234 MICROSCOPIC AND CULTURAL STUDY OF BACTERIA 
2. A 2 per cent neutral sodium oleate solution in distilled water. 
3. Rabbit red blood cells, washed free from serum and made up to 
the original volume with salt solution. 
(1) Add to 94 cc. of agar melted and cooled to 45° C, (2) 5 cc. of 
sodium oleate solution and (3) 1 cc. of sterile rabbit red blood cells. 
Pour into sterile Petri dishes, allow to harden and inoculate. 
Media for the Cultivation of Anaerobic Bacteria.— Anaerobic bacteria 
develop best in media containing highly organized nitrogen. Pep- 
tones and lower degradation products of the decomposition of pro- 
teins are decidedly less favorable to anaerobic cultivation than muscle, 
brain, milk or gelatin proteins. The "sterile tissue"^ medium of 
Theobald Smith is a very satisfactory medium, but difficult to prepare 
in a state of unquestioned sterility. Von Hibler' has used a brain 
medium composed of finely minced sheep or calves brains to which is 
added one-fourth the volume of plain nutrient broth, with excellent 
results. The putrefactive anaerobic bacteria induce blackening after 
a few days, and all bacteria capable of fermenting galactose (a carbohy- 
drate constituent of brain) produce more or less gas in the medium. 
Miss Robertson'^ substituted beef hearts for the brain medium of 
von Hibler with equally good results. In this medium the carbohy- 
drate is chiefly glucose, derived from the cleavage of glycogen, and 
from the glucose which is present in muscle. The same blackening 
takes place when putrefactive anaerobes are present, provided the 
degree of acidity formed from the initial fermentation of the glucose 
is not inhibitory to further development. Some organisms, as Bacillus 
welchii, produce a distinct red color of unknown composition after a 
few days' growth in this meat medium.^ 
Holman^ has modified the Robertson medium as follows: Fresh 
meat (beef heart or ordinary beef muscle) is freed from fat and tendons, 
finely ground in a meat chopper, and mixed with an equal volume of 
water. The mixture is heated to the boiling-point, with continuous 
stirring to permit of a finely divided heat coagulum, then made prac- 
tically neutral to phenolphthalein. The medium is placed in tall, 
narrow test-tubes, in a layer at least 2 inches deep, and sterilized 
in the autoclave for two hours.'' Incubation is practised at 37° C. for 
two days, and a second autoclaving follows for one hour. The medium 
is then tested for sterility. It is necessary to heat the medium for 
at least one hour in the Arnold sterilizer immediately before using, fol- 
lowed by rapid cooling to incubator temperature to remove all traces 
1 Centralbl. f. Bakteriol., 1890, 7, 502. 
2 Ibid., 1899, 25, 513. 
^ Jour. Pathol, and BacterioL, 1915-1916, 20, .327. 
4 Henry: Ibid., 1916-1917, 21, 344. 
'•< Jour. Bacteriol., 1919, 4, 149. 
» The dense coagulum is not readily penetrated by heat; a prolonged sterilization is 
absolutely essential to kill the very resistant anaerobic spores that may be present in 
the center of meat coagula. Failure to observe this precaution is responsible for the 
admixture of Bacillus sporogenes and other putrefactive anaerobes whose spores are 
killed with great difficulty. 
