128 CONN, HARDING, KLIGLER, FROST, PRUCHA AND ATKINS 

 PREPARATION OF MEDIA 



Beef -extract broth shall have the following composition: 



Beef-extract 3 grams 



Peptone 5 grams 



Distilled water 1000 cc. 



Beef-extract agar shall be of the same composition plus the ad- 

 dition of 12 grams of oven-dried agar or 15 grams of commercial 

 agar. Beef-extract gelatin shall be of the same composition, with 

 the addition of 120 grams of Gold Label gelatin, or 100 grams of 

 some brand of gelatin (such as "Bacto-gelatin," or United States 

 Glue Company gelatin) having greater jellying power. These 

 media are to be made up according to the directions given by the 

 committees of the American Public Health Association on water 

 analysis and on milk analysis (1916, 1917), except that white of 

 egg may be used for clarification if desired. It is recommended, 

 however, that instead of using phenolphthalein in adjusting the 

 reaction of these media the simpler and more accurate procedure 

 be adopted of adjusting to the neutral point of brom thymol 

 blue. 1 Bring the media to such an acidity as to turn this indicator 

 a distinct grass-green (neither yellow green nor blue green). This 

 color indicates approximately "true neutrality," i.e., a hydrogen- 

 ion concentration between pH = 6.6 and pH = 7.4. Another 

 equally satisfactory method of adjusting media to this hydrogen- 

 ion concentration is to bring them to such an acidity as to cause 

 the first faint trace of permanent pink to appear with phenol red. 2 



Sugar broths. Just before sterilization 1 per cent of the re- 

 quired carbohydrate is to be added to beef-extract broth. Other- 

 wise proceed as for sugar-free broth. Adjust reaction with brom 

 thymol blue or phenol red. 



Plain gelatin. Proceed as for beef-extract gelatin, but omit 

 beef-extract and peptone. Clarify with white of egg. 



Nitrate broth. For routine work add 0.1 per cent KN0 3 to the 

 above formula for beef-extract broth. Routine nitrate agar 

 should contain 0.1 per cent KN0 3 added to the ordinary formula 



1 Use a 0.04 per cent solution of brom thymol blue in 95 per cent alcohol. 



2 Use a 0.02 per cent solution of phenol red in 95 per cent alcohol. 



