V49-10 MANUAL OF METHODS FOR PURE CULTURE STUDY 



Relation to Free Oxygen 



In relation to free oxygen, organisms are generally classified as strict 

 aerobes, facultative anaerobes, or strict anaerobes. A fourth group 

 of microaerophiles may also be recognized. None of these distinc- 

 tions is clear-cut; but the following method gives a rough grouping of 

 bacteria in regard to their oxygen requirements. 



Agar shake culture affords a good routine method of determining 

 the oxygen requirements of an organism. A tube of deep agar medium 

 containing glucose or some other available carbon source, is inoculated 

 while in fluid condition at 45°C. with an inoculum not too heavy to 

 permit discrete colonies, rotated to mix the inoculum with the medium, 

 and cooled. Some bacteriologists prefer to pour or pipet the inocu- 

 lated medium into another sterile tube to insure thorough mixing. 



Upon incubation, strict aerobes will be found to grow upon the 

 surface and in the upper layers only; microaerophiles will grow best 

 just a few millimeters below the surface; facultative anaerobes will 

 grow throughout the medium; and strict anaerobes will grow only 

 in the depths, if at all. 



Action on Nitrates 



Nitrate reduction should be indicated by complete or partial dis- 

 appearance of nitrate accompanied by appearance of nitrite, am- 

 monia, or free nitrogen. As quantitative nitrate tests are too time- 

 consuming for routine pure culture work, one must ordinarily be 

 satisfied with tests for the end-products only. 



The following routine procedure is recommended: Inoculate into 

 nitrate broth and onto slants of nitrate agar (containing 0.1% KNO3 

 plus beef extract and peptone as usual). Test the cultures on various 

 days as indicated on the Chart. On these days examine first for gas 

 as shown by foam on the broth or by cracks in the agar. Then test 

 for nitrite with the following reagents. 



1. Dissolve 8 grams sulphanilic acid in 1 liter of 5 N acetic acid (1 part glacial 

 acetic acid to 2.5 parts of water), or in 1 liter of dilute sulphuric acid (1 part concen- 

 trated acid to 20 parts water). 



2. Dissolve 5 grams a-naphthylamine in 1 liter of 5 N acetic acid or of very dilute 

 sulphuric acid (1 part concentrated acid to 125 parts water). Or dissolve 6 ml. of 

 dimetliyl-a-naphthylamine in 1 liter of 5 N acetic acid. This latter reagent has re- 

 cently been recommended by Wallace and Neave (1927), and by Tittsler (1930) as it 

 gives a permanent red color in the presence of high concentrations of nitrite. 



Put a few drops of each of these reagents in each broth culture to 

 be tested, and on the surface of each agar slant. A distinct pink or 

 red in the broth or agar indicates the presence of nitrite. It is well 



