For this episode we host two guests. Corbin Schuster and Zoltan Varga from the Zebrafish International Resource Center at the University of Oregon join us to talk about why zebrafish are an ideal model organism for genetic studies and to tell us about some of the challenges of creating and maintaining thousands of genetically unique lines of the animal. The career corners part of this conversation is also super inspiring and insightful.
The use of model organisms in science dates to ancient Greece and represents an important way humans have progressed our collective understanding of biology and disease. We’ve probably all heard of using mice, rats, or even Drosophila melanogaster (i.e., fruit flies) in the lab, but zebrafish have surged in their use as a model organism. Zebrafish are small, more cost effective to maintain, have a fast generational lifecycle, and have clear embryos that enable direct observation of their development. As is the case with any model system used across the globe, standards matter and help make results transferrable to other studies. This is where our guests come into the picture.
For this unique episode we’re joined by both Corbin Schuster and Zoltan Varga of Zebrafish International Resource Center (ZIRC) at the University of Oregon. In their roles, they help raise and maintain over 12,000 genetically unique zebrafish lines for use in studies across the globe. We learn about the health monitoring they conduct on a regular basis to maintain their colony and support their own and partner studies using their zebrafish. The conversation touches on:
As always, you’ll also get to know our guests on a more personal level. We learn about how they each got into this line of work, what they love about it, and how they both value human relationships and helping their communities. Through the audio alone you’ll get a sense of the positive, kind, and collaborative attitudes that have helped them both be successful in their careers.
Visit the Absolute Gene-ius page to learn more about the guests, the hosts, and the Applied Biosystems QuantStudio Absolute Q Digital PCR System.
Cassie McCreary00:00
Ready to split some gills?
Jordan Ruggieri00:00
Yes, let's split some gills.
Cassie McCreary00:16
Welcome to Absolute Gene-ius, a new podcast series from Thermo Fisher Scientific. I'm Cassie McCreary.
Jordan Ruggieri00:22
And I'm Jordan Ruggeri. And for today's episode, we welcome two Gene-iuses to our tank full of magical zebrafish, Dr. Zoltan Varga and Dr. Corbin Schuster.
Cassie McCreary00:33
Zoltan holds a PhD in cell biology and neuroscience from the University of Basel in Switzerland. Since 2004, he has been the director of the Zebrafish International Resource Center (ZIRC) at the University of Oregon. Corbin Schuster is a member of the Yakama Nation and a recent PhD graduate in microbiology from Oregon State. He's worked with Zoltan at ZIRC as a postdoctoral researcher for the last year.
Jordan Ruggieri00:57
So sit back, relax and enjoy a little school with our two zebrafish experts.
Cassie McCreary01:03
I 'cod' hardly believe that!
Jordan Ruggieri01:06
Oh, that's a good one!
Cassie McCreary01:07
I tried. That one was a little ‘fishy.’
Jordan Ruggieri01:13
All right. Zoltan and Corbin, thank you so much for being our Absolute Gene-iuses today and joining the podcast. We're really, really thrilled to have you here. We were talking before about zebrafish, and you know, the facility that you have set up for zebrafish. Can you talk a little bit about what is it that you actually do at this facility when it comes to zebrafish? And now how's everything set up?
Zoltan Varga, PhD 01:37
So the Zebrafish International Resource Center was built in 20, well, in 1999. It started and was finished in 2000. And we have recently expanded the footprint in 2020 to 2022. The purpose of the building was to house zebrafish from mutagenic screens that were done in, in 1995. And we were built to house about 500 fish lines. And in the meantime, we have expanded that collection to over 12,600. And we have 46,000 zebrafish that we use roughly. That is approximately 55% coverage of the zebrafish genome with on average two to, three to four alleles per locus. And then the other leg on which the Zebrafish International Resource Center stands is that we have health services. The large colony that we have also requires extensive health monitoring. And this is where the DNA sampling comes in, which Corbin established for us last year. He was a postdoc at ZIRC, and he brought environmental DNA sampling to the portfolio of our health monitoring. There are so many lines, we can't keep them all alive. A lot of them are imported into the quarantine room and they're cryopreserved right away. And we can't do health checks on them until we recover them. And so then we have to either raise them in the quarantine room, or we have to raise them in the facility. And to ensure that at least one of the pathogens currently is out of, not in the system or not introduced, the environmental sampling has been developed by Corbin to facilitate that. Makes the health monitoring approach faster. And we can be more proactive.
