666 



CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 



extract, the sod. chloride, and the 

 calcium chloride, ^xhis addition 

 was found by trial to be necessary 

 in order to give a final medium of 

 pH = 7.0 to 7.2. Variations in the 

 mode of manufacture from that 

 here given would demand trials to 

 determine the exact amount of addi- 

 tional alkali required. ^Pound to 

 be the requisite amount by trial. 

 ^By keeping the agar at a tempera- 

 ture at which it remains fluid and 

 allowing time for sedimentation, 

 undissolved matter may be collected 

 at the extremity of the cone. This 

 extremity may be cut off and rejected 

 after the agar is allowed to solidify. 

 In practice the undissolved material 

 may be left in the agar, for it collects 

 untimately in very small amount in 

 the butt of the sloped agar medium 

 and does not at all inferfere with 

 its transparency. ^At higher tem- 

 peratures the medium is apt to 

 acquire a permanently brown colora- 

 tion. 60n this weight taken in 

 conjunction with the volume of 

 nutrient bouillon converted into 

 agar is based the calculation of the 

 amount of water which it is neces- 

 sary to add to the powder in order 

 to give the medium required for use. 

 (21) Weigh out a quantity of dried powder 

 sufficient for the purpose in hand. 



Note: This may be an amount to 

 make a single test tube of medium 

 or several tubes. If the requisite 

 amount is weighed out with pre- 

 caution into each test tube sepa- 

 rately, or roughly measured out, the 

 sterilization required, if the test 

 tubes are themselves sterile, need 

 not under ordinary circumstances 

 be more than is required to dissolve 

 the agar in the water. As an exam- 

 ple of the amount of powder required 

 we may take it that, say 1,600.0 cc. 

 of filtrate was obtained from 

 2,000.0 cc. of unfiltered bouillon and 

 that in dried powder this repre- 

 sented 128.0 g. Then, we recollect 

 that the original bouillon is double, 

 strength, it would require an addi- 

 tion of 1,600.0 cc. water to 64.0 g. of 



powder to produce a single strength 

 medium. As a percentage this 

 would be given as 4.0% of powder 

 to be added to the water. 



(22) Add the requisite amount of water. 



(23) Place in an autoclave for 30 minutes 

 at 120°C. to dissolve the powder. 



(24) Distribute the liquid nutrient agar 

 in test tubes in amounts of 7 to 

 10.0 cc. 



(25) Sterilize in the autoclave 30 minutes 

 at 120°C. or in the steamer for 

 20 minutes on three successive days. 



(26) Slope the tubes. 



References: Harvey and Iyengar (1921-22 

 p. 365), Vardon (1923-24 p. 429). 



2064. Harvey and Iyengar's Desiccated Bile 

 Salt Trypsinized Mutton Agar 



Constituents : 



1. Tryptic digest of 



mutton 1000.0 cc. 



2. Agar 60.0 g. 



3. Sodium taurocholate. 5.0 g. 



4. Neutral red (1.0%) ... 2.0 to 5.0 cc. 



5. Lactose 10.0 g. 



Preparation : 



(1) Prepare a tryptic digest of mutton. 



(2) Adjust (1) to pH = 8.0. 



(3) Cut up agar fiber into small pieces. 



(4) Add (3) to (2). (6.0% by weight.) 



(5) Autoclave at 120° for an hour to 

 melt the agar. 



(6) Filter thru cotton, wool and muslin 

 into a tin receptacle. 



(7) Cut the agar out of the receptacle 

 and into slices. 



(8) Pass slices thru meat mincing ma- 

 chine with a finely perforated out- 

 let disc. 



(9) Spread minced nutrient agar on 

 metal or other type of trays. 



(10) Dry in hot air oven or any other 

 convenient way. 



(11) Store powder in sterile glass stop- 

 pered bottle. 



(12) Dissolve 40.0 g. of (11) and 5.0 g. of 

 3 in 1000.0 cc. distilled water by 

 heating in the steamer. 



(13) Prepare fresh 1.0% solution of 

 neutral red. 



(14) Add to (12) 0.2% to 0.5% of (13) 

 and 1.0% lactose. Dissolve the lac- 

 tose by gentle heating. (Lactose 



