virology_ isolation & cultivation of bacgteriophage

8/17/2019 Virology_ Isolation & Cultivation of Bacgteriophage

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counting the number of plaques formed on the

seeded agar plate and multiplying this by the dilu-tion 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 show-

ing fewer than 30 PFUs are too few to count(TFTC).

PrincipleThis 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-infectedbacterial cell.

The procedure requires the use of a double-

layered 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 confluent 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 (seeFigure 39.1). Each plaque can be designated as aplaque-forming unit (PFU) and used to quanti-

tate the number of infective phage particles in the

culture.

The number of phage particles contained in

the original stock phage culture is determined by

Cultivation andEnumerationof Bacteriophages

EXPERIMENT

39

LEARNING OBJECTIVE

Once you have completed this experiment,

you should be able to

1. Perform techniques for cultivation and

enumeration of bacteriophages.

Figure 39.1 Plaque-forming units (PFUs)


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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 five 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 100°C to melt the agar.

Cool and maintain the melted agar at 45°C.

3. With 1-ml pipettes, aseptically perform a 10-

fold serial dilution of the provided phage cul-

ture 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 cul-

ture 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 prepa-

ration. Swirl the plate gently and allow to

harden.

5. Using separate Pasteur pipettes and 1-ml

sterile pipettes, repeat Step 4 for the tryp-

tone broth phage dilution tubes labeled 10-5

through 10-8 to effect the 10-6 through 10-9

tryptone soft agar overlays.

6. Following solidification of the soft agar over-

lay, incubate all plate cultures in an inverted

position for 24 hours at 37°C.

Procedure Lab Two1. Observe all plates for the presence of plaque-

forming 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.

MaterialsCultures24-hour nutrient broth cultures of Escherichia

coli B and T2 coliphage.

MediaFive 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.

EquipmentBunsen burner, waterbath, thermometer, 1-ml

sterile pipettes, sterile Pasteur pipettes, mechani-

cal pipetting device, test tube rack, and glassware

marking pencil.

Procedure Lab OneTo 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.

CL IN ICA L A PPLICAT ION

Identification of Pathogenic Bacteria

Bacterial viruses (bacteriophages) are very com-mon in all natural environments and are directlyrelated to the number of bacteria present. Theyare 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 specificabout which bacteria they attack, a process calledphage typing is used in clinical and diagnosticlaboratories for the identification of pathogenicbacteria.

AT THE BENCH


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Experiment 39 267

1 ml 1 ml 1 ml 1 ml 1 ml 1 ml 1 ml 1 ml 1 ml

Phagestock

Performa 10-foldserial dilution.

10–1

10–2

10–3

10–4

0.1 ml

Tryptone broth tubes (9 ml each)

0.1 ml 0.1 ml 0.1 ml 0.1 ml

10–5

10–6

10–7

10–8

10–9

10–5

10–6

10–7

10–8

10–9

10–5

10–6

10–7

10–8

10–9

Add two dropsof E. coli B cultureto each tube.

Mix and pour.

Overlay of tryptone soft agar

Tryptone hard agar

Tryptone soft-agar tubes

PROCEDURE

Figure 39.2 Dilution procedure for cultivation and enumeration of

bacteriophages


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Experiment 39: Lab Report 269

Phage Dilution Number of PFUsCalculation:

PFUs : Dilution FactorPFUs/ml of Stock

Phage Culture

10-5

10-6

10-7

10-8

10-9

Review Questions1. 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 infec-

tion to one that is lytic.

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

infection.

Observations and Results

Name:

Date: Section: Lab Report

EXPERIMENT

39


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270 Experiment 39: Lab Report

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. The release of phage particles from the host bacterium always oc-

curs 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 re-

verse pinocytosis. Regardless of the mechanism of release, most infected

cells die, while other viruses may escape the cell without damaging the host

cell. Explain.


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(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 in-

fecting the susceptible E. coli B host cell.

PrincipleIsolates of bacterial viruses (bacteriophages) can

be obtained from a variety of natural sources, in-

cluding soil, intestinal contents, raw sewage, and

some insects such as cockroaches and flies. Their

isolation from these environments is not an easy

task because the phage particles are usually pres-

ent 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 filter.

