STAINING METHODS 21 



Dorner's Method — Snyder's Modification 

 Snyder (1934) 



Staining schedule: 



1. Prepare a dried smear on a slide, and cover with a small piece of 

 blotting paper. 



2. Saturate blotting paper with freshly filtered ZiehPs carbol fuchsin. 



3. Allow to steam 5-10 min, keeping paper moist by adding more staining 

 fluid. 



4. For neat preparations, decolorize instantaneously with 95 per cent 

 ethyl alcohol (but omit this step if the organisms do not hold color well) . 



5. Wash with tap water. 



6. Apply a drop of saturated aqueous nigrosin (or Dorner's fluid), and 

 spread evenly. 



7. Allow slide to dry quickly with gentle heat, without prior washing. 

 Results: Same as with original method, but this modification proves 



applicable to some bacteria (e.g., Bacillus subtilis) that are difficult to 

 stain by Dorner's technic. 



Conklin's Modification of Wirtz Method 

 Wirtz {1908); Conklin {1934) 



Staining schedule: 



1. Make smears as usual and fix by heat. 



2. Flood slide with 5 per cent aqueous malachite green, and steam for 10 

 min, keeping slide flooded by addition of fresh staining fluid. 



3. Wash 30 sec in running water. 



4. Counterstain 1 min with 5 per cent aqueous mercurochrome. 



5. Wash in running water. 



6. Blot dry, and examine. 



Results: Spores, green; rest of cell, red. Trouble is sometimes experi- 

 enced with the green fading after the slides have stood a few days. 

 Apparently this is the result of an alkaline reaction and can be prevented 

 by treating the slides in acid before making the smears. (The alkalinity 

 may be due to an invisible film of soap or washing powder.) 



SPORE STAINING— ALTERNATE PROCEDURE 



Bartholomew and Mittwer's "Cold" Method 



Just as recent work is showing that heat is not necessary in making an 

 acid-fast stain, it is proving that it may also be eliminated from spore 

 staining, in which a very similar principle is involved. The following 

 modification of the Wirtz method by Bartholomew and Mittwer (1950) is 

 a good illustration: 



