202 DYEING 



is particularly close-textured and therefore difficult to penetrate 

 (e.g. mitochondria). 



The effects of fixation on dyeing have already been mentioned 

 in chapters 5 and 6, under the headings of the eight primary 

 fixatives selected for separate description. A general review of the 

 subject will be given here. 



These effects can be studied by experiments on proteins. 

 Seki ^^^ fixed small pieces of egg-white in various ways; he either 

 dyed them whole or made paraffin sections. Fibrin films are very 

 suitable for work of this sort; they are chemically uniform and 

 require no sectioning.*'^ The effects of fixation on the dyeing of 

 tissues has been investigated by several workers.^^^' ^^'^' *^^'*"^'^^ 

 Whether proteins or tissues be used, the material may be exposed 

 to a mixture of a basic and an acid dye,^^^ or to basic and acid dyes 

 separately; the pH of the dye-solution may be controlled. ^^^ 

 Instructions for carrying out simple experiments on the effects of 

 fixatives on dyeing are given in the Appendix (p. 325). 



For studies of this kind it is important to avoid cells (such as 

 nerve-cells) that have much RNA in the cytoplasm, for this would 

 colour strongly with most basic dyes and therefore mask the effect 

 of fixatives on the reactions of dyes with the cytoplasmic proteins. 

 Strongly basic protoplasm, such as that of mammalian red blood- 

 corpuscles, is also unsuitable. One needs an example of typical 

 cytoplasm and typical chromatin. Seki *^^ chose the skin of the 

 frog and mammalian kidney. The convoluted tubules of the latter 

 organ and the spermatogonia and spermatocytes of mammals are 

 particularly suitable. ^^ The late spermatids and spermatozoa them- 

 selves are unsuitable not only because of the absence of unspecial- 

 ized cytoplasm, but also because of the basic nature of the nuclear 

 material. 



Certain possible sources of error should be noted. The intensity 

 of dyeing is often different at different depths in a piece of tissue. 

 It is best to use small pieces of nearly uniform size and to compare 

 cells in the middle of each piece. Some fixatives shrink the cyto- 

 plasm strongly. In its shrunken condition it may give a false im- 

 pression of taking up a lot of dye, when in fact the same amount of 

 dye, spread over the cytoplasm of an unshrunken cell, would look 

 quite pale. It is desirable that quantitative methods should be 

 introduced into work of this kind. This has already been done in 

 studies made with fibrin films. *"^ 



