18 MANUAL OF MICROBIOLOGICAL METHODS 



one should never give the gram reaction of an unknown 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 phe- 

 nomena deserve consideration. (1) Henry and Stacey (1943) and 

 Bartholomew and Umbreit (1944) have shown that gram-positive 

 organisms can be made gram-negative 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 misinterpreta- 

 tion. 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 PROCEDURES 



Ziehl-N eelsen Method 

 Ziehl (1882); Neelsen (1883) 



Staining schedule: 



1. Stain dried smears 3-5 min with Ziehl's carbol fuchsin (page 12), 

 applying enough heat for gentle steaming. 



2. Rinse in tap water. 



3. Decolorize in 95 per cent ethyl alcohol, containing 3 per cent 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 

 page 13. 



6. Wash in tap water. 



7. Dry, and examine. 



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



Gross' ''Cold'' Method 



Of recent years an effort has been made (see Darrow, 1948; Gross, 

 1952) to eliminate the necessity of applying heat during the fuchsin stain- 

 ing so as to simplify the technic and to avoid ''messy" preparations. 

 Such procedures seem to have justified themselves and can be recom- 

 mended for pure culture work ; whether or not they are reliable for diag- 

 nostic purposes would require detailed comparison in actual use, and to 

 the committee's knowledge no such comparison has been made. Gross' 

 method is as follows : 



Preparation of basic fuchsin solution : 

 Add 25 ml of a stock 4 per cent alcoholic basic fuchsin solution to 75 ml of 



6 per cent aqueous phenol. 



