Chapter 6

Isolation of Novel Large and Aggregating Bacteriophages

Philip Serwer, Shirley J. Hayes, Julie A. Thomas, Borries Demeler,
and Stephen C. Hardies

Abstract
Viruses are detected via either biological properties such as plaque formation or physical properties. The
physical properties include appearance during microscopy and DNA sequence derived from community
sequencing. The assumption is that these procedures will succeed for most, if not all, viruses. However,
we have found that some bacteriophages are in a category of viruses that are not detected by any of these
classical procedures. Given that the data already indicate viruses to be the “largest reservoir of unknown
genetic diversity on earth,” the implied expansion of this reservoir confirms the belief that the genome
project has hardly begun. The first step is to fill gaps in our knowledge of the biological diversity of
viruses, an enterprise that will also help to determine the ways in which (a) viruses have participated in
evolution and ecology and (b) viruses can be made to participate in disease control and bioremediation.
We present here the details of procedures that can be used to cultivate previously undetectable viruses
that are either comparatively large or aggregation-prone.

Key words: Agarose gel, dilute, agarose gel, structure of, bacteriophage propagation, microbial
diversity, microbial genomics.

1 Introduction

Bacteriophages are viruses that infect and often kill host bac-
teria. Bacteriophages are a major driving force in many aspects
of ecology (1) and their killing of infected host cells is usu-
ally accompanied by bursting (lysis) of the infected cells. Lysis
causes a decrease in the turbidity of a bacterial suspension. This
decrease in turbidity both was and is used to assay the pres-
ence of a bacteriophage particle (2, 3). During infection, viru-
lent bacteriophages replicate in an infection/lysis cycle that does
not stop until bacteriophage-resistant host mutants emerge. In

Martha R. J. Clokie, Andrew M. Kropinski (eds.), Bacteriophages: Methods and Protocols, Volume 1: Isolation,
Characterization, and Interactions, vol. 501, 
C 2009 Humana Press, a part of Springer Science+Business Media
DOI 10.1007/978-1-60327-164-6 6 Springerprotocols.com

55
56 Serwer et al.

contrast, lysogenic bacteriophages only sometimes kill and cause

lysis. Lysogenic bacteriophages also have an alternative life cycle
in which they suppress their own host-toxic genes and do not kill
the host cell. Instead, they co-replicate with the host, often by
physically integrating the bacteriophage genome (prophage when
integrated) with the genome of the host (4).
Lysis-based assays for bacteriophages are typically either
(a) single-plaque assays in which a supporting gel maintains a
zone of host cell lysis next to a more turbid zone of intact host
cells or (b) liquid clearing assays in which an entire liquid bacte-
rial culture loses turbidity. The uses of these two assays for isolat-
ing new bacteriophages are discussed in the excellent reviews of
Adams (2) and Carlson (3) and also in Chapters 2, 4, and 5
in this volume. We will focus here on some recent, nonclassical
variations of previous procedures. These variations appear to have
the potential for expanding the range of those viruses that can be
characterized via propagation and purification. Much room exists
for doing this given the “great plaque count anomaly” whereby
the number of viruses detected by microscopy is minimally 2–3
orders of magnitude higher than the number culturable (1, 5).
The highest bacteriophage densities are in soil and aquatic sedi-
ments (6,7), sometimes in the absence of any bacteriophages that
can be cultured with classical procedures (7).

