MICROSCOPY, CHEMICAL ANALYSIS, VOLUME MEASUREMENT 431 



Method of Anfinsen et al. for Microscopy and Analysis on the 

 Same Tissue Section 



In the procedure of Anfinsen et al, frozen sections are cut in the 

 cryostat of Linderstr0m-Lang and Mogensen, and the sections are 

 allowed to stand in the cryostat until dry. Faster drying can be 

 effected by the use of a dehydrating agent, with or without vacuum. 

 (With phosphorus pentoxide as the desiccant, 20 fi sections of retina 

 were completely dehydrated within 1-1.5 hr. at —20° at atmospheric 

 pressure, or within 15-20 min. in vacuo.) A nonaqueous solvent is 

 then used for mild staining to minimize displacement or solution of 

 tissue constituents. (A mixture of 1 vol. of 40 milligram per cent 

 methyl violet in absolute alcohol to 50 vol. xylol was used.) The 

 stained section is washed with xylol, transferred to a slide, and flat- 

 tened with a cover slip for visual examination or photomicrography. 

 After this the cover slip is removed, excess xylol is absorbed on filter 

 paper, and the section is allowed to dry in the air. Then the dry sec- 

 tion may be employed for chemical determination. The preceding 

 treatment was found to have no significant effect on the peptidase 

 or diphosphopyridine nucleotide in rat liver or the cholinesterase in 

 rat brain. In some instances it should be possible to examine the 

 sections directly without staining. 



C. VOLUME OF IRREGULARLY SHAPED, 

 SMALL BIOLOGICAL SAMPLES 



The measurement of volume as a means of defining the quantity 

 of a biological sample has already been discussed (page 423). Holter 

 (1945) developed a colorimetric method for measuring the volume 

 of irregular objects of the order of 0.01 to 1.0 /xl. such as the amoeba 

 Chaos chaos. The simple method of drawing an amoeba into a capil- 

 lary of known diameter, measuring its length, and computing its vol- 

 ume, cannot be used with irregular organisms which do not fill the 

 lumen of the capillary. 



In Holter's method, the object is drawn into a capillary tube of 

 known diameter which is wide enough to avoid deformation of the 

 object, and some dye solution of known concentration is taken into 

 the capillary with it. The total length of the object plus dye is meas- 



