Mutations--Preexisting or Acquired?
Author: George L. Morse
Woodrow Wilson Biology Institute
Exposing a culture of E. coli to virulent bacteriophage results in a few surviving bacteria. These variants breed true and can be accounted for in one of two ways--either the E. coli mutated in response to the virus (acquired mutation) or the mutation preexisted. This activity explores the reasoning and experimental approaches to determining a solution to this problem.
Target age/ability Group:
High school/average and above
The debate over the adaptation hypothesis (environmentally caused variation) and the spontaneous mutation hypothesis (preexisting mutants) was addressed by Luria and Delbruck (1943), Newcombe (1949), and Lederberg (1952). These scientists believed in spontaneous mutations and devised methods to study the problem. A variation of the Newcombe spread is presented in this paper.
If mutations occur in response to contact with a virus, then a Petri dish (inoculated with bacteria) that is spread prior to introducing a phage and a Petri dish that is unspread prior to introducing a phage should have the same number of mutants. On the other hand, if the spread contains more mutants than the unspread dish, the mutants must have preexisted in the culture. Spreading simply moves the cells around, so if there are five mutants in one colony, they are spread over the area of the plate, each forming its own colony of phage resistant/immune bacteria.
The Newcombe spread involves culturing E. coli in a Petri dish for a few hours and then replica plating it onto two Petri dishes that have been spread with phage. One of these plates is then respread. The other plate is left unspread. Both plates are incubated at 37 degrees C overnight. Experimental evidence shows that there can be as much as a 50X increase in colonies on the respread dish.
It is possible to reproduce this experiment in the high school laboratory by using virulent lambda bacteriophage (lvir) and a strain of K12 E. coli such as YMC. The E. coli are spread on a plate of LB agar (5 X 105 cfu per plate) and incubated at 37 degrees C for a few hours to allow the formation of small colonies (these are not visible at this point). About 15 minutes prior to replica plating this dish, two other LB plates are spread with l vir (108 per plate). These plates are used to replica plate the original dish. This is accomplished by placing a piece of Whatman #3 filter paper on a 9 cm cylinder (anything will do--ust so that it is smaller than the bottom of the Petri dish) and pressing the inoculated plate against it. This plate is removed and each viral plate is pressed against the filter paper. One of these plates is respread. Both plates are incubated overnight and the results observed.
I. State the problem - are mutations preexisting or acquired?
II. Display transparency 1
A. Students complete the if...then statements.
- If...then statements create a common format for the presentation of hypotheses
a.If bacteria develop resistance to phage as a result of exposure to the phage then both dishes will have the same number of colonies present after one day of incubation.
b. If bacteria have preexisting mutations for resistance to the phage then the respread dish will have more colonies than the unspread dish after one day of incubation.
III. Display transparency 2
A. Interpret the results
- The resistance/immunity is due to preexisting mutations within the bacteria.
- If acquired mutations occurred, they should occur at the same frequency in each plate.
Note: the spontaneous mutation rate for E. coli is about 1 in 10-7 per cell division.
B. The flaw of the experiment, as presented to the students, is the lack of a control.
- One replica plate is needed that is not respread and is not sprayed with phage.
- One replica plate is needed that is respread and is not sprayed with phage.