1908.] 



Diffusion into Living Cells. 



99 



its Index of Diffusion (fx) may be defined as the sum of its constituents 

 which delay diffusion subtracted from the sum of constituents which 

 accelerate diffusion added to the quantity of stain contained in the jelly. 



The Coefficient of Diffusion (cf) of a cell is that index of diffusion plus the 

 time and temperature required to cause staining of the nucleus, or staining of 

 the cytoplasm in unnucleated cells (e.g., red corpuscles), when the specimen is 

 prepared by a standard method. 



Standard Method of Preparation. — This consists in : (1) Mixing the cells 

 with a neutral solution containing 3-per-cent. sodium citrate and 1-per-cent. 

 sodium chloride.* If blood is experimented with, it is mixed with an equal 

 volume of the solution. In the case of bacteria and other cells, the mixture 

 is made as convenient.^ (2) The mixture is then placed on a cover-glass, 

 which is inverted and allowed to drop flat on a film of agar jelly containing 

 Unna's stain and salts, and which, after boiling, has been allowed to set on a 

 slide. Since the surface of the film is convex, the solution spreads to the 

 periphery of the cover-glass, leaving the cells gently pressed out between the 

 glass and jelly, and this affords an excellent means of examination by the 

 microscope.^ Only the cells in the centre of the preparation should be 

 examined. 



It is stipulated that the jelly contains stain, but the amount of stain added 

 to the agar may be variable. The chemical nature of methylene blue may or 

 may not affect diffusion. The point is difficult to determine accurately, but 

 in this procedure it is of little importance, because the stain employed is 

 always the same, namely, Unna's polychrome methylene blue (Grubler). On 

 the other hand, it is obvious that the more concentrated it is — that is, the 

 more stain there is in the 10 c.c. of jelly, the more rapidly, ceteris paribus, 

 will the cells stain. It is also obvious that the effect of an increase of the 

 concentration of the stain can be neutralised by the addition of one or several 

 factors which delay diffusion. Consequently I also measure the stain in 

 units, so that an increase of its concentration by 1 unit can be neutralised 

 by the addition of 1 unit of a factor which decreases diffusion. § 



* Merck's reageDts have been used throughout these researches. 



t The mixture of the cells with this solution is merely used as a vehicle to keep them 

 alive. As the solution spreads to the periphery of the cover-glass, it does not materially 

 influence the diffusion of stain from the agar, a point which has been tested experi- 

 mentally by rendering it alkaline. The cells, however, should not be kept in it longer 

 than necessary. 



X The suggestion of mixing stain with the jelly was made to me, as already noted 

 elsewhere, by my brother, Prof. Ronald Ross. 



§ Unna's polychrome methylene blue is only supplied in solution, which is standardised. 

 It cannot be made in a powder. 



H 2 



