The Randomness of Mutation and the Nonrandomness of Selection
"Max is rather silent, but - to spend the days chewing on a problem, and writing and erasing things on
the blackboard with him, is terribly exciting. He is unusually cultured by American standards. You know,
most American scientists are duds; they never have read a sensible book."
-Salvador Luria on Max Delbrück. In Horace Freeland Judson's Eighth Day of
Creation. 1979.
Objectives
After completing this exercise you should be able to:
1.
2.
Interpret Luria and Delbrück’s “fluctuation test”.
Explain the random nature of mutations.
Summary
Genetic mutation is the ultimate source of all new allelic variation. However, the question
arises as to whether that mutation is a response to external stimuli or whether spontaneous mutants
are already present at very low frequency in a population. The experiment detailed below is a
derivation of an experiment carried by Salvador Luria and Max Delbrück in 1943 which they referred
to as the “fluctuation test”. Luria reasoned that if mutations occurred spontaneously, then the
mutations would occur at different times in different cultures.
Materials
Serratia marcescens
5 50 ml conical centrifuge tubes
Luria-Bratania Broth
1 LB plate
40 LB plates with 25 mg/ml
Streptomycin
6 1 ml pipets
Pipet aid
Bunsen burner
Hockey stick
Beaker of alcohol
Sharpie
Procedure
Streak S. marecescens for isolation using aseptic technique on a nutrient agar plate and a nutrient
agar + 50 mg/ml streptomycin plate. Cultivate the plates at room temperature for 48 hours. Examine
your plates after 48 hours. You must have well isolated colonies on the nutrient agar plate to continue the
experiment. If you do not have well isolated colonies you must repeat the isolation procedure. Transfer
one of the colonies from the nutrient agar plate to a flask containing 50 ml of nutrient broth. Place the
flask in a RT shaking incubator for 48 h.
Each group will receive 5 50 ml conical centrifuge tubes. Number each of the numbers. Four of the
tubes will contain 5 ml of LB broth which, does not contain an antibiotic (streptomycin). The fifth tube
will contain 20 ml of LB broth also lacking an antibiotic. Note the volume of the first 4 tubes is
equivalent to the volume of the fifth tube. Using Aseptic technique inoculate each tube with 0.1 ml of S.
marcescens culture. Place the tubes in a shaker incubator at 37C and grow overnight. Using aseptic
technique inoculate a LB plate, lacking antibiotics, with 0.1 ml of the S. marcescens culture.
After an overnight incubation, aseptically transfer 0.1 ml of culture from one of the four 5 ml tubes to
5 plates of LB agar contains 25 mg/ml Streptomycin. Flame a hockey stick and evenly spread the culture
on the plate. Repeat for the remaining three tubes containing 5 ml of culture. You should have a total of
twenty plates at this point – 5 for each culture. Aseptically transfer 0.1 ml of culture from the final tube
containing 20 ml of culture to 20 LB streptomycin plates. Be sure and mark each plate with the number of
the tube, which was used to inoculate the plate. Place the plates in the 37C incubator and grow
overnight. The next day count the red/pink colonies on each plate. Record your results on the table
below.
Tube No.
1 (5 ml)
2 (5 ml)
3 (5 ml)
4 (5 ml)
5 (20 ml)
No. colonies
Plate 1
No. colonies
Plate 2
No. colonies
Plate 3
No. colonies
Plate 4
No. colonies
Plate 5
Answer the following questions and turn them in next lab period along with your primary data.
1.
2.
3.
4.
Why are the tubes incubated on a shaking platform?
What is the purpose of inoculating the LB plate lacking antibiotics? What do you expect to see
on this plate?
Why are the cultures inoculated on plates containing an antibiotic?
Was there more variability in the number of colonies per plate from the tubes containing 5 ml of
culture than from the tube containing 20 ml of culture? What is the meaning of these results?