1.1 Nonclassical In the case of isolated eukaryotic double-stranded DNA viruses,

Bacteriophage and long genomes have been found with complexity far beyond
Host Isolation: anything expected, based on what is needed for virus-specific
Long-Genome Viruses events. These events include virus DNA replication, transcription,
capsid assembly and DNA packaging. The longer genomes are
313–415 Kb long in the case of phycodnaviruses (8, 9) and
1,200 Kb in the case of mimivirus (9, 10).
The second longest viral genome is the genome of bacterio-
phage G which infects Bacillus megatherium originally reported
at 500 Kb (11), but sometimes at ∼670 Kb. Given that bacterio-
phage G was isolated over 40 years ago, one expects other compa-
rable long-genome bacteriophages to have been isolated by now.
However, that is not the case. The next longest bacteriophage
genome is, to the authors’ knowledge, the 316.675 Kb genome
(unpublished data) of Pseudomonas chlororaphis bacteriophage
201φ2-1, isolated in ref. (12) with the procedures described here.
The 201φ2-1 genome is followed by the 280 Kb genome of Pseu-
domonas aeruginosa bacteriophage φKZ (13) and then by sev-
eral genomes in the 200–280 Kb range, including the genome
of bacteriophage 0305φ8-36 (below) and the genomes of several
bacteriophages related to Escherichia coli bacteriophage T4 (14).
The shortage of long-genome bacteriophage isolations suggests
that (a) classical isolation procedures will have to be modified in
order to detect and propagate the long-genome bacteriophages
 Isolation of Bacteriophages 57

and (b) at least part of the reason for the great plaque count
anomaly is the absence of suitable isolation procedures in the past.
In fact, bacteriophage G itself had a nonclassical isolation and
also a nonclassical history of post-isolation propagation. Bacte-
riophage G originally was a contaminant discovered by electron
microscopy of a preparation of bacteriophage α (15). The α host
did not propagate G. A host for G was subsequently found by
trial and error (15). We originally received bacteriophage G via
the Fangman laboratory and have always had difficulty in grow-
ing G in liquid culture. Plate stocks in 0.7% agar gels were used
for our early studies of the physical properties of G and its genome
(16). Subsequently, at one point, we were unable to retrieve activ-
ity in agar gels from any of our stocks of bacteriophage G. We also
could not find anyone else who had an active preparation. Had we
not changed our procedures, even bacteriophage G might today
have only historical significance. The key change in procedure was
the use of dilute agarose gels for single-plaque propagation.

1.2 The Structure of The rationale for propagating G in dilute agarose gels was that
Agarose Gels increase in the radius of the gel’s effective pore (PE ) was needed to
permit greater diffusion during plaque formation. The evidence
was that 2 × PE of the classical 0.4–0.7% agar gels was compara-
ble to the size of bacteriophage G. Bacteriophage G is a myovirus
with a DNA-containing outer shell that has a diameter of
140–160 nm and a tail that has a length of 400 nm (15).
The estimate of the PE of agar gels was based on the follow-
ing background information. Agar is a mixture of related linear
polysaccharides with a backbone of agarobiose. Agar polysaccha-
rides are derivatized with negatively charged groups to an extent
that varies among different polysaccharide chains both within one
agar preparation and among the various sources of agar. Agar is
a major component of the intercellular “connective tissue” of red
algae. Agarose is derived from agar by purification of the agar
components with the least derivatization and electrical charge
[reviewed in ref. (17)]. Thus, the working assumption was that
the PE of agar gels was close to the PE of agarose gels at any given
gel concentration and conditions of gelation. According to one
set of studies in which two independent measures of PE were com-
pared (18), the 2 × PE of a 0.7% gel of the least purified agarose
is ∼260 nm [HEEO agarose in Table II of ref. (17)]. A 2×PE
of 260 nm is less than 1.8× the average diameter of the bacterio-
phage G DNA-containing outer shell and about 0.65× the length
of the G tail. Thus, restriction of G diffusion by a 0.7% agar gel is
a reasonable assumption.
This assumption was confirmed by determination of the
dependence of plaque size on gel concentration for G. The
plaques of G converted from < 1 mm to > 0.5 cm in diameter
58 Serwer et al.

as the concentration of a supporting agarose gel changed from

0.4 to 0.1% (19).
Potentially useful for the propagation of long-genome bac-
teriophages are the observations that the value of PE increased
as the temperature of gelation increased (20) and as the ionic
strength during gelation increased (21). That is to say, a very
simple practical way (tested below) to increase PE during single-
plaque propagation is to increase the temperature of the gelation
of the plaque-supporting gel, independent of the temperature of
bacteriophage propagation in this gel.

