One of the more vital considerations to remember when working with C. difficile spores is that their hydrophobic and anionic properties allow them to adhere to many surfaces, including pipet tips and polypropylene. When working with spore stocks, it is important to pipet up and down several times, especially when making dilutions, to ensure as few spores as possible are left behind. The use of polypropylene materials, such as conical tubes, is sufficient when working with high concentrations of spore stocks (greater than 107 colony forming units (CFU)/ml) for a short period of time; however, for long-term storage of high concentration spore stocks and when diluting spores to a concentration of 106 CFU/ml or less, the use of glass [15 (link)] or Teflon-coated tubes [18 ] is highly recommended to prevent loss and obtain reproducible spore counts. Finally, the use of 1% BSA in final spore preparations decreases clumping of spores, prevents loss and facilitates accurate enumeration [15 (link)].
While it is not necessary to use phase contrast microscopy to track sporulation, as described in Section 3.1, the use of this method is a reliable and efficient way to determine the progress of sporulation without removing the plates from the anaerobic chamber and interrupting an experiment. This technique allows for the accurate enumeration of sporulation efficiency in various conditions and at different time points before embarking on the more time-consuming spore isolation and purification protocol.
While it is not necessary to use phase contrast microscopy to track sporulation, as described in Section 3.1, the use of this method is a reliable and efficient way to determine the progress of sporulation without removing the plates from the anaerobic chamber and interrupting an experiment. This technique allows for the accurate enumeration of sporulation efficiency in various conditions and at different time points before embarking on the more time-consuming spore isolation and purification protocol.