Jordan Ruggieri03:33
Yeah, makes sense. That's a huge number of lines. Corbin, what are you what are you doing on your end? Do you do house lines as well?
Corbin Schuster, PhD 03:41
It's nowhere near the capacity of was that we have there at the Zebrafish International Resource Center. I think our max capacity is about 1,200. I sort of focus in assay development, as Zoltan mentioned. So, it's working trying to develop environmental assays for zebrafish pathogens. And so I don't need necessarily a big capacity enabled to do the validation and the development of these assays. So having a smaller system really works to really focus in on culturing some of these pathogens in vivo and then being able to develop these assays.
Jordan Ruggieri04:14
You know, obviously you are zebrafish experts. That's, that is for sure. But why are zebrafish so important to the scientific community?
Zoltan Varga, PhD 04:23
Zebrafish are essentially a almost entirely Eugene, Oregon invention by the virtue of George Streisinger who was a virologist here at The U of O. He wanted to expand his research into vertebrates, and he was looking for a vertebrate model that lent itself as easily to genetics as viruses did. And so he was also hobby aquarist and he knew about zebrafish from the pet store trade, and he established them in the 70s in his lab. It's a great model for doing genetics. George Streisinger was interested in, as far as I understand, in the genetics on one side, but also the neuroscience. And then so at the Institute of Neuroscience, several researchers picked up the baton and continued zebrafish. And they exploited zebrafish for its, one for the genetic amenability. But also because as an as an embryo, it is transparent. It grows fast. And you can see single cells forming tissues, tissue layers, and the organs. The research community has expanded zebrafish into virtually any biomedical research field. And there's hardly any university that doesn't use zebrafish in some biomedical application.
Cassie McCreary05:45
It's crazy to me how we've gone from somebody, you know, being interested in this stuff and looking at like zebrafish in a pet store, to now where we're at. It's like such a huge leap in a relatively short amount of time, like when you think about it, pretty wild.
Zoltan Varga, PhD 06:00
It is, it is it's, we're talking about 40 years, maximum 50 years.
Jordan Ruggieri06:06
It's awesome. So I know you had mentioned that, you know, one of the benefits is the transparency, especially in early development. What are some other benefits that zebrafish have as a model organism?
Zoltan Varga, PhD 06:18
They have a relatively short generation time. So in about three to four months, you can breed a new generation, and then one litter one batch of eggs, has enough embryos that you can do very, very easily genetics. I mean, typically, it's at least 100 eggs. It's so much easier if you have a batch and you have 25% mutations right in front of you. And then the other part of it is that very recently, you know, with CRISPR Cas mutagenesis, we came away from regular forward genetics, to reverse genetics, where we can target specific genes and ask what the phenotypes are. And with CRISPR Cas genetic editing, this became very, very straightforward. Previously, it was kind of like a random approach, and you could only identify mutants that actually had a phenotype. Now, if there is a mutated gene, you can now really look thoroughly with the help of other model organisms make predictions about the phenotype, investigate particular cell types, particular gene expression patterns, then really hon in on a specific gene much better than that used to be possible with the forward genetics.
Jordan Ruggieri07:28
Absolutely incredible. Corbin, I think this might be more on your end. But I want to talk a little bit about how you maintain the zebrafish as well, and what that entails. You mentioned something about looking at the different pathogens, that that they might be exposed to. What exactly do you look for there? And why is it important that you understand some of the pathogens they may carry?
