CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 



499 



(3) Add 20.0 g. Merck's brown peptone 

 and 20.0 g. agar. 



(4) Steam (time not specified). 



(5) Cool and add the whites of 10 eggs to 

 clarify. 



(6) Resteam and filter. 



(7) Neutralize the egg albumin with HCI 

 and then render the medium alkaline 

 with XaOH. 



Sterilization: Not specified. 

 Use: General culture medium. Smith cul- 

 tivated Pseudomonas campestris (Pam- 

 mel) on the medium and reported that the 

 colonies developed moderately fast. 

 They were circular or nearly so, thin and 

 well defined margin pale yellow wet and 

 shining. Streaked cultures tended to 

 spread. In plates or streaks crystals of 

 ammonium magnesium phosphate formed 

 after several days. 



(a) Committee A. P. H. A. (1901 recom- 

 mended the following medium to be 

 used for bacterial counts in water 

 analysis. 



(1) Boil 15.0 g. thread agar in 500.0 cc. 

 water for half an hour and make 

 up weight to 500.0 g. or digest for 

 10 minutes in the autoclave at 

 110°C. Let this cool to about 

 60°C. 



(2) Infuse 500.0 g. lean meat 24 hours 

 with 500.0 cc. of distilled water in 

 refrigerator. 



(3) Make up any loss by evaporation. 



(4) Strain infusion thru cotton flannel. 



(5) Weigh filtered infusion. 



(6) Add 2.0% Witte's peptone. 



(7) Warm on water bath, stirring till 

 peptone is dissolved and not allow- 

 ing the temperature to rise above 

 60°C. 



(8) Neutralize. 



(9) To 500 .0 g . of the meat infusion add 

 500.0 cc. of the 3.0% agar, keeping 

 the temperature below 60°C. 



(10) Heat over boiling water, (or 

 steam) bath for 30 minutes. 



(11) Restore loss by evaporation. 



(12) Titrate, after boiling one minute 

 to expel carbonic acid. 



(13) Adjust reaction to -|-1.0% by add- 

 ing normal hydrochloric acid or 

 sodium hydrate as required. 



(14) Boil 2 to 5 minutes over a free 

 flame, stirring constantly. 



(15) ]\Iake up loss due to evaporation. 



(16) Filter thru absorbent cotton and 

 cotton flannel, passing the filtrate 

 thru the filter until clear. 



(17) Titrate and record the final 

 reaction. 



(18) Tube in 7.0 cc. quantities. 



(19) Sterilize 15 minutes in the auto- 

 clave at 110° or for 30 minutes in 

 streaming steam on 3 successive 

 days. 



(20) Store in the ice chest in a moist 

 atmosphere, to prevent evapo- 

 ration. 



(b) Committee A. P. H. A. (1905) recom- 

 mended the same method of prepara- 

 tion as in 1901, but sterilized the 

 medium by heating for 5 minutes at 

 120°C. or for 30 minutes in stream- 

 ing steam on 3 successive days. 



(c) Committee A. P. H. A. (1905) pre- 

 pared a basal medium as given under 

 variant (b) above and added 10.0 g. 

 of glucose or lactose just before 

 sterilization. The reaction is then 

 adjusted to neutral to phenolphthal- 

 ein. If medium is to be used in tubes 

 add sterilized azolitmin solution 

 (amount not specified) just before 

 final sterilization. If medium is to 

 be used in Petri dishes, add sterilized 

 azolitmin solution (amount not speci- 

 fied) just before the medium is to be 

 poured into the plate. 



(d) Committee A P. H. A. (1909) recom- 

 mended that the medium be adjusted 

 to 4-1 5 instead of +1.0 as in variant 

 (a). Sterilize in the autoclave for 

 one hour at 15 pounds pressure or in 

 streaming steam for 20 minutes on 

 3 successive days. To prepare lac- 

 tose or glucose litmus agar, add 1.0% 

 lactose or 1.0% glucose to the medium 

 just before sterilization. The reac- 

 tion is adjusted to neutral to phenol- 

 phthalein. Add the sterilized azo- 

 litmin to the medium just before 

 sterilization if the agar is to be used 

 in tubes. If to be used in plates, 

 add the sterilized azolitmin solution 

 to the Petri dishes when pouring 

 plates. 



