Isolation and Culturing
Culturing
- Make sure the hood has been on UV for at least 30 minutes before plating and clean hood surfaces
- Use scalpel or plug puncher to remove agar from the edge of the original culture’s growth, transfer to new plate
- Parafilm both plates back up, label the new plate with the specimen type, media type, date, and initials
- Place the new culture in the 25°C and the old culture in the tall incubator
Isolation From Fresh Poop
- Collect poop from specimen bag and place it into a 2mL tube
- Vortex the tube before plating
- Begin plating under the hood, ensure to Ethanol the area first
- Flame tweezers in the microincinerator, open a large plate and place a large filter paper on top of the TWA, gently flatten as needed. If the filter is not completely stuck down/partially moist, slightly moisten the filter with DNA/RNA free water.
- Pipette ~100mL of poop in 3 separate locations on the filter paper and spread in each area using the pipette tip.
- Clean sides of plate with Ethanol, parafilm it closed, label plate (media used, specimen name, initials, date, and time) and place on metal rack or in plastic box on top of the Brandt -20 (make sure the TWA and filter side is on the bottom with PDA on the top)
- Check every ~24 hours for sporulation
- If sporulation has occurred, perform a regular solid culture transfer from the area of sporulation into a fresh PDA plate
Liquid Sporulation Culture
- Make small YPSS plates of specimen at least 1 week prior to liquid culturing
- Autoclave and UV components - mason jars, mason jar tops
- Under the hood, ethanol rim of mason jar and jar top, then place jar top on mason jar and tape the top and jar together
- Pour about 150ml of PDB in through the top opening
- Ethanol sides of culture plate, then place plate in the top opening
- Cover top fully with aluminum foil, then tape the foil down and write the specimen name, date, type of liquid media, and initials on the tape