212 THE AMEKICAN MONTHLY [September, 



cultures, or other germ containing material, or in sections of animal 

 tissues that have previously been hardened in alcohol. 



There are two preparations, the weak (Koch's) and the strong (LofF- 

 ler's) . The weak solution is prepared as follows : Concentrated alco- 

 holic solution of methylene blue, i c.cm., 10% solution of potassium 

 hydrate, 2 c.cm., distilled water, 200 c.cm. 



The strong solution is the one to which special reference is made. 

 It is prepared by taking concentrated alcoholic solution of methylene 

 blue, 30 c.cm., 1% solution of potassium hydrate, 1 c.cm., distilled 

 water, 99 c.cm. 



Cover-glass preparations are sufficiently stained in this solution in 

 from 1 to 5 minutes, and sections in from 3 to 20 minutes, according to 

 the tissue. Epithelial cells, nuclei, granules of granular corpuscles and 

 white blood corpuscles are stained as readily as the germs themselves, 

 while connective tissue fibres are very slightly stained, and the red blood 

 corpuscles are not stained at all with this agent ; thus, in a section of 

 stomach or intestine stained with it the mucous membrane, together 

 with the bacteria, will be stained a deep blue color, while the sub- 

 mucosa and muscular coat will be very feebly tinted. 



If the preparations should, at any time, be overstained, they can be 

 readily decolorized by a momentary immersion in a weak (^ of 1%) 

 solution of acetic acid. Methylene blue is much less liable to overstain 

 preparations than other basic aniline dyes, and it is consequently to be 

 used if, for any reason, decolorizing agents should not be employed. 



The technique in the use of this stain is very simple, and differs in no 

 way from that of ordinary staining fluids. After staining, sections 

 should be washed in weak alcohol, then transferred to stronger, and, 

 finally, cleared in turpentine, xylol, or cedar oil, and mounted in bal- 

 sam. Cover-glass pi-eparations are washed in water, and allowed to 

 dry in the air until completely desiccated, when they are also ready for 

 mounting:. 



Gram's Method of Staining. — Among the different staining meth- 

 ods employed in studying micro-organisms the one introduced by Dr. 

 Gram is very useful. In his method the bacteria are stained a deep 

 blue while the surrounding tissue is colorless. The technique of the 

 method is, according to Friedlasnder,* as follows : 



The stain employed is Ehrlich's solution of gentian or methyl-violet 

 in aniline water. This is prepared by shaking pulverized gentian or 

 methyl-violet with aniline water and allowing it to stand for 24 hours, 

 when it is filtered and the clear filtrate is then ready for use. The same 

 result will be obtained if 5 cc. of a saturated alcoholic solution of the 

 stain be added to 100 c.cm. of aniline water. 



The sections, previously hardened in alcohol, are placed in a watch- 

 glass containing 2 to 3 c.cm. of the staining fluid and allowed to remain 

 in it for from 3 to 5 minutes. They are then transferred, by means of 

 a section lifter, to a second watch-glass containing a solution of iodine 

 in iodide of potassium (iodine, 1 gram, iodide of potassium, 2 grams, 

 distilled water, 300 c.cm. The iodide of potassium is dissolved in the 

 water and the iodine added.) They are allowed to remain in this for 



* Microscopische Technik, p. 49. 



