Diplococci in Rabbit Liver 239 



bleaching with sodium thiosulfate. The writer does not usually do this, 

 but the treatment is insisted upon by Mallory in the description cited. 



The staining solutions used in this technique are simple to prepare 

 and relatively stable. The first solution is a 2.5 per cent solution of 

 phloxine in water. Two stock solutions are also required: one of 1 per 

 cent each of methylene blue and borax in distilled water, and the other a 1 

 per cent solution of azur II in distilled water. Five ml of each are added to 

 90 ml of distilled water for use. Differentiation is in Wolbach's resin 

 alcohol. 



It is difficult to get a sufficiently heavy stain in phloxine to withstand 

 the alkaline thiazine solutions used for counterstaining. Mallory recom- 

 mends that the sections be placed in a coplin jar of the solution in a 

 55° C oven and that they remain there for at least 1 hr. The coplin jar 

 is then removed from the oven and cooled before the sections are re- 

 moved; they should be stained a dense orange. If they have not yet ac- 

 quired this color, they should be returned to a paraffin oven for a further 

 period. If the sections are satisfactorily stained, the solution may be 

 poured off or the slides removed from it and briefly rinsed in water. 

 The purpose of this rinse is not to differentiate the eosin in the section but 

 to remove it from the glass. The slides bearing the sections are then 

 placed in the methylene blue-azur solution in another coplin jar for 5 to 

 20 min; the exact time varies according to the specimen that one is 

 staining and can only be determined by experiment. Mallory recommends 

 that the solution be freshly filtered onto each slide, rather than that the 

 solution be used in a coplin jar; but the writer has not found this nearly 

 as convenient, nor does it appear to be in any way obligatory. After the 

 slides have taken up sufficient methylene blue solution to appear bluish 

 rather than pinkish, they may be accumulated in water, before being 

 differentiated in the resin alcohol one at a time. This differentiation is 

 best conducted in a dish that is large enough to admit the slide in a 

 flat position. The slide is taken in a pair of angle forceps, dipped under 

 the surface of the solution, and waved gently backward and forward 

 for about 1 min. As it is being moved backward and forward in the 

 differentiating solution, the blue color will come off in clouds; it is much 

 easier to overdifferentiate than to underdifferentiate. 



The differentiation can be readily controlled by inspection under the 

 microscope, although it is not necessary to observe the bacteria, since the 

 nuclei have exactly the same staining reaction. Differentiation should be 

 stopped when the nuclei can be seen, under the low power of the micro- 

 scope, to be very deep blue, while the general background of the section 

 is pink. After a little practice the required color may be gauged without 

 examination under the microscope. 



These specimens are not permanent unless the alcohol and resin are 



