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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 ASHISH ASHISH ASHISH AGARWALAGARWALAGARWALAGARWAL (65)(65)(65)(65)

HARSH PURWARHARSH PURWARHARSH PURWARHARSH PURWAR (76)(76)(76)(76)

NETHI NETHI NETHI NETHI VAMSIDHAR (VAMSIDHAR (VAMSIDHAR (VAMSIDHAR (82)82)82)82)

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CONTENTS

� Abstract 03

� Introduction 03

� Aim of the Experiment 04

� Apparatus Required 04

� Procedure 04

� Observations 05

� Precautions 05

� Acknowledgements 06

� References 06

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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.

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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