PRECAUTIONS 101 



excess of alkali may cause death, staining of the nuclei, 

 liquefaction, and the loss of stain on the part of the 

 cells. 1 This may occur before a preparation can be 

 focused, in which case the cells appear unstained 

 and will refuse to stain, no matter how much stain or 

 alkali are tried. Therefore it is better to begin with a 

 low index of diffusion and to try tube after tube, each 

 containing a little more alkali, until staining is ob- 

 tained. Further, the amount of sodium bicarbonate 

 should not exceed twenty units, because, as has already 

 been pointed out in a former paper, 1 if added to excess 

 it may act as a neutral salt and delay diffusion. 



As regards Excess of Deficiency or Heat: A tem- 

 perature above 40 C. may allow the cells to diffuse 

 through the agar. 2 A temperature below 15 degrees 

 has not been experimented with, because, even at a 

 temperature of 20 C. it requires a minimum of 3 units 

 of stain to cause staining of the nuclei of leucocytes 

 in spite of the addition of a large amount of alkali, 

 for the alkali is not sufficient, per se, to cause the 

 cells to absorb sufficient stain to colour the nuclei 

 unless the stain is concentrated. 



As regards Excess of Time: A period of more 

 than half an hour has not been employed for fear 

 of death and liquefaction of the cytoplasm, for the 

 cells may die and become achromatic before there 

 has been time for sufficient stain to diffuse into them 

 to cause staining of the nuclei, in which case, of course, 

 the cells will never stain. 



As regards Excess of Stain: More than 10 units 

 of stain may cause precipitation of the agar as the 

 film cools on the slide, and the precipitate carries 



1 "On the Death of Leucocytes" (H. C. Ross, Journal of Physiology, 

 vol. xxxvii., p. 327, 1908). 



"The diffusions of Red Blood Corpuscles through Solid Nutrient Agar" 

 (H. C. Ross, British MedicalJournal, May 5, 1906). 



