MICB 3301 Lab Quiz #5 Experiment 15 Bacteriophage: Isolation and Titration

What agents infect bacterial cells?

Bacteriophages

What is a bacteriophage?

A fragment of genetic material (nucleic acid) with a protein

How does a bacteriophage infection of a host cell begin?

With the binding of the phage, usually via tail fibers to a receptor

The phage genetic material is then injected into the bacterial cell

In the cell the virus nucleic acid either ______________ or _____________

Converts all cell machinery over to the manufacture of new virus particles, causing lysis of the bacteria

or

Comes into equilibrium with the genetic material of the host by physically inserting its genome into the bacterial chromosome and becoming a prophage

Lytic or virulent bacteriophage

Causes cell lysis by converting all cell machinery over to the manufacture of new virus particles

Temperate or lysogenic bacteria

Comes into equilibrium with the genetic material of the host by physically inserting its genome into the bacterial chromosome and becoming a prophage

Define prophage

The latent form of the virus genome that remains within the host but does not destroy the host

What can the bacterium-phage model system be used to study?

Heredity

Can also be used to detect and differentiate strains of bacteria by how they respond to bacteriophages

Phage Typing

test for determining which phages a bacterium is susceptible to

Transduction

Mediated transfer of bacterial DNA between bacteria by bacteriophages

What is the source material for the isolation of E. coli bacteriophage?

Raw sewage

The free phage particles are small enough to pass through filters that will retain all bacteria present

What is the phage used in this experiment?

T phage

Virulent DNA phage with a lytic cycle

Phage or Viral titer definition

The concentration of infectious phage particles per milliliter of growth medium

How were T phage titers determined?

By making serial dilutions of a phage stock culture and then mixing the phage with its E. coli host in soft agar and layering the mixture on top of an agar plate

What are the general expected results

The bacteria will form a translucent "lawn" of growth covering the surface of the plate

The bacteriophage will form small, clear zones of clearing in the otherwise dense lawn of bacterial cells

Plaques

The zones of clearing caused by the bacteriophage

Results of localized areas of lysis of phage infected bacteria

What kind of bacteria and broth was used ?

E. coli strain β

Luria-Bertani (LB) broth with Mg

Phage attachment to receptors on the surface of bacterial cells is facilitated by the presence of what?

Magnesium ions

Describe the main procedure for day 1

Place the T phage suspension into the LB broth, then inoculate with E. coli strain β

Incubate at 37°C

Describe the procedure for day 2

Transfer the phage/E. coli culture into a microfuge tube and centrifuge for 5 minutes at 13,000rpm

Transfer the culture to fresh tubes, avoiding the pellet, and add chloroform

Pipette the culture supernatant with the T phage into tube containing sterile saline, Make dilutions

Add dilutions into agar mixtures, and add host bacteria ( E. coli) and pour onto plates

Incubate at 37°C

What is the purpose of the chloroform?

It will kill any contaminating bacteria without harming the bacteriophage

Does the host bacteria need to be present in order to detect a bacteriophage on a plate?

Yes