152 MANUAL OF MICROBIOLOGICAL METHODS 



magnesium ribonucleate. Thus gram-positive organisms can be arti- 

 ficially converted to gram-negative ones and then restored to their gram- 

 positive state. 



In addition to this fact, it is also true that many bacteria are neither 

 definitely positive nor negative; some organisms are gram-variable and 

 may appear either negative or positive according to conditions. Other 

 organisms contain granules which resist decolorization and may cause 

 misinterpretation. The importance of taking such variations into 

 account has been repeatedly emphasized. Such organisms should be 

 recorded as gram-variable rather than made to appear either positive or 

 negative by some modification of technic. To determine if an organism 

 belongs to this variable group, it is necessary that it be stained at two or 

 three different ages by more than one procedure. If an organism changes 

 from positive to negative or vice versa during its life history, this change 

 should be recorded, with a statement as to the age of the culture when the 

 change was first observed. It is often practical to record such an organ- 

 ism as prevailingly positive or prevailingly negative ; obviously, however, 

 this cannot be done without a very considerable series of determinations. 

 Tests must therefore be made after 1 and 2 days' incubation, sometimes 

 also in even older cultures. It must, moreover, be recognized that gram- 

 variahle organisms are not necessarily ones that show uneven gram stain- 

 ing; the latter should be recorded as staining unevenly, not as gram- 

 variable. 



The two methods at present recommended are the ammonium oxalate 

 method (Hucker) and Kopeloff and Beerman's modification of the Burke 

 technic. In the former the manipulation is more simple, but the latter 

 gives better results if the organism is growing in a medium that may be of 

 acid reaction (e.g., exudates) and is claimed to distinguish better between 

 true and false positive reactions. These two procedures are given in 

 Chap. II. 



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 distinctions 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 con- 

 taining glucose or some other available carbon source is inoculated while 

 in fluid condition at 45°C with an inoculum not too heavy to permit dis- 

 crete colonies, rotated to mix the inoculum with the medium, and cooled. 