Corbin Schuster, PhD 07:55
Well, one of the main pathogens that I worked with, there at ZIRC and as well as in my doctoral training, was an opportunistic pathogen. So it's Pseudoloma neurophilia that’s a microsporidia parasite. But for in regard to looking at disease phenotypes or disease, clinical disease signs in regard to a population of fish. They, with this pathogen there are particularly asymptomatic. So you wouldn't necessarily know that your population is infected until, you know, 10-11 months down the road, when yet, when that prevalence has, you know, peaked, and it's pretty, it's pretty obvious that your fish are pretty sick. So being able to be proactive in regard to being able to detect these pathogens is pretty important, especially when we're talking about reproducibility in different research studies. These pathogens can also impact the behavior of these fish. So these could have really confounding factors, or what we like to call non-protocol induced variation, in these research studies. So understanding how these pathogens interact with the host and interact in regard to a population, on the population scale, is very important for maintaining rigor and reproducibility in research studies. And so one of the obvious things that we do is, we obviously look for clinical manifestations of these different pathogens that we that we study. But we also couple these with, you know, like I worked very closely with Dr. Katie Murray, who was the attending veterinarian there at the Zebrafish International Resource Center. And so it's very important to be able to, or at least I find it important, to be able to couple, you know, traditional, traditional surveillance efforts such as histopathology and other types of efforts to be able to really be proactive in trying to eliminate these pathogens from zebrafish facilities. I believe the pathogen that I've worked with, Pseudoloma neurophilia it may have decreased now, but a few years ago, it was reported that this pathogen impacted about 50% of all research laboratories that reported to ZIRC. So understanding how these pathogens first, what the origin is, is important, but also understanding the, what is the genetic variation of these pathogens across zebrafish laboratories, is also something that I've been particularly interested in, especially as we find more microsporidia species to actually infect other types of fishes. So we, recently we found a microsporidia that is, after doing some gene sequencing and alignments, we determined that it was actually relatively closely related to the Pseudoloma neurophilia that I've been studying. I know, there's been some reports, in labs that I've talked to, when they, were they, they were they had it in the microscope, and they're saying, I'm pretty sure it's, you know, Pseudoloma, but it's not, it's not being detected by our qPCR assays. Then the qPCR assays, you know, as we want them to be, are very specific. So sometimes it can be 113 base pairs. And so that's a very small sequence and very specific to be looking at, in regard to pathogens, and if it's not being detected, why is that so? So that's what had me thinking about the genetic variation in these different laboratories.
Jordan Ruggieri11:06
Is there a way that you implement, you know, qPCR versus digital PCR? We love digital PCR here at Absolute Gene-ius. So how does digital PCR and qPCR kind of fit into your workflow as you're looking at these pathogens?
Corbin Schuster, PhD 11:20
The nice thing with qPCR is it's essentially, we will build our qPCR assays. And that's what we will use to, you know, do detection of these pathogens for tissues and stuff, and in different sorts of tissues. Pseudoloma neurophilia specifically targets the central nervous system. So looking at the hindbrain, and like spinal cord tissue, or in the spinal column. And so it's really nice, that's sort of where we start, and then it's a very easy transition to take our qPCR assay and, you know, take it to the digital PCR platform. Um, it's essentially the same primers, a little different master mix, but, you know, we can take our primers and our probes and directly translate that over to digital PCR. Where I think the digital PCR has really complimented our qPCR efforts, has been in the fact that we can detect the pathogen at much lower concentrations than what we would have with the qPCR. Especially what we've seen with environmental sampling. We've used our qPCR assay for environmental samples, whether that be like tank tectritis or biofilms, or straight tank water, it was very inconsistent. We could detect it if the pathogen had a really high concentration in an environment, but oftentimes a Pseudoloma neurophilia, there's both extracellular so you know, outside of the cell, but it's an intracellular parasite. So there's also an intracellular phases, especially as that moves essentially through every bodily organ system until it finally reaches its, quote, final destination to the spinal cord and or, you know, the spinal column. So that movement, we've did transmission studies sort of looking at what does that look like, actually, for the detecting the parasite in the environment. And what we found was really, during those initial intracellular stages, it's not really, as it's moving through those organ systems within the host. There's some, you know, there's some gaps in detection. And so that gap in detection, you know, it's very, it's varied, it varies by how big your population is, how big of a, you know inoculum or a dose that the actual fish are exposed to. So being able to detect it at very low concentrations was very important. And the only way we were able to truly achieve that was with the digital PCR.
