neutralized solution, with the 

 white of an egg added, in the apparatus, and heat for half an 

 hour at two atmospheres pressure or for 45 minutes at 120 C. 

 Turn off the flame, and when cooled to below 100 C. open the 

 autoclave, and the agar medium will be found to filter clear 

 through a Chardin paper in fifteen to twenty minutes. 



Ex. 21. Repeat Ex. 18 and Ex. 19, using the above agar 

 medium instead of nutrient gelatine. 



2. Pure Cultures. After the bacteria have been 

 isolated, and the separate colonies allowed to grow on 

 the nutrient gelatine or agar medium, minute portions of 

 each distinctive colony are removed with a fine sterilized 



