STAINING METHODS iV46-13 



Spore Staining — Alternate Procedure 



SCHAEFFER-FULTON MouiKICATION OF WiRTZ MeTHOD 



Schacffer & Fulton (1933) 



Bacterial smears are made as usual and fixed in a flame. They are flooded with 5% 

 aqueous malachite green for 30 to GO seconds, and heated to steaming three or four 

 times. The excess stain is washed off in running water for about lialf a minute, and 

 0.5% aqueous safranin is added for about 30 seconds. The smears arc tiien washed 

 and blotted. The spores sliould be stained green, the rest of the cells red. 



Staining the Diphtheria Organism — Recommended Procedures 



Various special procedures have been devised for staining the 

 diphtheria 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 few seconds with either of the methylene blue solu- 



tions (i.e. Loeffler's, or dilute alcoholic) given on p. 6. 



3. Wash in tap water. 



4. Dry and examine. 



Results: Metachromatic granules, dark blue to violet; bacteria with- 

 out such granules, evenly stained. The picture varies a little ac- 

 cording 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 polychrome effect thus obtained to give better differ- 

 entiation; 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. 



Methyl green 0.20 g. 



Acetic acid (glacial) 1 ml. 



Ethyl alcohol (95%) 2 ml. 



Distilled water 100 ml. 



laybourn's modification 



Laybourn (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 of it. The latter is claimed to give deeper 

 staining of both granules an<l body of the cells, without lessen- 

 ing the contrast between them. 



3. Drain without washing. 



4. Treat 1 min. in a modified Lugol's solution (iodine, 2 g. ; KI, 3 g.; 



distilled water, 300 ml.). 



