Enriching samples for Diastaticus requires a 48-hour serial enrichment process using the FastOrange Yeast solution and copper sulfate additive. FastOrange Yeast solution provides a limit of detection of 1 cell/sample in 48 hours when followed by qPCR. A volume of 40 mL is recommended for sensitive detection. Less volume of FastOrange Yeast solution may be used, but this will result in decreased sensitivity. Note that the concentration of copper sulfate varies depending on whether you use the 40 mL FastOrange Yeast Enrichment Bottles or Broth. Diastaticus grows in aerobic environments, so lay your enrichment containers on the side to maximize air exposure when possible. All enrichment solution, copper sulfate additive, and sample volume should be transferred utilizing sterile technique.
Using the 40 mL FastOrange Yeast Enrichment Bottles:
- Add one tube of provided Wild Yeast Copper Additive per 40 mL Yeast Enrichment Bottle and gently swirl in a circular motion to mix. Add approximately 40 mL of sample to one FastOrange Yeast Enrichment Bottle with copper sulfate and incubate for 24 hours at room temperature (25 °C +/- 2 °C). Lay the bottle flat on its side and vent the sample to avoid pressure buildup.
- After 24 hours, transfer approximately 40 mL of the solution from the first bottle to a second, new 40 mL FastOrange Yeast Enrichment bottle, add another tube of copper sulfate, and incubate another 24 hours. Keep the bottle flat on its side and vented.
Using the 240 mL FastOrange Yeast Broth:
- Add one tube of provided Wild Yeast Copper Additive to a 240 mL FastOrange Yeast Broth bottle and swirl or invert to thoroughly mix. Aliquot your entire 240 mL FastOrange B Broth from the glass bottle into individual enrichment containers with your chosen volume. We recommend 40 mL for sensitive detection. Use sterile technique to transfer the solution. Aliquoted enrichment containers can be stored vertically at room temperature (max 25 °C), protected from light.
- Add your sample in a 1:1 ratio to one enrichment container containing copper additive and incubate the enrichment container for 24 hours at room temperature (25 °C +/- 2 °C)). Leave the bottle on its side and vent the sample to avoid pressure buildup.
- Transfer approximately half of the solution from the first container to a second, new sterile enrichment container containing the same volume of combined broth and copper sulfate additive. Incubate for another 24 hours, again making sure to keep the bottle on its side and vented.
After your sample has been enriched for 48 hours, proceed with analysis by qPCR.