A CUITlgUE OF CYTOCHEMICAL METHODS 235 



reagents have been most widely used, acetic acid-alcohol and neutral 

 formalin. The former has the advantages of conserving little but 

 nucleoprotein, and this in a state where fractions are readily lemovable; 

 but acetic acid-alcohol certainly introduces gross morphological artifacts 

 especially in lipid-containing structures such as mitochondria. Formalin 

 introduces less artifacts, but it does preserve many other substances in 

 addition to nucleoprotein and has a tendency to react vigorously with 

 proteins (French and Edsall, 1945) which may quite possibly lead to con- 

 siderable confusion in interpretation of staining and tests, or even of ultra- 

 violet absorption. 



The selection of the exact spot to be measured in the cell is also a diffi- 

 cult task. If the material is homogeneous over a considerable area, the 

 size of the sample is relatively unimportant. The Caspersson group, as a 

 rule, measures a very narrow cylinder (less than 1 /x- in area). This has 

 the merit of being so small that in most cases great variations of absorp- 

 tion across the field are unlikely. It has the disadvantage that the spot 

 may not be representative of any considerable part of the cell. All other 

 workers (e.g., Gersh and Bodian, 1943; Pollister and Ris, 1947; Leuchten- 

 berger, 1950; Swift, 1950; Lison and Pasteels, 1951; Fanijel, 1951) have 

 measured the transmission of larger areas, often entire nuclei. Some- 

 times these larger areas are fairly homogeneous, but more often the 

 absorbing material is to some extent concentrated in scattered masses 

 among which there is a sort of continuum of relatively lower absorption. 

 This heterogeneity introduces into the absorption measurements an 

 obvious unavoidable error (conveniently called the "distributional error") 

 which was first pointed out by Caspersson (1940a). This leads to lower 

 extinction values, never to higher. The error is best understood from 

 considering the actual conditions of measurement of two extreme con- 

 ditions, one where absorbing material is evenly distributed throughout an 

 area and another where the same amount of absorbing material is con- 

 centrated in one-half the area, the remainder being free of absorbing mate- 

 rial. Suppose the concentration in the first case is sufficient to cut the 

 light down 90 per cent, i.e., the transmission is 10 per cent and the extinc- 

 tion is 1.0. In the second case the light loss will be as follows: Since the 

 concentration of absorbing material in the dark half is doubled, its extinc- 

 tion becomes 2. This means that this half will transmit 1 per cent of the 

 light which falls upon it; and, since this darker portion is one-half the 

 total area measured, the amount of light passing through this absorbing 

 region to the photoreceiver is 0.5 per cent of the total light. The lighter, 

 nonabsorbing half, however, transmits all hght, which is one-half the 

 total amount; hence the reading on the instrument will indicate a trans- 

 mission of 50.5 per cent. The ec}uivalent extinction is 0.306, less than 

 one-third the true value (1.0) obtained when the material is evenly dis- 

 tributed. This case is an example of extreme heterogeneity which 



