500 



METHODS AND FORMULAS 



DS 23.30 



Demonstration of parasitic fungi in 



tissue scrapings using the technique 



of Chalmers and Marshall 1914 



All the methods for the diagnostic dem- 

 onstration of fungi in tissue scrapings are 

 very much the same, and combine partial 

 hydrolysis (clearing) of the removed epi- 

 dermal tissue in strong alkali 'uath the 

 staining of the fungi by a bacterial stain- 

 ing method, subsequent!}' differentiated in 

 aniline. The present method (DS 23.32 

 Chalmers and Marshall 1914) is one of the 

 easiest to use and has, therefore, been se- 

 lected for discussion. It is to be presumed 

 that scrapings from the surface of the 

 patient's skin will be removed bj- the 

 phj^sician, and it is recommended that 

 they be brought to the technician in a 

 ■watch glass. The technician should sort 

 over the material presented, and should 

 remove from it with fine forceps those 

 pieces which definitely present the appear- 

 ance of scales. These scales are then 

 placed in a 25 % solution of potassium h}-- 

 droxide at 40°C. for a few hours. The 

 scales must then have the alkali removed, 

 for which purpose it is best to use 15% 

 alcohol rather than water. The washing is 

 most easily done by picking up the scales 

 with a section lifter and passing them 

 through half a dozen watch glasses of 15% 

 alcohol, rather than by pouring off the 

 solution and replacing it with alcohol. It 

 does not matter how long the specimens 

 remain in alcohol. It is often a useful rou- 

 tine procedure to accumulate all of the 

 day's collected scrapings in separate jars 

 of alcohol, and to stain them the first 

 thing the next morning before additional 

 scales are received. When it is desired to 

 stain the material, each separate scale is 

 lifted from the 15% alcohol and placed in 

 the center of a chemically clean shde 

 where it is dried. If the scales have been 

 so softened that they cannot be lifted from 

 the fluid, it is only necessary to pour the 

 contents of the jar into a fingerbowl of 

 15% alcohol, and then to maneuver the 

 slide under the selected scale. This scale 

 is then held with a needle against the 

 shde, which is hfted from the alcohol, 

 leaving the scale stranded. The objection 

 to the stranding technique is that it is 



difficult to place more than one scale on a 

 slide, wlicreas if each scale can be lifted 

 and spread out, a dozen tj-pical scales 

 from one patient ma}' be stained on the 

 same shde. It does not matter whether 

 the slides are dried at room temperature 

 or on a warm table, but there is some risk 

 that, if the temperature is elevated too 

 far, the scale will curl off the shde as it 

 dries. 



A drop of Ehrlich's crystal violet (DS 

 23.211 Ehrhch 1882) is then placed over 

 the scales, and allowed to remain at room 

 temperature for about 20 minutes. It 

 should be looked at from time to time and, 

 if it shows signs of drying, further quan- 

 tities of crystal violet should be placed on 

 top. At the end of 20 minutes the crystal 

 \dolet is drained carefully from the scales, 

 which are not washed, and replaced with 

 several drops of Gram's iodine (Chapter 

 22, ADS 12.2) which is allowed to act for a 

 period of about 3 minutes. 



Differentiation in aniline is very simple, 

 for it is impossible to overdifferentiate. 

 The shde is placed at about a 45° angle in 

 a glass dish, and anihne is allowed to flow 

 over it from a pipet. If the drops are al- 

 lowed to fall from a height the scales may 

 become detached, therefore, the edge of 

 the pipet should be rested just above the 

 scales, and a slow and stead}' stream of 

 anihne allowed to flow over them. This 

 stream of anihne must be continued until 

 no further color comes away, by which 

 time the fungal hyphae Tvdll be perfectly 

 differentiated against a colorless back- 

 ground. They may be mounted in this 

 condition if desired, or, as suggested by 

 Chalmers and Marshall (DS 23.32 below), 

 counterstained for one minute with a 2% 

 alcoholic solution of eosin Y, which is 

 poured over the slide without removal of 

 the aniline. Aniline is then used exactly as 

 before to remove the excess eosin; the flow 

 of anihne is discontinued when no more 

 eosin comes away. The aniline is then 

 washed from the shde with xylene and the 

 specimens are mounted in balsam. Fungal 

 hyphae will be clearly stained bright blue 

 against a yellow background. The prin- 

 cipal purpose of the background is to en- 

 able one to find the scales under a low 

 power of the microscope before using the 



