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In this practical you will be using the fluctuation test to determine the mutagenicity of some common laboratory chemicals.  The Fluctuation test is carried out in liquid media, is more sensitive than the Ames test and allows mutation rates to be calculated. In this practical you will be assessing resistance to the antibiotic rifampicin which can be caused by spontaneous point mutations within the rpoB gene in E. coli.  The number of spontaneous rifampicin resistant mutants produced can be used to calculate the mutation rate.  The level of rifampicin used will allow only limited growth of the bacteria.

If there is a mutation giving rifampicin resistance more cells will grow and this can be detected by the addition of a pH indicator.

The pH indicator will show yellow when positive and negatives blue/green.

An increase in the number of positives compared to control cultures indicates that the test compound is mutagenic.

You will be using a miniaturised version of the test which is carried out in Microtitre plates. For each dose of each chemical 6 replicates should be set up.

You will calculate the mutation rate of E. coli grown in the presence of three potential mutagens, listed below.

: Incubation of E. coli with potential mutagens

•    You will work in groups of  5.

•    You will use E. Coli grown in one of two rifampicin concentrations (50 or 100μgml-1 depending on your group.

•    Each group will test three mutagens (either A, B or C), each at three concentrations (0.1, 1.0 and 10µg/mL).

•    You will be provided with the mutagen stock of 100 μgml-1 , you will need to make up stocks of 10μgml-1  and 1μgml-1  before you start.

•    Each group will also need to set up a control comprising the solvents used to dissolve mutagens (ie, either water or 14% ethanol).

•    The Table below gives the set up for each mutagen and other reagents. You need to add the volumes of each constituent and media to be combined to make up the 2 ml (2,000µL) total volume required. Mix each solution thoroughly.

•    Once the media is made up The 2 ml will be used to set up miniaturised versions of the tests using a microtitre plate.

•    For each mutagen concentration pipette 200 µl into 6 replicate wells and then incubate at 37oC for 48 hours as in the Table 2 below.

•    The cultures will then be stored in the cold and the following week between 12-1pm 10 μl of pH indicator (bromothymol blue).

•    Class results will later be pooled.