1756 Journal of Applied Microscopy 



all the oxygen present in the culture medium, and that in this way they make it 

 possible for the anaerobes to thrive in mixed cultures. Kedrowski attributes 

 this fact to another phenomenon. He holds that the conditions favorable for anae- 

 robic development in mixed cultures are not due to the assimilation of the oxj'gen 

 present in the nutrient medium by the aerobic species, but that the latter produce 

 substances which form a suitable medium for the anaerobes to grow in. 



Roux recommends the following method for mixed cultures : Boil a conven- 

 ient quantity of nutrient agar in a cotton plugged test tube and cool rapidly in 

 cold water. Immediately after the agar is solidified inoculate it with the anaero- 

 bic organism by means of a glass needle, then pour a small amount of melted but 

 not too hot nutrient gelatin into the tube. When this is congealed pour a few drops 

 of a bouillon culture of Bacillus subtilis upon the surface of the gelatin. Seal the 

 tube in the flame and place it in the incubator. B. subtilis grows very rapidly 

 upon the surface, forming a tough membrane and using up the oxygen in the 

 tube. In order to obtain material for anaerobic subcultures, the bottom of the 

 test tube is broken, avoiding the mixing of the two cultures. 



Fetizo (1891) used a similar method. He cultivated the bacillus of malignant 

 oedema successfully under aerobic conditions in agar and gelatin containing cul- 

 tures of Bacillus prodigiosus or Proteus vulgaris. 



Scholtz (1898) observed that anaerobes grow much more vigorously in a 

 medium containing a vigorous culture of an aerobic species. 



While it is evident that mixed cultures are a simple and generally successful 

 means for obtaining vigorous anaerobic growth, it is equally obvious that this 

 method is unsatisfactory where pure cultures of anaerobic bacteria must be 

 obtained. Therefore, the use of mixed cultures can serve only as an indirect 

 means in studying anaerobic bacteria. 



B. Chemical reducing agents. 



Glucose. Librius, in 188G, observed that the addition of glucose to nutrient 

 bouillon, gelatin or agar exerted a favorable influence on the development of 

 anaerobic species. This action has been explained by the fact that glucose in an 

 alkaline solution possesses an oxygen-reducing power. 



Novy (1893) successfully used and recommends media with the following 

 ingredients for the cultivation of anaerobes : 



1. Beef broth containing I/2 per cent, sodium chloride, 2 per cent, peptone 

 and 2 per cent, glucose. 



2. Beef broth as above plus 2 per cent, gelatin. 



3. 1-15 per cent, nutrient gelatin with the same constituents as under I. 



4. \y^ to 2 per cent, agar containing ^4 per cent, sodium chloride, 2 per 

 cent, peptone and 2 per cent, glucose. 



Hewlett s method : Fill a test tube two-thirds with 2 per cent, glucose agar and 

 steam immediately before inoculation to expel any traces of oxygen that may be 

 present in the nutrient medium. When the agar has set, inoculate well into the 

 depth of it. Carefully warm the tube over the flame, melting the superficial 

 layer of agar and filling the puncture made by the inoculation. Now flame the 



