104 Mr. H. C. Ross. On a Coefficient of [Dec. 9, 



Precautions. — As regards Life and Death : In a previous paper (3) it has 

 heen shown that the staining of the nuclei of leucocytes, when examined by 

 this method, is a sign of death, and that the nuclei of dead cells will stain, 

 ceteris paribus, before those of living cells. Consequently all the experiments 

 given in the present paper have been made with fresh normal cells, and in 

 the case of micro-organisms with cultures not more than 48 hours old. It 

 may also be mentioned that the liquefaction of the cytoplasm which occurs 

 after death materially alters the conditions of staining of leucocytes, and that 

 the cf of living blood cells falls gradually after the blood has been shed. The 

 fact has already been mentioned that in chronic wasting diseases the coefficient 

 of blood cells may be very low.* 



As regards Excess of Alkali causing rapid death and liquefaction of the 

 cytoplasm with consequent prevention of staining (achromasia) : The 

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

 liquefaction, and the loss of stain on the part of the cells (3, 6). This 

 may occur before a preparation can be focussed, in which case the cells 

 appear unstained and will refuse to stain, no matter how much more 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 obtained. Further, the amount of sodium bicarbonate should 

 not exceed 20 units, because, as has already been pointed out in a former 

 paper (3), if added to excess, it may act as a neutral salt and delay diffusion. 



As regards Deficiency of the Salts sodium citrate and sodium chloride: If 

 the jelly contains no salts, the blood lakes and the leucocytes are killed 

 outright. If it contains sodium chloride only, the cells are killed rapidly, and 

 the same may be said if sodium citrate only is employed (7). In examining 

 blood, therefore, the combination is essential. 



As regards Excess or Deficiency of Heat : A temperature above 40° C. may 

 allow the cells to diffuse through the agar (2). A temperature below 15° 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 



* I have found that the life of leucocytes of persons suffering from some chronic 

 diseases, when bathed in their own plasma, is considerably shorter than the life of 

 healthy persons' leucocytes bathed in their own healthy plasma (8). I have reasons for 

 believing that there is an association between this and a low cf. 



