BACTERIA. MEDIA 



30 



BACTERIA. MEDIA 



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 1^-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. 



Blood culture (Kracke). Add 500 

 gm. finely ground fat-free beef heart 

 muscle to 1000 cc. aq. dest. in ice box 

 over night. Press through 4 layers 

 gauze cloth, heat extract to boiling, 

 filter through small mesh wire gauze. 

 Add 250 gm. ground beef brain to 500 

 cc, treat in same way but do not filter 

 this suspension. Mix 800 cc. extract, 

 110 cc. suspension, 1 gm. sodium citrate, 

 10 gm. dextrose (Bacto), 10 gm. pro- 

 teose peptone (Difco), 2 gm. disodium 

 phosphate and 4 gm. sodium chloride 

 and place 50 cc. lots in tubes or flasks. 

 Autoclave 15 lbs., 20 min. 



Bile (For typhoid group). Combine 

 900 cc. ox bile, 100 cc. glycerol and 20 

 gm. peptone by heating over water 

 bath. Pour in bottles or small flasks 

 and autoclave. 



Brilliant green lactose bile. Dissolve 

 10 gm. peptone and 10 gm. lactose in 

 500 cc. aq. dest. add 200 cc. fresh ox bile, 

 or 20 gm. dehydrated ox bile dissolved 

 in 200 cc. aq. dest., the latter having 

 pH 7.4. Add 13.3 cc. 0.1% aq. brilliant 

 green and aq. dest. to make 1000 cc. 

 Filter through cotton, place in fer- 

 mentation tubes, sterilize after which 

 pH by potentiometer (not colorimeter) 

 should be 7.1-7.4. 



Le vine's eosin methylene blue agar 

 (Standard for water analysis). Dis- 

 solve 10 gm. peptone, 2 gm. K2HPO4, 

 and 15 gm. agar in 1000 cc. aq. dest. by 

 boiling. Add aq. dest. to compensate 

 for evaporation and distribute meas- 

 ured amounts in flasks. Immediately 

 before use to each 100 cc. add 5 cc. 20% 

 aq. lactose (sterile), 2 cc. 2% aq. eosin 



and 2 cc. 0.5% aq. methylene blue. 

 Mix, pour into Petri plates, harden and 

 incubate to prove sterility. 



Endo's (Standard for water analysis). 

 Add 5 gm. beef extract, 10 gm. peptone 

 and 30 gm. agar to 1000 cc. aq. dest. in 

 container and weigh. Boil till dis- 

 solved, restore lost weight with aq. 

 dest., place in vessel with straight walls 

 and autoclave 15 lbs., 15 min. Let agar 

 harden, remove en masse to clean paper, 

 cut away and discard debris from bot- 

 tom. Melt clean agar, make pH 7.8- 

 8.2, pour in 100 cc. or larger amounts 

 and autoclave 15 lbs., 15 min. To each 

 100 cc. of this stock agar add 5 cc. 20% 

 aq. C.P. lactose (sterilized by fractional 

 method), 0.5 cc. 10% basic fuchsin in 

 95% alcohol (from filtrate of super- 

 natant fluid having let stand 24 hrs.). 

 Mix carefully, pour into sterile Petri 

 dishes, let agar set at room tempera- 

 ture and harden over night in incubator. 

 Check sterility. 



Agar, sodium desoxycholate. Dis- 

 solve 10 gm. peptone in 1 Kg. water, 

 bring to pH 7.3-7.5 with sodium hy- 

 droxide, boil few minutes and pass 

 through filter paper. Add 12-17 gm. 

 agar. After soaking 15 min,, melt by 

 boiling. To each 1000 cc. add 6 cc. 

 1 N sodium hydroxide plus ferric am- 

 monium citrate, 2 gm. dipotassium 

 phosphate and 1 gm. sodium desoxy- 

 cholate. Titrate with phenol red in- 

 dicator to pH 7.3-7.5 and add 3 cc. 

 1% aq. neutral red. Sterilize in flowing 

 steam only sufficient to kill vegetable 

 cells (15 min. enough for tubes with 

 10-15 cc. medium). 



Selenite-F enrichment. Use mono- 

 sodium and disodium phosphates in 

 exact proportions which experiment 

 shows that with particular lot of pep- 

 tone and brand of sodium selenite will 

 give pH 7.0-7.1. Dissolve with heat 

 10 gm. these phosphates (anhydrous), 

 4 gm. this sodium hydrogen selenite 

 (anhydrous), 5 gm. peptone, 4 gm. lac- 

 tose in aq. dest. to make 1 Kg. Boil. 



Russell's double sugar agar. Mix 

 1000 cc. melted meat extract agar, 40 cc. 

 25% aq. lactose (sterile) and 4 cc. 25% 

 aq. glucose (sterile) and adjust to pH 

 7.2. Add 50 cc. 0.02% aq. phenol red, 

 filter if necessary, tube and autoclave 

 8 lbs., 25 min. Slant with deep butt. 

 Check reaction of medium with known 

 E. coli and E. iyphosa. 



Simmons' citrate agar. Dissolve 5 

 gm. sodium chloride, 0.2 gm. MgS04, 

 1.0 gm. (NH4)H2P04, 2.28 gm. sodium 

 citrate (2H2O) in 1000 cc. aq. dest. and 

 add 20 gm. agar. Heat to dissolve 

 agar, make pH 7.2, and add 10 cc. 1.5% 

 alcoholic bromthymol blue. Filter 



