Question

Regarding the Ames test for the mutagenic potential of chemicals:

1) Why do we use positive and negative controls?

2) Why do we do a serial dilution?

Thank you so much in advance!

Answer 1

AMES test was developed in 1970 by Bruce Ames to study the chemicals which can induce mutation in DNA strand. It is inexpensive easy and fast technique.

USE OF POSITIVE AND NEGATIVE CONTROL IN AMES TEST :

AME'S test uses Salomonella bacteria as a method to culture and form colonies with Mutagenic compounds which are being tested.

1.Here in case of AMES test a positive control consist of known mutagen capable of inducing mutagenicity ( ability to cause mutation ) in the bacteria enabling the cells to grow and form colonies. Hence called as positive control for AMES test.

2. Negative control uses specific strain of salmonella that cannot produce histidine. These are added to control plate and medium containing small amount of Histidine. A few colonies will form indicates reversal natural back mutation happened.

Histogram of AMES test shows positive and negative control with and without mix of mutagens for each bacterial strains of salmonella. Salmonella strains were used to study mutagenic activity of several pesticides.

2. We use serial dilution in AMES test to quantitatively determine the mutagenicity of a chemical which is tested which is directly proportionate to the number of colonies formed of bacteria.

The number of colonies growing on the growth medium indicates the number of revertants. In a true AMES test a variety of concentrations of each chemical would be tested to generate dose response curve. we will count the number of colonies formed which is known as Colony Forming Unit (CFU) and record observations of serial dilution test.

  
At different concentration of mutagens different colonies of bacteria will be formed on growth medium which are recorded relative to unit ML of dilution. In this way series of dilutions are performed relative to concentration factor of mutagen and CFU measurements are recorded based on dose response curve.