120 APPENDIX. 



GELATIN. AGAR. 



6. Add i % Witte's peptone and 10% Add 2 % of Witte's peptone. 



gold label sheet gelatin. 



7. Warm on water-bath, stirring till peptone and gela- 

 tin are dissolved and not allowing the temperature to 

 rise above 6o° C. 



8. Neutralize. 



9. To 500 gm. of the meat infusion 



add 500 cc. of the 3% agar, keeping 

 the temperature below 6o° C. 



10. Heat over boiling water (or steam) bath 30 minutes. 



11. Restore loss by evaporation. 



12. Titrate, after boiling 1 minute to expel carbonic acid. 



13. Adjust reaction to +1.0% by adding normal hydro- 

 chloric acid or sodium hydrate as required. 



14. Boil 2 minutes over free flame, constantly stirring. 



15. Make up loss by evaporation. 



16. Filter through absorbent cotton and cotton flannel, 

 passing the filtrate through the filter until clear. 



17. Titrate and record the final reaction. 



18. Tube, using 5 c.c. in each tube in the case of gelatin, 

 and 7 c.c. in the case of agar. 



ig. Sterilize 15 minutes in the autocla"v at no", or for 



30 minutes in streaming steam on three successive days. 



20. Store in the ice-chest in a moist atmosphere to pre- 



vent evaporation. 



Hill (Hill, 1899) has arranged the methods for prepa- 

 ration of broth, nutrient gelatin, and nutrient agar in 

 tabular form as given on the following page. 



For titration, the following method, suggested by 

 Fuller (Fuller, 1895), was adopted by the Public Health 

 Association Committee in 1897 (Committee of Bacteriolo- 

 gists, 1898). 



The medium to be tested, all ingredients being dis- 

 solved, is brought to the prescribed volume by the addition 

 of distilled water to replace that lost by boiling, and after 

 being thoroughly stirred, 5 c.c. are transferred to a 6-inch 

 porcelain evaporating-dish; to this 45 c.c. of distilled water 

 are added, and the 50 c.c. of fluid are boiled for three 

 minutes over a flame. One cc. of a .5 per cent solution 

 of phenolphthalein in 50 per cent alcohol is then added 



N 

 and the reaction is determined by titration with — sodium 



20 



