1908.] 



Diffusion into Living Cells. 



105 



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 pre- 

 cipitation of the agar as the film cools on the slide, and the precipitate carries 

 some of the stain down with it, vitiating the results, for it has been shown 

 that agar is not very soluble in cold stain (3). 



As regards Examination : The observation of cells floating near a bubble 

 under the cover- glass should be avoided. The fact that blood cells in such 

 a situation will stain before others has already been noted (3). I consider 

 this to be due to these cells floating in a small quantity of alkaline citrated 

 plasma collected round the bubble. 



Consequently the experiments have all been made within the compass of 

 the above restrictions. So far no cells, whether blood, bacteria, or other cells, 

 have been met with which would not give a coefficient of diffusion by this 

 method. It may also be advised that when unnucleated cells contain granules 

 in their cytoplasm, the staining of the granules gives a more constant rate 

 than the staining of the cytoplasm. By this Aeans it is seen that the cf of 

 the blood-plates is identical with that of the polymorphonuclear cells. 



The Construction of other Units. — It may be necessary to add ether 

 substances to the jelly to test their effect on cells. Eor instance, it may 

 be useful to try other salts, in which case their rate of antagonism to diffusion 

 must be found and a unit made. This may be done by comparing their 

 action with that of a unit of one of the other factors, after which the new 

 unit may be added to the equation. In the case of sulphate of atropine, it 

 was found that a tube of 10 c.c. of agar, which had a correct fx to cause 

 staining of lymphocytes in 10 minutes, but which also contained 

 0"013 gramme of sulphate of atropine, required the addition of 1 more 

 unit of alkali to cause the nuclei to stain in 10 minutes. Consequently 

 the unit of atropine sulphate may be said to be - 013 gramme. 



The Determination of the Coefficient of Diffusion of Leucocytes involves 

 Death. — Since the staining of the nucleus is the moment by which the cf of 

 leucocytes is obtained, and since the staining of the nucleus is a sign of 

 death (3), the cells are necessarily dead at the expiry of the time involved in 

 finding their coefficient. 



The Beconciliation of their Coefficient of Diffusion to Cells which may be 

 Alive at the Termination of the Time required. — It has been shown that 

 leucocytes will live for a considerable period and show amoeboid movement 

 with their granules stained (3, 5). If 1 digit is subtracted from their cf and 

 the jelly arranged according to such an equation, the granules but not the 



