The Ames/Salmonella Test: A Key to Our Research
Our success in detecting and identifying mutagens in cooked foods is made possible by the interplay of many different types of chemical analyses, including chromatography and mass spectrometry (Figure 3), and biological methods. The Ames test is an exquisitely sensitive biological method for measuring the mutagenic potency of chemical substances. The Ames test by itself does not demonstrate cancer risk; however, mutagenic potency in this test does correlate with the carcinogenic potency for many chemicals in rodents.
The test was developed in 1975 by Bruce Ames and his colleagues at The University of California at Berkeley. The Ames method is based on inducing growth in genetically altered strains of the bacterium Salmonella typhimurium. To grow, the special strains need the amino acid histidine. However, when the chemical agent (mutagen) that is being studied is given to bacteria, some of the altered Salmonella undergo mutations. Following a particular type of mutation, the bacteria can grow like the original "wild" (unaltered) strains without histidine. Because the mutant bacteria revert to their original character with regard to the nutrient histidine, they are called "revertants."
The Ames test yields a number--specifically, the number of growing bacterial colonies--which is a measure of the mutagenic activity (potency) of a treatment chemical. This value is often expressed as the number of revertants per microgram of a pure chemical (mutagen) or per gram of food containing that mutagen. Some pure mutagens result in hundreds of revertants per microgram, but many of the substances we have tested from cooked meat produce hundreds of thousands of revertants per microgram. For example, in one strain of bacterium, the PhIP mutagen results in about 2000 revertants per microgram, whereas another cooked food mutagen, IQ, results in 200,000. The illustration at the right shows how a food extruct is tested for its mutagenic activity.
In brief, a test begins by placing about 100 million Salmonella bacteria in a petri dish containing a nutrient agar lacking histidine. A few bacteria will spontaneously revert in the absence of mutagens. Counting these revertant colonies gives us a baseline against which to check the validity of our complex laboratory procedures. In a separate but essentially identical histidine-deficient petri dish, another batch of Salmonella bacteria are given a mutagen plus mammalian enzymes required for metabolism. (Adding such enzymes gives us a more realistic measure of the mutagenicity of a substance for mammals. The enzymes are typically supplied from liver cell extracts of rats given substances to increase levels of metabolizing enzymes.) Revertant bacteria grow into visible colonies. We simply count the colonies (equal to the number of revertants) after a standard time (48 hours) under standard growing conditions (37°C).
Different strains of altered Salmonella bacteria are available for the Ames test. The strains vary in sensitivity to specific mutagens. We used two strains, known as TA98 or TA100, for most of our recent work on fried beef and cooked grains. These strains were generously supplied by Bruce Ames.