FUNGI 



103 



FUNGI 



usual method is to mount the material 

 in an alkali — either sodium hydroxide 

 (NaOH) or potassium hydroxide 

 (KOH). The latter is preferable and 

 should be used in a 10-30% solution. 

 For rapid work 40% is employed but this 

 tends to swell and disintegrate the 

 fungi. A weak solution takes longer to 

 clear the skin. The skin usually clears 

 in 5 min. to 2 hrs. in concentrations of 

 10-30%. A little heat helps. Use sub- 

 dued light in order to avoid high lights. 

 The fungus is clearly discernible against 

 the irregular nondescript background 

 of skin which is usually clear. Dip 

 infected hairs taken from scalps, par- 

 ticularly those that are oily, in ether or 

 in alcohol (absolute alcohol is preferable 

 to 95%) for a moment in order to get rid 

 of the oil which often simulates spores 

 in shape and size. 



Adamson (H. G., Brit. J. Dermat., 

 1895, 7, 201-211, 237-244) has recom- 

 mended clearing with 5-10% KOH and 

 staining bj' the Gram method. Chal- 

 mers and Marshall (A. J. and A., J. Trop. 

 Med. Hyg., 1914, 17, 256-265, 289-291) 

 suggest soaking scales in 40% KOH for 

 some hours in a watch glass in an in- 

 cubator at 40 °C. Transfer specimens 

 to watch glass containing 15% alcohol for 

 30 min., remove to slide, allow alcohol 

 to evaporate and dry over flame; stain 

 with Anilin-Gentian Violet for 20 min. 

 Treat with Gram's Iodine for 3 min. ; 

 decolorize with anilin oil, 30 rain. ; stain 

 in concentrated alcoholic eosin, 1 min.; 

 wash off eosin with anilin oil or clove oil ; 

 treat with .xylol and mount in balsam. 

 Priestley (H., Med. J. Australia, 

 1917, 2, 471-475) recommends lacto- 

 phenol (lactic acid, 1 part; phenol, 1 

 part; glycerol, 2 parts, aq. dest., 1 part) 

 for clearing instead of 40% KOH; or 

 chloral hydrate crystals, 2 parts; lactic 

 acid, 1 part; phenol crystals, 1 part, 

 may be used. For thick material 

 Langeron suggests : chloral hydrate 

 crystals, 40 gm.; phenol crystals, 40 

 gm.; lactic acid (U.S.P.), 20 gm.; and 

 sodium salicylate, 10 gm. Slight heat 

 facilitates clearing. To stain. Priestley 

 recommends treatment with chloroform 

 to remove the fat; boiling, 2-3 min., 

 with formic acid; washing for a few 

 minutes in water and staining with 

 Sahli's methylene blue : after which the 

 tissue is to be washed, differentiated 

 with alcohol if necessary, dehydrated, 

 cleared and mounted in balsam. 



Bachman (R. W., Arch. Dermat. & 

 Syph., 1920, 1, 50-54) recommends the 

 following procedure : Place scrapings in 

 a drop of water on a cover slip, tease 

 thoroughly with a dissecting needle, 

 dry over a flame but do not scorch. 



Stain for 2 min.; decolorize in 95% al- 

 cohol, 15-30 sec; immerse in aq. dest., 

 15-30 sec. ; pour off excess, dry by heat, 

 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 



