In this episode, you’ll join us in gaining a better understanding of how microbiologists help with cannabis and hemp testing. If you’re curious about how this amazing plant is tested for threats like salmonella, mold and E. coli, this episode is for you!
Special Guest: Lauren Rudenko, PhD; Microbiologist of Eastern Analytical Labs
Lauren Rudenko, PhD, Microbiologist at Eastern Analytical Labs: [00:00:00] So, my name is Lauren Rudenko. I got my PhD from the University of Virginia in biology and I studied neuroscience at the time.
And my research history has been everywhere from plant biochemistry to neuroscience, and now I’m here and I’m working as a microbiologist. So yeah, so my background is in plant biochemistry, neuroscience and microbiology.
[00:01:00] And my job here is basically running the microbiology side of testing. And what that means is that it’s my job to make sure that there are no harmful pathogens in the products that we test.
What are some of the compounds you test for on a day-to-day basis?
Lauren: That kind of depends on the kind of material we’re testing. For plant material, you have to test for four basic categories. On the microbiology side, the first is total aerobic plate count, and that’s basically an overall picture of how much bacterial load is in the material.
The second thing we do is total yeast and mold, which is a similar thing but this time we’re looking for yeast and mold rather than just bacteria. And then we do a quantitative e-coli – again, same kind of thing. But this time we’re just looking for specifically e-coli and then we do pathogen identification tests.
[00:02:00] And for salmonella, we have an enrichment protocol because salmonella, if it’s present in very small amounts, might not show up on a quantitative plate count method. But it could be enough to cause damage. And so what we do is we do a couple of enrichment steps. So if the salmonella is in there, we’ll be able to find it and know that it could be dangerous. So that’s kind of the four categories.
What originally brought you into the Hemp & Cannabis Industry?
Lauren: Yeah. I kind of stumbled into it to be perfectly honest. I had a background in microbiology. I knew I wanted to be involved in medical research, medical testing. And it was always important to me that what I did would be able to help people.
And so when I was looking and deciding what I was going to do next, after I finished my grad work, I was kind of looking everywhere. I wasn’t exactly sure where I wanted to go. And then I kind of stumbled across this.
[00:03:00] I really liked the idea of doing this kind of medical testing and I don’t know, I just also liked the idea of helping to get something going. Kinda liked being able to be involved in the development of the methods.
What have been the biggest challenges in your specific role so far?
Lauren: There is definitely a learning curve to working in a medical testing lab versus an academic setting, right? So my history has been mostly in an academic research lab, and it’s very different, the kinds of ways you go about things, even the way you document things. Obviously, it’s extremely important that you document things well in any research setting.
But that traceability is so much more important and there is kind of a learning curve in learning the kinds of things you have to record and all of that. And in addition to that, I would say just with any new testing methodology you kind of have to work out the bumps in the method as you go.
[00:04:00] And you learn what materials work better and, and what processes cause issues that you wouldn’t think of. When I’m doing my dilutions for example we’re working with material that is, you know, when you grind it up into powder and you have all this little bits of leaves and flowers and stuff all in here, and then I have to pipette it.
The leaves are going to get stuck in my pipette or it, you know. So you do kind of have to learn the material that you’re working with because it’s very different from working with different matrices.
Welcome to the micro lab.
This is where the biology side of testing happens. So when samples come in here, we have four basic tests that we do. Three of them are quantitative methods, which we use plate counting type methods.
[00:05:00] And one is a pathogen enrichment protocol. So the first method that we do is a total aerobic plate count.
And that’s basically a measurement of generally how messy a sample is in terms of bacterial content. And so what we do is we take a sample, it gets homogenized into basically a powder. And then we do a serial dilution of that, and we played it on these petri films. And these petri films are just basically dehydrated growth media that bugs like bacteria used to evolve, and like to grow on.
And so once you’ve plated them in this dilution, you can actually calculate what the concentration of bacteria, yeast, mold, E. coli is in the original sample based on the results of that.
[00:06:00] So to just show you how these petri film methods work – here. So this is a couple of the different types of petri film we use here.
This is a rapid E. coli test. And you dispense right onto the petri film. And it has this little dam around the outside. Just take this fun little tool here, pop it down to spread the mixture across the petri film.
And now this media that is inside here will rehydrate and it has selected agents that make E. coli basically change and grow a different color than everything else. So you can tell how many colonies, or colony forming units we call them, of
[00:07:00] E. coli were in this sample. You do the same thing for your aerobic plate count, as well as your yeast and mold count. So these three methods are extremely similar in the way they’re actually done on the bench. But the difference is that these petri films have a different growth media that different things like to grow in.
