THE CYTOPLASM 207 



technic cannot often be checked by independent means. Until this unfortunate situa- 

 tion is resolved, therefore, it is imperative both to set up certain requirements de- 

 fining an adequate isolation procedure and to appraise critically the results obtained 

 in terms of the possible defects of the methods used. 



Obviously, it is much simpler to define than to devise an adequate method for the 

 isolation of a cell structure. It should provide the structure in unaltered form, in 

 homogeneous preparations, and in good yield. The term, unaltered, denotes the 

 absence of both morphological and biochemical changes. Thus it is difficult to believe 

 that a cytologically altered structure could possibly be biochemically intact. Since 

 cytological methods are practically the only means by which structures within living 

 cells have been characterized, however, these same methods must be relied upon in 

 an assessment of the integrity of cell structures isolated from their cellular environ- 

 ment. The use of cytological criteria of integrity has been criticized on the ground 

 that isolated cell structures that are morphologically intact may not be biochemically 

 intact." Mitochondria, for example, isolated in morphologically and cytologically 

 unaltered form, have been found less active in catalyzing certain enzymic reactions 

 than mitochondria isolated in an altered form (cf. footnote 8). This does not consti- 

 tute justification for disregarding the cytological observations and for substituting 

 biochemical criteria of integrity based upon enzymic activity, because it is quite 

 obvious that we are not as yet able to arrive at a definition of biochemical integrity. 

 Nevertheless, it must be admitted that the adequacy of cytological criteria is often 

 questionable, and it is therefore hoped that an answer to the difficult problem of de- 

 tecting physical and chemical alterations in isolated cell structures will result from 

 a continuous search for better methods and from the objective, critical evaluation 

 of extensive data based upon a diligent alertness for possible artifacts. 



The necessity for striving for homogeneity in the isolation of cell structures is 

 obvious. We must go even farther than this, however, for the term, homogeneity, can 

 only be used in a relative sense, and quantitative methods of analysis must therefore 

 be devised for determining the degree of homogeneity attained. This is an axiom in 

 the field of chemistry. In cytochemistry and in the absence of a better method, micro- 

 scopic examination has usually been employed in the past as the chief means of esti- 

 mating the purity of preparations of isolated cell structures. Unfortunately, cursory 

 microscopic examination is not in itself an objective, quantitative method and can 

 therefore only be used as a means of ruling out gross contamination. For this reason, 

 the assignment of a biochemical property to a cell structure can be safely made only 

 when that property is present in the isolated preparation in a relatively high concen- 

 tration, for example, in a concentration exceeding that in the whole tissue.'' ■^•*'^* This 

 principle is obviously arbitrary and will certainly have to be abandoned when im- 

 proved methods are available. 



It should be mentioned, however, that several quantitative methods are now 

 available that in certain instances permit an estimate of the extent of cross-contam- 

 ination between cell fractions. The amount of nuclear material in a preparation can, 

 for example, be determined by the diphenylamine or other colorimetric reaction for 

 DNA. Furthermore, the number of free mitochondria in fractions isolated in sucrose 

 solutions can be estimated by a direct counting procedure consisting essentially of 

 making a suitable dilution in hypotonic (0.125 M) sucrose and identifying and count- 

 ing the swollen mitochondria in a bacterial counting chamber under the phase micro- 



" A. L. Dounce, Cancer Research 11, 562 (1951). 



« G. H. Hogeboom, and W. C. Schneider, /. Biol. Chem. 186, 417 (1950). 