Jordan Ruggieri13:34
When you're actually developing assays, what exactly are you looking for? Are you creating primers and probes against these different pathogens? Are you know, are you actually doing any type of sequencing to look at all pathogens or all organisms in the water or how to talk to me a little bit about that development stage?
Corbin Schuster, PhD 13:56
Certainly, certainly. So, the primary thing, at least for the assays that I have worked with, has been to make them very specific and make them very sensitive. We do look at other pathogens that may be present within, you know, within a certain system, but we and we do sequencing, but we want that sequence that we are amplifying to be very specific to the type of pathogen that we're looking for. We don't want you know that any cross-reactants or any sort of things like that going on. So I would say that the primary thing that we're trying to make sure is that our assay is one sensitive, sensitive enough to detect a pathogen and then two, to make sure that it's specific enough that it's not cross-reacting with any sort of species, that other species that may be present in, you know, in the zebrafish population or there's you know, in a different fish species population. This can be challenging however, especially if we're looking you know, for speaking about like Mycobacterium which there's several different strains that may impact the zebrafish populations. So making assay that are very specific in regard to that without cross reactivity can be a bit of a challenge.
Jordan Ruggieri15:05
Zoltan, are you using digital PCR on your end as well, or any comments on how digital PCR is used, you know, in your space?
Zoltan Varga, PhD 15:15
So, Corbin taught us digital PCR, right? He, he helped us adopt the methodology. And now Katie Murray and Evan Loucks, are doing routine screens. And in the same way, it is of interest to do the environmental sampling. We know that the UV sterilization, the last step in the filtration system, is a very effective method to knock down microsporidia, Pseudoloma neurophilia specifically, but we can't be sure if it's, you know, it's 99% or 99.9%. And so, previously, we thought, “Well, this is endemic to zebrafish, there's nothing we can do about it.” Then we said, “Well, this is in the water system. Even if we clean up the fish population, there are niches in our water system like piping, like pumps, aquarium maybe or fish populations that shed the pathogen, we will never get rid of it.” And so with the environmental DNA sampling, now we can go in and go on a tank level and say, well, “We screened these in these tanks, the overall prevalence has been reduced from let's say, I don't know 10% to 1%.” But in addition to that, we can also go, and we can say, “Well, these are really old pipes. Let's sample them,” for example, biofilm that is something we still need to develop as a technology and see if we have any significant reservoirs of microsporidia spores in our system that we need to clean out. And so we can go step by step through our filtration system in addition to the fish population, and really try and eliminate the pathogen altogether.
Cassie McCreary17:03
Taking a quick break from our conversation to tell you about Applied Biosystems™ QuantStudio™ Absolute Q™ dPCR system.This instrument enables quantification of your targets without the need for standard curves in only 90 minutes. Digital PCR can be as simple as preparing your samples, loading onto the plate, and running the instrument.
Jordan Ruggieri17:23
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Cassie McCreary17:40
And Thermo Fisher Scientific has a suite of dPCR assays for applications like AAV viral titer quantification, liquid biopsy analysis, and wastewater surveillance. You can learn more at www.thermofisher.com/absoluteq or visit the Absolute Gene-ius webpage. Again, that's www.thermofisher.com/absoluteq or visit the Absolute Gene-ius webpage.
Jordan Ruggieri18:07
The Applied Biosystems™ QuantStudio™ Absolute Q™ dPCR system is for Research Use Only. Not for use in diagnostic procedures. Let's get back to our conversation.
Cassie McCreary18:20
Now I said it yesterday, so now I have to say it every time. Gentle peeps, welcome to Cassie's Career Corner! Zebrafish whispers step into my office. We're going to talk about your career, your backgrounds, your advice, some funny lab stories, so prepare yourselves because I have many fun questions for you. For both of you, how did you get to this point? Would you have said to yourself I really want to work with zebrafish.
