PREPARATION OF BACTERIOPHAGE STOCKS page 66

David B. Fankhauser, PhD

17 Feb 1993, latest revision 31 Dec 1995, 3 Jan. '97, 22 July '97, 19 July 98



Bacterial viruses have proven extremely valuable in the study of the mechanisms of DNA functioning. Their short life cycle (as short as 30 minutes) combined with their large number of progeny from each cell which is lysed (up to 400 phage particles/cell) allows the production of large numbers of progeny in a short amount of time.



EQUIPMENT:

sterile capped 13 x 100 mm tubes

37C hot block

sterile pipets or

micropipettor with sterile tips

sterile bubbler assemblies

aerator with humidifier flask

manifold with spaghetti tubing

spectrophotometer

clinical tabletop centrifuge

sterile capped 16 x 150 mm tubes



SUPPLIES:

sensitive host bacteria (E. coli B)

phage inoculant (108/mL)

sterile screw capped tubes

chloroform

tryptone soy broth*

tryptone soy agar plates*

tryptone soy top agar (0.75% agar)

sterile dH2O in repipetter

* Use LB medium for lambda phage

PREVIOUS NIGHT:



1. START INITIAL HOST CULTURE: The previous day, inoculate a small volume (about 1-2 mL) of nutrient broth or tryptone soy broth with a sensitive host (E. coli B for T4 phage). Grow ON at 37C in hot block without aeration.



NEXT MORNING: (NOTE: Read & graph A660 every 30 minutes during steps 2 & 3.)



2. PREPARE LOG-PHASE HOST CULTURE: Next AM, add 0.1 mL of the ON culture to about 4 mL of tryptone soy broth, and aerate at 37C in hot block until barely turbid (about 1 - 1½ hours, A660 = about 0.100, which is just visibly turbid).

3. INOCULATE WITH PHAGE: Add 0.05 mL of 108/mL desired strain of phage. Aerate at 37C until solution clears, about 1 - 2 hours. (Try ON, it might work?)

4. PURIFY PHAGE PROGENY: Spin down the bacteria for 10 min in a balanced clinical tabletop centrifuge at setting 5. Decant supernatant into screw-capped culture tube, add a few drops chloroform (~0.3 mL), shake, label with name of mutant phage, your initials, the date. Store at 4C in the refrigerator.

5. TITER THE PHAGE CULTURE: To titer, dilute 106, plate out 0.1 and 0.01 on tryptone agar using E. coli B as indicator bacteria, incubate ON at 37C. Count plaques and calculate total phage/mL in the suspension. (See protocol Titering of Phage Viruses.)