Titering your phage stock

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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.


phage titering

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.

Determine the time of lysis

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


Now, determine the lysis time using the vSPEC. You need do this measurement only once.   If you are paying attention, your "eyeball" measurement of the lysis time should be helpful in your vSPEC measurements.

Confused about the vSpec?
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Lab report part 1
  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 answerWhat factors contribute to error in your estimate of viral life cycle time?


Use Wikipedia | revised 20-Apr-2006