PREPARATION FOR MICROBIOLOGICAL EXPERIMENTS 139 



3. Exercise VIII 



Blood agar streptomycin plates. Prepare four 



l-liter batches in 2-liter flasks. Disperse 35 gm 



tryptose blood agar in 1 liter H2O. Autoclave 



30 min at 120°C. Cool to SOX. Add, per flask: 



5 ml sucrose, 20Tc, sterilized by heating 



15 min in boiling water, 

 5 ml streptomycin sulfate, 10 mg/ml, 

 sterilized by filtration, 

 20 ml sterile horse blood. 



Pour 120 plates, each containing about 30 ml. 



Growth medium for streptomycin-resistant 

 cells. Prepare three 1.5-liter batches in 2-liter 

 flasks. To 1.5 hters H2O add 



10 gm brain-heart infusion, 

 10 gm Difco yeast extract, 

 10 gm Casamino acids, 

 10 gm tryptone, 

 3 gm glucose. 



Adjust pH to about 7.5 with 10% NaOH. 

 Autoclave. 



Pneumococcal medium for competent strepto- 

 mycin-sensitive cells — basal. Dissolve : 



36 mg tryptophan, 

 200 mg cycteine-HCl, 

 12 gm sodium acetate, 

 30 gm Casamino acids, 

 51 gm K2HPO4 



in 6 liters H2O. Distribute: 200 ml in each of 

 sixteen 500-ml flasks, and 900 ml in each of 

 three 2-liter flasks. Plug. Autoclave 15 min. 

 Store at room temperature. 



Addition mix. Dissolve in 200 ml H2O the 

 following substances (this will make sufficient 

 mix for 4 liters of basal medium): 

 2gmMgCl2-6H20, 

 10 mg CaClo, 



100MgMnsd4-4H2O, 



0.8 ng biotin, 



0.8 mg nicotinic acid, 



0.8 mg pyridoxine-HCl 



0.8 mg thiamine- HCl, 



0.4 mg riboflavin. 



2.4 mg calcium pantothenate, 



2MgFeS04-7H20, 



2MgCuS04-5H20, 



2/igZnS04-7H20, 

 20 mg choline, 

 40 mg glutamine, 

 200 mg asparagine, 

 20 mg adenine, 



2 gm serum albumin (Armour Fraction V). 



Adjust pH to 7. Sterilize by filtration. 



Citrate-saline. Dissolve 6 gm NaCl and 20 

 gm sodium citrate in 700 ml H2O. 



Deoxycholate solution. Dissolve 5 gm deoxy- 

 cholic acid in 80 ml H2O by addition of 10% 

 NaOH to bring pH to about 7.5. Add water to 

 bring to 100 ml. 



Growth of streptomycin-resistant cells. Inocu- 

 late 2 drops of thawed culture of SR into each 

 of three flasks. Incubate without aeration for 

 15 hours at 37°C or until growth is maximal. 

 Refrigerate until 1-2 hours before class. Cen- 

 trifuge cells. Resuspend in 700 ml citrate-saline. 

 (If growth has been poor, resuspend in less 

 volume.) 



(Note: All cultures should be examined micro- 

 scopically in order to determine that they are 

 not grossly contaminated. It may be advisable 

 to grow cultures in duplicate to allow for those 

 discarded because of contamination.) 



Growth of competent streptomycin-resistant 

 cells. Add 5 ml addition mix and 1 ml auto- 

 claved 20% glucose to every 100 ml of basal 

 medium to form the complete medium. Inocu- 

 late with a drop or two of the frozen culture of 

 streptomycin-resistant cells and incubate as 

 above. Dispense convenient volumes into 

 sterile tubes for use in class. These must be 

 kept in ice. 



4. Exercise IX 



E. co// culture. Inoculate loopful of E. coli B 

 from agar slant into 20 ml nutrient broth. 

 Aerate at 37°C overnight. Inoculate 10 ml of 



