Methods of Staining in Bulk and Coitnter- Staining. 277 



about the size of the rat testis — thirty-six to forty-eight hours 

 will be required. After staining, remove the piece of tissue to 

 50 p.c. alcohol, to wash out the excess of colour, grade down, and 

 thoroughly wash out in running tap-water for a few hours, accord- 

 ing to the size of tissue. This washing out in water is important, 

 as greater clearness and density of the stain is obtained by this 

 means. Within certain limits, say six to twenty-four hours-, 

 according to the size of the piece of tissue, the longer the washing 

 the denser the stain. 



After washing out in water, the pieces are up-graded to absolute 

 alcohol, cleared with cedar-wood oil, embedded in paraffin -wax, and 

 sections cut 10 yu,. Sections are fixed to the cover-glass and treated 

 sccundem artem, then, if desired, counter-stained by immersion for 

 about ten minutes in erythrosine, not eosin — 



Erythorine .... 1 grm. 

 Alcohol .... 40 c.cm. 

 Water 160 „ 



previously down-grading into alcohol equivalent in strength to 

 that of the erythrosine stain ; washed in 30 p.c. spirit and up- 

 graded to absolute alcohol, cleared in cedar- wood oil, xylol, and 

 mounted in balsam or euparal, perhaps preferably in the latter 

 medium, as I am not yet in a position to say whether this stain 

 will keep so well in the former, although I must admit that balsam 

 gives the sharper image with this method of staining. 



Centrosomes that are as a rule easily demonstrated by Heiden- 

 hain's method are equally so by this latter method of staining iii 

 bulk. 



In many cases, counter-staining in bulk with light green, after 

 previously staining in bulk with caralum or htematoxylin, can be 

 carried out quite easily. 



Light green does not overstain, and therefore needs no differen- 

 tiation. All that is necessary after staining in bulk, as previously 

 described, and washing out, is to up-grade into alcohol of 50 p.c.,. 

 place the pieces of tissue direct into the alcoholic solution of light 

 green for at least twenty-four hours, wash out in methylated spirit 

 until no more colour comes from the tissues, dehydrate in absolute 

 alcohol, pass through cedar-wood oil, embed in paraffin, cut sections, 

 fix to cover-glass, dry, remove wax by cedar-oil, wash in xylol, and 

 mount in euparal. 



Some tissues, however, are of such density that the light green 

 only penetrates a very short distance, and prolonged immersion does, 

 not succeed in remedying this defect. 



In such cases, proceed to fix sections on the cover-glass, remove 

 the wax, and down-grade to methylated spirit, immerse for about 

 ten minutes in the alcoholic solution of light green, wash out in 

 methylated spirit, dehydrate, clear, and mount in euparal ; the 



