Budding Scientists collaborative: The Hunt for New Antimicrobials (Term 3, 2017)
This term we were looking at Antimicrobial Biodiscovery. We were looking to discover new antimicrobials which are chemicals made by microbes. Other microbes are developing resistance to the antimicrobial we use now, which is why we need to discover new ones. We focussed on actinomycetes, a type of bacteria found in dirt that produces antimicrobials.
Antimicrobials and actinomycetes (bacteria in fungal clothing)
What is an ANTIMICROBIAL?
ANTIMICROBIALS are the chemicals that organisms produce to kill microbes. Antibiotics are actually ANTIBACTERIALS which, as their name suggests, kill bacteria and bacteria ONLY. Fungi are not bacteria, so are only killed by ANTIFUNGALS.
What is an actinomycete?
Actinomycetes are bacteria that can be found in many different environments, e.g. water, both salt and fresh water. Another feature that makes them so interesting is that they can grow using hyphae, which makes them a bit like fungi. This was a feature that we tried to use to our advantage with our traps.
FUNGI VS BACTERIA
Who’s better? Fungi or Bacteria?
Well, what is a fungus? A fungus (aka mould) is a microbe and is found in many places. Some places could be spoilt food, damp areas and many more. A fungus has a very unique appearance. Most fungi look a bit fuzzy. Fungi come in many different colours and some fungi are even toxic. Anyway, enough about fungi, more about bacteria… Bacteria (aka germs) are microscopic and an individual cell can’t be seen by the naked eye. But bacteria like to grow together to make a colony which can be seen by the naked eye. The appearance of bacteria is different from fungi. Unlike fungi bacteria have more of a glossy look when growing on agar. The main bacteria we were interested in for this work was actinomycetes, which like fungi can produce antimicrobials. Actinomycetes almost try to role play as fungi for actinomycetes look a little bit fuzzy. Well, who would you choose as the best antimicrobial producers? The great fungi, the amazing bacteria or the “in-between” fantastic actinomycetes?
What we did
At the start of this term we set out to trap some actinomycetes from a number of different types of soils.
We experimented with several different traps that were specifically designed to capture actinomycetes. Most of these traps utilised a membrane that had such a small pore size that only the hyphae of these actinomycetes could grow or fit through. The first trap was adapted from a trap described in a scientific paper published in 2008 by Gavrish et al. “A trap for in situ cultivation of actinobacteria”. This trap did not work properly, as the plate became overrun by fungi. We then adapted the trap, in an attempt to fix this issue. However, our second trap was unsuccessful too. Eventually we decided to use a serial dilution to isolate some cultures directly onto starch agar and this worked! We finally had some actinomycetes to test for antimicrobial production!
How we did it
We taped a piece of foil over a metal washer and then pipetted starch agar into the hole in the metal washer. We placed a thin membrane over the agar. The membrane side went on top of the collected dirt samples, and had tiny microscopic holes so that the actinomyces hyphae could get through but not the thicker fugal hyphae.
Then we incubated our traps for one week, at 25 degree C. When we took our traps out to look at, they weren’t very successful. Most of the agar either shrunk or didn’t grow anything so we rethought it and designed a new trap, Trap 2.
As the first trap was fiddly and didn’t turn out well, we tried another version of the first trap. The agar in the first trap was not enough and dried out quickly, so we used more agar in the second trap. The foil lid for the first trap was fiddly to manoeuvre, so the second trap had the foil taped on. This helped us fold it over more easily. We filled it with agar. The thin membrane had microscopic holes that would hopefully only trap actinomycetes. We also added an anti-fungal called cycloheximide to prevent fungi growing.
We placed the traps on top of our dirt samples which were spread evenly over the agar plate. We then incubated the traps at 25 degrees C for one week. However, the second trap wasn’t that successful either and didn’t have actinomycetes. With our second trap, some of the agar still ended up having fungi around the edges on the outside. Either way, the isolation did not work and neither did the trap.
This term we used lots of different techniques such as patch plating, lawn plating and the dilution series, and had to master these.
What is patch plating?
Patch plating is when you draw up a grid on the bottom of your plate then number it and put the selected microbes you want to have in your grid (Figure 3).
To do a patch plate you need a marker, agar plate, toothpicks and a discard container.
What is lawn plating?
Lawn plating is when you get a swab and spread a microbe of choice (Figure 4). In our case it was different Tester bacteria.
To do a lawn plate you need a sterile cotton swab, an agar plate and a microbe of your choice.
FIGURE 5: WE COMBINED LAWN PLATING AND PATCH PLATES TO CHECK FOR ZONES OF INHIBITION TO SEE IF ANY OF OUR SOIL ISOLATES PRODUCED ANTIMICROBIALS.
What is a serial dilution?
The dilution series is when you get a substance, like soil, and put it in tubes and then put water in and then dilute it by making the soil less concentrated (Figure 6). We use this method because it can make sure that there are not too many microbes on each plate. From each dilution tube we took a sample out and put it on a spread plate.
You need a pipettor, tubes and diluent.
Lastly, we worked on our skills with isolating directly from soil using dilution series, spread plates, patch plates, lawn plates, lawn-patch plates and wet prep slides. We always worked to the best of our ability, and we finally saw results of antimicrobial production by actinomycetes!
What we found
In the beginning of this short journey we wanted to see the difference between wet, dry, shady and sunny dirt and the impact of that on actinomyces. Once we started our traps, we realised that it did! Some of our traps didn’t work too well because the dirt was too dry or too wet. In our first trap our agar dried up when we had dry soils and this caused the trap not to work well.
But we also discovered that having wet dirt did not produce as many actinomycetes. The shadiness or sunniness of the dirt did not really matter, but the wet or dryness of the dirt impacted the way the trap worked and the actinomycetes we were able to isolate.
In the end we isolated 10 different actinomycetes from three soil samples. The soils collected by Josh, Jemma and Dieter had the best results, but we all shared and saw some of these actinomycetes causing inhibition by producing antimicrobials against some other bacteria.
So, in conclusion, from this term, we now know what texture and place to collect from and learning new ways of creating traps and the best way to isolate actinomycetes. However, one thing stood out for all of us, was, how much fun it was to do this amazing and fun experience and educational short journey.
All content produced by The Budding Scientists collaborative of Aoife, Dieter, Eshwari, Jemma, Josh, Kieran, Lachlan, Natalia, Natasia, Pranav, Tom, Tori and Varun.