Preparation of Bacteriophage Stocks
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. Their growth is also an excellent model for how mammalian viruses infect and reproduce.
EQUIPMENT: (See bacterial growth)
sterile capped 13 x 100 mm tubes
37°C hot block
micropipettor with sterile tips
sterile bubbler assemblies
aerator with humidifier flask
manifold with spaghetti tubing
spectrophotometer
clinical tabletop centrifuge
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 10 mL repipetter
* Use LB medium for lambda phage
PROCEDURE:
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 overnight (ON) at 37°C in hot block without aeration.
NEXT MORNING:
2. PREPARE LOG-PHASE HOST CULTURE:
The next AM, using the same set up as you did for the bacterial growth curve, prepare a mid log phase culture of E. coli B by adding 0.1 mL of the ON culture to about 4 mL of tryptone soy broth, and aerate at 37°C in hot block. Aerate until it is barely turbid (A660 = about 0.100), about 1 – 1½ hours.
(NOTE: Read & graph A660 every 20 minutes during steps 2 & 3.)
2b. The mid-log culture should be barely turbid compared to uninoculated tryptone soy broth.
3. INOCULATE WITH PHAGE: Add 0.05 mL of 108/mL desired strain of phage to the mid-log culture. Aerate at 37°C until solution clears, about 1 – 2 hours. Continue reading & graphing the A660 every 20 minutes during step.(Try ON, it might work?)
4. PURIFY PHAGE PROGENY: The culture should lyse in a bout 1 to 1 1/2 hours after inoculation. Evidende of this will the a dramatic drop in the A660 in the culture.
Spin down the bacteria in the original culture tube for 10 min in a balanced clinical tabletop centrifuge at setting 5.
Decant the supernatant into screw-capped culture tube, add a few drops chloroform (~0.3 mL), shake, label with name of mutant phage, your initials and the date. Store at 4°C 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 37°C. Count plaques and calculate total phage/mL in the suspension. (For details, see protocol Titering of Phage Viruses.)
David Fankhauser 