FUNGI 



104 



FUNGI 



down to squeeze out any excess dye, 

 which is taken up by filter paper. Add 

 a drop of lactophenol to the edge of cover 

 slip and allow it to replace the cotton 

 blue which dries out. The stain may be 

 rapidly replaced by holding a bit of filter 

 paper at the edge of the cover slip op- 

 posite the lactophenol. The cell wall 

 stains lightly as compared with the 

 darkly colored central portion of the 

 fungus. The tissue elements also stain 

 light blue. 



Swartz and Conant (J. H. and N. F., 

 Arch. Dermat. & Syph., 1936, 33, 

 291-305) have modified the lactophenol 

 and cotton blue procedure. First put 

 a few scrapings in 5% aq. potassium 

 hydroxide, heat somewhat and wash in 

 water. Place material in a drop of the 

 combined cotton blue (0.5%) and lacto- 

 phenol. The fungi stain a darker blue 

 than the tissue cells. 



Schubert M., Dermat. Wchnschr., 

 1937, 105, 1025-1029) has modified the 

 Swartz -Conant technique. Soak the 

 scales in 2% KOH for 30 min. or until 

 they appear glassy and then wash in 

 aq. dest. 2-10 hrs. Transfer small 

 particles to a slide and add 1 or 2 drops 

 of following stain : cotton blue, 0.25 gm. ; 

 lactic acid, 10 gm.; phenol crystals, 10 

 gm.; and aq. dest., 20 gm. The fungi 

 appear dark blue while the epidermal 

 cells stain lightly. See also Berberian's 

 Method, 



2. Sputum, pus and exudates: Exam- 

 ine for fungi after mounting directly 

 on a slide after mixing in 20% KOH or 

 on stained smears. The latter are not 

 very satisfactory because smearing 

 tends to disturb the arrangement of the 

 cells but they are useful for detection of 

 mycelium. Many contaminating or- 

 ganisms are generally present in these 

 exudates unless material is secured from 

 fresh lesions opened aseptically. Sev- 

 eral examinations may be necessary since 

 the organisms in exudates are seldom 

 numerous. The hydroxide usually dis- 

 solves most of the tissue elements and 

 the fungi stand out as refractile bodies. 

 Several of the staining methods em- 

 ployed in the study of hair and scrapings 

 may be used. Of these, the methylene 

 blue and glycerin method is best but the 

 lactophenol-cotton blue technique is 

 likewise advised. 



3. Vesicles, blister fluid, spinal fluid 

 or urine: These can also be directly 

 examined. But vesicle, or blister, fluid 

 yields only a small amount of material 

 and for best results, the methylene 

 blue-glycerin method or the lactophenol- 

 cotton blue technique is advised. 

 Urine, or spinal fluid, should be con- 

 centrated by centrifugation before 



examination. The same staining pro- 

 cedures are advocated. See Blasto- 

 mycosis. 



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

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

 the following modification of the Pap- 

 penheim-Unna, Sr. method for staining 

 fungi in skin. Fix in absolute alcohol, 

 then run through the alcohols to xylol 

 and imbed in paraffin. Cut sections at 

 \Qn, 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 



