70 DIFGO MANUAL 



Ayers and Mudge^ devised a differential medium for milk control which was 

 essentially Nutrient Extract Agar, containing skim milk powder extractives. The 

 medium gave high counts when compared with Nutrient Agar because it was 

 well adapted for the growth of lactic acid bacteria. Black, Prouty, and Graham,^ 

 and Prouty^ gave a detailed method for the practical use of Bacto-Nutritive 

 Caseinate Agar and modified the medium by adding the brom cresol purple 

 indicator to the medium prior to sterilization, using 8 ml. of the indicator 

 solution per 1000 ml. of Nutritive Caseinate Agar. 



Bacto-Nutritive Caseinate Agar was developed in our laboratories to duplicate 

 the medium of Ayers and Mudge. Modifications of the original formula were 

 necessary, but the dehydrated culture medium yields results comparable with 

 the original. Peptonizers and acid formers are readily distinguished, which to- 

 gether with the total count, give a more complete sanitary knowledge of the 

 milk sample under investigation than is possible with plain agar media. In 

 determining the differential count, the plates are incubated for 48 hours, and 

 then a total count of all colonies is made. Next the strong acid forming colonies 

 are counted, these being distinguished by a white zone of precipitated casein 

 surrounding the colonies. The plate is then flooded with brom cresol purple indi- 

 cator solution (0.4 gram Bacto-Brom Cresol Purple, 7.4 ml. of N/10 sodium 

 hydroxide and 92.6 ml. distilled water) for five minutes, and a count made of all 

 acid forming colonies which are yellow against the purple background of the 

 medium. To determine the number of weak acid formers, the number of strong 

 acid formers is subtracted from the number of total acid forming colonies. The 

 plate is then flooded with dilute acetic acid (5 per cent solution), and the pep- 

 tonizing colonies, which are surrounded by a clear zone are counted. 



To rehydrate the medium, suspend 22 grams of Bacto-Nutritive Caseinate 

 Agar in 1000 ml. cold distilled water and keat to boiling to dissolve the medium 

 completely. Distribute in tubes or flasks and sterilize in the autoclave for 15 

 minutes at 15 pounds pressure (121°C.). The reaction of the medium will be 

 pH 6.5. 



One pound of Bacto-Nutritive Caseinate Agar will make 20.6 liters of medium. 



1 J. Bact., 5:565:1920. 'J. Dairy Science, 17:115:1934. 



s J. Dairy Science, 15:99:1932. 



BACTO 



THERMO AGIDURANS AGAR (B303) 



DEHYDRATED 



Bacto- Yeast Extract 5 g. 



Proteose Peptone, Difco 5 g. 



Bacto-Dextrose 5 g. 



Dipotassium Phosphate 4 g. 



Bacto-Agar 20 g. 



Bacto-Thermoacidurans Agar is recommended for the cultivation of Bacillus 

 thermoacidurans {Bacillus coagulans) , the organism causing "flat sour" spoilage 

 of tomato juice. It is prepared according to the formula described by Stern, 

 Hegarty and Williams^ for the isolation of this organism, and for its cultivation 

 in pure culture. 



For the detection of B. thermoacidurans Stern, Hegarty and Williams^ recom- 

 mend the plating of 1 ml. of tomato juice per 20 ml. of agar medium. They ob- 

 served that larger quantities of tomato juice exhibited an inhibitory effect on 

 the growth of the organism. Plates are poured with the sterile melted agar at 

 45-55 °C. and, following solidification, incubated at 55 °C. for 48 hours. 



