in an isotonic solution, the refractive index of which can be varied in 

 small increments. 



The phase change (0) or optical path difference introduced by an 

 object is expressed as 



= iit„-n^)t 



where Uo and «,„ are the refractive indices of the object and the mounting 

 medium, respectively, and / is the thickness of the object. Consideration 

 of this equation shows that if the refractive index of the mounting 

 medium is made equal to that of the object, the phase change will be zero 

 and the object will be invisible or have minimum contrast when viewed 

 with the phase-contrast microscope. When the refractive index of the 

 mounting medium is greater or less than that of the object, the object 

 will appear in dark or bright contrast, respectively. By varying the 

 refractive index, a value will be found which matches that of the cell 

 structure under study. Since the refractive index of the cell structure is 

 the same as that of the mounting medium at the matching point, the 

 structure will produce virtually no phase change and will show little or 

 no contrast with respect to its surroundings. 



Variation of the refractive index of the mounting medium by small 

 orders of magnitude is done most accurately with protein solutions, 

 which for every 1 per cent increase in concentration change their re- 

 fractive index by a specific amount. This value is called the specific 

 refraction increment (a) and is calculated to be about 0.00180 for most 

 proteins. Since protein accounts for most of the solid content of proto- 

 plasm, the specific refraction increment of protoplasm is considered to 

 be the same as that of protein or 0.00180. The refractive index of a 

 protoplasmic structure (n) is directly related to its concentration of 

 solids (mosdy proteins). This relationship is expressed by the formula 



n = Hg + a C 



where ng is the refractive index of the solvent, a the specific refraction 

 increment, and C the concentration of solids in grams per 100 ml of 

 solution. In the case of living cells, Us is taken as the refractive index of 

 water or a very dilute salt solution, and is calculated to be about 1.334. 

 The refractive index of the cell structure (n) is determined by immersion 

 refractometry and the value a is taken as being equal to that of protein 

 or 0.00180. The unknown C is calculated by the formula 



C = " - "" 



SURVEY OF CYTOLOGICAL TECHNIQUES / 233 



