DEHYDRATED CULTURE MEDIA 185 



is prepared according to the formula published in "Pure Culture Study of 

 Bacteria"^ of the Society of American Bacteriologists. 



For determining the ability of an organism to reduce nitrate to nitrite, tubes of 

 medium, prepared from Bacto-Nitrate Broth, are inoculated with a pure culture 

 of the strain under examination. The tubes are then incubated at 37°C. for 12 

 to 24 hours. The medium is tested for the presence of nitrites by adding a few 

 drops each of sulfanilic acid and a-naphthylamine reagent solutions. The sul- 

 fanilic acid reagent is prepared by dissolving 8 grams of sulfanilic acid in 1000 ml. 

 5N acetic acid. The a-napththylamine reagent consists of 5 grams of «-naph- 

 thylamine dissolved in 1000 ml. 5N acetic acid. A distinct pink or red color 

 indicates the presence of nitrite reduced from original nitrate. If an organism 

 grows rapidly and reduces nitrate actively, it is suggested that the test for nitrite 

 be performed at an early incubation period since the reduction may be carried 

 beyond the nitrite stage. The test must always be controlled by comparison with 

 an uninoculated tube of medium. 



To rehydrate the medium, dissolve 9 grams of Bacto-Nitrate Broth in 1000 ml. 

 distilled water. Distribute in tubes and sterilize in the autoclave for 15 minutes 

 at 15 pounds pressure ( 121°C.). The final reaction of the medium will be pH 7.0. 



One pound of Bacto-Nitrate Broth will make 50 liters of medium. 



1 Pure Culture Study of Bacteria, i2:Leaflet 11:8:1944. 



BACTO 



NITRATE AGAR (B106) 



DEHYDRATED 



Bacto-Beef Extract 3 g. 



Bacto-Peptone 5 g. 



Potassium Nitrate 1 g. 



Bacto-Agar 12 g. 



Bacto-Nitrate Agar is a dehydrated medium for use in testing nitrate reduc- 

 tion by bacteria. It is prepared according to the formula of the "Manual of 

 Methods" of the Society of American Bacteriologists. 



Nitrate reduction by microorganisms is a valuable criterion in the differentia- 

 tion and identification of various types of bacteria. Certain bacteria reduce the 

 nitrates to nitrites only, while others are capable of further reduction to free 

 nitrogen or even ammonia. Furthermore, many aerobic bacteria can grow under 

 anaerobic conditions in the presence of nitrates from which they derive their 

 oxygen. 



Tubes of sterile slanted medium are inoculated by streaking over the surface of 

 the slant, and stabbing into the butt. Incubate at 37°C. Examine the cultures on 

 various days for gas production which will be indicated by splitting of the agar. 

 Test for nitrates with sulfanilic acid and a-naphthylamine reagent solutions. The 

 sulfanilic acid reagent is prepared by dissolving 8 grams of sulfanilic acid in 1000 

 ml. 5N acetic acid. The a-naphthylamine reagent consists of 5 grams of c-naph- 

 thylamine dissolved in 1000 ml. 5N acetic acid. A few drops of each reagent 

 are put into the tube to be tested. A distinct pink or red color indicates the 

 presence of nitrite reduced from original nitrate. The test should always be con- 

 trolled by comparing with an uninoculated tube of the medium which has been 

 kept under the same conditions as the inoculated tubes. The evolution of gas in 

 a nitrate medium containing no sugar or fermentable substance is a definite indi- 

 cation of reduction to free nitrogen. 



To rehydrate the medium, suspend 21 grams of Bacto-Nitrate Agar in 1000 



