Couting CFUs

Measuring absorbance does not, however, tell us how many cells are actually alive in the culture.

A simple way to count the number of living cells is to exploit their ability to reproduce.  On an agar-coated petri dish, each viable cell will divide repeatedly, but its progeny will remain largely immobilized -- it will form of colony.

The cells of this colony are a clone of the original cell that landed on the agar surface.

By counting colonies, we get a direct estimate of the concentration of viable bacteria or more accurately the number of colony forming units - CFU per ml in the original culture.

To determine the number of CFUs per ml (CFU/ml) in a culture a small sample or aliquot of known volume is withdrawn from the culture and diluted into a known volume of sterile media.

Once diluted by a known amount, we can measure the number of CFU in a known volume of the diluted solution. 

To do this, we spread an aliquot of the diluted culture, generally between 10 to 200 µL,evenly over the surface of a LB-agar petri plate.

When incubated at 37 °C, the individual bacteria will grow up to form easily visible colonies within 18-24 hours.

Doing dilutions is one of the most difficult conceptual problems student have! 
Please think carefully about what your doing!   need a refresher? read this

Do you know the difference between an ml and µL? 
What does a 10^-3 dilution mean?  How would you set one up.

If too many bacteria are plated on a dish, the colonies grow together to form a continuous lawn.

Bacterial concentrations can be very high; that is why it is generally necessary to set up a dilution series.

  pipettes & pipettors tutorial

To do this, take an aliquot from your first dilution and dilute it again, then repeat the process.

This allows us to span a wide range of concentrations.

This process is illustrated here

It is difficult, tedious, and inaccurate to count more than ~300 colonies per plate, you can prove this to yourself if you want.

At the same time, counting too few colonies results in increasing inaccuracies due to sampling errors.

You need to determine the CFU/ml in the original culture, in large measure to prove that you understand how to do dilutions. 

If you plate out too many or too few cells, you can repeat the plating process, without having to set up a new dilution series

Do you know how to go from CFU per plate to CFU per ml in the original culture?  

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