T4 Bacteriophage

T4 Bacteriophage Replication, Titration

and Calculation Practical

Aims

* To infect bacterial cell cultures with a bacteriophage and observe the effects of virus infection by monitoring bacterial cell growth.

* To infect, establish and monitor bacterial cell growth.

* To use the spectrophotometer to monitor bacterial cell growth.

* To sample extracellular virus at a specific time point during the infection.

* To use the sample of infected cells with the virus for plaque assay.

* To choose the correct range for serial dilutions, and use them and mathematical equations to calculate virus tires.

* To understand virology concepts using the plaque assay to study and record infection of cells with a bacteriophage.

Methods

Practical 1:

* Add 3ml E. coli to 20ml LB broth in a conical flask; do this twice into 2 separate flasks.

* One flask is to be used as a control, label this flask. To the other flask add 50µl T4 bacteriophage.

* Take 1ml of culture from each flask and put it into separate cuvettes and take absorbance readings after blanking the spectrophotometer with 1ml LB broth.

* Put the foil lids back onto the flasks and place them on the orbital shaker.

* After 1 hour of incubation and then at 20-minute intervals, until you have monitored for 2 hours, repeat taking absorbance readings for both cultures after blanking the spectrophotometer. Record your results.

* After 60 minutes (our given time) of incubation remove 1ml from both cultures and place each culture into separate microfuge tubes.

* Use the bench microfuge to pellet the bacterial cells, spin at 6500rpm for 5 minutes.

* Discard the control sample once it had been microfuged.

* From the T4 infected tube remove 750µl into a bijou, label the bijou and give the sample to a member of staff to used in next weeks practical.

* Leave the T4 infected flask on the orbital shaker and a member of staff will take an overnight sample to be used...