Isolation & Purification Of Bacteriophage Particles From Soil Sample

Biology Project Report Title: Isolation & purification of bacteriophage particles from soil sample.

Indian Institute of Science Education & Research, Kolkata Semester-II 2008

Project Done By:ASHISH AGARWAL (65) HARSH PURWAR (76) NETHI VAMSIDHAR (82) (82) 1

CONTENTS
Abstract

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Introduction

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Aim of the Experiment

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Apparatus Required

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Procedure

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Observations

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Precautions

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Acknowledgements

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References

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Abstract In this project we isolated bacteriophage particles from the soil sample and purified them.

Introduction Bacteriophage:- Obligate intracellular parasites that multiply inside bacteria by making use of some or all of the host biosynthetic machinery. It is not capable of life outside a host. Its significance Models for animal & plant cell viruses Gene transfer in bacteria (Transduction) Medical applications Structure & Composition Head (Capsid):- Made of protein coat, which contains the genetic material (DNA or RNA) of the phage. Tail:- Also made of a protein and have a Contractile Sheath (Protein) over it. Contractile sheath helps in inserting the DNA into the host cell. Tail Fibres (Protein):- Helps in attaching the phage to the cell surface. A bacteriophage infects its host by first attaching itself on the bacteria surface and then inserting its DNA or RNA into the host cell. This is then followed by lysogenic and/or (as the case may be) lytic cycle resulting into the death or lysis of the cell. Now what are 'Plaques'? Plaque is a clear circular zone on agar surface resulting from bacterial lysis by the phage. It generally spreads over the soft medium i.e. top agar layer.

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Aim: To isolate different bacteriophages and purify them from soil samples(3) of different concentrations.

Apparatus Required: • • • • • • • • • • • •

Soil Sample Different bacterial species Luria Broth Agar Test tubes LB Plates Toothpicks Eppendorf tubes Micropipettes and tips Incubator Laminar Autoclave

Procedure:  



 

  

Get the soil from about 10cm deep. Mix it with sterilised water and prepare 3 such soil solutions of different concentrations. Prepare 9 LB plates with 1.5% agar (hard medium). Hard medium base (1.5% agar) Soft medium on top (Containing known bacterial species & soil solution (phages)). Mix the soil samples with each of the bacterial strains. Take 1 mL of the above mixture and mix it with the 0.75% agar (soft medium) and pour the medium on the plates. Incubate the plates for 48 hours at 27° C. Then look for the plaques on the plates. Pick up the plaques using a thin wire loop or a toothpick and repeat the above steps to purify the bacteriophages.

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Observations: We observed clear circular zones on the agar surface resulting from bacterial lysis by phages. These are called plaques. The phages collected from different plaques have been stored for further use.

Is it a plaque or air bubble? • • • •

Pick up the phages from a particular plaque. Mix them with the corresponding bacteria and soft agar media. Pour it on a petri plate and incubate at 30º C for 48 hours. Look for the plaques.

Purifying phages • • • •

Pick up the phages from a single plaque by a loop or a toothpick. Mix it with the soft medium. Repeat the procedure for a few times. The obtained phage particles are bacterio-specific.

Precautions: • • • •

Apparatus should be autoclaved before use. Don’t use a micropipette tip or a toothpick for more than once. Be careful while working in the laminar. Wear mask and gloves while working with harmful viruses or bacteria. 5

Acknowledgements We would like to produce my sincere thanks to the following faculty members and all my friends. • Dr. Tapas K. Sengupta • Dr. Tridib Ganguly • Dr. Sujata Guha •

Mr. Subhrojit Sen

I would also like to thank Mr. Sudhanshu for his great help.

References • http://www.google.co.in • http://www.wekipedia.org • Gene - VIII • Biology Concepts & Connections – By Campbell, Mitchell & Reece

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