GKAM'S STAINING OF SECTIONS. 541 



much. An excessive exposure to dye requires a corre- 

 sponding exposure to the decolorizing agents, and, as 

 a result, there ,is lack of differentiation. Hence, the 

 section should remain in the dye for as few minutes as 

 possible. 



Double staining by Gram's method. — This is an extremely 

 useful method but unfortunately it is not applicable to all 

 bacteria. A list of those bacteria which can be stained by 

 Gram's method is given on p. 289. The method is easy of 

 execution and, when properly carried out, it will give clean, 

 beautifully stained preparations. 



Fresh solutions of anilin-water gentian violet and of 

 iodine are prepared according to the directions given on 

 p. 288. The stain may be slightly warmed, but this is not 

 necessary. As stated above it is desirable not to overstain 

 too much.. 



The section is placed in the stain for 10 to 15 minutes. 

 It is then washed in water, or in anilin-water, to remove 

 the excess of dye. In this way, the formation of unsightly 

 deposits in the section, on subsequent contact with the iodine 

 solution, is avoided. The section is then placed in the 

 solution of iodine (Lugol's) for frogi 3 to 5 minutes. It is 

 now transferred to absolute alcohol, in which it is gently 

 moved about till most of the stain is removed. The sec- 

 tions should not be completely decolored, but should still 

 show a distinct violet color. 



It is now placed in very dilute eosin for about J^ min- 

 ute. Over-staining with eosin will impart to the tissue a 

 deep red color which will thus make an unfavorable con- 

 trast for the violet organism. It is preferable, therefore, 

 to stain with eosin so that the tissue has a light pink color. 

 Weigert's picrocarmin can be used instead of eosin. It 

 should be allowed to act for from 3 to 5 minutes. The sec- 

 tions may be first stained with picrocarmin and then sub- 

 jected to Gram's staining. 



