Bacteriophage Isolation
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Transcript of 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
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