12 SUBCELLULAR PARTICLES 



in the salivary chromosomes of Diptera, at certain stages of development local 

 bursts of activity occur in which the DNA content increases greatly in specific 

 chromosome loci. This has been shown now by three different techniques: two- 

 wavelength microspectrophotometric analysis (36, 40) of Feulgen staining by 

 Stich and Naylor (50), ultraviolet microspectrophotometry by Rudkin and Cor- 

 lette (46), and tritium-labeled radioautography by Ficq and Pavan(i3). 



The staining reactions for enzyme activity are generally incapable of true 

 quantitation. However, they can demonstrate intracellular localization, if the 

 organelles under question are preserved, if the enzyme activity is not totally in- 

 hibited, and if the product of the enzyme reaction is trapped at its site of forma- 

 tion because it is insoluble or because the site binds it strongly. A procedure cur- 

 rently under investigation in a number of laboratories holds much promise in 

 this area. In the 'freeze-substitution' procedures, tissue is rapidly frozen at low 

 temperatures ( — 170 to — 200°C) and then fixed in nonaqueous fluids like 

 osmium tetroxide in absolute acetone at low temperature ( — 70°C). It is known 

 that structure is excellently preserved by this procedure, and it is hoped that suf- 

 ficient enzyme activity will survive to make it useful for enzyme cytochemistry 

 on the intracellular and fine structure levels. 



In the past, our work has involved fixation at more usual temperatures, 2-5 °C. 

 Figure 10 illustrates the lead method for phosphatases as developed by Wach- 

 stein and Meisel (57). In this instance, the tissue was fixed in cold formol-calcium 

 and then cut with a routine freezing microtome. The substrate is ATP. The 

 enzyme, probably an ATPase (9, ^7,), liberates phosphate ions. These are trapped 

 by lead ions in the medium. The resultant lead phosphate is converted to black 

 lead sulfide by immersing the slide in ammonium sulfide. As figure 10 shows, 

 the bile canaliculi of liver are beautifully delineated by the precipitated reaction 

 product of the enzyme. 



By using brief fixation in cold osmium tetroxide and then incubating small 

 blocks of fixed tissue in the ATPase medium. Dr. Edward Essner (9) has been 

 able to visualize the reaction products in electron micrographs. Figure 1 1 is a 

 low-power electron micrograph. It demonstrates that all the reaction product 

 is within the canaliculus. By studying an area with little to moderate enzyme 

 activity, the reaction product can be localized in the cell membrane (fig. 12). 



When a similarly fixed liver block is incubated in the same medium with 

 adenosine-5 '-monophosphate substituted for ATP, 5'-nucleotidase activity is visu- 

 alized. This enzyme is also present in the bile canaliculi, but, in addition, it is 

 present in the microvilli of the Disse space. The damage to the Disse space 

 microvilli is a bit too extensive to permit the firm conclusion that the precipitate 

 is in the plasma membrane. If it is, then we may have an interesting enzymic 

 differentiation in different parts of the cell membrane. At the absorptive surface 

 exposed to the blood, ATPase activity is not demonstrable (with this incubation 

 time) but ^'-nucleotidase is. At the secretory surface exposed to the bile canaliculi. 



