MICROSCOPIC STUDY AND STAINING 121 



Sherman. 18 Wells' suggestion is that the iodin may render the cell 

 membrane impermeable to alcohol. 



Preparations are made on cover-slips or slides in the usual way. 



The preparation is then covered with an anilin gentian-violet 

 solution which is best made up freshly before use. 



The staining fluid is made up, according to Gram's original direc- 

 tions, 19 as follows: 



Five c.c. of anilin oil are shaken up thoroughly with 125 c.c. of distilled 

 water. This solution is then filtered through a moist filter paper. 



To 108 c.c. of this anilin water, add 12 c.c. of a saturated alcoholic 

 solution of gentian-violet. The stain acts best when twelve to twenty-four 

 hours old, but may be used at once. It lasts, if well stoppered, for three 

 to five days. A more convenient and simple method of making up the stain 

 is as follows: 



To 10 c.c. of distilled water in a test tube add anilin oil until on shaking 

 the emulsion is opaque ; roughly, one to ten. Filter this through a wet paper 

 until the filtrate is clear. To this add saturated alcoholic solution of gentian- 

 violet until the mixture is no longer transparent, and a metallic film on 

 the surface indicates saturation. One part of alcoholic saturated gentian- 

 violet to nine parts of the anilin water will give this result. This mixture 

 may be used immediately and lasts two to five days if kept in a stoppered 

 bottle. 



Cover the preparation with this; leave on for 5 minutes. Pour off excess 

 stain and cover with Gram's iodin solution for 2 to 3 minutes. 



Iodin 1 gm. 



Potassium iodid 2 gm. 



Distilled water 300 c.c. 



Decolorize with ninety-seven per cent alcohol until no further traces of 

 the stain can be washed out of the preparation. This takes usually thirty 

 seconds to two minutes, according to thinness of preparation. 



Wash in water. 



Counterstain with an aqueous contrast stain, preferably Bismarck brown, 20 

 dilute fuchsin or safranin. 



PALTAUF'S MODIFICATION. OF GRAM'S STAIN. 21 The staining fluid 

 as prepared by this modification possesses the advantage of retaining 



18 Sherman, Jour, of Infec. Dis., 12, 1913, 249. 



19 Gram, loc. cit. 



20 To make up Bismarck brown solution, prepare a saturated aqueous solution 

 of the powdered dye by heating. Cool and filter. Dilute 1 to 10 with distilled 

 water. 



21 Sharnosky, Proc. N. Y. Pathol. Soc., Oct., 1909, n. s., ix, 5, 



