There are a lot of steps involved in identifying bacteria. First, you need to get a sample, inoculate it, isolate it, and then break open the cells to extract the DNA from the cells (I will explain what these words mean. Don't worry). Once the DNA is collected, we amplify it via Polymerase Chain Reaction (PCR).
Steps Before PCR: Sample collection, Inoculation, Isolation, DNA extraction
Sample Collection
Here, we find a nice place to collect some soil (dirt) to analyze in the lab. Grab some extra soil for some yummy mud pies.
Inoculation
Inoculating is nerd talk for placing bacteria on a plate where it can grow; to do this, we mix the dirt with water because we want the yummy bacteria growing in the soil.
Soil is home to a lot of bacteria, so we move small amounts of soil solution to the following tube, and then we plate the samples from each tube on a plate (the image below will explain this better than I can). Serial dilutions
Look at all the colonies, yuck!
Isolation
After we get bacteria on plates, we want to remove the ones of interest and have them grow on their own plate. Now, we need to confirm that only one type of bacteria is present. Here's how to do it properly
DNA extraction
Once only one bacteria is present on the plate, we can take a sample and extract the DNA from it.
Cell harvest: our pure sample
Cell lysis: breaking of the cell membrane
Protein removal: exactly that
DNA binding-DNA elution: we have our DNA
POLYMERASE CHAIN REACTION
We now know the steps that precede PCR. Why do we do PCR? We do PCR because it is convenient because it can take a small sample of DNA and multiply it exponentially in a short period of time. I'M TALKING BILLIONS OF COPIES! Most of the time, PCR copies small sections of DNA that help distinguish this sample from others (like a fingerprint). For bacteria, this is the 16s gene.
Now that we have established why PCR is cool. Let's discover how it works! We'll start by mentioning the components involved. To perform PCR, you need a thermocycler, taq polymerase, and primers that attach to the region you want to copy. What the heck are those!? A thermocycler is like an instant pot --- you can just hit start, and it'll take care of the rest. A thermocycler adjusts the temperature automatically during PCR. Taq polymerase is a protein that is heat stable and replicates DNA. Primers are pieces of DNA that bind to the ends of the region we want to copy.
The three major steps in PCR are denaturation, Annealing, and Extension.
Denaturation
In this step, the strands of DNA separate by heat energy. In this step, the temperature is 96 °C. We can now primers can now attach.
Annealing
Primers bind specifically to the ends of the DNA strands. The temperature at this step is 55 °C. We are ready to copy.
Extension
Taq polymerase copies the DNA from the ends of the primers. The temperature during extension is 72 °C. We have completed one cycle of PCR. We had one DNA strand from our sample, and now we have two.
But wait, there's more
PCR can run about 30 cycles in a few hours, making over A BILLION COPIES! This can help forensic investigations (see, I told you PCR is cool) or bacterial identification; you might have a limited sample from a crime scene or a limited sample of a sneaky pathogen. But, with PCR a limited sample is all you need!
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