Titering your phage stock

Before you can use your phage stock for experiments, you need to determine how many infectious phage are present, its titer.

You have a 5 ml overnight culture of E. coli and three tubes of top agar.  Decide how to dilute your phage stock and how much of each dilution to add to the bacteria.

The buffer preserves virion structure and minimizes the absorption of virions to the walls of test tubes.

After an overnight incubation count the plaques.  As you proceed, record your data in your vDATAPAD; your vTA will be able to check your work.

Record your phage titer in your Notebook, you will need to use it again.

The virulent phage life-cycle:  The ability of bacteria to scatter light is drastically reduced upon lysis. As the bacteria in a culture are lysed, absorbance falls and the cloudy culture becomes clear.

We can use this clearing of the bacterial culture to estimate the phage generation time.

Start with an overnight culture of E. coli growing in LB.

Dilute the stationary phase culture to an A =0.45 or ~10⁷ bacteria/ml.

Infect the culture with an approximately equal number of T4 phage.

Set time that the phage and the bacteria are mixed to T=0.  NOTE: the clock starts at T = 20

1. Note your estimated of the original phage stock titer (and show your data).
2. Repeat the clearing experiment two times to check whether your estimate is reproducible. 
3. Use the vSpec to get an estimates of time to lysis (show your data)

Questions to answer:  What factors contribute to error in your estimate of viral life cycle time?