1.3 Objective and The major objective of the procedures described here is to isolate
Related Assumptions and propagate large and aggregating bacteriophages so that they
can be characterized in terms of the content and evolution of their
protein structure, and in terms of their interactions in the complex
systems in which they are found. Achieving this objective will also
provide materials for (a) understanding microbial ecology and (b)
managing bacterial growth by both phage therapy and phage pro-
phylaxis of infectious disease (22,23), among other practical appli-
cations (24,25,26). Virulent bacteriophages are preferred for ther-
apeutic purposes and they do not produce prophages with which
other bacteriophages can subsequently recombine. Therefore, vir-
ulent bacteriophages should undergo less horizontal gene trans-
fer than lysogenic bacteriophages (27). Horizontal gene transfer
interferes with establishing homology relationships (28, 29). The
most immediate objective, therefore, is to improve procedures to
find the long genome, virulent bacteriophages that we assume to
be present in environmental samples, but, somehow, are missing
in previous isolations.
The use of dilute agarose gels is the strategy presented here.
In addition to being effective for isolating long-genome bacterio-
phages, this strategy should also be effective for isolating bacte-
riophages that aggregate during plaque formation and, therefore,
behave during growth as though larger than they are. Aggre-
gating bacteriophages (and aggregating eukaryotic viruses) are
almost completely uninvestigated and may well have a major com-
ponent of the earth’s genome. The literature is curiously silent in
the area of aggregating viruses in general, although other infec-
tious agents (prions) are already known to typically be in an aggre-
gated state (30). Aggregating bacteriophages will be lost when
procedures such as filtration and centrifugal pelleting are used to
remove bacteria before assay. To the authors’ knowledge, all cur-
rently used procedures remove bacteria before either microscopy-
based or community sequencing-based assay for bacteriophages.
Bias against aggregating bacteriophages can also be present dur-
ing liquid enrichment culture, as illustrated by the observation
that the aggregating bacteriophage described below (0305φ8-
36) does not lyse liquid cultures, although it forms clear plaques.
 Isolation of Bacteriophages 59

Thus, at least some aggregating bacteriophages have been largely,

if not entirely, invisible to previous studies.

2 Materials

1. Petri plates containing 1% gelled sterile bottom agar dissolved

in a nutrient broth that contains the following: 10 g Bacto
tryptone, 5 g NaCl in 1000 ml water.
2. Molten agarose that contains the following (growth medium):
10 g Bacto tryptone, 5 g KCl in 1000 ml water with 0.002 M
CaCl2 added post-autoclaving.
3. An incubator held at 50◦ C for the long-term storage of
molten agarose.
4. A plating block with culture tubes (1.0 cm in inner diameter)
held at 50◦ C.
5. An overnight culture of host cells in growth medium. This
culture has been grown with aeration at room temperature
(25 ± 3◦ C) and then transferred to a sterile dropper bottle.
6. A solution of the fluorescent dye, DAPI (4 , 6-diamidino-2-
phenylindole) at 10 μg/ml.
7. Glass microscope slides and cover glasses.
8. Fluorescence microscope with filter set appropriate for DAPI
(U-MNU filter cube [Olympus], for example).
9. Centrifuge tube (40 ml), sterile glass pipettes and sterile
spatula.
10. Cryoprotecting solution: 10% Dextran (molecular weight =
10,000) in growth medium.
11. –70◦ C freezer.

