Webinar Q&A — Nested Isolation

Webinar Q&A — Nested IsolationIn a recent webinar, Dr. Alex Rodriguez-Palacios of Case Western Reserve University School of Medicine discussed the use of a new, low-cost caging system for conducting microbiome research in rodents. The speaker shared their experience with the NesTiso system and discussed:

  • How NesTiso makes gnotobiotic research simple and practical
  • How to inexpensively verify the germ-free status of your models
  • How the portability of NesTiso systems accelerate gnotobiotic research
Due to time constraints, some of the questions you submitted to our Q&A session went unanswered. We present here the full Q&A, as well as several poll questions posed during the webinar.

Nested Isolation Webinar Q&A

  • Q: How often do you change your cages in the NesTiso Colony?

Dr. Alex Rodriguez-Palacios (ARP): It depends on how many mice you have in the cage and how big the mice are. There are other factors that could affect the cage change rate as well. In our study, we averaged 2.5 mice per cage and changed cages every ten days.
  • Q: What were the most common contaminants you saw in your colony?

ARP: The most common contaminants are spore formers, typically from Bacillaceae because they are commonly found in the environment and feed. Spore formers can survive irradiation, so autoclaving food is an important step to decrease the risk of spore formers getting into the isolator from food. The next most common contaminant is fungi, followed by Staphylococcus, which can be found on the skin of people.
  • Q: Are people able to get grants using this non-traditional housing system?

ARP: We have applied for and received grants which have included the use of the NesTiso caging system. With the NesTiso paper published1, use of this innovative caging system should not reflect negatively on grant applications.
  • Q: We want to infect germ-free mice with a single pathogen. Is it possible to use the NesTiso caging system with a disposable inner cage?

ARP: One caution about disposable cages is that they are sterilized using irradiation and we know that irradiation is not always effective for killing spores.
  • Q: I am not a microbiologist; how can I interpret culture agar plates? What is real? What might be a plating mistake?

ARP: When looking at a plate, if you have a true contaminant, you should see bacteria growing on and around the streaking lines or the feces, but not outside of the lines. If you have a contaminated plate, you should see just a few colonies growing outside of the streaking lines.
  • Q: Did you measure CO2 and ammonia is these cages over time?

ARP: We did not measure CO2 because the evaporation rate of the moistened bedding showed that external aeration was as efficient in the inner static cage as it is in the standard static cage without ventilation, which has been extensively validated. We also showed that the increase of humidity due to the outer cage (extra layer of filtration) is only 5%, making animal density more relevant than the presence of the external cage.

We also determined the potential for induction of chronic hypoxia (low O2 over time) by measuring hematocrit and verified that there were no differences between NesTiso and Isolator conditions.

We did not measure ammonia because it does not occur in germ-free mice. If NesTiso is used for gnotobiotic experiments, the role of ammonia as irritant of the upper respiratory tract then would be important and will depend on the microbes studied.

If ammonia-producers are of interest, ammonia needs to be addressed in each particular case. Evaporation rate of bedding material, however, indicate that ventilation is adequate in this setting, especially if external aeration is used. Our animal density was on average about 2.5 mice/cage.
  • Q: I have problems where the plates seem as if they were autoclaved, but they show they were not. How do you assure your plates stay sterile?

ARP: We always use autoclave indicators to ensure the correct autoclaving conditions are met, in terms of time and temperature, such as chemical strips, autoclave tape indicators, and biological indicators. This is especially important when we prepare our autoclaved diets and NesTiso supplies.

Webinar Poll Questions

We asked five poll questions during the germ-free housing webinar. Here are the results submitted by our audience:

  • Q: How much would you like/need to start experiments using germ-free animals, e.g., mice?

82% of respondents stated they were very interested or extremely interested
  • Q: Do you have experience in basic microbiology?

96% of respondents have some or a lot of experience with microbiology
  • Q: Do you have access to an anaerobic chamber in your institution to grow anaerobic microbes from a fecal animal sample?

46% of respondents stated they have access to an anaerobic chamber, while 25% stated they do not have access. 30% were not sure if they have access to an anaerobic chamber.
  • Q: When was the last time you used an agar plate to culture microbes?

25% have used agar plates in the past twelve months, 36% more than one year. 16% have no experience culturing agar plates.
  • Q: Do you know how expensive it is to purchase a germ-free mouse?

77% of respondents know the price of germ-free mice
Rodriguez-Palacios, A., Aladyshkina, N., Ezeji, J.C., Erkkila, H.L., Conger, M., Ward, J., Webster, J., Cominelli, F., 2018. 'Cyclical Bias' in Microbiome Research Revealed by A Portable Germ-Free Housing System Using Nested Isolation. Sci. Rep. 8, 3801.

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