Cultivation and

Enumeration
of Bacteriophages

EXPER IME NT

39

LEARNING OBJECTIVE
Once you have completed this experiment,
you should be able to
1. Perform techniques for cultivation and
enumeration of bacteriophages.

Principle
This exercise demonstrates the ability of viruses
to replicate inside a susceptible host cell. For
this purpose, you will be provided with a virulent
phage and a susceptible host cell culture. This
technique also enables you to enumerate phage
particles on the basis of plaque formation in a
solid agar medium. Plaques are clear areas in an
agar medium previously seeded with a diluted
phage sample and a host cell culture. Each
plaque represents the lysis of a phage-infected
bacterial cell.
The procedure requires the use of a doublelayered culture technique in which the hard agar
serves as a base layer, and a mixture of phage and
host cells in a soft agar forms the upper overlay.
Susceptible Escherichia coli cells multiply rapidly
and produce a lawn of conuent growth on the
medium. When one phage particle adsorbs to a
susceptible cell, penetrates the cell, replicates, and
goes on to lyse other host cells, the destroyed cells
produce a single plaque in the bacterial lawn (see
Figure 39.1). Each plaque can be designated as a
plaque-forming unit (PFU) and used to quantitate the number of infective phage particles in the
culture.
The number of phage particles contained in
the original stock phage culture is determined by

Figure 39.1 Plaque-forming units (PFUs)

counting the number of plaques formed on the

seeded agar plate and multiplying this by the dilution factor. For a valid phage count, the number
of plaques per plate should not exceed 300 nor be
less than 30.
Example: 200 PFUs are counted in a 10-6
dilution.
(200) * (106) = 200 * 106 or 2 * 108
PFUs per ml of stock phage culture

Plates showing greater than 300 PFUs are

too numerous to count (TNTC); plates showing fewer than 30 PFUs are too few to count
(TFTC).

265

C L I N I C A L A P P L I C AT I O N
Identication of Pathogenic Bacteria
Bacterial viruses (bacteriophages) are very common in all natural environments and are directly
related to the number of bacteria present. They
are most prevalent in soil, intestines of animals,
sewage, and seawater. These viral particles have
played an important role in the development of all
types of viruses. Since many phages are specic
about which bacteria they attack, a process called
phage typing is used in clinical and diagnostic
laboratories for the identication of pathogenic
bacteria.

AT THE B E N C H

Materials
Cultures
24-hour nutrient broth cultures of Escherichia
coli B and T2 coliphage.

Media
Five each of the following per designated student
group: tryptone agar plates and tryptone soft agar,
2 ml per tube; and nine tryptone broth tubes, 9 ml
per tube.

Equipment
Bunsen burner, waterbath, thermometer, 1-ml
sterile pipettes, sterile Pasteur pipettes, mechanical pipetting device, test tube rack, and glassware
marking pencil.

Procedure Lab One

To perform the dilution procedure as illustrated in
Figure 39.2, do the following:
1. Label all dilution tubes and media as follows:
a. Five tryptone soft agar tubes: 10-5, 10-6,
10-7, 10-8, 10-9.

266

Experiment 39

b. Five tryptone hard agar plates: 10-5, 10-6,

10-7, 10-8, 10-9.
c. Nine tryptone broth tubes: 10-1 through
10-9.
2. Place the ve labeled soft tryptone agar tubes
into a waterbath. Water should be of a depth
just slightly above that of the agar in the tubes.
Bring the waterbath to 100C to melt the agar.
Cool and maintain the melted agar at 45C.
3. With 1-ml pipettes, aseptically perform a 10fold serial dilution of the provided phage culture using the nine 9-ml tubes of tryptone.
4. To the tryptone soft agar tube labeled 10-5,
aseptically add two drops of the E. coli B culture with a Pasteur pipette and 0.1 ml of the
10-4 tryptone broth phage dilution. Rapidly
mix by rotating the tube between the palms
of your hands and pour the contents over the
hard tryptone agar plate labeled 10-5, thereby
forming a double-layered plate culture preparation. Swirl the plate gently and allow to
harden.
5. Using separate Pasteur pipettes and 1-ml
sterile pipettes, repeat Step 4 for the tryptone broth phage dilution tubes labeled 10-5
through 10-8 to effect the 10-6 through 10-9
tryptone soft agar overlays.
6. Following solidication of the soft agar overlay, incubate all plate cultures in an inverted
position for 24 hours at 37C.

