200 MICROSCOPIC AND CULTURAL STUDY OF BACTERIA 



+2.0. Dissolve 24 grams agar in one liter of water, boiling steadily 

 until complete solution is attained. Add to the meat infusion broth, 

 boil for ten minutes and clarify with eggs in the usual manner. Filter 

 and sterilize. 



Meat Extract Agar. Meat extract agar is made by substituting 

 meat extract solution for meat infusion; otherwise the process is the 

 same. The medium must be clarified with eggs. 



Glycerin Agar. Five per cent, of glycerin is added to meat infusion 

 agar immediately before filtration. The reaction for cultivation of the 

 tubercle bacillus should be +1.0 acid to phenolphthalein. Tubercle 

 bacilli do not thrive in media neutral to phenolphthalein. 



Loffler's Blood Serum. Add one part by volume of 1 per cent, 

 nutrient dextrose broth 1 to three parts of clear, hemoglobin-free beef 

 or sheep serum, and distribute in test-tubes. The tubes are placed 

 in a Koch's serum inspissator or in specially designed racks in an auto- 

 clave in an inclined position to produce a slanted surface, and slowly 

 heated to 80 C. This temperature is maintained until the medium 

 is firmly coagulated. The temperature is then raised to 95 or 100 

 C., and maintained for an hour on each of three successive days, or to 

 115 in the autoclave, and maintained for one hour. The medium is 

 opaque and white and the surface is smooth and should be free from 

 a metallic lustre when viewed by reflected light. The lustre indicates 

 an accumulation of salts, which are inimical to the growth of many 

 bacteria. 



Coagulated Serum. 2 Clear blood serum from the dog, sheep, cow. or 

 other animal, preferably sterilized by filtration through Berkefeld 

 filters, and with or without the addition of glycerin, is placed in test 

 tubes and slanted and coagulated in a serum inspissator at a tempera- 

 ture of 75 to 80 C. An exposure of one hour to this temperature on 

 each of six successive days is necessary to insure sterility. The medium 

 should be translucent, free from bubbles, and firm. 



Hiss Serum Water Media. Hiss 3 has recommended a serum water 

 medium for the cultivation of pneumococci and similar organisms. 

 It is prepared in the following manner : 



Sheep or beef serum, 4 clear and free from hemoglobin, is added to 

 water in the proportion of one volume of serum to three of water. 



1 If the liquefaction of blood serum by bacteria is to be tested, sugar-free broth must 

 be used in place of dextrose-broth. 



2 Theobald Smith, Tr. Am. Phys., 1898, xiii, 417. 



3 Jour. Exp. Med., 1905, vii, 223. 



4 It is advisable to sterilize the serum by passage through an unglazed porcelain filter. 



