112 MICROSCOPIC METHODS. 



instead of alcohol (p. 107). Other modifications have been 

 introduced, of which only Kiihne's need be mentioned. 



Kilhne's Modification of Grant's Method. 



1. Stain for five minutes in a solution made up of equal parts of 

 saturated alcoholic solution of crystal-violet ("krystall-violet ") and 

 I per cent solution of ammonium carbonate. 



2. Wash in water. 



3. Place for two to three minutes in Gram's iodine solution, or in 

 the following modification by Kiihne : 



Iodine 2 parts 



Potassium iodide .... 4 parts 

 Distilled water . . . .100 parts 



For use, dilute with water to make a sherry-coloured solution. 



4. Wash in water. 



5. Decolorise in a saturated alcoholic solution of fluorescein (a 

 saturated solution in methylated spirit does equally well). 



6. Dehydrate, clear and mount. 



Contrast Stains. With these methods, especially that 

 of Gram's, it is often advantageous, after decolorisation, to 

 counterstain the tissues with a dye different in colour from 

 that retained by the bacteria. 



Lithia carmine or alum carmine may be used for contrast-staining 

 in Gram's method. The sections here are stained first with the 

 contrast, and then treated by Gram's method. (This is the most 

 satisfactory procedure. ) 



In the case of the following stains the contrast-staining is not 

 carried out till the tissues have been subjected to the bacterial stain 

 and decolorised as far as necessary. 



Safranin. Stock solution is a I per cent solution of safranin 

 dissolved in equal parts of methylated spirit and water. For use, 

 dilute one part with five of water, and stain for thirty seconds. 



Bismarck -brown. Stock solution saturated solution in equal 

 parts of alcohol and water. Stain for from two to three minutes. 



Both safranin and Bismarck-brown are excellent nuclear stains, and 

 in cases where Gram's method has been used, colour most of the 

 organisms which are left unstained by the violet. 



Eosin may be used as a i-iooo watery solution, and applied for 

 about a minute. It is a good ground stain, but does not bring out the 

 nuclei. 



