138 APPENDIX A 



Pneumococcal strains are best propagated by 

 growing an inoculum in the medium described 

 in part E-3 of this outline, supplemented with 

 ^ volume of fresh yeast extract. (Fresh yeast 

 extract is prepared by crumbling 1 pound of 

 fresh yeast in 1 liter of water, bring to a boil, 

 cooling, centrifuging, and sterile-filtering the 

 supernatant.) The culture is grown at 37°C 

 until visibly turbid; ^ volume of sterile glycerol 

 is added, and the culture is frozen at — 20°C. 

 Such frozen cultures retain their viability for 

 three months to a year. Competent cultures of 

 streptomycin-sensitive cells to be transformed 

 are grown in the same fashion; they retain 

 optimal transformability for a week or two. 



Bacteriophage must be procured from labora- 

 tories doing research on bacteriophage. Most 

 universities, medical schools, or research insti- 

 tutes could either supply the virus or else suggest 

 where it could be obtained. The virus is propa- 

 gated by addition of a sample to a culture of 

 E. coli B in logarithmic growth at a density of 

 about 10^ cells/ml. Incubation is continued 

 until the culture lyses. The resultant phage sus- 

 pension may be kept sterile by addition of a 

 drop of chloroform. (All chloroform must be 

 removed by aeration before using the virus.) 

 The concentration of particles may be deter- 

 mined by the method described in Exercise IX 

 or, more accurately, by the agar layer technique. 

 For details of this technique and other useful 

 information on bacteriophage properties and 

 handling, see M. H. Adams, Bacteriophages, 

 New York, Interscience Publishers, Inc., 1959. 



E. DETAILS OF PREPARATION* 

 1. Exercise VI 



Agar plates. Disperse 350 gm of nutrient 

 agar in 10.5 liters HoO in six 2-liter Erlenmeyer 

 flasks. Plug. Autoclave 40 min. Let flasks 

 cool to about 60°C. Pour layer equivalent to 

 30 ml into each of 300 petri dishes. Store 

 plates at room temperature. 



Broth. Dissolve 40 gm of nutrient broth and 

 24 gm NaCl in 5 liters HjO. Add a squirt of 

 antifoam. Distribute: 200 ml in each of thirteen 

 500-ml flasks, 800 ml in a 2-liter flask containing 

 a stone aerator, 300 ml in a 500-ml flask. Plug. 

 Autoclave 20 min. 



3% H2O2. Dilute 30 ml of 30% H2O2 

 (Superoxal) with 270 ml H2O. 



10% hydroxylamine. Dissolve 10 gm hy- 

 droxylamine hydrochloride in 50 ml H2O. Add 

 sufficient 10% NaOH to give pH 7. Add water 

 to give a total volume of 100 ml. 



Blood. Dilute 5 ml defibrinated horse blood 

 with 95 ml NaCl, 0.85%. Refrigerate. 



Cultures. Inoculate loopful of Senatia 

 marcescens from agar slant into 800 ml of 

 broth. Incubate at 37°C with gentle aeration 

 for 10-20 hours. Culture should be heavy. 



Transfer 3 ml with aerator to 300-ml broth 4 

 to 6 hours before class begins. Aerate at 37°C. 

 Divide up the rest of the culture into flasks to 

 be used as the "old culture." Refrigerate these 

 until class begins. Just before class, distribute 

 the "young culture" (transfer made 4-6 hours 

 earlier) in sterile fashion. 



2. Exercise VII 



Agar plates. 



plates. 



See E-1 above. Prepare 500 



For a class of 100 students. 



Broth. See E-1 above. Two 200-ml portions 

 with aerators for growing cultures of Serratia 

 marcescens and Bacillus megatherium are needed. 



Cultures. Inoculate broth with a loopful from 

 agar slants of Serratia marcescens and Bacillus 

 megatherium and grow the two cultures over- 

 night at 37°C with aeration. {Note: When 

 grown at temperatures over 30°C, Serratia 

 marcescens may not develop its characteristic 

 red pigment.) 



Alkaline methylene blue. Dissolve 1 gm 

 methylene blue in 100 ml of 95% alcohol. Add 

 300 ml 0.01% KOH. 



