Chapter 16

Measurement of the Bacteriophage Inactivation Kinetics

with Purified Receptors
Andrew M. Kropinski
Abstract
Practical methods are described for studying the interaction between bacterial viruses and their surface
receptors.
Key words: Adsorption, neutralization, inactivation, receptor, T4, M13, lipopolysaccharide, outer
membrane protein, flagella, teichoic acids, pili.

1 Introduction
Bacteriophages bind to all possible cell surface receptors including pili [M13, D3112, F116] (1, 2), flagella [, SP3, PBP1]
(3, 4, 5), lipopolysaccharide (LPS) [T7, P22], surface proteins
[T1,T5, , AR1] (6), teichoic acids [SP50, 25] (7), and capsules [K29, K1F, H4489A] (8, 9, 10, 11). In certain cases, such as
T4, two receptors are used (12). These observations make phages
extremely useful tools for selecting receptor-deficient mutants,
and for characterizing strains for specific receptors. An example
of the latter would be the use of phages in typing systems.
Rather than using viable cells, receptor studies have been also
carried out with cell extracts (13), cell walls preparations (14, 15),
purified lipopolysaccharide (16,17), and complexes of outer membrane proteins with LPS (12, 15). In many cases the phages bind
irreversibly to their isolated receptor resulting in inactivation. This
can be tested in the following manner which is optimized from
our studies of phageLPS interactions (17, 18, 19, 20).
Martha R. J. Clokie, Andrew M. Kropinski (eds.), Bacteriophages: Methods and Protocols, Volume 1: Isolation,
C 2009 Humana Press, a part of Springer Science+Business Media
Characterization, and Interactions, vol. 501, 
DOI 10.1007/978-1-60327-164-6 16 Springerprotocols.com

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2 Materials
1. 1.0, 0.1, and 0.01 ml micropipetters such as Finnpipette or
Eppendorf pipetters (Fisher Scientific). These should be periodically recalibrated.
2. Sterile tips.
3. Sterile capped 13 100 mm test tubes.
4. Two sterile 125 ml Erlenmeyer flasks.
5. Heating block or waterbath set at 48 C.
6. Waterbath shaker set at the growth temperature of your bacterium.
7. Visible spectrophotometer set at 650 nm.
8. Overnight culture of your bacterium grown in medium of
choice supplemented with 110 mM CaCl2 , subcultured and
grown to mid-log phase (Note 1).
9. Phage diluted in growth medium (plus Ca2+ ) to a titer of
13 105 . Prewarm to the assay temperature just prior to
the experiment.
10. Bucket or styrofoam box containing crushed ice.
11. Agar plates and overlays (Chapter 7).

3 Methods
1. Set up a rack containing 12, 13 100 mm glass test tubes,
and add carefully 1.6 ml of distilled water or buffer to the
first tube (Note 2).
2. Add 0.9 ml of water or buffer to remainder of the tubes.
3. Number the tubes 1 through 11, and the last tube C.
4. Add 0.2 ml of LPS to the first tube so as to achieve a final
concentration of 200 g/ml. The stock solution of LPS is
therefore 1.7 mg/ml.
5. Mix, and using a new pipette tip transfer 0.9 ml from tube
1 to tube 2. Mix and continue to make doubling dilutions to tube number 11.
6. Discard 0.9 ml from tube 11.
7. Add 0.1 ml of phage preparation, diluted in broth or buffer,
to each tube so as to achieve a final titer of 3 103 pfu/ml
(Note 3).
8. Place the tubes in a waterbath or heating block at the desired
temperature.
9. After an incubation period of 1 h remove 0.1 ml from each
tube to molten overlay medium, seed with host cells and pour
onto plates.

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159

10. After appropriate incubation count and record the plague

numbers, and calculate the percentage of phage neutralized
at each concentration of LPS.
11. Plot the data on two or three cycle semilog paper (or in a
software package using a log scale) with the final concentration of LPS on the log scale and the number of plaques on
the linear scale. From this you can easily calculate the PhI50
i.e., the concentration of LPS which inactivates 50% of the
phage.

4 Notes
1. The medium that which you use to grow your bacterium and
propagate your phage. If you are starting a new project I
recommend that you use the medium recommended for the
propagating the host bacterium (see for example American
Type Culture Collection (ATCC at http://www.atcc.org/) or
Deutsche Sammlung von Mikroorganismen und Zellkulturen
(DSMZ at http://www.dsmz.de/). N.B. many phages require
110 mM divalent ions (such as Ca2+ or Mg2+ ) for optimal
adsorption and the medium should be supplemented accordingly (21, 22).
2. We noted that dilutions of LPS in broth had an inhibitory
affect on the PhI50 value.
3. This assay is optimized for phages which produce small plaques
in which accurate counts of 300 plaques per plate can be easily
made. It is far more difficult to accurately count the number
of phages, such as T7, which produce large plaques.

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