colloidal mill. The nuclei are isolated by repeated centrifugation, either 

 in a mixture of known density or in a density gradient of organic sub- 

 stances such as ether and chloroform, which are immiscible with water. 

 While this method is more time-consuming and gives a lower yield of 

 nuclei than more conventional methods, it has the distinct advantage of 

 permitting isolation without excessive loss of water-soluble substances. 

 The main disadvantages of the method are the possible inactivation of 

 certain enzymes as the result of freezing and the loss of lipid-soluble 

 substances because of the use of lipid solvents during homogenization 

 and centrifugation. 



The advent of the electron microscope has made it possible to study 

 the fine structure of the various subcellular fractions and to correlate 

 this information with the biochemical activities known to be associated 

 with each of these fractions. A number of important correlations have 

 been made using this kind of approach. For example, the microsomal 

 fraction has been identified with certain membranes and granules of the 

 endoplasmic reticulum (see Chapter 3, Figure 3-26), and the enzymes 

 concerned with electron transport and oxidative phosphorylation have 

 been found associated with the membranes of the mitochrondrion, es- 

 pecially the cristae. 



MICROMANOMETRIC METHODS 



While autoradiography, microspectrophotometry, and specific cyto- 

 chemical staining are the most widely used and sensitive of the micro- 

 methods commonly employed by the cytologist for the study of cell 

 function, they are by no means the only ones available for the analysis 

 of metabolic activity in the intact cell. A micromethod which has had 

 only limited use in the past but which is highly quantitative, is the 

 manometric analysis of gas volume changes caused by single cells. The 

 micromanometric methods found most useful because of their high sen- 

 sitivity are: (1) the capillary respirometer method and (2) the Cartesian 

 diver method. Both of these techniques permit the measurement of gas 

 volume changes as small as 0.1 u\ per hour. 



The capillary respirometer used by Brachet (1949) to measure the 

 oxygen consumption of a single frog egg is shown in Figure 11-38. This 

 instrument consists of a chamber to hold the specimen and a capillary 

 which carries an index drop and is attached to the chamber. The gas 

 volume is measured with a microscope by observation of the changes 

 in level of the index drop. The detail of the Cartesian diver is shown 

 in Figure 11-39. It consists essentially of a container in which there is 



SURVEY OF CYTOLOGICAL TECHNIQUES / 261 



