This is a follow up to this post, looking at what things are growing on our plates (and hopefully out of our samples). We didn’t use antibiotics in our PDA growth media, so we expected some bacterial growth as chances are high that they would outgrow the fungi.
We were not disappointed, many of our samples showed both bacterial and fungal growth, unfortunately they also showed a lot of contamination. We didn’t have access to a laminar flow hood during the plating phase so we just used a still air hood (basically a clean chemical hood with the blower turned off to minimize air flow.
We are chemists, remember, not really microbiologists, though we are expanding our skill-set and knowledge fast!
Either way, not all of our samples were contaminated, and our surface sterilization protocol seemed to work very well on leaves, but not on the bark of stems. This makes more sense, as bark is much rougher and has more nooks and crevices for spores or bacteria to hide and escape the bleach or ethanol, especially since a wetting agent was not used.
Notice here in the control plate (sorry for the glare) that the leaf press after surface sterilization showed no contamination, whereas the press of the bark sample even after surface sterilization showed a few fungal colonies. This gave some credit to our sterilization protocol, but we have to re-think how we will deal with stem and bark samples in the future. The most likely way will be more agitation, a longer soak time, and a wetting agent.
Above are my two stem samples, the only samples I plated that showed some fungal growth on or around the excised piece of plant matter. The leaf samples I plated have only shown yeast/bacteria. I admit, having never done this before, I was quite happy to see anything growing at all, but I was especially excited to see what looked like two distinctly colored mycelia growing out of the sample on the right. That third one with the green center looks to be contamination. As for the sample on the left, that may also be growing from something that wasn’t killed during surface sterilization.
One thing I have learned is…no longer am I going to plate the brown bark/stem itself, but rather remove that and plate the soft woody tissue right below it.
Here are the samples a few days later.
I cannot say for sure whether anything that has grown was endophytic, which is what I was hoping for, or if everything was just contamination or unsterilized surface organisms. Either way, I am amazed at how beautiful some of these fungal colonies look, as well as the brilliant colors produced by some of the bacterial or yeast colonies on some other plates.
Take for example my control plate. After a while whatever was growing on the “stem” side decided to spread across to the “leaf” side, leaving behind what looks like a sun exploding with some planets nearby and a sprinkling of stars in the background.
Anyone can pick up a book or search the internet and figure out that the diversity of microorganisms in our environment (and on us) is immense, but there is something about actually growing the organisms yourself, and finding things you never intended to find, which made the whole process really gratifying and exciting.
I may not switch to becoming a full-time microbiologist, but I am definitely starting to look at the world through new eyes, because now I want to sample and culture every plant, leaf, flower, doorknob & cellphone I see – to figure out what is growing on or in it!
Here are a few extra photos of some leaf samples that showed no growth, and one that showed a lot of growth…of something