NO. 3596 ENZYME METHOD — TAYLOR 5 



Preparing Stock Solutions 



1. Saturate distilled water with sodium borate powder. Mix thor- 

 oughly and allow to settle until supernatant is clear. A large volume 

 of the solution can be mixed well in advance and used as needed. 

 When preparing a new supply, break the caked borax at the bottom 

 of the container and add sufficient new powder to insure saturation. 



2. Prepare a solution of one-half to one percent potassium hydroxide 

 in distilled water. 



3. Prepare the enzjmie buffer solution by mixing three parts of the 

 clear upper liquid from the saturated sodium borate solution (1) with 

 seven parts distilled water if the temperature of the saturated borate 

 solution is in the range of 22° to 27°C (about room temperature). 

 This concentration (30 percent saturated borax solution) is estimated 

 to be approximately 0.9 to 1.0 gram sodium borate per 100 ml. of 

 the solution at 25°C. 



If bacterial contamination takes place in the clearing process at 

 this strength and the original borate solution (1) is fuUy saturated, 

 the amount of saturated borax may be increased slightly in new solu- 

 tions. The solubility of borax in water varies materially with the 

 temperature. The following quantities of saturated borate should be 

 used in preparing enzyme buffer solutions when the saturated solu- 

 tion is at the temperature indicated, based on the saturation rate 

 ("Handbook of Chemistry and Physics"). — 10°C: use 6}^ to 7 parts 

 saturated borate solution and 3 to 3)^ parts distilled water for 10 of 

 solution; 15°C: 5 to 5K parts borate; 20°C: 3}^ to 4 parts borate; 

 30°C: 2K parts; 35°C: 2 parts; 40°C: 1 part borate in 6 of solution; 

 45°C : 1 part borate in 8 of solution. 



4. Mix the following solutions of glycerin and potassium hydroxide 

 solution (2) or distilled water: 



a. 40 percent glycerin and 60 percent potassium hydroxide. 



b. 70 percent glycerin and 30 percent potassium hydroxide. 



The Method 



In the following step-by-step process it is necessary to avoid con- 

 tamination of specimens and materials in order to curtail undesirable 

 organic growth in the solutions. Do not expose specimens to high 

 temperatures. A maximum of 30°C is recommended for most fishes. 

 Slightly higher temperatures should not damage warmblooded verte- 

 brates and those that have lived in a hot environment. 



1. KiU specimens in 10 percent formalin (about 4 percent formal- 

 dehyde) and leave in this solution for one week to 10 days. This 

 fixing solution consists of one part by volume of concentrated formal- 



