n„-18 MANUAL OF METHODS FOR PURE CULTURE STUDY 



with the smaller percentage of agar is preferred by some for seeded 

 plates as an aid in securing discrete colonies.) ' An alternate formula 

 is as follows: 



Proteose peptone 20 g. Na2HP04 2 g. 



Glucose 1 g. Sodium thioglycollate 1 g. 



Agar 20 g. (or 7.5 g. for subsurface colonies) 



Distilled water 1000 ml. 



Adjust 7.6 pH. If used for subsurface colonies, clarify medium 

 by filtration through paper using reduced pressure. 



Use: Satisfactory for routine purification and colony study of 

 pathogenic types. Convenient to prepare, since the ingredients are 

 usually available and for fresh meat extracts are not needed. 



Yeast infusion glucose agar. Prepare yeast infusion as follows (although other 

 methods, sometimes preferred, are equally satisfactory): Obtain fresh yeast (starch- 

 free if possible) from a fermentation company and add 10% by weight to several liters 

 of tap water. Autoclave for 3 hours or more. Allow cells to settle by standing for 

 several days at room temperature. Remove liquid infusion by syphon or with the 

 Sharpies centrifuge. Sterilize the liquid, after removal from the cells, in screw-capped 

 bottles and store indefinitely. For plating medium add 0.5% glucose and 2.0% agar. 

 Adjust to 7.0 pH; sterilize for 20 minutes at 15 pounds pressure. (Note: An equally 

 satisfactory, but considerably more expensive, basal medium may be prepared from 

 dehydrated yeast extract, adding 0.5% yeast extract to distilled water). 



Use: Recommended as plating medium for butyric-butyl group. 



Peptone-try ptone-glucose agar. If a source of yeast for the preparation of yeast in- 

 fusion is not readily available, the following plating medium may be substituted which 

 is only slightly less satisfactory than the one above. 



Peptone 0.5% Glucose 0.5% 



Tryptone 0.5% Agar 2.0% 



Adjust 7.0 pH before sterilization. (The medium is improved by the addition of 

 100 ml. of liver infusion, if available). 



Use: A satisfactory plating medium for the butyric-butyl group, calling for ingre- 

 dients which are usually available. 



3. MEDIA FOB DETERMINATION OF PHYSIOLOGICAL REACTIONS 



Sugar-free Base for Qualitative Fermentative Reactions'. Two 

 basal media for use in anaerobic fermentation reactions are given 

 here. Certain general directions are necessary: Indicators should 

 be used to test reaction after incubation or on small samples with- 

 drawn during incubation; they should not be incorporated in the 

 medium, as many anaerobes reduce them to their leuco form. The 



^Some workers have used a meat infusion broth or other medium which has been 

 rendered sugar-free by fermentation with Escherichia coli or Clostridium perfringens 

 This seems unnecessary at the present time as most species will grow quite well in one 

 or the other of the media suggested here. If a particular strain should not grow well 

 in the basal medium plus glucose, it is probable that some needed nutrient is not 

 present. For these, as with fastidious aerobes, ascitic fluid may be added, though this 

 will rarely be necessary. 



For quantitative studies on fermentation of the sugars the usual problem requires a 

 base medium suitable for the butyric-butyl group. Perhaps the most generally useful 

 basal medium is yeast water infusion prepared according to the method discussed for 

 yeast infusion glucose agar. 



