114 MICROSCOPIC METHODS 



stain to 1 c.c. water (the mixture being well shaken). (Sometimes the 

 water is made alkaline by the addition df one drop of 1 per cent, potassium 

 eai-bonate to 10 c.c. water.) (3) Stain for fifteen minutes (a longer period 

 is often desirable, even twenty-four hours). (4) Wash in brisk stream of 

 distilled water. (5) Drain with filter-paper, dry, and mount in Canada 

 balsam. 



Neissers Stain, —(a) The following is the original method introduced 

 by Neisser as an aid to the diagnosis of the diphtheria bacillus. Two 

 solutions are used as follows : (a) 1 grm. methylene-blue (Griibler) is 

 dissolved in 20 c.c. of 96 per cent, alcohol, and to the solution are added 

 950 c.c. of distilled water and 50 c.c. of glacial acetic acid ; (b) 2 grms. 

 Bismarck-brown (vesuvin) dissolved in a litre of distilled water. Films 

 are stained in (a) for 1-3 seconds or a little longer, washed in water, 

 stained for 3-5 seconds in (6), dried, and mounted. The protoplasm of 

 the diphtheria bacillus is stained a faint brown colour, the granules a blue 

 colour. Cultures should be grown on a* serum medium and examined 

 within 24 hours. Satisfactory results are not always obtained in the case 

 of films prepared from membrane, etc. 



(b) The following is Neisser's modified cresoidin method : — 



1 . Stain films for a few seconds in a mixture of solutions A and B, 



two parts of the former to one of the latter. 



A. Methylene-blue 

 Absolute alcohol 

 Glacial acetic acid . 

 Distilled water 



B. Crystal-violet (Hbchst) . 

 Absolute alcohol 

 Distilled water 



2. Wash in water, and 



3. Stain in cresoidin solution (1 : 300) for a few seconds (the cresoidin 



should be dissolved in warm water and the solution then 

 filtered). 



4. Wash in water, dry, and mount. 



Instead of cresoidin the following solution of erythrosin may be used 

 with advantage: Saturated alcoholic solution of erythrosin, 20 parts; 

 saturated watery solution of picric acid, 90 parts ; add to the mixture 

 precipitated calcium carbonate to excess ; allow to stand for a time, 

 shaking at intervals ; filter. 



Sabouraud's Method for Staining Trichophyta. — Remove the fat from 

 the hair or epithelial squames with chloroform. Place in a test-tube 

 with 10 per cent, formol, and warm for two or three minutes till.ebullition 

 commences. Wash well in distilled water, and stain for one minute in 

 Sahli's blue, which is made up as follows : — 



Distilled water 40 parts. 



Saturated watery methylene-blue. . . 24 ,, 

 5 per cent, solution of borax in water - . 16 ,, 



Mix the constituents. Allow to stand for a day, and filter. After 

 staining, wash in water, dehydrate with absolute alcohol, clear in xylol, 

 and mount in balsam. 