Corbin Schuster, PhD 18:48
I will say I definitely grew up with like fishing. Fishing was sort of something that, like a pastime, with my father. So we definitely went fishing like almost every weekend. But it wasn't until undergrad that I really got interested in zebrafish and this was my junior, my junior year. So I did an internship with the National Institutes of Health. And we were looking at how high fat diets may impact different neurological signaling. And this was using mice, this was a really large, large scale mouse project. And it was during that experience that I was like I definitely don't want to work with mice ever again. So after that experience, I came back to my undergraduate institution and we started, I started working with my undergraduate advisor on a zebrafish project where we were actually looking at spinal cord regeneration in zebrafish, and that's where I really became super fascinated by the model. And I started to pursue graduate opportunities that really revolved around I'm using zebrafish as a model organism to understand different biomedical pathways.
Cassie McCreary19:56
Excellent. And how about you, Zoltan?
Zoltan Varga, PhD 19:59
Well, I was born at a very young age, and immediately realized that I like animals. I was probably eight or nine when I started collecting tadpoles and watch them grow up, and then set them free when the summer came around. So that was the very first thing. And then the other part was that my grandfather on my dad's side was a farmer. My parents were refugees from Hungary. Every summer, we visited my parents in Hungary. I grew up in Germany, but we visited them in Hungary, and I spent essentially six weeks on the farm and was exposed to, you know, cattle, delivering babies, horseback riding, bringing in the hay, or whatnot. And so with this, that was also a huge exposure to animals in general and to agriculture, specifically. From then on, it just came naturally that biology became my preferred class in middle and high school. And when I was finished with high school, I immediately applied for biology in Basel in Switzerland, and I stuck with it. And so as the next step after that, after the PhD, I was then, I joined Monte Westfield's lab, and I was looking at patterning of the neural plate, and how the neural plate forms the forebrain. And that was in zebrafish. And, and to this day, to this day, if I sit down to get a chance to sit down with the microscope, I'm just at awe, you know, at the aesthetic of a fish embryo. Right. And that starts with the egg and the cell divisions. And that after 24 hours, we have something that actually looks like a little fish with a yoke. It's just amazing.
Jordan Ruggieri21:46
It's an awesome story.
Cassie McCreary21:48
I love that. Yeah. For both of you, it's very evident that you have this kind of immense passion around science and this, this work that you're doing and the work that you have done. And so what is it about science, as a whole but also just like the specific things that you've studied that just, what is it that makes you so passionate about it, or that really has like, done it for you that you're you decided to go this route?
Corbin Schuster, PhD 22:12
I guess for me, so I grew up with a tribal background. So I grew up very immersed in tribal culture. And I'm actually the first PhD in microbiology for my nation.
Cassie McCreary22:24
Amazing!
Corbin Schuster, PhD 22:25
So for me, I'm, I'm very driven by my community and being able to come back to my community and recruit students, you know, to increase representation in science. And so that's one of my, I guess, my overarching goals and priorities that really drives me forward. Especially like when things you know, the experiments aren't working, and that, you know, it gets frustrating, one of the best things is persistence, and being able to remember that one of the bigger goals that I'm here to hopefully do is to really increase that representation, especially with indigenous folks and in science and STEM. In, you know, in the professional realm in general. So, that's one of my primaries, I would say, primary passions. Um secondary, I really love microbes. So being able like, Zoltan mentioned, looking at the microscope, I could spend all day, literally all day, at the microscope, looking at different slides and looking at different, you know, I do have a bias towards parasites. I really, my PhD advisor, Dr. Michael Kent, he definitely stoked the fire with me with my passion with parasites. And so being able to work with someone who was very enthusiastic about the different sorts of things that we're looking at and shared passion for different, you know, different fish species and aquaculture species, was really something that I felt like, it really came full circle for me. And it's really driven sort of those things. So being able to bridge I guess, my passion to be able to train the next generation of indigenous folks out of our community, and then be able to really pursue what I, what I've been interested in, which is microbiology.
