In this video, we're going to begin our lesson on replica plating. Replica plating is a specific technique that's used to indirectly select for non-selectable mutants, which are mutants that cannot be directly selected for. Examples of non-selectable mutants are auxotrophs. Replica plating involves a series of 3 steps that we have numbered down below, 1, 2, and 3. The numbers that you see here in the text correspond with the numbers that you see down below in the image as well. That's important to keep in mind as we move forward.

In the very first step of replica plating, you need to plate both the auxotroph mutants as well as the non-mutant prototrophs. You plate these auxotrophs and prototrophs onto what's known as a master plate. Then you take the master plate and physically press the master plate onto sterile velvet fabric. The velvet fabric will then contain the auxotrophs and prototrophs. If we take a look at our step number 1 down below in our image, notice it's showing the master plate in orange. The master plate contains both the auxotrophs, which are in purple here, as well as the prototrophs, which are in blue here. After growing both the auxotrophs and prototrophs onto the master plate, you will physically press the master plate onto this sterile velvet. That leaves a colony imprint on the velvet. You can now see the colonies on this velvet, as shown here.

After the first step, you move on to the second step. In the second step of replica plating, use the velvet fabric to inoculate 2 replicate agar plates. These 2 replicate agar plates are going to be, one a nutrient agar plate, and the other a minimal media. The nutrient agar contains additional growth factors that allow auxotroph mutants to grow. The minimal media contain only the bare minimum needed for the prototrophs to grow. The auxotrophs will not be able to grow on the minimal media. If we take a look at our step number 2 down below right here, notice that you take a nutrient agar plate and physically press it down onto the velvet to transfer the colonies over to the nutrient agar plate. Do the same with the minimal media plate, pressing it down onto the velvet and, again, transfer the colonies onto the minimal media.

This takes us to step number 3, where the plates will be incubated and then observed. The prototrophs, which are non-mutants, will be able to grow on both the nutrient agar plate, as well as the minimal media plate. However, the auxotroph mutants will only be able to grow on the nutrient agar plate, and the auxotrophs will not grow on the minimal media. If we take a look at our image down below, what you'll notice is that the nutrient agar plate will contain both prototrophs as well as the auxotrophs, and so both of these will grow as seen here on the nutrient agar. However, on the minimal media which lacks additional growth factors needed for the auxotrophs, only the prototrophs grow. The missing positions, these little dotted circles that you see here, represent the missing auxotrophs. By the positions of these missing auxotrophs, you can trace them back to these positions in the nutrient agar. Then you know that these colonies here growing on the nutrient agar must be the mutant auxotrophs.

At this point, you could select these mutant auxotrophs. They have been indirectly selected for by replica plating, creating these 2 replicate plates. This here concludes our brief lesson on replica plating and how it can be used as a technique to indirectly select for non-selectable mutants such as auxotrophs. We'll be able to get some practice applying these concepts as we move forward, so I'll see you all in our next video.