Bacteriophage Isolation

Bacteriophage

Obligate intracellular parasites that multiply inside bacteria by making use of some or all of the host biosynthetic machinery.

Highly specificCan reproduce in great number

Bacteriophage

SignificanceModels for animal cell virusesGene transfer in bacteriaMedical applications

Identification of bacteria - phage typingTreatment and prophylaxsis???

Examples:T4

Lambda

Composition and Structure

CompositionNucleic acid

Genome sizeModified bases

ProteinProtectionInfection

Structure (T4)SizeHead or capsidTail

Tail

Tail Fibers

Base Plate

Head/Capsid

Contractile Sheath

Infection of Host Cells

Irreversible attachment

• Adsorption– LPS for T4

Nucleic acid injection

Sheath Contraction

Types of Bacteriophage

Lytic or virulent phage: Phage that can only multiply within bacteria and kill the cell by lysis. (e.g., T4)

Lysogenic or temperate phage: Phage that can either multiply via the lytic cycle or enter a quiescent state in the bacterial cell.

Important steps in the Isolation of Bacteriophage

Enrichment

To increase the number of cells for the virusEnrichment medium

Culture mediumRaw sewage – source of Virus / bacteriophageHost cells (E. coli, P. aeruginosa)

Incubate at 37 C for 48 hours

Enrichment

During incubation, phage in the sewage sample capable of binding to E. coli will replicate in and eventually lyse the bacteria.

This step serves as a means of amplifying the phage that can infect the prepared bacteria.

Eclipse Early genes Phage DNA

synthesis Late genes

Intracellular accumulation

Lysis and Release

LYTIC CYCLE

Centrifugation10 minutes at 2500rpm.

This centrifugation step should cause the bacteria and other cell debris to form a pellet at the bottom of the tube.

Separating the host cells from virus

Virus (supernatant) - Enriched phage prep’n. Bacteria (pellets) - discarded

Heating of supernatant

Killing other host cells / bacterial cell debris Viruses – resistant to heating Enriched phage preparation can be stored in

the refrigerator until use.

Seeding

Preparation of bacterial lawn for the virus specific for the host.100 microliters of an overnight E. coli growth

onto the center of a 60 mm Petri plate containing tryptic soy agar.

With a bent glass rod, spread the bacteria over the plate.

Place 100 microliters of your enriched phage prep in the center of the plate.

Incubate at 37°C overnight.

Bacteria

Phage

+Phage

Result

Because thousands (or maybe millions!) of bacteria were spread onto the plate, there will be no isolated colonies. Rather a confluent lawn of bacteria will cover the agar.

Phages mixed with the bacteria will infect the bacteria, undergo a lytic cycle to propagate, leaving the agar surface seemingly clear due to lysed cells.

Clearing (plaques) / Viral Plaques

The phage will infect and kill the bacteria in the center of the plate, creating a visible region of no bacteria - a plaque.

www.bch.msu.edu/bchug/web/bch472/472lm3.htm

Photographs

PFU – Plaque forming unitsMeasures infectious particles in a given sample.Count the number of PFUs on a series of

plates:

Calculate the number of PFU per mL of phage stock

For example: 37 plaques were observed.

Hence, (37 plaques / 0.1 ml) x 106 dilution = 3.7 x 108 PFU / mL of original phage stock

Medical Applications of Phage“I strongly believe phage could become an

effective antibacterial tool” - Carl Merril, Chief of the Laboratory of Biochemical Genetics, National Institute of Mental Health, NIH.

“It might be another string on the bow, such that when (conventional antibiotics) fail, here’s something that has a chance of working. But it’s not going to be a panacea” - Joshua Lederberg, Sackler Foundation Scholar at The Rockefeller University

Reassessment of Medicinal Phage Spurs Companies to Study Therapeutic UsesAmerican Society for Microbiology News 64:620-623, 1998

end