TITERING OF BACTERIAL VIRUSES

©David B. Fankhauser, Ph.D.,
Professor of Biology and Chemistry
             University of Cincinnati Clermont College, 
Batavia OH 45103
equipment to
dilution phage
stock

This page has been accessed Counter times since day month year. 
17 Feb 1993, rvsd 31 Dec '95, 23 July '96, 22 July '97, 12 Aug 02, 11 Aug 03
pour seeded top agar with
aliquot of phage over
a prewarmed plate.

Related protocols:    Preparation of Phage Stocks
                               Commonly Used Media for Phage Growth
                               Agar Overlay Technique

 When an individual bacterial virus grows in a bacterial host suspended in a top agar lawn, its progeny infect and lyse the surrounding host cells.  This causes the appearance of a "hole" or plaque in the otherwise homogeneous bacterial lawn. Since each plaque represents a single virus, the number of viruses in the aliquot added to the plate is equal to the number of plaques which appear.

Here is the experimental plan:
EQUIPMENT: SUPPLIES: 
sterile capped 16x150 mm test tubes
sterile capped 13x100 mm tubes
sterile pipettes, 0.1 mL and 1.0 mL
hot block, 45°C
vortex
Bunsen Burner
37°C incubator melted top agar, about 60°C 
phage culture to be titered
sensitive host bacteria grown ON in TSB
 (such as E. coli B)
sterile dH2O in 10.0 mL repipet
pre-warmed to 37°C tryptone soy agar plates
 (or LB agar plates for lambda phage)
 

 
PROTOCOL:
 

THE PREVIOUS NIGHT:
1. Inoculate about 3-4 mL (for thirty plates) of nutrient broth or tryptone soy broth with a sensitive host (E.g.: E. coli B).  Grow ON at 37°C in hot block.  (Aeration is not mandatory.)
THE DAY OF ASSAY: 
Equipment for dilutions:   10 uL phage stock into 10 mL
              
2. Prepare a dilution of the phage such that there are about 103 particles per mL (usually a dilution of 106: 10 uL into 10.0 mL, repeat a second time).
Add 0.1 mL down the side   The bacteria are on top:
            
3. Set up seeded top agar:
  a. Pipet about 2 mL of melted agar into sterile capped 13x100 mm tubes in 45°C hot block.
  b. Pipet about 0.1 mL host bacteria (E. coli B) using a 1.0 mL pipet into melted agar (down the side of the tube is OK).
  4. Add phage: Pipet 10 uL or 100 uL of 10-6 phage into the host-inoculated tube (deliver with care, just below surface of agar).
5. Vortex to mix.
6. Pour out and distribute over a pre-warmed agar plate, immediately tilt back and forth to evenly distribute before it begins to gel. Let sit undisturbed until gelled.
6. When gelled (1-2 minutes), invert plates, incubate ON at 37°C.
THE NEXT DAY:
7. Count all plaques, record data, calculate phage particles/mL in the original culture.

To do the calculation of number of phage particles per mL, apply the following formula:

 phage/mL  = (number plaques/plate) x (1/mL plated) x dilution factor

For example, if you got 347 plaques when you plated out 0.1 mL of a 106 diluted phage suspension,
the titer is 3.47 x 109/mL.



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