Spore Preparation Protocol for Enrichment of Clostridia from Murine Intestine

Materials and Reagents

1. Sterile 1.5 ml microcentrifuge tubes (Eppendorf, catalog number: 022363204 )
   
2. 15 ml tubes
   
3. Female (male mice may be used if necessary) C57BL/6 mice aged 8-12 weeks (THE JACKSON LABORATORIES)
   
4. Compressed CO₂ gas in cylinder (AirGas)
   
5. Chloroform (Sigma-Aldrich, catalog number: 288306 )
   
6. Sterile PBS pH 7.4 (Reference 2)
   

Equipment

1. Sterile necropsy instruments (operating scissors, tweezers and forceps) to avoid contamination
   
2. Shaker with 200 rpm capacity and 37 °C setting (or inside 37 °C room)

Procedure

1. Without pre-charging the chamber, place the animal(s) in the chamber and introduce 100% carbon dioxide (whenever possible euthanize animals in their home cage). Open the CO₂ tank or valve regulator to initiate flow of gas. A fill rate of about 10% to 30% of the chamber volume per minute with carbon dioxide, added to the existing air in the chamber should be appropriate to achieve a balanced gas mixture to fulfill the objective of rapid unconsciousness with minimal distress to the animals. Wait approximately 3-5 min for animal to stop moving or breathing. Maintain CO₂ flow for a minimum of 1 min after respiration ceases. Death was confirmed by cervical dislocation.
   
2. Open the abdominal cavity using operating scissors to remove the cecum. Using operating scissors, keep the cecum intact by carefully excising to not lose any contents.
   
3. Open the cecum with operating scissors and add the cecal contents to a 1.5 ml microcentrifuge tube on ice.
   
4. Take cecal contents and dilute 1:10 in PBS (w/v). Add chloroform in a final concentration of 3% (v/v).
   
5. Incubate the cecal contents/chloroform mixture by shaking at 200 rpm at 37 °C for 30 min.
   
6. Allow the chloroform to settle to the bottom of the tube at room temperature (approximately 20 min).
   
7. Remove top aqueous layer to recover the spores and add to a sterile 1.5 ml tube. Be careful not to take any chloroform that has settled to the bottom of the tube.
   

Data analysis

Real-Time PCR using Clostridia specific primers were used for subsequent analysis of log CFU of Clostridia in feces of mice that received spores isolated by this protocol (Rivera-Chávez et al., 2016). Fold changes of ratios (mRNA levels) were transformed logarithmically prior to statistical analysis. An unpaired Student’s t-test was used to determine whether differences in fold changes between groups were statistically significant (P < 0.05).

Notes

1. Expect 100 to 200 mg of cecal contents per mouse.
   
2. It is very important to collect the cecal contents as quickly as possible once mouse has been euthanized to ensure maximum recovery of spores.
   
3. Use sterile technique to not contaminate cecal contents during transfer into a sterile 1.5 ml tube.
   
4. If pooled cecal contents from multiple mice will be prepared, then 15 ml tubes could be used to accommodate greater volumes.
   
5. Isolated spores should be used fresh (same day of isolation) to ensure maximum viability of spores. We do not recommend storing spores.
   

Acknowledgments

This protocol was adapted from Momose et al., 2009. This work was supported by Public Health Service Grants AI096528 (A.J.B.), AI112949 (A.J.B.), AI103248 (S.E.W.), AI112241 (C.A.L.), OD010931 (E.M.V.), and AI060555 (E.M.V. and F.R.-C.).

References

1. Momose, Y., Maruyama, A., Iwasaki, T., Miyamoto, Y. and Itoh, K. (2009). 16S rRNA gene sequence-based analysis of clostridia related to conversion of germfree mice to the normal state. J Appl Microbiol 107(6): 2088-2097.
   
2. Phosphate-buffered saline (PBS) (2006). Cold Spring Harb Protoc.
   
3. Rivera-Chávez, F., Zhang, L. F., Faber, F., Lopez, C. A., Byndloss, M. X., Olsan, E., Xu, G., Velazquez, E. M., Lebrilla, C., Winter, S. E. and Bäumler, A. J. (2016). Depletion of butyrate-producing Clostridia from the gut microbiota drives an aerobic luminal expansion of Salmonella. Cell Host Microbe 19: 443-454.
   
4. Yano, J. M., Yu, K., Donaldson, G. P., Shastri, G. G., Ann, P., Ma, L., Nagler, C. R., Ismagilov, R. F., Mazmanian, S. K. and Hsiao, E.Y. (2015). Indigenous bacteria from the gut microbiota regulate host serotonin biosynthesis. Cell 161(2): 264-276.