SUBJECT: Lab on antibiotic resistance
DATE: 3/96
We are just finishing a week that featured experiments with bacteria. One
of these was a test of the susceptibility of various species of bacteria
to
several different types of antibiotic. The results are interesting enough.
Yet, we here more and more about the imminent end of the "Age of
Antibiotics." Does anyone out there know of any good experiments involving
the development of antibiotic resistance among bacteria that might be
suitable for intro-level courses? I am aware of Michael Dennison and Corey
Goldman's exercise from Vol. 15 of the ABLE Proceedings concerning
adaptation of yeast to copper. But how about bacteria and antibiotics?
Anyone?
Yours,
Michael Dini
Texas Tech Univ.
y3mld@ttacs.ttu.edu
There are a number of labs on the TRANSFER of antibiotic resistance, via
bacterial conjugation. Fischer Education sells a kit. Haven't tried
it though.
-Bob Moss
MOSSRE@WOFFORD.EDU
Wofford College
Michael,
We do an introductory lab on antibiotic resistance and mutation
caused by UV radiation. Basically, prepare duplicate nutrient agar plates
and divide the plate into four quadrants. Pour a top layer of soft agar
to produce a lawn of bacteria (we use E. coli, but you could substitute
as desired). Allow to solidify and place four discs (3 antibiotic
and distilled water control) onto the plates in the appropriate areas (not
too near the intersection). We use penicillin (affects Gram positive),
ampicillin (Gram positive and negative) and chloramphenicol (protein
synthesis). Expose one plate to short UV for about 30 seconds and use the
other as a control.
Follwing incubation there will be clear zones of inhibition around
the antibiotic discs where the bacteria have been killed. What the
students are to look for is colonies growing in these zones (on the
UV treated plate) which might be resistant to the antibiotic they
would normally be susceptible to (indicated by the clear zone). Although
the chance of finding an induced mutation is very small it does get the
students to think about mutations (in terms of the chance of one
being beneficial) and how one could know/test if a colony in the
clear zone was actually antibiotic-resistant or just due to a diffusional
problem/contamination.
If you would like more details or a copy of the exercise let me know.
----------------------------------------------------------------------
Mike Weber
Carleton University
Department of Biology
1125 Colonel By Drive
Ottawa, Ontario
Canada
K1S 5B6
Email address: mweber@ccs.carleton.ca
Yes, you have to use an antibiotic for which resistance is
conferred by mutation of a chromosomal gene rather than
acquisition of a plasmid. Jeffrey Miller in "Experiments in
Molecular Genetics" lists Nalidixic acid (topoisomerase gene),
Rifampicin (some ribosomal protein or other), Ampicillin (at 10
ug/ml), Kasugamycin, Streptomycin and Spectinomycin for
starters.
The procedure is simply to inoculate a plate containing
antibiotic at suitable concentration quite heavily with sensitive
bacteria and see what grows. The only trick is to pick the
correct concentration of antibiotic that provides selection
without killing everything. Also the frequency of mutation is
different for different antibiotics. For streptomycin the
frequency is low, so you need to spread a heavy lawn of cells,
e.g. 0.1 ml of an overnight broth culture. Possibly the
composition of the medium might also have some effect on the
action of different antibiotics. These unknowns can be used to
challenge students with elements of the experimental design, or
if you want it to work first time get the cookbook procedure
from Miller's book.
You could have fun by picking the mutants that grow and testing
their minimum inhibitory concentration for the antibiotic, compared
with the starting "wild type" strain. The procedures for doing
this are in all the standard microbiology lab manuals.
----------------------------------------------------------------------
Walter Ogston ogston@hobbes.kzoo.edu
Department of Biology Phone: (616)337-7010
Kalamazoo College Fax: (616)337-7251
Kalamazoo, MI 49006-3295