Ask A Scientist Molecular Biology Archive

name         Will B.
status       student
age          17

Question -   I have been experimenting with the effectiveness of
freezing bacteria, Lactobaccilus acidophilus in particular, as a
sterilization method.  I've been using a spectrophotometer to take
readings before and after freezing to calculate growth.  The results, as
suspected, have shown that the longer a sample is frozen, the less growth
it will show after incubation.  However, I would like to be able to take
more exact measurements.  I also want to expand to different bacterium
and compare their responses to freezing periods.  Do you have any
suggestions or guidelines in either of these areas?  Any assistance would be appreciated.
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Dear Will,
A more exact approach would be to count the number of
colony-forming units (CFU) before freezing, after
freezing, and after growth incubation for a
standardized period of time. I do not know if you have
the facilities to do this. What you would need is a
(large) number of agar plates to grow your bacteria,
sterile phosphate buffered saline solution (PBS),
sterile tubes and micropipettes.
Briefly, for each time point you want to measure
(before freezing, after freezing, after incubation)
you'd take a sample from your culture and dilute it
stepwise 10-fold down to, say, expected 1000 CFU/ml.
If you put 100 microliters of this on a plate, you'd
see 100 colonies growing. Since you can't predict
precisely how many CFU are in your broth, you plate
out 10 or 100 times more, and 10 or 100 times less, of
the expected correct dilution. In that way you can be
sure that one of your plates has a sufficient nr. of
colonies to be counted. Thus, you need 3 or 4 plates
(each with 100 microliters of different dilutions) for
each sample you want to determine. Count the countable
dilution plate and work back how many CFU were in your
original sample. It is a lot of work, but very
precise.

A way to minimize agar plates (if that is your
restriction) is to make the dilutions, but only put 10
microliters of each dilution on a plate, and not
spread it over the complete plate. Instead, let each
drop form a 'tear' by tilting the plate. In that way,
you have less colonies on a smaller surface, and you
can put several tears on one plate, provided they
don't touch each other. It is less precise, but still
better than photometric measurement: with the latter,
you also measure dead cells, where as CFU only gives
you numbers for viable organisms.

Good luck,
Trudy Wassenaar
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