So this particular one has a growth media that is friendly to yeast and mold and not as friendly to bacteria. So this one is going to show me how many yeast and mold colony forming units are in my sample. Whereas this one is E. coli. So once you have plated them and let them sit for long enough for the agar to rehydrate, then you turn around here and you put them into these growth chambers. So we have our incubators here.
[00:08:00] We keep incubators at several different temperatures because different kinds of bugs like to grow at different times. E. coli, for example, grows at approximately 35 degrees. It’s pretty close to our body temperature. And there’s a reason for that. And yeast and mold actually grows pretty well at just about ambient room temperature.
So there we go. Now those will grow and they grow for between 18 and 72 hours, depending on the type of petri film and the method that you’re using. And after that time, you pull them out and you take a look at how many colonies there are. So basically these are the different kinds of organisms that we’re looking at.
This is an aerobic plate count petri film. And you can see that the bacterial colonies grow as little red dots. So once I have all of my samples on petri films and they’ve had enough time to grow, then I come over here and I count how many dots are there. That
[00:09:00] number is how many colony forming units were in the original volume that I put on the plate and I can use that information to calculate based on the dilution how many colony forming units are in the sample. Similarly these are the petri films that we looked at just now that were showing yeast and mold. So as you can see, it looks completely different. This is a sample that has yeast in it, and they grow as these little blue colonies on these petri films.
And a mold grows like this in this kind of spreading pattern. Now, obviously these are samples that have only yeast or only mold when you have an actual yeast and mold sample, it’s a mixture of things. Sometimes it looks kind of nasty, but you can count those and figure out what the load of yeast and mold is.
[00:10:00] Again, as kind of a general picture of how clean the sample is. This one is the E. coli petri film that we looked at and the E. coli petri film you can see there’s some red dots there that are red and some that are green, the E. coli colonies show up as green on this petri film. So it’s really easy to see if there’s E. coli contamination in your sample based on this petri film.
So the fourth test that we do on our samples is to identify contamination with salmonella. And so for that process we have a few steps. The first step is called a non-selective enrichment step: put it in a very friendly medium that all sorts of bacteria like to grow in and give it 24 hours to just kind of recover from the trauma of being, you know, ground up in a commercial blender.
And then we go ahead and move it to what’s called the selective enrichment step. This is a media that salmonella is really
[00:11:00] happy in, but a lot of other bacteria don’t like to grow in. We go through those two steps and then we plate the sample on selective agar. These are agar plates that have media in it that again, causes different kinds of bacteria to grow and grow different colors.
So just to demonstrate how we plate these [here’s] a couple of demo plates that I was going to make, so we use these sterile inoculating loops. You can also use flame denoculating loops.
[00:12:00] And go ahead and streak that on there. And then we do what’s called an isolation streak. And what’d you do with an isolation streak is you just take each streak with the new wand and get it just a little bit in the previous streak and take it into some clean agar. So the purpose of that is where I put a lot of bacteria at one side of the plate, it will be impossible to look at an individual colony. So I just kind of spread the sample bit by bit so that I can get an individual colony.
And that allows me to take it on to more specific tests. Later on if we have something that looks like it’s a potential colony of salmonella, we’ll take that isolated colony and we’ll do some further tests on it to make sure of what it is. I’ll do the same thing on this one.
[00:13:00] And again, we just use a clean one for each step. That way we have a pretty good chance of having a single colony to look at at the end. We have a couple of different kinds of selective media that we use and that’s because salmonella can be a little tricky.
There are sometimes variant strains that don’t look like salmonella on selected media. And so if you use multiple kinds of selected media it increases your chance of actually being able to see traditional salmonella growth characteristics. So after that, you go ahead and again… So those will grow for about 48 hours. And after that, we can go over here and take a look at what those plates will look like after.
[00:14:00] What I just plated over there is the same as this plate right here. It’s called the HardyCHROM salmonella. And salmonella grows this pretty purple wild color on this particular agar. So if you see it’s got all of these, this particular sample has eight different samples that were all streaked on there as a test. And all of them came up positive for salmonella.
If you look at this one here, I plated a few different things as an experiment. I plated salmonella alone, as well as a mixture of salmonella with some other bacteria to see if I could see individual colonies coming up as salmonella in a mixed culture.
And so you can see here, the salmonella colonies are coming up as purple and the variations of E. coli are coming up as blue. So those blue E. coli variants are not positive for salmonella. The purple ones are.