IV46-10 MANUAL OF METHODS FOR PURE CULTURE STUDY 



9. Wash in tap water. 

 10. Dry and examine. 

 Results: Gram-positive organisms, blue; Gram-negative organisms, 

 red. This technic is claimed to have the advantage of not giving 

 false positives due to vacuolar bodies that resist decolorization by 

 other Gram-staining procedures. 



INTERPRETATION OF THE GRAM STAIN 



A word of caution is necessary as to the interpretation of the Gram 

 stain. The test is often regarded with unjustified finality because 

 organisms are generally described as being either Gram-positive or 

 Gram-negative. Many organisms, however, actually are Gram- 

 variable. Hence, one should never give the Gram reaction of an un- 

 known organism on the basis of a single test. He should repeat the 

 procedure on cultures having different ages and should use more than 

 one staining technique in order to determine the constancy of the 

 organism toward the stain. Two phenomena deserve consideration. 

 (1) Henry & Stacey (1943) and Bartholomew and Umbreit (1944) 

 have shown that Gram-positive organisms can be made Gram-nega- 

 tive by treatment with ribonuclease, and that their Gram-positive 

 reaction can be restored subsequently by treatment with magnesium 

 ribonucleate. (2) Some organisms have granules which resist 

 decolorization and which may cause misinterpretation. Such 

 observations show that the Gram stain does not always give a clear 

 cut reaction and that the results must be interpreted with care. 



Acid-fast Staining — Recommended Procedure 



ziehl-neelsen method 

 Ziehl (1882); Neelsen (1883) 



Staining schedule: 



1. Stain dried smears 3-5 min. with Ziehl's carbol fuchsin (p. 5), 



applying enough heat for gentle steaming. 



2. Rinse in tap water. 



3. Decolorize in 95% ethyl alcohol, containing 3% by volume of 



cone. HCl, until only a suggestion of pink remains. 



4. Wash in tap water. 



5. Counterstain with one of the methylene blue solutions given on 



p. 6. 



6. Wash in tap water. 



7. Dry and examine. 



Results: Acid-fast organisms, red; others, blue. 



AciD-FAST Staining — Alternate Procedures 



Fluorescence Method 

 Richards and Miller (1941) 



Although this method is not of special importance in pure culture work, special 

 mention should be made of it because of the amount of attention now given to it in 

 diagnostic work. Its real advantage is that it can be used with relatively low magnifi- 

 cation, and the large fields that can be examined assure positive diagnoses in cases 

 where the numbers of tubercle organisms are few. 