Procedure Lab Two

1. Observe all plates for the presence of plaqueforming units that develop on the bacterial
lawn.
2. Count the number of PFUs in the range of
30 to 300 on each plate.
3. Calculate the number of phage particles per
ml of the stock phage culture based on your
PFU count.
4. Record your results in the chart in the Lab
Report.

PROCEDURE
Perform
a 10-fold
serial dilution.

1 ml

Phage
stock

1 ml

101

1 ml

102

1 ml

103

1 ml

104

1 ml

105

1 ml

106

1 ml

107

1 ml

108

109

Tryptone broth tubes (9 ml each)

0.1 ml

0.1 ml

0.1 ml

0.1 ml

0.1 ml

Add two drops

of E. coli B culture
to each tube.
10

10

10

10

10

Tryptone soft-agar tubes

Mix and pour.
Overlay of tryptone soft agar
Tryptone hard agar

105

106

107

108

109

Figure 39.2 Dilution procedure for cultivation and enumeration of

bacteriophages

Experiment 39

267

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EXPER IME NT

39

Name:
Date:

Lab Report

Section:

Observations and Results

Phage Dilution

Number of PFUs

Calculation:
PFUs : Dilution Factor

PFUs/ml of Stock
Phage Culture

10-5
10-6
10-7
10-8
10-9

Review Questions
1. Discuss the effects of lytic and lysogenic infections on the life cycle of the
host cell.

2. Discuss the factors responsible for the transformation of a lysogenic infection to one that is lytic.

3. Distinguish between the replicative and maturation stages of a lytic phage

infection.

Experiment 39: Lab Report

269

4. In this experimental procedure, why is it important to use a hard agar with a

soft agar overlay technique to demonstrate plaque formation?

5. Explain what is meant by plaque-forming units.

6. Determine the number of PFUs per ml in a 10-9 dilution of a phage culture

that shows 204 PFUs in the agar lawn.

7.

270

The release of phage particles from the host bacterium always occurs by lysis of the cell and results in the death of the host. Animal
viruses are released by either the lysis of the host cell or exocytosis, a reverse pinocytosis. Regardless of the mechanism of release, most infected
cells die, while other viruses may escape the cell without damaging the host
cell. Explain.

Experiment 39: Lab Report

EXPER IME NT

Isolation of Coliphages
from Raw Sewage

LEARNING OBJECTIVE
Once you have completed this experiment,
you should be able to

40

(T2, T4, and T6) differ from the T-odd phages in

that the former vary in size, form, and chemical
composition. All of the T phages are capable of infecting the susceptible E. coli B host cell.

1. Isolate virulent coliphages from sewage.

C L I N I C A L A P P L I C AT I O N
Phage Therapy

Principle
Isolates of bacterial viruses (bacteriophages) can
be obtained from a variety of natural sources, including soil, intestinal contents, raw sewage, and
some insects such as cockroaches and ies. Their
isolation from these environments is not an easy
task because the phage particles are usually present in low concentrations. Therefore, isolation
requires a series of steps:
1. Collection of the phage-containing sample at
its source.
2. Addition of an enriched susceptible host cell
culture to the sample to increase the number
of phage particles for subsequent isolation.
3. Following incubation, centrifugation of the
enriched sample for the removal of gross
particles.
4. Filtration of the supernatant liquid through a
bacteria-retaining membrane lter.
5. Inoculation of the bacteria-free ltrate onto a
lawn of susceptible host cells grown on a soft
agar plate medium.
6. Incubation and observation of the culture for
the presence of phage particles, which is indicated by plaque formation in the bacterial
lawn.
In the following experiment, you will use this
procedure, as illustrated in Figure 40.1, for the
isolation of Escherichia coli phage particles from
raw sewage. Most bacteriophages that infect
E. coli (coliphages) are designated by the letter T,
indicating types. Seven types have been identied
and are labeled T1 through T7. The T-even phages

Phage therapy is the therapeutic use of bacteriophages to treat pathogenic bacterial infections. It is
mainly used in Russia and the Republic of Georgia
and is not universally approved elsewhere. In the
West, no phage therapies are authorized for use on
humans, although phages for killing food poisoning
bacteria (Listeria) are now in use. They may also be
used as a possible therapy against many strains of
drug-resistant bacteria.

