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Intended Use
For the detection and cell counts of Dekkera/Brettanomyces (Brett) in juice or wine samples.Brett tends to settle on the bottom. Sample from the bottom or mix before taking sample.
Remove Parafilm from edges of each plate before you start. (OPTIONAL place at room temperature after removing Parafilm for at least 30 minutes to allow evaporation of water droplets that normally form during cold storage.)
Procedure
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Apply 5 drops (50uL) in circular pattern directly to agar using supplied dropper
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Add 1 tube worth of supplied glass beads,cover plate.
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With plate flat on the table, slide/shake plate back and forth about 10 times.
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Remove top and invert plate over waste container to dispose of beads.
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Incubate plates at 25ºC–30ºC (room temperature is okay) in supplied self-sealing incubation bags.
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After 4-14 days (up to 20) look for cheesy white or green colonies. Record the number of colonies. look/smell for colour change and phenolic odor confirmation of Brett. Optional: send suspected positive plates to Lebrun Labs for laboratory confirmation.
Interpretation
This media is very selective for Brettanomyces and is enriched to assure relatively fast growth of this slow-growing organism.
Consider being positive if:
1. Colony growth is detected between 4-14 days. Colony appearance is intially cheesy, whitish-yellow later turning green.
2. The agar colour changes from blue to green (low cell number) or yellow (high cell number); can occur within 4-14 days.
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3. A phenolic medicinal or leathery odor is detected when opening the bag/plate. Can occur in 48 hours or longer. Suggested: Compare odor to Brett sensory set (sold separately).
If single colonies develop, multiply by 20 to get colony forming units (CFU) per ml. If color change and development of phenolic odor is rapid and a haze develops over the entire plate, your sample is TNTC (To Numerous To Count) and contains over 10,000 CFU/ml. Brett phenolic metabolites taint the wine when Brett is at a high concentration.
If you get a TNTC result:
1. Immediately filter or otherwise sanitize the wine
2. Repeat the assay with serial 1:10 dilutions to determine exact cell concentration and confirm the extent of Brett contamination.
3.Retest after remediation to make sure effective.
Precautions
To avoid cross contamination of the winery environment, it is recommended that after the test is complete 10% bleach or 70% alcohol is applied to the plate and beads prior to disposal.
Minimize the time that the top is off from the dish. Work in a draft free environment.
Avoid directly touching the agar medium.
Seal plates within supplied self-sealing bags to reduce plate drying and enhance phenolic odor detection.
Storage Store refrigerated between 2-8ºC.
Shelf life: 4 months with refrigeration, 2 months without refrigeration.
Lebrun Labs LLC
5475 E. La Palma Ave. Suite 206
Anaheim, CA 92807
Office (949) 246-0339, Lab (714) 345-4689, Fax (714) 779-1902
http://lebrunlabs.com
sales@lebrunlabs.com
Identification and Control of Brettanomycesin the Vineyard and Winery
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