117 



c. c. of recently prepared 1 percent neutral red solution are added. The 

 medium is then tubed and sterilized for 15 minutes on three successive 

 days. 



(j) Aesculin Agar. (After Eyre) (B. coli and allied organisms 

 give black colonies surrounded by black halo.) 



Measure out 400 c. c. distilled water into a tared 2-liter flask. 

 Weigh out 



Agar 15 grams 



Peptone 10 grams 



Sodium taurocholate 5 grams 



and make into a thick paste with 150 c. c. distilled water. 

 Add this paste to the distilled water in the flask. 



Dissolve the ingredients by bubbling live steam through the mixture. 

 Weigh out 



Aesculin 1.0 gram 



Ferric citrate 0.5 gram 



and dissolve in a second flask containing 100 c. c. distilled water. 



Mix the contents of the two flasks adjust the weight to the calculated 

 medium figure (in this case 1031.5 grams) by the addition of distilled 

 water at 100 C. 



Clarify with egg and filter. 



Tube and sterilize as for nutrient agar. 



(k) A Simplified Eosine Methylene Blue Agar. (Levine) 



Distilled water 1000 c. c. 



Peptone (Difco) 10 grams 



Dipotassium phosphate (K 2 HP0 4 ) 2 grams 



Agar : 15 grams 



Boil ingredients until dissolved and make up any loss due to evapora- 

 tion with distilled water. 



Place measured-quantities (100 or 200 c. c.) in flasks or bottles, and 

 sterilize in the autoclave at 15 pounds pressure for 15 to 20 minutes. 



Just prior to use add, to each 100 c. c. of the melted agar prepared 

 as above, the following: 



Lactose, sterile 20% solution .5 c. c. or 1 gram dry substance 



Eosine yellowish 2% aqueous sol. 2 c. c. 



Methylene-blue 0.5% aqueous sol. 2 c. c. 



Pour medium into perti dishes, allow them to harden, and inoculate 

 by streaking on the surface. 



There is no adjustment of the reaction and filtration of the medium 

 is not necessary. 



V. SPECIAL MEDIA FOR DIFFERENTIATION OF THE COLI 

 AND AEROGENES SECTIONS. 



A. Clark and Lubs Medium. (Standard Methods A. P. H. A., 

 1920). 1. To 800 c. c. of distilled water add 5 grams of Proteose-Pep- 

 tone, Difco., or Witte's Peptone (other peptones should not be substituted), 

 5 grams c. p. dextrose, and 5 grams dipotassium hydrogen phosphate 