Zoltan Varga, PhD 24:05
For me, it is the realization of, you know, there's something unknown to discover every day. Sometimes it's just you step out the door, and its sunshine, or a little flower or something like that. But it's amplified. You know, when you look at science, because you have the capacity to learn something new every day some you discover something new, that you didn't know about, and I'm just fascinated, you know, being able to come to work. I mean, I really feel blessed in the sense that I can come to work and every time I walked through the door, I can ask myself, “Well, what will this day bring?” And it always inevitably gives something wonderful. The other part of me is, I really enjoy being there for other people. I like helping people. And I think, in the position where I'm now I can combine these two things. And it really, you know, is fulfilling?
Cassie McCreary25:05
Wow! Both those responses just gave me like the warm fuzzies. That was so nice from both of you. Thank you. To you both, being is that you're at different points in your careers and everything else, what's a piece of advice that you would give to somebody just starting out or even your younger self, like when you're, you know, what, what is a key piece of like career advice you would give?
Zoltan Varga, PhD 25:26
You know, before I start with the advice giving, I have to realize how much, much more complicated things are for young scientists today. It seems to me that, as a biology student, I was never trained in, you know, working with people. I was never trained in administration. I was never trained with budgeting. And so all these things had to be added, just when they needed to be added. And that's fine. But if I would give an advice to somebody who wants to become in academia or in technology and in a company, you know, a biologist, and work as a biologist, I would tell them that this is also coming their way. And you better not neglect those things, especially people skills, especially caring for other people.
Corbin Schuster, PhD 26:23
For me, it would be don't cross things off the list. For me, I when I was when I started my undergrad, I was like, oh, I just need to get in, get out, go get a job. I never thought that I would be you know, pursuing, one would be a professor, a university professor, but neither did I think that I would have pursued a graduate degree. So for me, it was just really don't cross things off the list. You never know what sort of opportunities will arise and will be, you know, essentially will pave a way in regard to your careers, something that you've never imagined.
Cassie McCreary26:59
Enjoy the ride, don't cross things off the list. I love that. Corbin, you are now a professor. How is it that you arrived at these decisions? Or like how is it that you decided this is what I want to try, or this is what I want to because at some point, you kind of come to a crossroads right? So, how is it that you want to open about making your decision?
Corbin Schuster, PhD 27:19
For me, the decision to pursue, you know, a faculty position at a university really revolved around that, that that first priority I was talking about in regard to increasing representation in science. I'm faculty at a both a Hispanic-serving and Native-serving institution here in Washington State and so our population is 90% people of color. And so being able to work with a community that I feel like, you know, has brilliant people, and maybe it has some barriers or whatever it may be that we could essentially break those barriers down and get them to the places that they deserve to be and in the positions, they deserve to be, leading our community in these different realms. One of the things that I worked with a little bit within my doctoral program was we were doing COVID, COVID-19 trace testing. And so we were testing across the state of Oregon. It really inspired me in regard to that community that, what sort of Zoltan was talking about, was that caring for other people in that community and building community it really is what it is. That's sort of what got me really interested in with the community that I'm currently serving. That community was hit hardest at some point in the pandemic, with COVID-19. And so it's really inspired me to try to train individuals to be in those leadership positions. And for people who actually care and you know, came from these communities, is really important to me.
Cassie McCreary 28:55
Awesome. If you're feeling bold and brave, and funky and fresh. Are you willing to share with us your biggest lab oops, moment? And then if you're, I mean if you're not, that's fine too. Sharing your most like proud lab moment or even career moment as a whole.
Zoltan Varga, PhD 29:14
I don't think I had a big the biggest lab oops, once, but I had a biggest presentation oops.
Cassie McCreary29:24
Ah, okay, that works.
Zoltan Varga, PhD 29:25
And part of it is rooted that of course, by my primary language, my native language is German. And then the other thing is that I am sometimes a little absent minded. And so actually the second international zebrafish meeting I was at, somebody asked me about a procedure and how to do it. I can't remember what that was. The answer entailed, “Give it to the technician, and she will do something with it.” And there was this gasp going through the audience, you know that I made a, how should I say, a sexist remark. Because all technicians are she's. And it was just it was just, you know, I just misspoke. But I felt so bad afterwards. And sure enough, one of my colleagues who is very, you know, dedicated to these issues came to me afterwards and said, “How could you?” And that was right after the question meeting, she was already up at the podium and was talking to me, and that was, that was certainly my worst moment.
