The autoradiograph is examined microscopically, using either the 

 phase-contrast or the light microscope. Frequently it is necessary to stain 

 cells in order to determine whether incorporation of the isotope has 

 occurred in a particular type of cell. Staining of the specimen is most 

 conveniently done before application of the emulsion film; however, 

 some fading of the stain may occur during the developing process. The 

 Feulgen stain works well in this respect as it is not appreciably affected 

 in the photographic processing of the autoradiograph. 



The autoradiographic technique has made a number of contributions 

 to our knowledge concerning basic cell metabolism. This is especially 

 true in those cases where autoradiography has been used in combination 

 with other cytochemical methods such as microspectrophotometry, en- 

 zyme digestion, and specific cytochemical staining (e.g., Feulgen stain- 

 ing). Autoradiographs of oocytes obtained with the use of C^^-labeled 

 glycine have shown the nucleolus to be a site of active RNA and protein 

 metabolism. This same radioisotope has been used to study rates of 

 turnover of cell populations in the circulating blood of mammals. The 

 use of tritium-labeled thymidine has made it possible to investigate 

 chromosomal DNA metabolism (see Chapter 4, Figure 4-24), cell turn- 

 over times, and DNA metabolism in specific bands of dipteran salivary 

 chromosomes during different stages of larval development (Figure 

 4-23). Tritium-labeled cytidine, a precursor of both DNA and RNA, 

 has been utilized in the study of nucleic acid metabolism in cell struc- 

 tures during regeneration. The changes in nucleic acid and protein me- 

 tabolism during meiosis in plants have been investigated using orotic 

 acid — C^^ glycine — C^*, cytidine — W, and thymidine — H^, The ra- 

 dioisotope, adenine — C^^, has also been used to follow changes in 

 nucleic acid metabolism during animal spermatogenesis. 



ISOLATION OF CELL COMPONENTS 



The techniques which have probably contributed most to our under- 

 standing of cell function are those developed by the biochemist for the 

 separation of the cell into its component parts so that they may be 

 analyzed separately to determine their specific activity or function. The 

 isolation of cell components is accomplished simply by disrupting the 

 cells of a tissue in a suitable medium (homogenization) and then spin- 

 ning down the resulting suspension or homogenate at different speeds 

 of centrifugation. As a result of differential centrifugation, cells are 

 separated into four general fractions: (1) nuclear, (2) mitochondrial, 

 (3) microsomal, and (4) soluble or supernatant (Figure 11-37 (a) 



SURVEY OF CYTOLOGICAL TECHNIQUES / 259 