3 Methods

Isolation of bacteriophages from environmental water typically

requires that initial propagation be started either immediately in
the field or, in any case, in a time that is less than the time for
the sample to change its microbiology. However, we have found
that isolation of bacteriophages from dry soil samples can be per-
formed months to years after taking the sample and storing it
in a laboratory cabinet. Our soil samples were dry and obtained
in Southern Texas, including the King Ranch (Kingsville, Texas),
where ground temperatures reach 49–60◦ C in the summer under
intense irradiation from the sun. Thus, soil-associated bacterio-
phage stability in the laboratory is not a surprise. The proce-
dure below produces new bacteriophages at an average rate of
60 Serwer et al.

1 bacteriophage per host, per 2–3 attempts made with 1–5 g of

soil. The soil used was not precisely quantified.
Thus, the isolations are not part of a rigorous study of soil
ecology. Considering the absence of aggregating bacteriophages
from past studies of soil microbial ecology, no rigorous and com-
plete study of soil microbial ecology has, apparently, yet been
performed.

3.1 De novo 1. Remove molten agarose solution from the oven and pipette
Bacteriophage 4 ml of molten agarose into each culture tube in the plating
Isolation block at 50◦ C. The agarose concentration is typically either
0.1% or 0.15% to increase PE and optimize the growth of both
large and aggregating bacteriophages (Notes 1 and 2).
2. Place a soil sample on the gelled agar in a Petri plate. This is
usually done with a sterile spatula, but can also be done by
pouring.
3. Place 4 drops of overnight culture in the culture tube with
4 ml molten agarose and mix.
4. Pour the host cell/molten agarose mixture over the soil sample
and rock the Petri plate gently.
5. Place the Petri plate in location for overnight incubation, usu-
ally on a laboratory bench. Once placed, Petri plates with
dilute agarose gels should not be disturbed for about 10 h
because of the fragility of the gels.
6. Incubate the Petri plate, typically for 16 h, though some bac-
teriophages have taken up to 48 h to make a visible plaque.
Moving the Petri plate even after 16 h should be done with
care if agarose gels more dilute than 0.25% are used. Other-
wise the agarose gel may slide and distort. Petri plates should
not be inverted if a gel this dilute is used.
7. Observe the Petri plate and scan for clearing within a turbid
bacterial lawn, i.e., potential bacteriophage-induced clearing.
Take a photograph. Examples are in Figs. 1a and 2a of ref.
(12). The cleared areas will be tested for the presence of bacte-
riophages by the procedure in the next section. Bacteriophages
are not the only source of clearing (Notes 3 and 4). The larger
the PE of the agarose gel, the larger the bacteriophage that can
be detected by this procedure

3.2 Single-Plaque 1. Touch a sterile needle to the surface of a cleared zone from
Propagation, Cloning, the de novo experiment of Section 3.1. Bacteriophages, if
and Storage present, adhere to the needle.
2. Stab the needle 3–10 times into the center of the bottom agar
in a new Petri plate.
3. Place 4 drops of overnight host culture in a culture tube
with 4 ml of molten agarose at 50◦ C and mix. Pour
the molten agarose near the edge of the Petri plate to
minimize uncontrolled turbulence around the inoculated
 Isolation of Bacteriophages 61

bacteriophages. Allow the molten agarose to flow over the

entire plate. The intent is to produce fluid flow that just
partially spreads the inoculated bacteriophages. The targeted
result is a single-plate dilution series. That is to say, one area of
the incubated plate is confluent and other areas have progres-
sively fewer plaques, eventually single isolated plaques that are
used for single-plaque cloning and further propagation.
4. Gently rock the Petri plate to increase the spreading of bacte-
riophage particles. This aspect has some experience-based art
in it. If rocking is too great, the spreading will be sufficient to
make the plate confluent after incubation.
5. Incubate the plate overnight at room temperature.
6. Examine the plate and photograph. If the original clear area
had bacteriophage particles, this second Petri plate should have
areas of both confluent lysis and single plaques [Figs. 1b and
2b in ref. (12)].
7. To further purify bacteriophages, steps 1–6 are repeated, but
the inoculum now becomes a single plaque from the previous
cloning. Cloning is continued until single-plaque cloning and
propagation has been accomplished three times.
8. For freezing and storage, an entire plaque is removed and
placed in a screw cap freezer vial that has cryoprotecting solu-
tion added in an approximately 10× volume. Then the vial is
placed in the –70◦ C freezer. Storage is done in quadruplicate
in at least two different freezers (For further details on storing
see Chapter 16).
9. The procedures of 1–8 are slightly modified for preparative
propagation and storage in liquid at 4◦ C (Note #5).

