302 TITRIMETRIC METHODS 



2. Place 50 fA. of the metaphosphoric acid soln. in a 250 /xl. tube 

 and add the sample (care must be exercised to prevent oxidation of 

 the material prior to extraction with the acid). For work on tissue 

 sections, the practice has been to freeze the tissue solid immediately 

 upon removal from the source, and fresh-frozen sections (20-40 /x 

 thick, 4-5 mm. diameter) are prepared and transferred individually 

 as they are cut to separate tubes containing the acid extractant. If 

 titration cannot be made at once, as in the case of serial-section 

 titrations, the tubes may be kept in a freezing mixture until time for 

 titration. 



3. Titrate with the standardized dye soln. using a microburette 

 in which the mercury does not come into contact with the dye soln. 

 The end point is reached when the color of the imknown persisting 

 for 5 sec. matches that of the color standard placed beside it. Employ 

 active stirring throughout the titration. 



AMYLASE 



The Linderstr0m-Lang and Holter (1933a) method (page 296) 

 for the determination of reducing sugars was used by Linderstr0m- 

 Lang and Engel ( 1937) for the measurement of amylase activity in 

 tissue sections of barley. Holter and Doyle (1938) employed the 

 method for studies of amylase in protozoa. 



In the barley work the enzyme was extracted with M/15 phos- 

 phate buffer (pH 5.3) and 7-8 ix\. extract was added to a similar vol- 

 ume of substrate solution consisting of 1.5% soluble starch in phos- 

 phate buffer of the same pH. After an appropriate digestion period 

 at 40°, the increase in reducing sugar was estimated. In much the 

 same manner the protozoan enzyme was measured. The phosphate 

 buffer (pH 6.0) extract was added to a 2% starch substrate con- 

 taining 2 % sodium chloride. 



The relative proportions of enzyme extract and substrate solution, 

 the length of the digestion period, and the pH of the buffer must be 

 dictated by the requirements of the particular case. 



PROTEOLYTIC ENZYMES 



Titrimetric methods for the determination of proteolytic enzymes 

 in small samples of biological material, such as microtome sections 



