FUNGI 



135 



FUNGI 



4. Skin: Unna, Jr. (P., Dermat. 

 Wchnschr., 1929, 88, 314-321) advises 

 the following modification of the Pap- 

 penheim-Unna, Sr. method for stainins 

 fungi in skin. Fix in absolute alcohol, 

 then run through the alcohols to xylol 

 and imbed in paraffin. Cut sections at 

 10/x, stain with pyronine-methyl green 

 (pyronine, 9 parts; methyl green, 1 

 part; 96% alcohol, 90 parts; glycerol, 

 100 cc; 0.5% phenol to make 1000 cc), 

 5-10 sec; rinse in water; dry with 

 absolute alcohol; and mount in balsam. 

 Fungi, rubin red; leukocytes, green to 

 blue green ; nuclei of cells of basal horny 

 layer of the epidermis, red. 



Fungi in tissue can be easily stained 

 by Iron-Hematoxylin and eosin. The 

 fungous elements take the hematoxylin 

 stain nicely, although some difficulty 

 may be encountered in distinguishing 

 spherical cells or spores from tissue 

 elements. The Gram method of stain- 

 ing for bacteria has been used with a 

 measurable amount of success since 

 fungi are, in general, gram -positive. 



Unna's Alkaline Methylene Blue 

 (Unna, P., Monatsh. f. prakt. Dermat., 

 1891, 13, 225-237, 286-311), although 

 recommended for staining plasma cells 

 and as a general stain in combination 

 with phloxine or eosin, has been espe- 

 cially adapted for staining organisms in 

 the stratum corneum. It consists of 

 methylene blue, 1 gm.; potassium 

 carbonate, 1 gm.; and aq. dest., 100 

 cc. The solution stains better after 

 ripening for a week or two and should 

 be diluted 1 to 10 or 1 to 5 before use. 



Malcolm Morris (Mallory, F. B. and 

 Wright, J. H., Pathological Technique, 

 Philadelphia and London, 1924, p. 175) 

 in staining various parasites of the skin, 

 avoids the use of potassium hydrate. 

 Place skin in ether, or in 1:1 alcohol- 

 ether; stain for 5-30 min. in 5% gentian 

 violet in 70% alcohol. Then pass 

 through iodine solution, 1 min.; anilin, 

 or anilin plus 2-4 drops of nitric acid; 

 anilin; and xylol (2 changes) to balsam. 



5. Other tissues: A number of methods 

 listed for staining Bacteria in tissue 

 can be used successfully for fungi. 

 Mallory's Connective Tissue stain is 

 good for Cryptococcus hominis in brain 

 tissue. Fungus cells, red ; thick mucoid 

 capsules, light blue. TheGram-Weigert 

 staining method is also excellent. 

 Organisms, deep violet; nuclei, blue to 

 violet; connective tissue, red. 



Actinomyces in sections may be 

 stained successfully with Alum-Hema- 

 toxylin followed by strong eosin. Mal- 

 lory (p. 279) lists 2 methods of which 

 the following gives good results with 

 paraffin sections of formalin or Zenker 



fixed tissue. Stain in alum-hematoxy- 

 lin, 3-5 min.; wash in water; stain in 

 a 2.5% a,q. phloxine or 5% aq. eosin, 

 15 min. in the paraffin oven; wash in 

 water; stain in Anilin Crystal Violet 

 (try Stirling's), 5-15 min.; wash in 

 water; treat with Gram's Iodine solu- 

 tion, 1 min.; wash in water and blot 

 with filter paper; differentiate in 

 several changes of anilin until no more 

 color comes off; rinse in several cliangea 

 of xylol and mount in balsam. The 

 branched organisms stain blue while 

 the hyaline sheaths ("clubs") become 

 pink to red. 



The Hotchkiss-McManus or Periodic 

 Acid-Schiff Stain (Kligman, A. L. and 

 Mescon, H., J. Bact., 1950, 60, 415-421) 

 may be used to find fungi in tissue or 

 sections. Tissue fixed, embedded in 

 paraffin and sectioned in usual manner. 

 After deparaffinizing, sections rinsed 

 in absolute alcohol; washed in distilled 

 water; immersed in 1% aq. periodic 

 acid C.P. (Eimer & Amend) 5 min.; 

 washed in tap water, 10 min.; placed in 

 Schiff reagent, 10 to 15 min. ; transferred 

 to 1 normal HCl, 5 min.; washed in 

 tap water, 10 min.; counterstained with 

 1% aq. light green, for sec. ; dehydrated, 

 cleared and mounted. Fungi stain red 

 to purple. Tissue cell nuclei do not 

 stain. 



Schiff reagent prepared as follows: 

 Dissolve 0.5 gm. basic fuchsin (Cole- 

 man & Bell) by passing over it 100 cc. 

 boiling water; cool to 50°C.; filter and 

 add 10 cc, 1 normal HCl and 0.5 gm. 

 potassium metabisulfite dry reagent 

 (Eimer & Amend) to filtrate. Solu- 

 tion becomes colorless to pale straw 

 colored by standing in dark overnight. 

 Add 0.25 to 0.5 gm. activated charcoal, 

 shake well and filter immediately to 

 completely decolorize. Refrigerate in 

 tightl}^ stoppered bottle. 



For smears or nails: Use thin smears 

 and fine nail scrapings. Material made 

 to adhere to slide with egg albumen. 

 Coplin jar or drop bottle technique 

 employed. Immerse material in 95% 

 alcohol, 1 min.; cover with 5% aq. 

 periodic acid, 3 minutes; wash in run- 

 ning water. 2 min.; stain in Schiff re- 

 agent, 5 min.; rinse in running tap 

 water, 1 min.; dehydrate in 95% then 

 absolute alcohol and two changes 

 xylol, 1 min. each. Mount in clarite. 

 See Polysaccharides. 



After the fungi have been successfully 

 cultivated on the various mediums 

 recommended (Moore, M., Arch. 

 Dermat. & Syph., 1936, 34, 880-886) 

 they can be examined microscopically 

 by transferring part of the growth with 

 a sterile platinum or nichrome wire to 



