Microbiology, part 38: Genetics - Replica Plating & Ames Test

Hi, I'm Cathy with Level Up RN. In this video, I will be doing a brief review of replica plating and the Ames test. And at the end of the video, I'm going to give you guys a little quiz to test your understanding of some of the key facts I'll be covering, so definitely stay tuned for that. And if you have our Level Up RN microbiology flashcards, go ahead and pull out your flashcards so you can follow along with me. Replica plating is a technique used to identify bacterial mutants. Specifically, it is used to identify auxotrophs, which are bacteria that are unable to synthesize a required nutrient due to a genetic mutation. A wild-type bacterial cell is a cell that does not have the mutation. A wild-type cell is able to grow on a medium that lacks the nutrient because it can synthesize it itself. However, since an auxotroph cannot synthesize the nutrient, it will be unable to grow in a medium that lacks the nutrient. Let's take a look at an example of a replica plating procedure. In this particular example, the nutrient that the auxotroph cannot synthesize is histidine. Bacterial cells are first removed from a master plate using sterile velvet. This sterile velvet is then pressed on to a nutritionally complete plate, so a plate containing histidine, as well as a nutritionally incomplete plate, so a plate lacking histidine. Colonies are placed in the exact same position on the two plates for easy comparison after incubation. Next, we incubate the plates. And after incubation, you can see that the auxotrophic mutant was unable to grow on the plate that was lacking histidine. In order to study this auxotroph further, researchers can obtain a sample of the colony at the same location on the nutritionally complete plate.

Next, we have the Ames test, which is a screening test used to determine if a specific chemical can cause mutations in bacteria. If it does cause mutations, it may also be carcinogenic, which means cancer-causing. Let's take a look at an example of an Ames test. The Ames test uses a mutated strain of salmonella that is unable to synthesize histidine. During the test, two plates are prepared without histidine, an experimental plate and a control plate. Bacteria that are exposed to the chemical in question are placed on the experimental plate, and bacteria that are not exposed to the chemical are placed on the control plate. Both plates are then incubated. If the chemical is mutagenic, meaning it can cause mutations, it will cause some of the bacteria on the experimental plate to gain the ability to synthesize histidine, which means that the bacteria will grow despite the fact that there is no histidine in the medium, which is an indication that they are mutants. And you can see in this example that we have a lot of bacterial growth on the experimental plate. The number of colonies on the experimental plate as compared to the control plate indicates the degree to which the chemical is mutagenic.

All right. It's quiz time, and I have four questions for you. Question number one. What do you call bacteria that are unable to synthesize a required nutrient due to a genetic mutation? The answer is auxotrophs. Number two. In replica plating, auxotrophic mutants are unable to grow on the nutritionally incomplete medium. True or false? The answer is true. Number three. Which test screens chemicals to see if they can cause mutations in bacteria? The answer is the Ames test. And number four. If a chemical is mutagenic, it is more likely to be carcinogenic. True or false? The answer is true. All right. That's it for this video. Hope you did great with that quiz, and I hope you found this video to be helpful. Take care and good luck with studying.

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