124 CYTOLOGICAL TECHNIQUE 



The refractive index of tlie medium must be related to the 

 means adopted to obtain contrast in the image produced by the 

 microscope. An example will make this clear. Let us imagine that 

 a piece of tissue has been fixed by formaldehyde, and that all 

 lipids, carbohydrates, and nucleic acids have been dissolved out 

 of it. The specimen will consist mainly of protein, and this, in its 

 fixed condition, will generally have a refractive index of about 

 1-536.^^ If the fixed tissue is now thoroughly permeated by a 

 colourless, transparent fluid of the same or very nearly the same 

 refractive index, such as methyl salicylate,^^ the whole object dis- 

 appears and no image of it can be obtained by the microscope. 



A fluid that has this eff'ect is often called a 'clearing agent', but 

 the term is misleading. The fixed proteins of the tissue are trans- 

 parent and colourless, and require no clearing. To render the 

 tissue as a whole transparent, it is only necessary to fill all the 

 spaces intervening between the proteins with a transparent, 

 colourless fluid of the same refractive index. There is then no 

 diff'raction of light at the surfaces of the protein, and therefore no 

 image of it can be formed. The protein is unaff"ected. Methyl 

 salicylate no more 'clears' the proteins than a transparent, colour- 

 less fluid of the same refractive index as glass 'clears' a glass 

 object immersed in it. 



To produce contrast and thus prevent invisibility caused in the 

 way just described, one may dye the tissue and then mount in the 

 same medium as before. The object becomes visible because light 

 of certain wave-lengths is reduced in intensity by the dye attached 

 to the proteins, but passes freely through the mounting medium. 

 The fact that the latter has the same refractive index as the pro- 

 teins is now helpful, because the complete homogeneity of protein 

 and medium in this respect is favourable to the production of an 

 optically perfect image. 



Alternatively one may leave the object undyed, but render it 

 visible by mounting it in a fluid that diff'ers slightly from the pro- 

 teins in refractive index. The specimen should then be examined 

 by phase-contrast microscopy. To get the best efl'ect, the diff"er- 

 ence in refractive indices should be small. The retardation or 

 advancement of the light that passes through the object in relation 