3.3 Screening for 1. Use a pipette to dissect a bacteriophage plaque. Place the dis-
Aggregating sected contents in a glass tube.
Bacteriophages 2. Add growth medium and dice/homogenize the contents of
the plaque. Gels of 0.1–0.2% agarose behave as a quasi-liquid
when this is done.
3. Add 0.05× amount of the DAPI solution and mix.
4. Place 2–5 ml on a glass microscope slide and place a cover glass
on the stained extract.
5. View in the fluorescence microscope [see, for example, refs.
(31, 32) and Chapter 8]. Aggregates are sometimes large
enough (multi-μm dimensions) to be obvious. The larger
aggregates sediment at 1 g and, therefore, are found at the
surface of the slide, not near the cover glass. Smaller aggre-
gates are also usually obvious based on size, fluorescence inten-
sity, and resolution of more than one particle. Bacteriophage
0305φ8-36 was over 99% aggregated when observed with this
procedure (32). Further work on characterizing bacteriophage
aggregates by fluorescence microscopy is in ref. (32). Note #6
62 Serwer et al.

describes characterization by centrifugation of more purified

0305φ8-36 aggregates.

4 Notes
1. Dilution of agar gels (to 0.3%) has also been found to be
one of several ways to increase plaque sizes (33, 34). We used
agarose, rather than agar, primarily because agarose gels were
stronger at any given concentration. Thus, concentrations as
low as 0.1% were readily accessible and we have had success
with agarose concentrations as low as 0.075%.
2. To further increase the PE of the upper layer agarose gel used
for bacteriophage propagation, a possible procedure is raising
the temperature of gelation without changing the temperature
of bacteriophage propagation (Section 1.2). This procedure
was tested for an aggregating bacteriophage, 0305φ8-36
(genome length = 218.948 Kb) by (a) pouring host cell-
containing, molten 0.1% agarose in several different Petri
plates (the host was Bacillus thuringiensis), (b) gelling the
agarose at different temperatures, (c) inoculating bacterio-
phage particles with a stab (four separate stabs per plate) and
(d) forming plaques with all plates at the same temperature, in
thermal contact with each other. The result was that the plaque
radius decreased as the gelation temperature decreased from
42◦ C (Fig. 6.1a) to 37◦ C (Fig. 6.1b), 25◦ C (Fig. 6.1c)
and 4◦ C (Fig. 6.1d). The same result was found when this
experiment was done by conventional dilution and plating.

Fig. 6.1. Effect on plaque diameter of the temperature of agarose gelation before
growth of bacteriophage 0305φ8-36. Host cell-containing molten agarose was added
to a Petri plate previously equilibrated at either (a) 42◦ C, (b) 37◦ C, (c) 25◦ C, or (d) 4◦ C.
The plates were then placed at room temperature (25 ±3◦ C) and inoculated after the
agarose gelled. The plates were then incubated in thermal contact with each other for 8
h at room temperature.
 Isolation of Bacteriophages 63

