68 MEDICAL MYCOLOGY 



making the smear. Hufschmitt, Sartory and Meyer (1931) advocate the 

 Moeller method which is slightly different from Maneval's procedure. Treat 

 smear from 10 seconds to 5 minutes in 1% sulphuric acid; wash, stain with 

 carbolfuchsin, heating for 1 minute; differentiate with 5% sulphuric acid for 

 5 seconds ; wash, counterstain with aqueous methylene blue for 3 minutes. 



Buschke and Harry (1923) recommend the Schumacher method. Fix, 

 stain for 1 minute in carbol-methylene blue, rinse with distilled water, stain 

 for 11/2 minutes, while slowly moving the slide, with 1% phosphin (diamido- 

 phenylacridin). Spores also stain with Ziehl-Neelsen acid-fast procedure if 

 the sulphuric acid is replaced by 1% nitric acid alcohol. 



Maneval (1929) suggests the following modification of Gutstein's pro- 

 cedure for staining vegetative cells. Fix smear with heat, stain with 5% 

 tannin for 2 minutes, then with safranin or 1% methylene blue, or stain with 

 carbol-methylene blue (5% carbolic acid plus 1% methylene blue) or methy- 

 lene blue; treat with 5% tannin for 2 minutes, wash, and counterstain with 

 safranin. 



It is often very easy to find spores just by making mounts in glycerin 

 and using some dye, such as crystal violet, or lactophenol preparations. Most 

 of the dye preparations will stain vegetative mycelium. 



Stains for Fungi in Skin. — Unna, Jr. (1929) advises the following modi- 

 fication of the Pappenheim-Unna, Sr. method for staining fungi in skin. Fix 

 in absolute alcohol, then run through the alcohols to xylol, and embed in 

 paraffin. Cut sections about 10/a thick; stain with pyronine-methyl green 

 (pyronine 9 parts, methyl green 1 part, 96% alcohol 90 parts, glycerol 100 c.c, 

 0.5% phenol to make 1000 c.c.) for 5-10 seconds; rinse in water; dry with 

 absolute alcohol, and mount in balsam. The fungi will be rubin red, leuco- 

 cytes green to blue green. (N.B. The cells of the basal homy layer of the epi- 

 dermis will have red nuclei by this method.) 



Fungi in tissue can be stained easily by the usual iron-alum hematoxylin 

 and eosin procedure. The fungus elements take the hematoxylin stain rather 

 nicely, although some difficulty may be encountered in distinguishing spheri- 

 cal cells or spores from tissue elements. The Gram method of staining for 

 bacteria has been used with a measurable amount of success since fungi are, 

 in general, gram-positive. 



The formula of Malcolm Morris (Mallory and Wright 1924, p. 175) for 

 staining various parasites of the skin avoids the use of hydrate of potash. The 

 skin is placed in ether or in a mixture of alcohol and ether, equal parts, stained 

 for 5-30 minutes in a solution of 5% gentian violet in 70% alcohol; iodine 

 solution, 1 minute ; anilin, or anilin plus 2-4 drops of nitric acid ; anilin ; 

 xylol ; xylol and balsam. 



Stains for Fungi in Other Tissues. — A number of methods listed in Mal- 

 lory and Wright (1924) for staining bacteria in tissue, as well as various 

 types of cells, have been used successfully with fungi. Mallory 's anilin blue 

 stain (p. 118) has been used very nicely for Cryptococcus histolyticus (Torula 

 histolytica) in brain tissue. The Gram-Weigert staining method (p. 288) is in 



