What is the Ames Test?

In the science article we read discussing the airborne mutagens in fried meats there were many procedures that lacked a detailed explanation, and instead, referred us to other articles that explained the procedure more in depth. I felt I would do my part in explaining one of those procedures in order to save us the hassle of looking up the reference article.

On page 823 under the heading Mutagenicity the author of this article described the test they used to determine the mutagenicity of each substance tested. The author references Ames et al., 1975, and this was an article that was published to demonstrate a remarkable way of testing possible mutagens at a very low cost. What the Ames test does is use a strain of Salmonella typhimurium where every gene is normal except the gene for producing histidine (His). The His gene is mutated; therefore, the strain is unable to produce its own histidine, a vital compound needed for the survival of the bacterium. The bacteria is spread out in a petri dish as a lawn, or a homogeneous spread, upon a minimal glucose agar gel. This means that the gel only contains glucose, which will be used as the bacteria's energy source. However, since the gel does not contain histidine and the bacteria can not produce its own histidine (because that gene is mutated) the bacteria is unable to grow and reproduce, but the bacteria is capable of producing all other essential nutrients because none of those genes are mutated. Next, the substance being tested is placed in the center of the lawn, the petri dish is closed, and the dish is left to sit. After the allotted time has passed any growth that has occurred in the dish is recorded because this means that if the bacteria were able to grow than a "reverse" mutation must have occurred to allow for His production. A reverse mutation is when an already mutated gene mutates again back to its functioning self.

One colony of growth is called a revertent, and it represents one bacterium on the original lawn that underwent a reverse mutation. You may say but wait, a colony contains millions of bacteria so why does it only represent one bacterium from the lawn. The answer is because bacteria reproduce by binary fission, which means one cell splits equally into two identical cells. Therefore, if you extrapolate the colony backwards to its original cell you will find that only one mutation occurred to allow the colony to grow.

There are many observations that can be made from the pattern of growth on the plate. The first, is the shear number of colonies which is directly correlated to the mutagenicity of the substance tested. This was what the researchers of this article measured in there experiments and reported as revertents. A second observation can give you an idea of the toxicity of the substance. This observation is called the halo effect. When you place the substance in the center of the dish many times bacterial growth will not occur directly next to the substance, but instead a halo will form around the substance because it is too concentrated to allow for bacterial growth. Therefore, the thicker the halo the more toxic the substance is, and as we discussed in class today there is a difference between mutagenicity and toxicity. For example, cyanide gas is toxic but not mutagenic.

The Ames test has proven an invaluable tool in testing the mutagenicity of substances, but it is important to note that not all substances that test positive with the Ames test will cause mutations in the human genome. However, this is a low cost and effective way to determine possible mutagens. The remarkable thing about this test is it is so much more reliable than using genotypically normal bacteria and seeing how many bacteria die. First, because it is much harder to determine how many cells actually died because they are so small and numerous. And second, because it would be difficult to say with certainty that the bacteria died as a result of the substance being tested or some other variable beyond the researchers control.