3. In addition to providing a lawn for bacteriophage growth, the

host bacteria in the molten agarose also suppress the growth
of endogenous bacteria that are in the soil sample. If problems
are encountered with endogenous bacteria overgrowing the
lawn, the first solution to try is increasing the concentration of
the host bacteria in the molten agarose.
4. As suggested in Note 3, new hosts can be isolated if the host
bacteria in the molten agarose are omitted from the proce-
dure for isolating bacteriophages. Omitting the host bacte-
ria releases repression of the growth of endogenous bacte-
ria of the soil sample. The pattern of endogenous bacterial
growth varied, though it always included bacteria eventually
growing to either confluence or near confluence. In one case,
a swarming bacterium overgrew the Petri plate to form a
lawn, except in restricted areas where colonies formed. Some
colonies were surrounded with a zone of clearing (for example,
arrow #1 in Fig. 6.2). The turbid continuous lawn (arrow #2)
was formed by a sometimes swarming B. pumilus, as deter-
mined by single-colony propagation, followed by sequencing
of the 16 s ribosomal RNA gene (12). When plated on the
B. pumilus, the zone indicated by arrow #1 in Fig. 6.2 pro-
duced a B. pumilus-clearing B. subtilis, not a bacteriophage.
The B. subtilis was also identified by single-colony propaga-
tion, followed by sequencing of the 16 s ribosomal RNA gene.
The competition between the B. pumilus and the B. subtilis
was replicated with single-colony purified strains (not shown).
The colonies indicated by arrows #3 and #4 in Fig. 6.2 were
formed by B. thuringiensis and B. megaterium, respectively.
The B. pumilus and B. subtilis strains from this plate have been
used to isolate bacteriophages.

Fig. 6.2. Isolation of hosts. The procedure for bacteriophage isolation in Section 2.1
was performed with the host cells omitted from the molten agarose. The Petri plate was
incubated for 20 h at 25◦ C and then photographed.
64 Serwer et al.

5. For growth on a preparative scale, the procedure of steps 1–

8 is repeated with the following changes: (a) The stabs used
for bacteriophage inoculation are located throughout the bot-
tom agar so that incubation and plaque formation results
in confluent lysis. (b) After incubation, the entire agarose
layer is removed with a sterile spatula and placed in a cen-
trifuge tube with growth medium for extraction of bacte-
riophage particles. After further purification of bacteriophage
particles, the optimal buffer used for short-term storage can-
not be known in advance. A good place to start is the inclu-
sion of 3% polyethylene glycol (PEG; molecular weight in the
1,500–8,000 range) to stabilize (35) the bacteriophage parti-
cles. Higher PEG concentrations may cause precipitation. In
addition, magnesium cation should be present, as found for
various bacteriophages over a period of over 50 years. Mag-
nesium cation at a concentration of 0.05 M is a good place
to start. An advantage of in-gel preparative growth is that
aerosol production is dramatically reduced in comparison to
preparative growth in liquid culture. Aerosols can create cross-
contamination problems (33).
6. To obtain an independent test of 0305φ8-36 aggregation after
purification by buoyant density in a cesium chloride density
gradient, analytical ultracentrifugation was used. Detection
was performed by SYBR green staining and use of fluores-
cence detection optics previously described (36). The resulting
profile (Fig. 6.3) has sedimentation coefficients ranging from
350 to 1200, a confirmation of aggregation. For comparison,
the same analysis is shown for bacteriophage T7 (Fig. 6.3), a

Fig. 6.3. Analysis of aggregation by analytical ultracentrifugation with fluorescence

detection. The following were subjected to centrifugation at 3000 rpm, 20◦ C in a
Beckman XLA analytical ultracentrifuge with fluorescence detection optics (36): bac-
teriophage 0305φ8-36 (filled circles) and bacteriophage T7 (empty squares). Data pro-
cessing was performed by the procedures in ref. (37).

non aggregating bacteriophage that produces a mostly homo-
geneous sedimentation pattern with an average sedimentation
coefficient of ∼450 s.
Acknowledgments
We thank the following for support: The National Institutes of
Health (GM24365 and GM069757), The Welch Foundation
(AQ-764) and The Robert J. Kleberg Jr. and Helen C. Kleberg
Foundation.
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