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Bacteriophage Isolation

Bacteriophage Isolation

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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

  20. end