Moving on from our hypotheses, we need to get a better feel for the tools that we can use in our experiments. The advantages of using bacteria like E. coli is that they are clonal organisms, they have a very short generation time, and they are easily cultivated. So let's put these characteristics to work.
First, we need to go back the opening post. As I stressed, the entire experiment started with a single bacterium that formed a single colony. What we do next is transfer this single colony to some liquid culture and let them multiply overnight. This will give us a ton of bacteria to work with, and they will still all share a common ancestor. However, these bacteria have been accumulating mutations as they replicate so we will have a genetically diverse population over these many generations that have occurred since we founded that original colony with a single bacterium.
As noted in the post above, E. coli don't have legs. When a founding bacterium is placed on a plate the descendants stay right next to the founder. Think of it like the children of farmers using the land adjacent to their parent's farm. As long as you keep the founders far enough apart you will get nice separate colonies like those in the picture above. However, if you put the founders close together you will get a "lawn" of bacteria where the borders of one colony butt up against the colony next to it. What you get is a continuous layer of bacteria much like your lawn of grass at home, and this plate of bacteria will now be referred to as the
master plate. The master plate will contain colonies founded by genetically diverse descendants of a single bacterium.
Now we will introduce replica plating. This process can be described as bacteria stamping where our bacterial lawn serves as our ink pad. What you do is take your round pad that is just a little smaller than the diameter of the plate and press it down on the bacterial lawn.
Once you have "copied" the master plate you stamp it onto the
replica plates making sure to record the orientation of the stamp so you can keep track of where the bacteria came from on the original master plate.
For this experiment, we create a master plate on media without antibiotic. We then replica plate onto media containing antibiotics.
What predictions do each of these hypotheses make?
1. Random mutations: If resistance is due to mutations that occur prior to antibiotic exposure then they will already exist on the master plate, and they will be clonal. Therefore, we should find resistant colonies on the replica plates at the same position on each replica plate since they came from the same position on the master plate.
2 and 3. Non-random mutations and/or resistance induction: Since each bacteria has the same chance or same mechanism of producing resistance we should NOT see colonies appear at the same position on each of the replica plates.
What are the results? We see colonies at the same position on each of the replica plates. This is consistent with random mutations, and inconsistent with non-random mutations or induced resistance.
Any questions?