BACTERIA. MEDIA 



34 



BACTERIA. MEDIA 



titrate to pH 7.4, tube or flask, and 

 autoclave 15 lbs., 15 min. 



Meat infusion agar. Add 20 gm. agar 

 to 1000 cc. Meat infusion broth and 

 continue as in making meat extract 

 agar, pH to 7.4. 



Gelatin, Nutrient. Add 120 gm. 

 gelatin to 1000 cc. meat extract broth 

 in double boiler, weigh, dissolve by 

 heat, titrate to pH 7.4 and add aq. dest. 

 to make original weight. Add 1 egg 

 clarified by mixture with small volume 

 of aq. dest., heat slowly till egg is 

 coagulated, filter through cotton and 

 sterilize filtrate in 10 cc. portions in 

 tubes in Arnold 20 min. 3 successive 

 days. 



Huntoon's hormone. Add 500 gm. 

 fresh finely ground beef heart, 10 gm. 

 peptone, 5 gm. sodium chloride, 1 

 whole egg, 20 gm. agar (Bacto) to 1000 

 cc. aq. dest. in enamel-ware dish, heat 

 and stir constantly. Make pH 8. 

 Cover, place in Arnold 1 hr. Remove, 

 separate clot from sides and return to 

 Arnold l|-hr. Remove, let stand in- 

 clined, room temperature, 10 min. 

 Remove clear part and filter it through 

 fine wire sieve into tall cylinders. Al- 

 low to stand 15-20 min. and skim off 

 fat. Clear further by passing through 

 glass, or asbestos wool, or by centrifug- 

 ing. Tube in 10 cc. lots, sterilize in 

 Arnold 30 min. on 3 successive days. 



Glucose agar. Add 10 gm. glucose 

 to 1000 cc. meat extract or meat in- 

 fusion agar and dissolve by slowly 

 heating. Adjust pH to that of original 

 agar. Pour in tubes, or flasks, and 

 sterilize in Arnold 3 successive days. 



Blood agar. Add 5-10% of sterile 

 defibrinated blood (preferably horse) 

 to meat infusion or meat extract agar 

 which first has been melted and cooled 

 to 45°C. Pour into plates or into tubes 

 and slant, then incubate to prove 

 sterility. 



Chocolate blood agar. Add 5% of 

 sterile defibrinated blood to meat in- 

 fusion agar at 50-55°C. mix avoiding 

 bubbles, slowly increase to 75 ^C. Pour 

 into plates, or into tubes and slant, 

 then incubate to prove sterility. 



Serum agar. Add 100 cc. sterile 

 normal horse serum to 1000 cc. melted 

 meat infusion agar, pour into plates, 

 or tubes, and slant, then incubate to 

 prove sterility. 



Liver infusion agar (for Br. abortus). 

 Mix 500 gm. ground beef liver with 500 

 gm. aq. dest. in cool place 24 hrs., strain 

 through cheesecloth and collect 500 gm. 

 resulting infusion (1). Add 20 gm. agar 

 and 500 gm. aq. dest. and autoclave 

 15 lbs. pressure, 30 min. (2). Dissolve 

 10 gm. peptone and 5 gm. sodium chlo- 



ride in No. 1, beef infusion (3). Add 

 aq. dest to 2 and 3 combined to make 

 up weight lost by evaporation, adjust 

 pH to 7 and cool to 50°C. Add 10 gm. 

 egg albumin (first dissolved in 10 cc. 

 aq. dest.), heat to lOO^C. l|-hrs., strain 

 through fine wire sieve, filter through 

 clean glass wool, adjust pH to 7, tuiae 

 in 15 cc. lots and autoclave at 15 lbs., 

 30 min. When required melt and pour 

 plates, or make slants. 



Trypagar. Put 500 gm. fat free, 

 finely ground beef or veal ground in 

 1000 cc. aq. dest. in container adding 

 20% aq. NaOH until slightly alkaline to 

 litmus. Cook at 75°C., 5 min., cool to 

 37°C. and add 0.5 gm. trypsin (Bacto). 

 Incubate 37.5°C., 5 hrs. If trypaniza- 

 tion is complete 5 cc. liquid -f 5 cc. in 

 NaOH + 1 cc. dil. aq. CuS04 will give 

 pink color. If not incubate again 1 hr. 

 and re-test. When complete, slightly 

 acidifj' with glacial acetic acid, slowly 

 bring to boiling point and hold 15 min. 

 Filter through wet paper, add 20 gm. 

 agar and 5 gm. sodium chloride. Dis- 

 solve agar with heat, clear with an egg, 

 adjust to pH 7.6 and autoclave 15 lbs., 

 15 min. 



Veal infusion brain broth (for Strep- 

 tococci and anaerobes). With large 

 bore pipette insert about 50 cc. ground 

 fresh calf brain in bottom 200 x 25 mm. 

 tube and add 35 cc. veal infusion broth 

 pH 7.6. Autoclave 15 lbs., 20 min. 

 Remove 10 cc. test reaction, pH 7.4- 

 7.6 being satisfactory, if a change has 

 taken place adjust to pH 7.6 and esti- 

 mate from titration of this 10 cc. 

 amount needed to bring to this figure 

 bulk and correct the whole. Fill tubes 

 with similar amounts, then incubate at 

 37'^C. to prove sterility. 



Robertson's (for Anaerobes). To 

 500 gm. ground fat, fascia and blood 

 vessel-free fresh beef heart, add 10 gm. 

 peptone and 1000 cc. aq. dest., bring to 

 boil and adjust to pH 8. Continue sim- 

 mering l|-hrs. and again adjust reac- 

 tion. Separate broth from meat, place 

 former in flasks, autoclave 15 lbs., 15 

 min. Dry meat on filter paper in oven 

 56 ""C. 48 hrs. Place desired amounts 

 of meat plus 10 cc. broth in tubes. 

 Autoclave cool, remove broth and re- 

 titrate. Adjust to desired pH, finally 

 fill tubes same quantity meat and broth 

 and autoclave 15 lbs. 30 min. Final 

 pH should be 7.4-7.6. 



Calcium carbonate broth (for Pneu- 

 mococci). Dissolve 10 gm. glucose in 

 1000 cc. meat infusion broth by heating, 

 make pH 7.6. Place clean marble chips 

 (CaCOs) in bottom of tubes pour in 

 broth, sterilize in Arnold 15 min. 3 suc- 

 cessive days. 



