180 THE TECHNICS OF PHASE MICROSCOPY 



Several series of liciuids of ^'arying refracti^'e index are available com- 

 mercially or may be made from directions given in the literature. They 

 should not dissolve or alter the specimen, should be colorless or nearly so, 

 and should ha^■e low vapor pressures so as not to change with age. 

 Some useful materials are listed in Table lYA. However, a change in 

 temperature will usually change the refractive index, and it is desirable 

 to keep temperature records to 0.1° C along with the observations. 

 Solutions of a desired index may be made by mixing suitable mate- 

 rials according to the formula V^Nj: = FiA^i + 12-^2, where Vj; is the 

 volume, N:c is the index desired, and the right-hand terms are the vol- 

 umes and indices of the materials to be mixed. Such mixtures will not 

 be overly accurate. To determine the exact refractive index it should 

 be measured on a refractometer, or if solids of known index are available 

 they may be put in the liquid and its index discovered (Chamot and 

 Mason, 1939). Micro methods for determining refractive index have 

 been proposed by Kirk and Gibson (1939), Alber and Bryant (1940), 

 and Jelley (1949). Groat (1941) recommends tissue sections of 50-^ 

 thickness for the determination of their refractive index. When the 

 material is sectioned in paraffin the latter should be removed with 

 xylene and several rinses made with the test liquid to remove the 

 xylene before the index of the tissue is measured. 



Most of the standard index series are oils or oily liquids. Therefore 

 many materials must be dehydrated before their index may be measured. 

 The dehydrated specimen often has a different index owing to concentra- 

 tion, or possibly to loss of materials extracted by the solvent, or to 

 other chemical action occurring during the dehydrating process. 



Three factors should be considered in choosing a mounting medium, 

 assuming that it otherwise has no adverse effect on the specimen: (1) 

 If color effects are to be avoided the medium should have about the 

 same dispersion as the specimen; if optical dispersion staining is desired 

 then the dispersions should have optimal relations to each other (Section 

 3.5). (2) Some materials make specimens appear more transparent 

 than do others, even though the optical path differences are the same 

 (e.g., tissues cleared in organic phosphates rather than in paraffin base 

 liquids), and this difference may be important in examination with the 

 microscope (Downs, 1943). (3) Other chemicals transparentize a speci- 

 men (as in making parchment) or have an effect, like that of phthalates, 

 on paper and some fabrics. Dibutylphthalate is often so used in 

 microscopy. 



These well-known methods for microscopy are especially important 

 for phase microscopy in which smaller path and intensity differences 

 are clearly visible. 



