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Bacteriophage Isolation

Bacteriophage Isolation. Bacteriophage. Obligate intracellular parasites that multiply inside bacteria by making use of some or all of the host biosynthetic machinery. Highly specific Can reproduce in great number. Bacteriophage. Significance Models for animal cell viruses

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Bacteriophage Isolation

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  1. Bacteriophage Isolation

  2. Bacteriophage • Obligate intracellular parasites that multiply inside bacteria by making use of some or all of the host biosynthetic machinery. • Highly specific • Can reproduce in great number

  3. Bacteriophage • Significance • Models for animal cell viruses • Gene transfer in bacteria • Medical applications • Identification of bacteria - phage typing • Treatment and prophylaxsis??? Examples: • T4 • Lambda

  4. Head/Capsid Contractile Sheath Tail Tail Fibers Base Plate Composition and Structure • Composition • Nucleic acid • Genome size • Modified bases • Protein • Protection • Infection • Structure (T4) • Size • Head or capsid • Tail

  5. Infection of Host Cells • Irreversible attachment • Adsorption • LPS for T4 • Sheath Contraction • Nucleic acid injection

  6. 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.

  7. Important steps in the Isolation of Bacteriophage • Enrichment • To increase the number of cells for the virus • Enrichment medium • Culture medium • Raw sewage – source of Virus / bacteriophage • Host cells (E. coli, P. aeruginosa) Incubate at 37 C for 48 hours

  8. 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.

  9. LYTIC CYCLE • Eclipse • Early genes • Phage DNA synthesis • Late genes • Intracellular accumulation • Lysis and Release

  10. Centrifugation • 10 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

  11. 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.

  12. 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.

  13. Phage Bacteria + Phage

  14. 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.

  15. 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

  16. Photographs

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

  18. 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

  19. 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 Uses American Society for Microbiology News 64:620-623, 1998

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