22 MANUAL OF MICROBIOLOGICAL METHODS 



Staining schedule: 



1. Fix the smear by passing through a flame 20 times. 



2. Stain 10 min with saturated aqueous malachite green (i.e., about 7.6 per cent), with- 

 out heat. 



3. Rinse with tap water for about 10 sec. 



4. Stain 15 sec in 0.25 per cent aqueous safranin. 



5. Rinse, blot, and dry. 



Results are the same as with the Conklin modification. 



STAINING THE DIPHTHERIA ORGANISM— RECOMMENDED PROCEDURES 



Various special procedures have been devised for staining the diph- 

 theria organism in such a manner as to render it distinctive in appearance 

 by differentiation of its characteristic metachromatic granules. 



Staining with Methylene Blue 



Staining schedule: 



1. Prepare smear as usual, and fix with gentle heat. 



2. Stain for a feAV seconds with either of the methylene blue solutions 

 (i.e., Loeffler's or dilute alcoholic) given on page 13. 



3. Wash in tap water. 



4. Dry, and examine. 



Results: Metachromatic granules, dark blue to violet; bacteria without 

 such granules, evenly stained. The picture varies a little according to 

 which of the two methylene blue solutions is employed. The Loeffler 

 formula gives purplish shades of staining because of the oxidation of 

 methylene blue caused by the alkali. Some users consider the poly- 

 chrome effect thus obtained to give better differentiation; others think 

 the metachromatic granules show more sharply with the clear blue of the 

 unpolychromed dye. 



Albert's Diphtheria Stain 

 Albert {1920) 



Toluidine blue 0.15 g 



]\Iethyl green 0.20 g 



Acetic acid (glacial) 1 ml 



Ethyl alcohol (95 %) 2 ml 



Distilled water 100 ml 



Laybourn's Modification 



Lay bourn (1924) has modified the Albert stain by replacing the methyl 

 green with an equal amount of malachite green. 

 Staining schedule: 



1. Make smears as usual, and fix with gentle heat. 



2. Stain 5 min in either Albert's staining fluid or Laybourn's modification 



