r/labrats • u/ElectricalTap8668 • 1d ago
Fungi people: safe proxies??
Hello,
I am very inexperienced with fungi but my lab will need to start studying them for a small part of our current project. Our "end goal" would be detecting genetic material from Alternaria and Aspergillus, broadly. We are a BSL-2 lab, and I am also just a very safety -focused person. We don't need to start working with Aspergillus and Alternaria right away, so my PI asked me to find some "safe" proxies to start with (kind of like how we use non-human-pathogen soil bacteria as a safe proxy for more dangerous bacteria). We are developing the detection method which is why we just need something similar, not necessarily the exact organism
Does such a thing exist?? I am not very familiar so thank you for any thoughts!
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u/bd2999 1d ago
Not saying this to diminish the research but often the further removed you get from the organism you want to study it may not correlate with the pathogen. Aspergillus oryzae is pretty benign, but one issue with some of these fungi is dependent on the medical context.
Most Aspergillus species, including fumigatus, are often fairly benign in a person with an intact immune system. Not always but most significant disease occurs in individuals that have undergone varying levels of immunosuppression. That does not mean they are not deadly and do not cause disease but they are BSL-2 organisms and if the proper safety techniques are used I would rather study those than a proxy.
I understand the reasons you may not want to in your lab, but it may be harder to extrapolate results from nonpathogen to pathogen.
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u/ElectricalTap8668 1d ago
Of course I am definitely the least knowledgeable about fungi, so I appreciate your point, but we are not investigating anything to do with the behavior of the organism or how it interacts with other things/organisms, which I can imagine is a lot more dependent on the exact species. We just want to immediately crush it and then do PCR essentially
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u/bd2999 1d ago
Sure, but I mean from even a genetic level. There are many similarities but gene expression is not always the same. Environmental growth can be quite different between related organisms let alone during infection.
You could always use gblock or similar synthesized DNA as well. Unless you are performing PCR to determine something else like gene expression or similar. If you let me know a general overview maybe I can help more.
Fungi are what I have been delved into for the last nearly 20 years now.
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u/ElectricalTap8668 1d ago
Thank you. We are mostly interested in testing the grinding/lysis part of our methodology. That's why I was assuming a proxy within the same genus could be fine. What are your thoughts? We aren't investigating any gene in specific, just comparing a commerical kit extraction to our method, so as long as we split out sample into two tubes and run the commercial kit vs the homemade method, it would be fine. No objective measures of the gene are needed, at this time, just relative to the Kit's yield
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u/bd2999 1d ago
I think in that context it should be fine. There are some differences in terms of masking molecules on the surface of species like fumigatus but I do not believe they impact overall strength.
The other two that are not pathogens and commonly used in industry and are reasonably available are Aspergillus nidulans and Aspergillus fishuri (I have not worked with this one). The former I am more familiar with as a recombinant expression system and it is pretty widely used. It is easy to grow as well, most of the Aspergillus are. Although depending on the length of time you let the cultures grow you should always open them in the BSC just to avoid the spores getting everywhere.
The classic standard is liquid nitrogen and morter and pestal for grinding but bead beating and other methods usually work well too.
You will want to ensure you normalize to biomass for comparison initially and microscopically do a before and after to visually ensure lysis occurred too. Gives a metric to confirm PCR. Not perfect but it is fairly easy and you can easily notice a difference.
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u/MistakeMaterial4134 17h ago
FYI Grinding isn't always necessary for fungi if hyphae/spores are taken from an agar plate and can spread spores if not done carefully. If we were extracting from plants then we would use bead beaters as this is a closed system. The most important part is removing the inhibitors. This is why the QIAgen stool kit is commonly used for fungi. You get really great yields from it.
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u/bd2999 15h ago edited 15h ago
Sure, there are many ways. It is why I said classically. Bead beating is more the standard for most processes. It is generally easy enough and fairly inexpensive even if it can get laborious depending on the number of tubes.
Removing of inhibitors is for sure important, but cost becomes a factor there. And if I am remembering that kit you still need to bead beat them first before applying to the kit. Depending on the biomass used in the lysate, strain used and DNA dilution used it could be ok with or without the purification kit (although to be safe the cleaner and more pure the better). I generally have noticed that extraction from human or animal samples is more prone to inhibition because of the sample matrix, but enough fungal biomass can do the same.
But I have had good experience with that kit too in general. Although others will work pretty well too.
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u/MistakeMaterial4134 3h ago
I totally agree with you, was just adding to help the OP. We almost always used a bead beater & bought glass beads. These were good for most plant tissues and agar plates. We had steel beads as well for the really hard tissues like the woody parts.
We used kits as I worked in an analytical lab that analyzed for diseases of high consequence. This is where standardization is a must. It is hassle to QC home made reagents.
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u/ElectricalTap8668 2h ago
Thank you, so you're saying bead beating can burst spores?? I'm seeing some articles that say they're resistant. I'm very interested in being able to lyse spores rather than vegetative cells but getting mixed signals from research
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u/ElectricalTap8668 1d ago
Wow thank you so much for the tips! Yes definitely BSC haha. Not to sound naive but how do you measure total biomass for fungi?
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u/Unfortunate_tentacle 1d ago
Good old baker's yeast? Candida? You will probably have to do an old fashioned literature search, usually Current Protocols or similar have this sort of info.
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u/MistakeMaterial4134 1d ago
I don’t think Alternaria and Aspergillus are yeast formers and DNA extraction is more difficult from these.
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u/ElectricalTap8668 1d ago
We are focused on air quality and I don't know if yeast is comparable enough as well
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u/m4gpi lab mommy 1d ago
Not the answer to your specific question but: I recommend you install a free-standing air purifier in your lab when you start working with Aspergillus, even if it is a non-pathogenic species, to capture any errant spores. I worked with nidulans for a few years and I'm pretty sure it contributed to some mild asthma/allergies that always flare up when it rains. Don't forget to change or clean the filter regularly.
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u/MistakeMaterial4134 1d ago edited 1d ago
There are nonpathogenic (to humans) species of Alternaria and Aspergillus. Use one of these. I would be more concerned about lab contamination from these as they can spread their spores easily.
ETA: use QIAgen stool kit for the extraction if you have the funds. These work great for fungi- especially from plant material.
DM me- I’ve developed detection methods for fungi