22 STAINING REACTIONS OF BACTERIA 



with increasing ease as molecular weight increases, at least up to a certain poin*. 

 Molecular weight alone has little to do with cell permeability except as a limiting 

 factor.'^ 



MECHANISM OF STAINING 



The theory of the molecular structure of coal-tar dyes, which I have stated, while 

 perhaps not certainly established in all details, accounts for all the known facts, and 

 its adoption has been so fruitful that it is now generally accepted. About the actual 

 mechanism of staining, on the other hand, there is by no means general agreement. 

 Much of the discussion has been devoted to a debate as to whether the process is 

 chemical or physical. This seems peculiarly futile since a rigorous definition of chem- 

 ical and physical processes is difhcult if not impossible to formulate and since the two 

 processes are ultimately one. Even the ardent adherents of one or the other theory 

 are usually forced in the end to acknowledge the possible identity of the two. 



If a chemical process be defined as a reaction between two substances in which a 

 new chemical substance is formed and a physical process as a reaction between two 

 substances in which no new chemical substance is formed, it is clear that both proc- 

 esses occur in staining. A dyestuff as a whole may enter into and be deposited upon a 

 tissue or cell by a process which Michaelis describes as "insorption," in which case the 

 coloring matter may be subsequently extracted by any chemically indifferent solvent. 

 On the other hand, a dye may become chemically united to the cell protoplasm by the 

 formation of a salt, and in such a case the color can be removed only by agents like 

 free acids which are capable of decomposing salts. It is certain that Ehrlich's dia- 

 grammatic conception of chemoceptors to account for selective staining can no longer 

 be held. Many observations in recent years point to the fact that other properties of 

 the molecule than its structural formula (upon which the Ehrlich theory laid so much 

 stress) are concerned and are probably of greater importance. 



There is good reason to believe that chromophilic protoplasm, so far as it is di- 

 rectly stainable (that is to say, without mordants), is amphophilic or amphoteric, i.e., 

 it possesses basic (amido-) and acidic (carboxyl-) groups side by side in the molecule. 

 It has therefore the structure of amido-carbonic acid. The chemical processes of 

 staining go on best the more auxophoric groups there are in the dye and the greater 

 the number and the adequacy of chromophilic groups in the material to be stained. 



The physical processes of staining consist of surface attraction, osmosis, diffusion, 

 adsorption ; and the factors which promote these processes are proper size of the dye 

 molecule and adequate pore-volume of the material to be stained. 



It cannot be said that a completely satisfactory description of the chemistry of 

 bacterial staining can be given. This is perhaps not strange considering the enormous 

 complexity of the chemical structure of dyes, the complicated structure of bacteria, 

 the difficulties of chemical analysis of bacterial bodies which are constantly changing 

 their composition during life, and the minuteness of the microchemical reactions on 

 which we have to depend. 



One thing essential for the process of staining bacteria is water. Water-free, al- 

 coholic solutions of dyes will not stain dehydrated bacteria, nor will water-free alcohol 



'Lillie, R. S.: General Cytology, p. 167. Chicago: University of Chicago Press, 1924; Jacobs. 

 M. H.: ibid., p. 99. 1924. 



