Bacteriophage Isolation

EXPERIMENT 12 GROUP 2

Virus
Genetic elements that replicate within the host cell.

Can

replicate independently of a host cells chromosomes but independently of the cells themselves Confer new properties on host cells Difficult and time-consuming to study among plants and animals.

Virus
Possess an extracellular form that allows for easy

transmission GENERAL PROPERTIES:

Possess EXTRACELLULAR state (virion) Capable of INFECTION Can have either DNA or RNA or both, single stranded or double stranded 20 300 nanometers

Bacteriophage
Viruses that infect bacteria

Protein coat + genetic material

Infection = invasion of genetic material DNA invasion may result in LYSIS or LYSOGENY

Obligate intracellular parasites that use host cell

machinery to reproduce.

Bacteriophage
Consist of: NUCLEIC ACID PROTEIN

Bacteriophage
Transduction of genetic material may be: Generalized Specialized

LYSIS
Metabolism of the host cell is reoriented to the

synthesis of new viral DNA and protein coat to produce mature phages.

LYSIS
Upon completion, result in bursting of the cell to

release new and mature phages.

LYSOGENY
Viral DNA integrates with that of the host

lysogenic bacteria Most virus genes are not expressed. Virus genome replicated in synchrony with host chromosome DNA damage may trigger eventual lysis phase to result in the occurrence of the lytic cycle

LYSOGENY

OBJECTIVES
DESCRIBE the bacteriophage

PERFORM and UNDERSTAND techniques in the

cultivation of the bacteriophage QUANTIFY bacteriophage titer

Methodology

Enrichment
25 ml sewage water Centrifuge at 3,000 rpm for 10 minutes

20 ml filtered supernatant + 20 ml double strength T broth

Filter using 0.22 m or 45 m membrane filter

Enrichment
Supernatant T broth mixture

Incubate for 24 hours at 35 C.

1 2 ml overnight culture of E. coli in TSB

Isolation of bacteriophage
Centrifuge enriched sewage at 3,000 rpm for 1 minutes (2x)

Filter using 0.22 m or 45 m membrane filter

15 ml filtered sewage + 5 ml chloroform

Separate sewage and place in sterile tube

Allow mixture to stand for 5 minutes in room temp.

Isolation of bacteriophage
0.3 ml E. coli overnight culture 1 drop sewage water + molten soft NA

5 drops of sewage water + molten soft NA

Molten soft NA (control)

Overlay to prepared NA plates and allow to solidify

Incubate for 24 48 hours at 35C

Observe and count clearing in the lawn of bacteria