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Sampling

Take samples from bottles/tanks 24h pre-inoculation (domestic fermenters have sampling ports).

Risk of contamination: use professional skills + strict aseptic procedures.

Sampling containers: sterilized 500ml Erlenmeyer flasks/test tubes (pre-prepared with sterilized agar medium).

Procedure: 2 people required (1 for flame protection, 1 for collecting liquid from tank’s sterilized bottom outlet); keep cotton plug under flame protection.

Incubate sample at 30°C for 1-2 days; discard all tank medium if contamination is found.

Sensory & Odor Testing

Observation (every 12h, 24h post-inoculation):

Color: Normal medium = pure (pale yellow/orange-yellow/light brown), no turbidity, fades gradually.

Clarity: Clear/transparent = abnormal.

Mycelial balls: Obvious burrs, discernible (no turbidity); uniform in size/quantity; after 5min standing, neither float nor settle (clear boundary with medium).

Smell (at air release valve): Normal = sweet medium aroma fades to light fresh scent; alcoholic/sour/foul odor = spoilage.

Plate Culture Test

Aseptically pour sample onto pre-prepared plate, seal, incubate at 30°C (3 replicates/sample).

Result: Colonies other than Flammulina velutipes = contaminated; only F. velutipes mycelium (no smooth cheesy liquid) = normal.

Microscopic Examination

Method: Apply small amount of liquid culture to slide, observe mycelium.

Two types:

Direct examination: Easy to observe bacteria.

Gram staining: Distinguishes stained structures (cell wall, nucleus).

Normal F. velutipes mycelium: Distinct septa + numerous lock-like joints.