Isolation of Bacteriophages

LECTURE 10:

Viro102:Bacteriophages & Phage Therapy3 Credit hoursAtta-ur-Rahman School of Applied Biosciences (ASAB)

• Bacterial viruses are generally present in natural habitats where bacteria are present.

• In this lecture we will discuss how to isolate and detect bacteriophage specific for E. coli in a wastewater sample.

Bacteriophage Isolation

The Procedure below

uses a pre-incubation of the wastewater sample with an

E.coli host strain to enrich, or amplify, the population of

phage

Subsequently we

use a plating technique to detect and

count specific phages from the enriched

sample

The plating technique

is referred to as "plaque assay” and involves seeding a

“lawn” of host bacteria

with a small volume of sample

containing phage

When phage seeded on

the lawn infect and lyse the host

cells, they produce a plaque within the

confluent opaque lawn of bacteria.

Phage isolation

Setting Up Enrichment1. In a sterile 125 ml flask, combine 5 mL of wastewater with 5.0

mL of 10Xphage broth media and 1.0 mL of E. coli strain culture.

2. Incubate overnight in a 37°C shaker with vigorous agitation.

10 mL of wastewater+

1.0 mL of media+

1.0 mL of E. coli

Plug it and incubate

At 37 °C

Culture Incubator, 37°C

Dilution and Plating

3. Transfer 1 mL of the overnight enrichment culture to a 1.5mL microcentrifuge tube.

4. Add one drop of chloroform to the microcentrifuge tube and mix by finger vortexing or inversion.

• The chloroform disrupts cell membranes to facilitate release of free phage from infected cells that have not yet lysed.

1ml of Over night

culture

Add 1dropof

chloroform

Vortex it

5. Centrifuge the micro-centrifuge tube at maximum RPM for 5-10 min.

• The goal is to collect insoluble cell debris into a pellet. Phage are too small to sediment at the low gravitational force generated in a micro-centrifuge and will remain suspended in the supernatant.

6. Repeat the centrifugation step by transferring the uppermost layer (supernatant) into a fresh micro-centrifuge tube.

• centrifuge again at maximum rpm for 5-10 min. (We are trying to remove

all bacteria and cell debris from the supernatant.)

Centrifuge

Transfersupernatant

to fresh tube

• The first microcentrifuge tube is contaminated with chloroform, and should be discarded in the appropriate hazardous waste container.

7. Transfer the supernatant of the second spin to yet another fresh microcentrifuge tube.

• Discard the second microcentrifuge tube as regular waste.

Serial Dilution of Phage Enrichment

8. Using autoclaved distilled water as the diluent, perform a dilution series of the supernatant (in steps of 10-2) to a total dilution of 10-6.

• The phage idealy should be diluted in TMG (Tris Mg Gelatin) Buffer, not the usual 0.9% NaCl that you use for E. coli.

• TMG Buffer contains Mg++ that stabilizes some phage virions and facilitates attachment to the host cell outer membrane.

• Save the remaining sample at 4°C.

Serial dilution

Plaque Assay for Phage Detection

10. Add 0.1 ml of E. coli host culture to all the plating tubes

11. Add 0.1 ml of the appropriate phage dilution to their respective tubes.

• 10-1 dilution of phage sample + host bacteria• 10-2 dilution of phage sample + host bacteria• 10-4 dilution of phage sample + host bacteria• 10-6 dilution of phage sample + host bacteria

• Gently mix the tubes and incubate for 10-15 minutes at 370C.

This pre-incubation allows the phage to attach to the host cells more effectively than in the agar.

• Add 2-3 mL melted top agar to each of the tube.

0.1 ml E.COLI+

0.1 ml of Phage dilution

Incubatefor 10-15

Min.

Add 2-3 ml

of meltedTop agar

• Mix the tubes and immediately pour the contents of each tube onto the appropriate plate.

• Immediately rotate the plate gently so that the melted agar completely and uniformly covers the surface.

• Let the plates be allowed to solidify .

• Incubate the plates upside down at 37°C overnight before observing plaques next day.

Plaque Assay for Phage Detection

Incubate at37 °C

for 24 hoursand observe

the results

Plaque formation, Results• Examine plates (a dissecting microscope is helpful) for

evidence of zones of clearing, or plaques, within the lawn of bacteria.

• The size and appearance of plaques may vary, at least in part because there are different types of phage present in the sample.

• Note particularly the difference between clear and turbid (murky) plaques.

• Turbid plaques may result when temperate phage establish lysogeny in some of the infected host cells.

• Clear plaques, on the other hand, indicate that the phage is lytic, or virulent (i.e. non-lysogenic).

B3 plaques LK1 plaques

Thanks!!