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Fecal Transplant to Gnotobiotic Mice

Background

This protocol is for conducting fecal transplant to gnotobiotic mice. The starting material can be feces or cecal contents. For highest transplanation efficiency, a euthanized whole mouse (or clamped cecum) can be transfered into the anaerobic chamber for preparation of the inoculum.

Materials

  • Fecal Transplant Media (BHI CHVR + 20% Glycerol, see Media Recipes)
  • 1mL sterile syringes (BD309659)
  • Gavage Needles (VWR 20068-636)
  • Sterile 1.5 mL Cyrovials with internal threads and washer
  • Sterile 15 mL Conicals
  • 100 µm cell strainers (Fisher 08-771-19) Optional
  • Scale capable of ~10mg accuracy
  • Appropriate sterilant (i.e. CLIDOX or ExSpore)

FMT Preparation

Note: all supplies/media need to be sterilized and moved into anaerobic chamber at least 24h ahead of time.

  • Weigh ~200 mg of either feces/cecal contents in anaerobic chamber
  • Resuspend 10% w/v (i.e. 2mL) in transplant media
  • Vortex well to mix
  • Option A: Leave standing for ~10 minutes to let solids settle OR Option B: pass mixture through 100µM cell strainer (note, this may remove some fungal components of community).
  • Transfer cleared solution to cryovial and seal. Optional: crimp-top vials can be used in place of cryovials; however, it is unclear if the additional oxygen exclusion is worth the effort.
  • Either take immediately to gnotobiotic facility or freeze at -80˚C.

FMT Administration

Note: Both the gave needles and syringes should have been sterilized in advance by ethylene oxide treatment. If unavailable, use sterilant and carefully flush syringe and needle with drinking water before drawing inoculum.

  • Prepare sterilant (i.e. Exspore using a 1:4:1 base:water:activator ratio).
  • Submerge entire cryovial in sterilant and transfer into exchange port of gnotobiotic isolator
  • Fog gnotobiotic port and wait ~40 minutes (or appropriate contact time) to externally sterilize inoculum.
  • Draw entire volume into syringe and tap out air bubbles
  • Scruff mouse and administer 100µL/mouse by oral gavage.
  • Examine mouse 15 minutes after gavage to ensure injury has not taken place, if there are suspicions: refer to animal protocol.
  • Stable colonization usually occurs within 7 days for complex communities or 3 days for synthetic communities.