Quantitation in Histochemistry 23 



( quantitative determination of potassium in tissue sections ) , 

 but further investigations will be required before the value 

 of this procedure is established. 



For such approximate estimations, model slides serve as 

 standards, and the Coujard technique o£Fers a simple ap- 

 proach. The principles of the method v^ill be illustrated by 

 describing its application to the quantitation of histochem- 

 ical reactions for enzymes.'^ 



First of all, a highly active enzyme preparation is made 

 according to one of the accepted methods, and its activity is 

 assayed accurately. Serial dilutions by a factor of 2 are made 

 with a suitable diluent, such as a 1 per cent gelatin or gum 

 acacia solution. Standard slides are prepared as follows: 

 carefully cleaned microscopic slides are coated thinly with 

 egg-white glycerol, just as for histological purposes. They 

 are subsequently heated over a Bunsen flame until com- 

 pletely dry. This pretreatment will prevent the running of 

 ink when the marks are made. With a clean steel pen, marks 

 are made on the slides, using the serial dilutions as ink. To 

 avoid confusion, the dilution fraction can be used as a mark 

 for each "ink." Every slide will carry the marks of the entire 

 dilution series. The slides are dried, fixed for a few hours in 

 alcohol or acetone, coated with thin (about 0.1 per cent) 

 collodion, and washed. A number of them are incubated, to- 

 gether with the same number of regular histological slides, 

 in the substrate solution. At intervals (e.g., 5, 10, 20, 40 

 minutes, etc. ) one tissue slide and a corresponding standard 

 are removed from the incubating mixture, and the color is 

 developed. The pair of slides in :which the tissue structure in 

 question first shows up in the shade chosen (for simple in- 

 spection, preferably black; for colorimetric measurement, 

 any shade lighter than black) is used for comparison. The 

 mark made with the lowest dilution and showing in the 

 shade chosen will have approximately the same activity per 

 unit area as the histological detail in question. It is important 



7. Gomori, G.: Exper. Cell Research, 1:33, 1950. 