AT T HE BE NCH

Materials
Cultures
Lab One: 5-ml 24-hour broth cultures of E. coli
B and 45-ml samples of fresh sewage collected
in screw-capped bottles. Lab Two: 10-ml 24-hour
broth cultures of E. coli B.

Media
Per designated student group: Lab One: One 5-ml
tube of bacteriophage nutrient broth, 10 times
normal concentration. Lab Two: Five tryptone agar
plates and ve 3-ml tubes of tryptone soft agar.

Equipment
Lab One: Sterile 250-ml Erlenmeyer ask and
stopper. Lab Two: Sterile membrane lter apparatus, sterile 125-ml Erlenmeyer ask and stopper,
125-ml ask, 1000-ml beaker, centrifuge, Bunsen

271

PROCEDURE

Pour in several
centrifuge tubes
and centrifuge

Remove tubes
and decant
supernatant
into a 125-ml beaker.

sewage sample
at 2500 rpm
for 20 minutes.

Filter supernatant
through membrane
filter into vacuum flask.

E. coli B enriched sewage

sample in 250-ml flask

Flask and membrane

filter apparatus

Centrifuge

Bacteria-free phage filtrate in vacuum flask

1 drop

2 drops

3 drops

4 drops

5 drops

Add 0.1 ml
to tubes 15
of molten
soft tryptone
agar.

E. coli B
culture

3
Plates of hard tryptone agar

Figure 40.1 Procedure for isolation of coliphages from raw sewage

272

Experiment 40

burner, forceps, 1-ml sterile disposable pipettes,

sterile Pasteur pipette, mechanical pipetting device, test tube rack, and glassware marking pencil.

Procedure Lab One

Use disposable gloves. It is essential to
handle raw sewage with extreme caution because
it may serve as a vehicle for the transmission of
human pathogens.

Enrichment of Sewage Sample

1. Aseptically add 5 ml of bacteriophage nutrient broth, 5 ml of the E. coli B broth culture,
and 45 ml of the raw sewage sample to an appropriately labeled sterile 250-ml Erlenmeyer
ask.
2. Incubate the culture for 24 hours at 37C.

Procedure Lab Two

3. Pour the supernatant solution through a sterile membrane lter apparatus to collect the
bacteria-free, phage-containing ltrate in the
vacuum ask below. Refer to Experiment 50
for the procedure in assembling the lter
membrane apparatus.
4. Melt the soft tryptone agar by placing the ve
tubes in a boiling waterbath and cool to 45C.
5. Label the ve tryptone agar plates and the
ve tryptone agar tubes 1, 2, 3, 4, and 5,
respectively.
6. Using a sterile 1-ml pipette, aseptically add
0.1 ml of the E. coli B culture to all the molten
soft-agar tubes.
7. Using a sterile Pasteur pipette, aseptically add
1, 2, 3, 4, and 5 drops of the ltrate to the respectively labeled molten soft-agar tubes. Mix
and pour each tube of soft agar into its appropriately labeled agar plate.
8. Allow agar to harden.
9. Incubate all the plates in an inverted position
for 24 hours at 37C.

Filtration and Seeding

Procedure Lab Three

1. Following incubation, pour the phage-infected

culture into a 100-ml centrifuge bottle or
several centrifuge tubes and centrifuge at
2500 rpm for 20 minutes.
2. Remove the centrifuge bottle or tubes, being
careful not to stir up the sediment, and carefully decant the supernatant into a 125-ml
beaker.

1. Examine all the culture plates for plaque formation, which is indicative of the presence of
coliphages in the culture.
2. Indicate the presence (+) or absence (-) of
plaques in each of the cultures in the chart in
the Lab Report.

Experiment 40

273

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EXPER IME NT

40

Name:
Date:

Lab Report

Section:

Observations and Results

Drops of Phage Filtrate

Plaque Formation
(+) or (-)

Based on your observations, what is the relationship between the number of

plaques observed and the number of drops of ltrate in each culture?

Review Questions
1. Why is enrichment of the sewage sample necessary for the isolation of
phage?

2. How is enrichment of the sewage sample accomplished?

Experiment 40: Lab Report

275

3. How are bacteria-free phage particles obtained?

4.

276

Why must you exercise caution when handling raw sewage

samples?

Experiment 40: Lab Report