A CRITIQUE OF CYTOCHEMICAL METHODS 207 



putcd from in vitro (l;it;i tor a number of naturally colorc^d .substances 

 commonly found within cells [\\\i\ foi- some speeitic stains and tests for 

 proteins and nucleic acids (columns 1, 2, and 3, Tables G-l, 2). The 

 sources of the \alues from which computations were carried out are indi- 

 cated in column 5.' 



These values in Tables 6-1 and 2 are not claimed to be necessarily close 

 approximations of any physical constants of the intracellular substance. 

 They are almost certainly subject to considerable revision as more is 

 learned of the effects on absorption of high concentrations and of special 

 intermolecular associations within the cell (see p. 215 and Chaps. 1 and 5, 

 this volume). In the meantime, they are useful relative values from 

 which the possibilities of seeing, or measuring photometrically, an intra- 

 cellular substance under the microscope can readily be estimated; they 

 are likewise the only method of translating results of intracellular absorp- 

 tion measurements into the familiar values of chemical analysis, and in 

 radiation experiments they can serve as the basis for an approximate esti- 

 mate of the amount of energy absorbed per cell or cell part. Since the 

 errors introduced by high concentrations and other special conditions 

 within the cell tend, as a rule, to reduce the specific extinctions, it seems 

 fairly safe to assume that the values in column 3 are maximal and those 

 in column 4 are minimal. The great usefulness of the cytological stand- 

 ard, {E\)a, arises from the fact that of itself extinction is a direct measure 

 of the number of molecules in the absorbing path and can be used as such 

 when neither concentration nor thickness of the absorbing area is known. 

 From the standard {E\)lt '""'* the amount per total area of the part of 

 the cell measured (^4) can be computed simply as {E\)a-A {A being meas- 

 ured in square microns). Likewise, because extinction times area is the 

 equivalent of amount, it can be used in simple arithmetical compu- 

 tations to compare compositions of cytological objects in purely arbitrary 

 amounts (see Swift, 1950). 



So far W'C have been considering photometric methods with measure- 

 ment in a more or less restricted spectral region, a procedure which, some- 

 what paradoxically, is often called colorimetry. .When measurements are 

 made at many wave lengths, a picture is obtained of the etTect of the 

 absorbing substance on the light, which expresses in objective data the 

 phenomena which cause the visual sensation of color. Such data are 

 often plotted as absorption curves, with some measure of relative light 



^ The cytological standard, {E\)ll ^"'"^ is computed as follows, from a cuvette 

 standard, e.g., for deoxyribonucleic acid (DXA) where {E2i4)l',o^ "" is 20. It is 

 given that 20 is the E^hi of 1 yu^ of a standard solution in a thickness of 1.0 cm, lO"* m 

 (for extinction depends on thickness and concentration and is independent of area). 

 Each cubic centimeter of cuvette standard contains 1.0 mg of DX.\; 1 cc is 10'^ fx^, and 

 the volume of a mass which is 1 m^ in area and 1 cm thick is 10^ n^. This volume 

 of the standard solution then contains lOVlO'^, or lO"**, mg of DNA. Therefore, 

 (Ei,,)lV'"'"^ is 20/102 or 0.200 (Table 6-1). 



