FUNGI 



134 



FUNGI 



and mount in balsam. Spores and 

 mycelium, blue; scrapings, yellow. 

 The dye is sat. ale. gentian violet, 2.5 

 parts; aq. dest., 17.5 parts; orange G 

 solution, 9 parts; acetic acid, 1 part; 

 95% ale, 5 parts. The orange G solu- 

 tion is orange G, 2 parts; 95% ale, 20 

 parts; water, 80 parts. Decolorize with 

 10-20% KOH. 



The hydroxide method of examination 

 is simple and often rapid, but unless 

 used by one familiar with it the results 

 may be misleading. There is danger of 

 confusion with structures which Becker 

 and Ritchie (J. W. and E. B., Arch. 

 Dermat. & Syph., 1940, 22, 790-802) have 

 indicated as resembling yeast cells. 

 These artifacts may be removed by 

 treating the material progressively with 

 absolute alcohol, ether, absolute and 

 95% alcohol. They have been termed 

 'mosaic fungus' and have been reported 

 by Greenwood and Rockwood (A.M. and 

 E. M., Arch. Dermat. & Syph., 1930, 

 21, 96-107) as degenerate fungi. In 

 fact they are cholesterol crystals. The 

 use of dyes eliminates in great measure 

 such artifacts. However, the use of 

 dyes is not practical with thick sections 

 for which recourse must be had to the 

 hydroxide method. 



When the scrapings or scales are thin, 

 or when sputum, pus or exudate are ex- 

 amined, a 1% aq. methylene blue and 

 glycerin can be used as follows : One 

 drop of the 1% solution of methylene 

 blue is placed on a clean slide and the 

 material is stirred within it, allowed to 

 stand for approximately 2 min. when a 

 clean cover slip is placed over the mix- 

 ture and pressed down to flatten out the 

 material and to express the excess solu- 

 tion. The superfluous stain is taken up 

 by filter paper. A drop of glycerin is 

 then placed along one edge of the cover 

 slip and allowed to seep under, dis- 

 placing the stain and giving a clear back- 

 ground to the stained material. The 

 fungus appears bright blue. 



The lactophenol-cotton blue technique 

 was developed in the French labora- 

 tories using the formula of Amann (J., 

 Zeit. Wiss. Mikr., 1896, 13,18-21). Lac- 

 tophenol consists of phenol crystals, 

 20 gm.; glycerin, 40 gm.; lactic acid, 

 20 gm. andaq. dest., 20 gm. Cotton blue 

 (anilin blue, China blue) is a mixture of 

 the trisulphonates of tri-phenyl para- 

 rosanilin (C.I. 706) and of di-phenyl 

 rosanilin. Place a drop of the cotton 

 blue (0.5% aq.) on the slide ; stir up the 

 material within it and allow to stand for 

 about 2 min. Add cover slip and press 

 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 hydro .xide 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. 