5. Inoculation of the bacteria-free filtrate 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 in-

dicated 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 identified

and are labeled T1 through T7. The T-even phages

Isolation of Coliphagesfrom Raw Sewage

EXPERIMENT

40

LEARNING OBJECTIVE

Once you have completed this experiment,

you should be able to

1. Isolate virulent coliphages from sewage.

CL IN ICA L A PPL ICAT ION

Phage Therapy

Phage therapy is the therapeutic use of bacterio-

phages to treat pathogenic bacterial infections. It ismainly used in Russia and the Republic of Georgiaand is not universally approved elsewhere. In theWest, no phage therapies are authorized for use onhumans, although phages for killing food poisoningbacteria (Listeria) are now in use. They may also beused as a possible therapy against many strains ofdrug-resistant bacteria.

AT THE BENCH

MaterialsCulturesLab 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.

MediaPer designated student group: Lab One: One 5-ml

tube of bacteriophage nutrient broth, 10 times

normal concentration. Lab Two: Five tryptone agar

plates and five 3-ml tubes of tryptone soft agar.

EquipmentLab One: Sterile 250-ml Erlenmeyer flask and

stopper. Lab Two: Sterile membrane filter appara-

tus, sterile 125-ml Erlenmeyer flask and stopper,

125-ml flask, 1000-ml beaker, centrifuge, Bunsen


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272 Experiment 40

E. coli B

culture

PROCEDURE

Plates of hard tryptone agar

1 2 3 4 5

1 2 3 4 5

1 drop 2 drops 3 drops 4 drops 5 drops

Pour in severalcentrifuge tubesand centrifuge

sewage sampleat 2500 rpmfor 20 minutes.

Filter supernatantthrough membranefilter into vacuum flask.

E. coli B enriched sewagesample in 250-ml flask

Bacteria-free phage filtrate in vacuum flask

Add 0.1 mlto tubes 1–5

Remove tubesand decantsupernatantinto a 125-ml beaker.

Centrifuge Flask and membranefilter apparatus

of moltensoft tryptoneagar.

Figure 40.1 Procedure for isolation of coliphages from raw sewage


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Experiment 40 273

3. Pour the supernatant solution through a ster-

ile membrane filter apparatus to collect the

bacteria-free, phage-containing filtrate in the

vacuum flask below. Refer to Experiment 50

for the procedure in assembling the filter

membrane apparatus.

4. Melt the soft tryptone agar by placing the five

tubes in a boiling waterbath and cool to 45°C.

5. Label the five tryptone agar plates and thefive 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 filtrate to the re-

spectively labeled molten soft-agar tubes. Mix

and pour each tube of soft agar into its appro-

priately labeled agar plate.

8. Allow agar to harden.

9. Incubate all the plates in an inverted position

for 24 hours at 37°C.

Procedure Lab Three1. Examine all the culture plates for plaque for-

mation, 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 inthe Lab Report.

burner, forceps, 1-ml sterile disposable pipettes,

sterile Pasteur pipette, mechanical pipetting de-

vice, test tube rack, and glassware marking pencil.

Procedure Lab One

Use disposable gloves. It is essential to

handle raw sewage with extreme caution becauseit may serve as a vehicle for the transmission ofhuman pathogens.

Enrichment of Sewage Sample

1. Aseptically add 5 ml of bacteriophage nutri-

ent broth, 5 ml of the E. coli B broth culture,

and 45 ml of the raw sewage sample to an ap-

propriately labeled sterile 250-ml Erlenmeyer

flask.

2. Incubate the culture for 24 hours at 37°C.

Procedure Lab Two

Filtration and Seeding

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 care-

fully decant the supernatant into a 125-ml

beaker.


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Experiment 40: Lab Report 275

Drops of Phage Filtrate 1 2 3 4 5

Plaque Formation

(+) or (-)

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

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

Review Questions1. Why is enrichment of the sewage sample necessary for the isolation of

phage?

2. How is enrichment of the sewage sample accomplished?

Observations and Results

Name:

Date: Section: Lab Report

EXPERIMENT

40


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276 Experiment 40: Lab Report

3. How are bacteria-free phage particles obtained?

4. Why must you exercise caution when handling raw sewage

samples?

virology_ isolation & cultivation of bacgteriophage

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