Department of Energy Argonne National Laboratory Office of Science NEWTON's Homepage NEWTON's Homepage
NEWTON, Ask A Scientist!
NEWTON Home Page NEWTON Teachers Visit Our Archives Ask A Question How To Ask A Question Question of the Week Our Expert Scientists Volunteer at NEWTON! Frequently Asked Questions Referencing NEWTON About NEWTON About Ask A Scientist Education At Argonne Bacteria Resistance
Name: Prudence
Status: Student
Age: 19
Location: N/A
Country: N/A
Date: N/A 


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?



Replies:
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



Click here to return to the Molecular Biology Archives

NEWTON is an electronic community for Science, Math, and Computer Science K-12 Educators, sponsored and operated by Argonne National Laboratory's Educational Programs, Andrew Skipor, Ph.D., Head of Educational Programs.

For assistance with NEWTON contact a System Operator (help@newton.dep.anl.gov), or at Argonne's Educational Programs

NEWTON AND ASK A SCIENTIST
Educational Programs
Building 360
9700 S. Cass Ave.
Argonne, Illinois
60439-4845, USA
Update: June 2012
Weclome To Newton

Argonne National Laboratory