Cassie McCreary30:34
How about you, Corbin? What's your biggest oops? Or do you think you just haven't come across it yet?
Corbin Schuster, PhD 30:38
No, I came across it. So we were doing our transmission studies, we had already developed the assay for Pseudoloma neurophilia . And now we wanted to just sort of see what the, what it looks, what the pathogen detection would look like, you know, over a set period of time with a certain population. So we had a population of about just under 600 fish. And we, it was a really big tank. We, I had to pull parasites essentially out of brains, brains, and spinal cords, out of I don't even remember how much fish at this point, I'd have to go through my lab journal, but it was a lot of fish. And because the concentration of parasite within each fish it varies quite a bit. So it can be anywhere from one spore to you know, up to 15,000 spores that are found within a single fish. So I had to pull, I had to pull tons of spinal cords and brains. And then we had the lyse those and essentially purifying, extract the parasite out of those tissues. Once I finally got the extract the purified spores out of there, I had to quantify that and then determine how much a spore that we wanted to actually inoculate our population with so we can do our transmission study. But at that time, this is where math is very, very, very important.
Cassie McCreary31:53
Oh no.
Corbin Schuster, PhD 31:53
At that time, I had messed up on essentially the math when I was calculating well, how much of the inoculant that I needed to actually give the entire population. And so I gave the inoculation out of the calculation that I used. But we were wondering why it didn't seem like the fish were getting very infected. About a week later, I redid the math and everything and determined, “Oh crap.” I was actually off, you know, where we inoculated the fish with was below the actual minimal detection limit, or the minimal infectious dose that was calculated at the time. But it all worked out, it all worked out, because we did determine the fish, that there was a new minimal infectious dose that we could report with. And then the transmission study actually turned out very successful, we were able to see, you know, sort of just exactly what sort of patterns were occurring within a big population at a low dose. And what's actually more interesting with that, even though it was a mistake, that turned out to be beneficial. That was probably more representative of a natural exposure, right? So, I'm giving fish a large high dose concentration of a parasite, versus a really low minimal amount is probably what you would probably see within a zebrafish facility anyway. And so we were very pleased with those results, and actually published on some of those results, even though it initially turned up as a, you know, as a minor mistake.
Cassie McCreary33:25
How about your best or proudest moment so far, Corbin?
Corbin Schuster, PhD 33:29
One of my proudest moments was actually in our development of a sensitive assay for Pseudoloma neurophilia. That process actually took quite a bit longer than I had initially thought I got myself into. I remember at some points, that would get it really inconsistent detection, where we're wondering what was going on. And so like, one of the proudest moments is really everything sort of came together in regard to because the, the assay is actually sort of a multi-step sort of thing. So we, the spores are very hardy, and all that DNA is essentially encased within that tiny little spore. And so they're very resistant to disruption. And so being able to disrupt that spore so we can actually detect the environment and the DNA was very important. So we essentially determined that we needed to add a sonication step. So we actually sonicated water samples to release that DNA into the environment. So being able to develop the assay was definitely one of the proudest moments. I remember going running into my PhD advisor’s office like, “Hey, we finally got this, and you know, it's actually consistent.” We, we essentially went through, I essentially went through every single digital PCR machine. Now at ZIRC I've used, you know, the Thermo Fisher system with the Absolute Q. And so I've sort of had my experience with every sort of system, and they're all a little different. I was very pleased with how quick the workflow was with the Absolute Q which made, sort of made our, that assay and those multiple steps that we have to do to get you know, actually get detection with water samples more efficient.
Jordan Ruggieri35:06
That was Corbin Schuster and Zoltan Varga of the Zebrafish International Resource Center at the University of Oregon. Thank you so much for joining us for today's episode of Absolute Gene-ius. Stay curious and we'll see you next time.
Cassie McCreary35:20
Just like the tuna fish sandwich said, “I've got a feeling we're not in can-sas anymore.” Ah man, these are great. Lobsters would get along a lot better with other shellfish if they weren't always trying to lobster things up. Weirdest one on the list!