Determine the safest dye to use in the lab based on the results obtained from ames test for each mutagen

Question

Scenario

You are in charge of a new forensics laboratory and you need to decide the best method to conduct DNA analysis. DNA analysis is conducted by gel electrophoresis, which allows the technician to sort DNA fragments by size. Since DNA is not coloured, a dye that can bind to DNA must be used to visualize the fragments. However, dyes that bind to DNA tend to be mutagenic. Therefore, you must use the Ames Assay to determine which of the provided dyes are safest for laboratory use.

You have been provided with the following protocol:

Materials

• 4 Ames agar plates

• 95% ethanol

• Sterile, distilled water

• Salmonella typhimurium (His-ve)

• Sterile filter disks

Mutagens

• Ethidium bromide (10 µg/ml)

• EvaGreen (10 µg/ml)

• SYBR Green (10 µg/ml)

Method

• Sterilise the spreader and forceps with 95% ethanol.

• Place 2-3 drops of bacterial culture on each plate and spread well with a sterile spreader.

• Using the sterile forceps, place a sterile filter disk on the center of each plate.

• Add a drop of sterile water to the filter paper on the control plate, a drop of ethidium bromide to another plate, a drop of EvaGreen to another plate, and a drop of SYBR green to another plate. The sterile water is used as a negative control for the experiment.

• Incubate the plates at 37°C for 48 hours

After incubation, the plates appeared as below:

1) water - 5 counts

2) EvaGreen - 5 counts

3) Ethidium bromide- 11counts

4)SYBR geeen1 - 28 counts

Use the above findings to identify the safest dye to use in the laboratory and give the reason for your answer and Present the results using tables or graphs.

Answer 1

The Ames test seeks to identify mutagens, by seeing if the compound is able to increase the number of mutations in bacteria. This is done by growing His- Salmonella, which require Histidine to grow. Even with sterile water added, a small number of bacteria will naturally mutate to no longer require histidine to grow. That is, their DNA mutates to regain the ability to synthesize their own histidine. However, when mutagens are added, the amount of DNA mutations will increase dramatically, and so too will the number of colonies that appear on the plate. The more bacteria regain their independence from histidine, the more counts will be observed on the plate with the mutagen.

Here, Ethidium Bromide and SYBR Green are clear examples of mutagens, with EtBr containing over double the amount of counts as control water at 11, while SYBR Green over 5 times as many. However, EvaGreen has the exact same number of counts as the control water with only 5 counts. This means that EvaGreen is not a mutagen, and is therefore the safest dye to choose to work with.