10 A MANUAL OP BACTEEIOLOGY 



5. Neutralize. If method B has been used, neutralization is 

 often unnecessary, but the reaction should be tested in every 

 case. For research work the method described in Appendix E 

 is more accurate and should be used. For student work the 



following method is satisfac- 

 tory: Add to the hot bouil- 

 lon a few cubic centimeters of 

 a normal solution of sodium 

 hydrate. Stir thoroughly with 



Fig. 7. Method of making an agar slant a glass rod. Test the reaction 



by touching the end of the 



wet stirring rod to a strip of neutral litmus paper. Continue 



the addition of small quantities of sodium hydrate solution until 



the litmus paper shows only a faint pink color. 



6. Boil for five minutes and restore water lost. 



7. Add 0.5-1.5 cc. normal hydrochloric acid solution. For 

 ordinary work 1.0 cc. acid is employed. 



8. If the precipitate in the bouillon is mealy, filter through 

 folded, moistened filter paper ; if not, the bouillon must be cooked 

 again until the precipitate becomes mealy, and then filtered. 



9. Place the bouillon in plugged tubes or flasks and sterilize 

 twenty minutes at 120° C. in the autoclave. 



Exercise 9. Preparation of Nutrient Gelatin 



1. Measure 500 cc. sterile bouillon, made according to Exer- 

 cise 8, into a clean beaker or pitcher. 



2. Add Gold Label gelatin. In cold weather a quantity equal 

 to 10 per cent of the bouillon used is ample, but in warm weather 

 15 per cent gelatin should be used. 



3. After weighing the vessel and its contents, place it in the 

 Arnold sterilizer or on a steam bath until the gelatin is melted. 



4. Neutralize as in Exercise 8 ; then boil five minutes. 



5. Filter through absorbent cotton while hot. Much clearer 

 gelatin may be obtained if the solution, before being filtered, is 

 allowed to cool somewhat and the white of an egg added, then 

 cooked again until the egg white is completely coagulated before 

 filtering. Fill test tubes to a depth of 4-5 cm. 



