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C. difficile Spore Preparation

Background

This protocol is for C. difficile spore collection. The isolation and purification of C. difficile spores are necessary to understand and study the mechanism of germination and more importantly, infection! Please make sure to perform this procedure in a biosafety hood/anaerobic chamber whenever the bottle/tube/culture needs to be opened in order to minimize the spread of aerosols. C. difficile is a spore-forming anaerobe! Use bleach to sterilize any spills since ethanol is not sufficient!

To induce sporulation in C. difficile, plate C. difficile on BHIS plates (see growth media recipes) and incubate in the anaerobic at 37˚C for 3-7 days.

Note: the hydrophobic and anionic nature of C. diff spores make them stick to the pipette tips very well so make sure to mix well during each transfer (1).

Materials

10X PBS: Dissolve 25.6g of Na2HPO4·7H2O, 80 g NaCl, 2 g KCl, and 2 g KH2PO4 in 800mL of water and bring up the total volume to 1000 mL. Filter sterilize or autoclave for 20min and store at room temperature.

1X PBS: Add 100mL of 10X PBS into 900mL of water. Filter sterilize or autoclave for 20min and store at room temperature.

10% w/v taurocholate sodium: Dissolve 100mg of taurocholate sodium into 1mL of water. Filter sterilize and freeze to storage.

5% w/v Cysteine: Dissolve 2.5g (Sigma 168149) in 50mL of water. Filter sterilize and store in room temperature. This solution tends to crash out very easily so it’s recommended to scale down based on the final volume.

BHIS (Brain Heart infusion + Yeast Extract): Mix water, BHI, Agar, yeast extract, and autoclave on liquid 20 cycle. Cool and add cysteine and 10% taurocholate sodium. Ingredients (for 400 mL): BHI (14.8 g or final conc. of 37g/L), Agar (6 g or final conc. of 15g/L), yeast extract (2g or final conc. of 5g/L), 5% cysteine (4mL of 5% w/v cysteine or final conc. of 0.05% w/v), 10% taurocholate sodium (4mL of final conc. of 0.1% w/v).

Spore collection (Biosafety cabinet):

  • Add 1.5 mL of 1X PBS to each plate and gently scrape off the bacterial lawn using a spreader or a loop.
  • Tilt the plates and transfer the PBS+cell mixture to a 15 mL conical tube and suspend the mixture in 8.5 mL of PBS. Vortex briefly to mix.
  • Incubate the mixture at 4˚C for 24hr aerobically.

Spore purification (Biosafety cabinet):

  • Centrifuge the mixtureat 6000g for 5 minutes on the Beckman Coulter JS5.3
  • Decant the supernatant and resuspend the pellet in 1mL 1X PBS and transfer to a microcentrifuge tube. (use the screw-top microcentrifuge tube to prevent aerosols and spills)
  • Centrifuge the mixture at 16,100 g for 2 minutes at 4oC. Repeat this wash step for 3 times.
  • Heat the mixture at 60˚C water bath for 20 minutes to kill any remaining vegetative cells.
  • Wash the mixture with 1 mL 1X PBS for 5 times (Centrifuge the tube for ~2 minute at >10,000g and discard supernatant) and resuspend the final spore pellet in 1.0 mL 1X PBS.

QC: Spore enumeration (anaerobic chamber):

  • Count the spore yield by performing serial dilutions and platting on BHIS+0.1% TCA or TCCFA plates (see growth media recipes).
  • Expected yield ~ 1e7 spores/mL

References:

  • Melissa Weldy, Clayton Evert, Peter I. Dosa, Alexander Khoruts, Michael J. Sadowsky, Convenient Protocol for Production and Purification of Clostridioides difficile Spores for Germination Studies, STAR Protocols, Volume 1, Issue 2, 2020, 100071, ISSN 2666-1667, https://doi.org/10.1016/j.xpro.2020.100071.
  • Fimlaid KA, Jensen O, Donnelly ML, Francis MB, Sorg JA, Shen A. Identification of a Novel Lipoprotein Regulator of Clostridium difficile Spore Germination. PLoS Pathog. 2015 Oct 23;11(10):e1005239. doi: 10.1371/journal.ppat.1005239. PMID: 26496694; PMCID: PMC4619724.
  • Fimlaid KA, Jensen O, Donnelly ML, Francis MB, Sorg JA, Shen A. Identification of a Novel Lipoprotein Regulator of Clostridium difficile Spore Germination. PLoS Pathog. 2015;11(10):e1005239. Published 2015 Oct 23. doi:10.1371/journal.ppat.1005239
  • Edwards A.N., McBride S.M. (2016) Isolating and Purifying Clostridium difficile Spores. In: Roberts A., Mullany P. (eds) Clostridium difficile. Methods in Molecular Biology, vol 1476. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6361-4_9
  • Heeg D, Burns DA, Cartman ST, Minton NP (2012) Spores of Clostridium difficile Clinical Isolates Display a Diverse Germination Response to Bile Salts. PLOS ONE 7(2): e32381. https://doi.org/10.1371/journal.pone.0032381
  • Edwards, A. N. & McBride, S. M. Isolating and Purifying Clostridium difficile Spores. Methods Mol. Biol. 1476, 117–128 (2016).