Ask A Scientist© Molecular Biology Archive

name       prudence
status     student
age        19

Question - i'm a second year b.sc student. we have done three
experiments whereby each was a follow up of the previous one. in one of
the experimets we had to plate out some of the E.coli cells onto the
Luria Agar plate and incubate them overnight at 37 degrees celcius. we
then had to count the number of colonies produced.
Then we had do plate out some E.coli cells onto rifampicin plates and
also incubate overnight at 37 degrees celcius. What i would like to know
is that we had to count does that mean. I mean
what is so special about the rifampicin that the
cell could be resistant?  And how do the cells become resistant to
rifampicin? I would also like to know what "mutation frequency" means. How is it
calculated?

Dear Prudence,

Rifampicin is an antibiotic that inhibits protein
synthesis. It is a bacteriocid that will kill bacteria
because without protein synthesis life is not
possible. Bacteria that can grow in the presence of
rifampicin have found a way to evade the killing
action of the antibiotic: they are resistant to it.
Bacteria can become resistant to an antibiotic because
they mutate easily and frequently (there are other
ways to become resistant as well). Mutation is a
random process. Most mutations will not have any
effect, several would have a beneficial effect if the
proper selection was applied, others are lethal. The
mutations that lead to rifampicin resistance allow
those bacteria to grow despite the presence of the
antibiotic. These form the colonies on your plate with
rifampicin. If you count the colonies, you have your
number of 'rifampicin resistant mutants'. The
frequency of mutation can be calculated when you
compare the number of these mutants with the number of
original bacteria, the ones that grew on Luria plates.
You have to take into account what dilution was plated
out on both plates. I'm sure you can do those
calculations now that you understand what you did.

What kind of mutations lead to rifampicin? to answer
that you have to know how the antibiotic works. In the
case of rifampicin it prevents the ribosomes from
binding to messenger RNA, and thus they cannot produce
protein. Frequently, mutations occur in the ribosomal
RNA (ribosomes consist of RNA and ribosomal protein)
and if such mutations occur in the right place they
prevent the binding of rifampicin. That renders the
antibiotic ineffective. Other antibiotics work
differently, but whatever their action, bacteria
manage to become resistant sooner or later. It needs
time, and selection.

If you want to read about mutations (their important
role in evolution is nicely illustrated by bacteria
because they mutate so fast), about antibiotics, and
about resistance, visit the Virtual Museum of Bacteria
at www.bacteriamuseum.org. The topics I mentioned you
can find in the subject category 'how we fight
bacteria', in the exhibit 'antibiotics'. There is also
a subject category 'evolution' which explains some
basic principles of mutations. Have fun!

Trudy Wassenaar,
Curator of the Virtual Museum of Bacteria
=======================================================
Rifampicin is an antibiotic that should be toxic to your wild-type cells.
The rifampicin in the agar medium would have killed the cells that lacked
some sort of resistance to it, which would have arisen as a mutation.

The mutation frequency would be the fraction of cells that contain the
rifampicin resistance gene. (There is some uncertainty in exactly what you
are measuring here, because it is possible, even likely, that more than one
possible mutation could confer resistance.) Your best estimate of this rate
is the number of colonies on the rifampicin plate divided by the number of
colonies on the neutral plate. This assumes, of course, that the two plates
differ only in the fact that one contains the antibiotic, and that they were
both coated in exactly the same way.

Richard E. Barrans Jr., Ph.D.
Assistant Director
PG Research Foundation, Darien, Illinois
========================================================
This is an experiment to demonstrate that there is diversity in every
population, which is important to the survival and evolution of any species.
For example, when E. coli are subjected to an antibiotic such as Rifampicin,
this represents a change in their environment. If all organisms in the
environment are sensitive to the change, then the entire population will be
wiped out. But if there are a few members in the population that are
resistant to the effects of the antibiotic and can survive long enough to
reproduce, the population will go on. The reason there are a few resistant
organisms is because their DNA contains some kind of different version of a
message (gene) that allows the organism to fight the effect of the
antibiotic. This is a mutation in the DNA.  So its not the Rifampicin that
is special, its the bacteria that are able to survive the effects. This
creates a health care problem because if the all of the bacteria that are
sensitive to the drug are killed, and the only bacteria to survive are not
affected by the drug, the resistant bacteria will have more room to grow and
all you'll have are resistant bacteria. The antibiotic becomes less
effective toward that type of bacteria over time. I'm not sure how your
instructor wants you to calculate mutation frequency. There are many ways to
do this. I'm sure the directions are in your lab somewhere!!


Van Hoeck
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