Dr. William Weiss is the Director of Pre-Clinical Services at University of North Texas in Fort Worth, working to develop animal models in infectious disease for the evaluation of new and novel therapies in antibacterial, antifungal and antiviral research. He has been using a Don Whitley Scientific DG250 and an A35 anaerobic workstation for about 2 decades.
Read more about his research on novel antibiotics for the treatment of Clostridium difficile infection here.
Q: What manner of specimens are you working with and which bacteria do you cultivate in the anaerobic workstation?
A: Here in the Pre-Clinical Services group, we are essentially a contract research organization, and the majority of our work in the A35 anaerobic workstation involves C. difficile. We are carrying out various research projects for different pharmaceutical companies, and biotechnology companies, here in the US, as well as Europe and South America. There are various experimental models for reproducing C. difficile disease, for example, the “gold standard” model involving the hamster, and there is also one that involves mice.
Basically, in this model, spores from C. difficile are introduced into the animal, and they colonize in the gut. C. difficile spores come from the environment and are ubiquitous, but because they are held in check by the normal gut flora, they never proliferate and they never cause disease. It’s only when patients are treated with broad spectrum antibiotics that healthy bacteria are destroyed and C. difficile then proliferates and causes disease. That’s why often, people going into the hospital and receiving multiple antibiotics can develop C. difficile disease.
Q: So what is the role of the A35 workstation in your C.diff. research?
A: The model we are using to test novel therapies designed to combat this disease depends strongly on the A35: we grow the C. difficile culture by streaking the frozen cultures onto appropriate media, incubate them in the A35 chamber at 0% oxygen, and then harvest the vegetative cells and treat them to form spores, which we store. The spores are then introduced into the animals, the animals are treated with a broad-spectrum agent, they develop the disease, and we try different forms of therapy. The end-points in terms of efficacy are increased survival, as well as the amount of C. difficile that can be found in the fecal pellets of the hamsters, or in the contents of the cecum. We process the fecal pellets or a cecal sample and plate it, then we place those plates inside the chamber to incubate for about 48 hours. An animal that might have the disease could have 6 logs of C. difficile in their gut, whereas one that has seen successful treatment, such as with vancomycin, the gold standard right now, might be reduced from 6 logs to 2 logs or even less. All that incubation is being done inside the A35.
Q: The A35 isn’t your only workstation though, tell us about that.
A: With C. difficile being a larger problem than it used to be, we are seeing a real uptick in the number of companies interested in it, thus explaining our need for the A35 workstation. The C. difficile in the environment is very hardy, because it can form spores, but in order to count the spores, they have to germinate into the vegetative state, and that’s what you need the workstation for. Prior to the A35, we had the smaller DG250 model, and at the time, when we were just working with some bacteroides, not a lot, it sufficed. But when we started working with C. difficile, the amount of studies and the type of studies we did really expanded. So at that time, we contacted Microbiology International since we needed a larger workstation in order to accommodate all those studies. The A35 is now our second Whitley workstation, but we are still using the DG250, too. We’ve converted the DG250 to a microaerophilic environment, because we do Helicobacter pylori work in there. It’s a facultative anaerobe, so when we need to grow up plates or process samples, we use that smaller workstation with a 5% oxygen mix.
Q: How were you achieving anaerobic conditions before you purchased the DWS anaerobic workstations?
A: We used to use anaerobic jars, but the seals give out on those jars over time, the sachets are pretty expensive, and the volume of samples we work with is tremendous, maybe a few hundred plates at a time. The jars just won’t do for that. Jars also aren’t as accessible as the workstation is: you have to open the jar, then you see that the plates have to incubate further, so a new sachet has to be added and the jar is closed up again. Whereas with the workstation, lab personnel can just look at all the plates from the outside, and say, “Well, we’re going to incubate a little further” or “Let’s take them out and count them now.”
Q: What prompted your decision to switch from gas pack jars to the Don Whitley Scientific anaerobic workstation?
A: The moment comes when the lab has so many jars that it becomes unmanageable, and you run out of incubator space to cultivate those jars. Then you really do need a chamber. For us, one of the big reasons to buy a larger workstation was when we started hygiene surveillance projects. We did a project for the local Dallas/Fort Worth hospital administration here, where they swabbed many types of surfaces in the hospital, such as bed rails, countertops, doorknobs, and brought the swabs to us for detection of C. difficile. This required those swabs being cultured in bags, so there was a large volume of media that went into the workstation to incubate for several days. The project entailed a second phase in the same hospitals, when a thorough cleaning and repeat swabbing were carried out, and our lab re-checked for C. difficile counts. So, with hundreds of bags, each with 30 ml of medium, incubating for several days, jars would have been unmanageable. Even the A35 was full, and that kind of surveillance with lots of samples would never be possible with jars.
Q: How would you rate your workstation’s gas consumption?
A: On a routine basis, going in and out of the sleeves, through the port doors, with plates coming in and out through the interlock, the gasses last very well for the A35. Even with our heavy usage, when a study is going on with lots of plates, the 50 L mixed gas tank will last three and a half weeks or more.
Q: What is your maintenance and cleaning schedule and do you find that easy to perform?
A: We mainly clean the workstation through the sleeves, spraying everything down and then wiping it clean. With C. difficile we don’t want any kind of cross-contamination so we prefer not to take off the front panel. It isn’t even necessary though: unless you are being sloppy and spilling samples, there should not be contamination.
Q: Do you confirm that the workstation is always sufficiently anaerobic?
A: We monitor the anaerobic atmosphere using resazurin strips, breaking open a new one every time we enter the workstation. The new anaerobic conditions monitoring system might be a useful backup system for the resazurin strips, which can dry up and go bad.
Q: Is the workstation simple to use and intuitive, or did you require a lot of training in your facility?
A: The workstation is easy to train on, in terms of initial instruction on evacuating and filling the sleeves before opening the ports and using the interlock. It is not a hard learning curve at all.
Q: Do you find it easy to work in the workstation and is workflow in the chamber efficient and timely?
A: Our users work in the chamber less than 20 minutes at a time, so even if we have a lot of plates, we’ll trade off people. We routinely have upwards of 250 plates inside the chamber and there’s still plenty of room to move around. They find it convenient and not uncomfortable, even with the warm atmosphere inside the chamber.
Q: How has it been working with Microbiology International for almost 2 decades?
A: I’ve known Bill and Kevin at Microbiology International since the late ‘90s, as I bought my first chamber when I was working at Wyeth. It worked well, they were always good in terms of support, and we always catch up at the MI booth at scientific meetings. Microbiology International are very responsive and we can usually get things fixed, and we don’t then have any issues from the repair.
Thank you for your time, Dr. Weiss!