Understanding Mutation and Selection Using the Fluctuation

Test
Emily Cribas
Hypothesis. Mutations arise spontaneously and do not depend on selection (by
the environment)
Prediction. All tubes will have a higher number of mutations, therefore, there
will be a significantly high variance of mutations between each tube and overall,
as a class.
Treatment and Controls. 100 L of E. coli and 5 mL of nutrient broth were
individually prepared and placed in 5 separate tubes. After this,100 L of T4
bacteriophage was spread onto 5 LB plates each. 50 L of each bacterial culture
was added to each plate after the phage completely dried, and incubated at 37 C
for 18 hours and then placed in 4 C afterwards.
As a control, a common culture of bacteria was used, as well, for the entire
class to use to quantify sampling error (limited pipette accuracy, non homogenous
bacteria, lost cells, etc.) and compare the variance from these plates to those of
the individually prepared plates. If the variance between the two is very different,
mutations arise spontaneously and are not induced.
The mutants that formed after were mixed with top agar and placed on another
LB plate. The same was done with the original wild type strain to support the idea
that what grew on the plates were actually mutants and not something else (contamination). Serial dilutions for T4 bacteriophage (up to 107 ) were conducted
and each were placed in part of each plate to see how resistant the strains were
versus the original bacteria.
Results. Individually, my plates displayed the following mutant colonies:
Table 1. My Results
Individual Culture Results
Plate
1
2
3
4
5

Common Culture

# Colonies

Plate # Colonies

N/A
66
51
153
42

1
2
3
1

287
258
109

The following equation was used to determine how much the number of colonies
varied per plate as a class (a more accurate measure)
Variance Equation:
(X )2
N
Where X is the individual measurement, is the (class) average, and N is the total
number of plates used.
Based on this equation, the results of the variance for the common culture and
individual cultures were calculated using excel. The results are:
Class Results
Variance Common Culture Variance Independent Culture
7,350.97
16,078.67
After the wild type and mutant strains were replated, the first LB plate with
the normal bacterial strain only grew on the 107 T4 dilution, while the mutant
plate showed that the bacteria grew everywhere.
Conclusions. The discrepancy between the variance values of the common culture and the individual cultures, as a class, is quite large. Variance for the independent cultures was expected, but if the mutations were induced, they would
resemble the variance values for the common culture and the number of colonies
should not differ by much. However, the variance was much higher (more than
twice as large), meaning that the difference in mutants per plate could not be
attributed to just sampling error and the mutations were not induced, but arose
spontaneously.
Replating the wild type and the mutant strain on an LB plate afterwards indicated that the growth from the other plates were indeed mutant strains, seeing as
they were resistant to the bacteriophage that wild type was not, unless exposed
to very diluted phage.
Individually, I didnt get any colonies on my first plate, most likely because of
a fundamental mistake when setting up the plates. If done again, I would have
waited longer for the bacteriophage to absorb into the LB plate surface for each
plate to make sure the phage sufficiently infected the bacteria, which could have
reduced my number of colonies in total or resulted in non mutant bacteria to be
seen on the plate as well.

Key Question. One of the key questions the results from this experiment answers is the following:
Some bacterial strains have much higher mutation rates than other strains.
What would be the benefits to the bacteria of a high mutation rate? What would
be the downside to a high mutation rate?!
Having a high mutation rate is beneficial if the mutations favor survival and
enhance the species ability to grow and reproduce. They have the opportunity
to evolve faster and can develop resistance faster as well, as with HIV. However,
a high mutation rate can have hugely detrimental effects as well. For example, a
virus or bacteria with a specific genome could develop mutations in their sequence
that could lead to a loss of function of essential replicative proteins that could
potentially lead to its death.