BACTERIA. MEDIA 



36 



BACTERIA. MEDIA 



agar and neutralize to litmus about pH 

 6.8. Mix 150 cc. defibrinated beef blood 

 and 150 cc. in 1 A'^ KOH and steam in 

 Ai-nold 30 min. Add this to blood agar 

 in proportion of 3 to 7. Pour Petri 

 plates, let harden uncovered (but pro- 

 tected by paper) placing strips sterile 

 filter paper between dish and protection 

 to take up ammonia and moisture. 

 Incubate 15 hrs. at 37°C. before use. 



Carbohydrate broth (for fermenta- 

 tion tests). Inoculate 1000 cc. infusion 

 broth with active E. coli and incubate 

 18 hrs. at 37.5°C. Boil few minutes to 

 kill organisms. Put in large mortar 

 20-30 gms. purified talc. While grind- 

 ing add broth and thoroughly mix. 

 Pass through wet filter paper till clear. 

 Titrate and adjust pH to 7.3. Weigh 

 broth and add 1% of desired ferment- 

 able substances dissolved in a little hot 

 water. Then add 45 cc. 0.04% aq. 

 bromcresol purple per liter. Sterilize 

 in Arnold 20 min. on 3 successive days, 

 or autoclave 7 lbs., 10 min. 



Lactose broth (Standard for water 

 analysis). Add 0.5% lactose to nu- 

 trient extract broth and adjust reac- 

 tion to pH 6.4-7. Autoclave 15 lbs. 

 15 min. restricting total heat exposure 

 to 30 min. 



Clark and Lubs. Dissolve 5 lbs. 

 each of peptone, de.xtrose and dipo- 

 tassium phosphate in 1000 cc. aq. dest. 

 using heat. Filter through paper, add 

 water lost, tube in 10 cc. lots, sterilize 

 in Arnold 20 min., 3 successive days. 



Bendick's saccharose peptone-water 

 (for Vibrio comma). Add 1 gm. an- 

 hydrous sodium carbonate to 1000 cc. 

 peptone solution neutralized to phenol - 

 phthalein. Boil, filter and to filtrate 

 add 5 gm. saccharose -f 5 cc. sat. phe- 

 nolphthalein in 50% alcohol. Tube 

 10 cc. lots, sterilize in Arnold 15 min., 

 3 successive days. 



Dunham's peptone solution (for indol 

 test). Dissolve 10 gms. bacto-tryp- 

 tone (Difco) + 5 gm. sodium chloride 

 in 1000 cc. aq. dest. with heat. Make 

 pH 7.6 and filter if necessary, tube 10 cc. 

 lots, autoclave 15 lbs. 15 min. 



Nitrate broth (for nitrate reduction 

 test). Dissolve 10 gms. peptone -f 1 

 gm. potassium nitrate (nitrite -free) in 

 1000 cc. aq. dest. (ammonia-free) with 

 heat. Filter through paper, tube 10 cc. 

 portions and sterilize in Arnold 20 min., 

 3 successive days. 



Bromcresol purple milk. Remove 

 cream and heat remainder in cylinder 

 in Arnold 20 min. Again skim off fat 

 and to each liter remaining add 40 

 cc. 0.04% aq. bromcresol purple. Tube 

 10 cc. lots, sterilize in Arnold, 20 min., 



3 successive days. Prove sterility by 

 incubation, 



Loefiler's (for C. diphtheriae) . Col- 

 lect beef blood in large glass vessels 

 and let clot without moving. Loosen 

 clot from wall with sterile glass rod and 

 place in refrigerator. To 3 parts clear 

 serum removed by pipette add 1 part 

 meat infusion broth containing 1% 

 glucose pH 6.8-7. Mix by stirring, in- 

 spissate on slant raising temperature 

 gently to approximately 85°C. Main- 

 tain temperature till coagulated firmly. 

 Sterilize in Arnold 20 min., 3 successive 

 days, paraifinize cotton plugs and test 

 sterility. 



Hiss' serum-water (for fermentation 

 tests). Add 3 parts aq. dest. to 1 part 

 clear serum, mix, heat in Arnold 15 min. 

 Add 1% desired carbohydrate dissolved 

 in small quantity hot aq. dest. Add 

 50 cc. 0.02% aq. bromthymol blue to 

 each 1000 cc, tube, sterilize in Arnold 

 20 min., 3 successive days and prove 

 sterility by incubation. 



Glycerol agar. Add 30 cc. pure 

 glycerol to 1000 cc. melted infusion 

 agar, adjust to pH 7.2, tube, autoclave 

 15 lbs., 15 min. and slant. 



Petroff's (for M. tuberculosis). In- 

 fuse 500 gm. beef or veal in 500 cc. 

 15% aq. glycerol. After 24 hrs. put in 

 sterile press and collect extract in 

 sterile vessel. Place washed eggs in 

 70% alcohol, 10 min. Take out with 

 sterile tongs, flame and remove con- 

 tents to sterile vessel. To 2 parts egg 

 add 1 part meat extract. Add 1% alco- 

 holic gentian violet to make final con- 

 centration 1:10,000. Mix and continue 

 as with Loeffler's medium. 



Cystine blood agar (for P. tularensis). 

 To 1000 cc. beef or veal infusion broth 

 add 15 gm. agar, 10 gm. peptone and 

 5 gm. sodium chloride. Autoclave 

 15 lbs., 15 min. Before use add 1 gm. 

 cystine (or cystine hydrochloride) and 

 10 gm. glucose. Dissolve by heating in 

 Arnold and sterilize 30 min. Cool to 

 50°C. add 50 cc. sterile horse blood, 

 tube aseptically in 10 cc. lots, slant and 

 incubate to prove sterility. 



Noguchi's leptospira medium. Com- 

 bine sterile 80°C. 0.9% aq. sodium 

 chloride, 100 cc. fresh rabbit serum, 

 100 cc. 2% aq. agar (melted pH 7.4) and 

 10-20 cc. rabbit hemoglobin (1 part 

 blood, 3 parts aq. dest.). Tube asepti- 

 cally in 10 cc. lots. Incubate to prove 

 sterility. 



Tryptone glucose extract milk agar. 

 Combine 15 gm. agar, 3 gm. beef ex- 

 tract, 5 gm. tryptone, 1 gm. glucose and 

 1000 cc. aq. dest. by boiling over free 

 flame. Make up volume lost with aq. 

 dest., adjust to pH 7, add 10 cc. skim 



