DEHYDRATED CULTURE MEDIA 53 



outlined against a uniformly blue background. The colonies vary from 0.4 to 0.8 

 mm. in diameter. 



The medium is rather sensitive to light, particularly direct sunlight, which 

 produces a decrease in the productivity of the medium and change in color from 

 deep blue to purple or red. It is recommended that the medium be prepared just 

 prior to use and when necessary to store the medium, it should be kept in the dark. 



To rehydrate the medium, suspend 20.6 grams of Bacto-Brilliant Green Bile 

 Agar in 1000 ml. of cold distilled water and heat 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 final reaction of the medium 

 will be pH 6.9. 



One pound of Bacto-Brilliant Green Bile Agar will make 22 liters of medium. 



* J. Am. Water Works Assoc, 27:108:1935. 2 Standard Methods for the Examination 



of Water and Sewage, 9th Edition: 228: 1946. 



BACTO 



TRYPTONE (B123) 



DEHYDRATED 



Bacto-Tryptone in a 1 per cent concentration is specified in "Standard Methods 

 for the Examination of Water and Sewage"^ for the performance of the indole 

 test, and is also specified in "Standard Methods for the Examination of Dairy 

 Products,"^ for use in preparing media for the standard plate count. 



A complete discussion of Bacto-Tryptone, and other uses of this peptone, is 

 given on page 260. 



The test for indole recommended in "Standard Methods" is that of Kovacs^ 

 which is also recommended in "Diagnostic Procedures and Reagents"* of the 

 American Public Health Association. This test is performed by adding 0.2 to 0.3 

 ml. of Kovacs' reagent to 5 ml. of a 24-hour culture of the organism under in- 

 vestigation. A dark red color in the surface layer constitutes a positive test; the 

 original yellow color of the solution is a negative test when this reagent is used 

 for indole detection. Kovacs' reagent is made by dissolving 5 grams of p-dimethyl- 

 aminobenzaldehyde in 75 ml. of amyl alcohol and adding 25 ml. of concentrated 

 hydrochloric acid. 



Among the many other tests for the detection of indole, particular mention may 

 be made here of the Ehrlich-Boehme^ procedure, the Gore^ modification of this 

 reaction, and the Gnezda'^ technique. 



For the Ehrlich-Boehme test two solutions are necessary. Solution I consists 

 of 1 gram p-dimethylaminobenzaldehyde in 95 ml. of ethyl alcohol (95 per cent) 

 and 20 ml. of concentrated hydrochloric acid. Solution II is a saturated aqueous 

 solution of potassium persulfate. To about 10 ml. of the culture showing good 

 growth of the organism 5 ml. of Solution I are added, then 5 ml. of Solution II 

 are added, and the mixture is shaken well. A red color developing in five minutes 

 indicates a positive test for the presence of indole. Pure Culture Study of Bacteria^ 

 states that this test may be performed by first shaking up the culture with ethyl 

 ether and then adding Solution I, dropping it down the side of the tube so that it 

 spreads out as a layer between the ether and the culture fluid. The formation of 

 a purplish red color at the interface of the two liquids within 5 minute§ indicates 

 indole production. 



For the Gor^ test the same solutions used for the Ehrlich-Boehme test are 

 employed. The cotton plug, which must be of white absorbent cotton, used for 

 plugging the culture tube is moistened with four to six drops of Solution II and 



