Antibiotics are essential tools for fighting bacterial infections. The process for discovering and creating them is quite a complicated process. Below I will detail some of the steps that an undergraduate student could take to discover their own antibiotic producing bacteria.
The first step is the actual collection of bacteria. Most samples are taken from soils, and it's a great idea to try to be creative and find a sample site that is unique and hasn't been used before! It's also important to ensure that the site you're collecting from has given you permission for soil collection!
Serial Dilution
Once you have collected your soils, you're going to need to perform serial dilution. Serial dilution involves using sterile water to progressively dilute your sample. I recommend diluting to the 10^-1, 10^-2.... all the way to 10^-5. Once you have your five samples, you can plate each of them on a large LB plate. Once you allow them to incubate and grow for 24 hours, you'll have grown your first bacteria!
Creating a Library Plate
The next step is to choose 16 different bacterial colonies that you're interested in. Try choosing some that have zones of inhibition or look unique. It's important to look for certain characteristics like texture, color, size, and shape in each of the colonies. Then carefully transfer those colonies you selected onto a separate library plate that you've created! Make sure to label each colony so you can remember which bacteria is which.
After your library plate incubates for 24 hours, it should look something like the image here. After your library plate is grown, you'll need to test the colonies against certain bacteria to see if your colonies inhibit their growth. The bacteria used are commonly known as ESKAPE bacteria, however, for safety purposes we will use ESKAPE safe relatives. ESKAPE includes several common bacterial strands that can cause various diseases.
Creating Screening Plates
Choose 2 different ESKAPE safe relatives to initially screen your colonies against. Create the 2 screening plates and incubate them again to observe your results. A screening plate will look identical to your library plate but will have a thin film of your chosen ESKAPE safe relative across its surface.
When viewing the results of the screening plates, look for areas in the ESKAPE safe relatives' film that appear to have been inhibited. The bacteria colonies that you see inhibiting the ESKAPE safe relative are producing antibiotics that are effective against those ESKAPE safe relatives. These will be the colonies that you will continue forward with.
Quad Streaks & Maintenance Lawns
The next step is ensuring that your colonies are truly isolated. To verify this, it will take a few new techniques. Using a loop, create quad streaks of the colonies that inhibited the ESKAPE safe relatives. Incubate these quad streaks for 24 hours.
After incubation, carefully pick a single colony of each isolate and create a maintenance lawn for each. After incubating the maintenance lawn for another 24 hours, create gram stains of each isolate and view them under a microscope. Under 100x magnification, you should be able to observe the morphology of your bacteria, as well as whether or not it is isolated. If the bacteria aren't isolated, then you'll need to start again with that isolate starting from quad streaking.
Further Testing
After each of your isolates are isolated, you need to test them against all ESKAPE safe relatives. This will give you a better idea of which of your selected isolates are the best antibiotic producers. It's important to carefully record your results throughout each step.
There are a couple more steps from this point, such as conducting biochemical testing, and glycerol stocking. Conducting various biochemical steps is important to determine certain biochemical traits of your bacteria, and glycerol stocking is important to preserve your bacteria in case it becomes an actual candidate for an antibiotic. Further steps involve DNA sequencing and sending the bacteria out for further testing.
Purpose
The discovery of antibiotics is extremely important for the future health of humanity. Bacteria constantly develops antibiotic resistance that makes antibiotics useless in infections. This article by the WHO (World Health Organizaton) details the current crisis, as well as what's causing it and what steps we need to take to combat it.
The WHO identifies antibiotic resistance as a major global health threat, responsible for approximately 1.27 million deaths in 2019. The primary cause of antibiotic resistance stems from misuse and overuse of antibiotics on humans, plants, and animals. It's a crisis that affects all regions and economic levels, especially middle-income countries.
Antibiotic resistance threatens to slow medical advances, making it harder to treat infections and increased risks in surgeries and cancer treatments. There is also a serious economic burden that is being incurred, with a projected $1 trillion increase in healthcare expenses by 2050.
To address this issue, it would require a coordinated global effort to reduce unnecessary use of antibiotics, as well as research into new ways to combat antibiotic resistance.
-jasen
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