Equipment and Methods for Concurrently Housing Germfree and Gnotobiotic Mice in the Same Room

Abstract

Here, we combined the use of 2 technologies that have not previously been used together-a positively pressurized isolator IVC (IsoIVC-P) and a modular isolator with integrated vaporized hydrogen peroxide (VHP) technology???to develop highly tractable and scalable methods to support long-term maintenance of germfree mouse colonies and the concurrent use of germfree and gnotobiotic mice in the same room. This space-efficient system increases the practicality of microbiome studies. Specifically, the exterior surfaces of microbially similar IsoIVC-P were sterilized by using VHP prior to opening the cages and handling the mice therein. This space-efficient system increases the feasibility of microbiome studies. After over 74 wk of experimentation and handling equivalent to more than 1,379,693 germfree mouse-days, we determined that the method and practices we developed have a weekly performance metric of 0.0001 sterility breaks per husbandry unit; this rate is comparable to the isolator 'gold standard.' These data were achieved without adverse incidents while maintaining an Altered Schaedler Flora colony and multiple gnotobiotic studies involving fecal microbial transplants in the same room. Our novel IsoIVC-P???VHP workstation housing system thus improves microbiome research efficiency, eliminates hazards, and reduces risks associated with traditional methods.

Figure 1.

(A) Aseptic workstation with front sash of VHP module open, showing (B) supplies for cage change (CC, clean IsoIVC-P; DC, dirty IsoIVC-P with mice; WB, sterile water; T, animal transfer tongs, T) and experimental manipulation (white arrows, syringes and PBS for oral gavage) and the pass-through windows to the material transfer device (MTD) and extension module (EXT). (C) Surface sterilization with hydrogen peroxide vapor of all supplies during a VHP loaded cycle prior to opening of a IsoIVC-P and mouse use.

Figure 2.

(A) Rack of IsoIVC-P (green arrow) and a 4 chamber VHP-equipped workstation (red arrow) were used to house and handle germfree and gnotobiotic mice. (B) The safety and efficacy of the 2 system components were confirmed first in the absence of mice by using biologic (green arrow) and chemical (red arrow) indicators of VHP placed inside and on the outside of the IsoIVC-P (foreground) and germfree shipper (background), (C) exposed to a QVHP loaded cycle, after which a (D) handheld low-level H₂O₂ monitor was used to confirm that VHP levels inside the module had degraded to less than 1 ppm. (E) Conditions inside and outside of the IsoIVC-P during a VHP loaded cycle indicated a VHP-sterilizing effect on the exterior surfaces of the IsoIVC-P, whereas the IsoIVC-P interior experienced only a modest increase in ambient temperature.

Figure 3.

Read counts of microbiota shotgun sequencing of fecal pellets of 16 representative mice (8 male mice [M1–6, 15, and 16] and 8 female mice [F38–41, 28, 29, 31, and 35]), prior to gavage (top) and after 1, 3, and 5 wk of weekly gavage of either PBS or SPF FMT. A monocontaminant, Paenibacillus macerans, in a single female mouse (F41) spread to members of the PBS female cohort handled during the same processing mode (F38–41) but not to members of the germfree PBS male cohort handled during a separate processing mode (M1–4) or to germfree colony mice housed nearby on the same rack and processed separately (data not shown).

Figure 4.

Read counts of microbiota shotgun sequencing of fecal pellets of mice gavaged with SPF FMT, showing that individual mice were colonized similarly (that is, no individual variability), regardless of whether they were housed in the same or a different IsoIVC-P (that is, no cage effect).

Figure 5.

During 12 VHP processing mode events, airborne live microbes were recovered by using (A) culture settle plates (red arrow) that had been placed in (B) an active air sampler (blue arrow). (C) Total airborne particles of 2 size thresholds were counted by using a light-scatter airborne particle counter (black arrow).

Figure 6.

Average airborne particle counts of 12 QVHP processing modes remained well below requirements for ISO 14644-1 Class 5 while mice were handled and gavaged for FMT.

Figure 7.

Read counts of microbiome shotgun sequencing of fecal pellets of representative mice after 6 wk of biweekly gavage of either sterile PBS (from left, lane 1), 2 unique SPF FMT (lanes 6 and 7), or ASF FMT (lane 5), and of germfree colony mice of either the BALB/c (lane 2), C57BL/6 (lane 3), or Swiss Webster (lane 4) lines housed on the same IsoIVC-P rack.

Figure 8.

Relative abundance of the fecal bacterial microbiota of mice that were gavaged for FMT of one of 3 distinct flora, either an Altered Schaedler Flora (ASF) slurry, or one of 2 SPF slurries derived from mouse colonies housed in separate vivaria on different campuses. The microbiota of these mice remained distinct through the end of the 6-wk study while mice were housed on the same rack.

Figure 9.

The percentage of mouse colony fecal specimens that were negative by PCR analysis for bacterial 16s rRNA and microbial cultures during the study (green circles) and the number of germfree IsoIVC-P (blue diamonds) and gnotobiotic IsoIVC-P (red triangles) housed during the study. Blue bars indicate the dates of germfree studies; red bars indicate the dates of gnotobiotic studies.

Figure 10.

The IsoIVC-P–VHP workstation housing system that we describe here was used for (A) production of germfree mouse colonies, (B) studies of tumor growth, (C) microsurgical procedures, and (D) inhalational anesthesia of germfree mice using procedures that incorporated in-line anesthesia gas filters (white arrow).