ZBrett Illustrated Directions

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Z-Brett Illustrated Directions

• For use with Z-Brett 24 Brettanomyces, 3hr Visual
  EIA
• Detection Reagent Kit

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Part 1

• SAMPLING SAMPLE PREP

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SAMPLING

• Collect 1mL of wine (or up to 50 mL - refer to
  Ultra Sensitive Detection Procedures). Sample
  from within an inch of the bottom of tank or
  barrel. Take two independent samples for
  duplicate testing.

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Tip

• Wash supplied bulb pipette between different
  samplesby rinsing with water 2 or 3 times.

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SAMPLE PREP

• (a) Centrifuge 1 mL of wine sample in 1.5 ml
  microfuge tube. Tubes should be balanced to avoid
  damaging the microfuge rotor.

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• (b) Remove supernatant using a bulb-pipette.
  Avoid disturbing any pellet at the bottom of the
  tube.

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• (c1) Add 1ml Decolorizer (A) to microfuge tube

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• (c2) Suspend cells by vortex mixing,tapping with
  finger, tapping on table or pipette/re-pipette
  5-10 times.

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• Repeat wash steps (a) to (c2) above once and then
  steps (a) to (b) one more time.
• (d) Suspend pellet in 40 µL of cell Suspension
  Buffer (B) (1 drop). Mix, then proceed to Test
  Procedure below.

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Part 2

• TEST PROCEDURE

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STEP 1 Apply Samples Dry

• (a) Mix samples immediately before spotting. it
  is very important to mix each sample immediately
  before application, since Brett settles quickly

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• (b) Apply 5 µL of each sample in duplicate to
  chip using a micro-pipette.

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• (c) Record Sample I.D. on the chip to identify
  wine locations

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• (d) cover chip with petri dish cover (or any
  other cover you select) to dry overnight (be
  careful moving chips after spotting to prevent
  spots from running or smearing)

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STEP 2 Destain Block

• For the following processing steps, the chip must
  be covered in liquid (i.e.10 mL). Place dried
  chip in Petri dish provided.

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• (a) Using measuring tube provided, pour 10 mL
  Destain/Blocker (C) carefully over chip, so that
  the chip is covered in liquid.

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• (c) Allow the chip to rest in Destain/Blocker
  (minimize bubbles) for about 15 minutes, mixing
  periodically by gentle shaking.

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• (b) After about 15 minutes, agitate the dish
  again rapidly from side-to-side until wine color
  diffuses away.

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Tip

• If strong color persists after 15 minutes,
  repeatedly squirt Destain/ Blocker solution with
  a disposable bulb-pipette (provided) onto the
  persistent stain(s) until only a very light (or
  no) color is present. Note that a light color
  will fade after processing.

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STEP 3 Antibody

• Add 6 drops of Anti-Brett (D) to the petri dish
  (containing Destain/Block chip)

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• and then add 6 drops of Conjugate (F)

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• Mix by hand periodically (2 or 3 times) over 30
  minutes.
• Discard all liquid
• from petri dish.

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STEP 4 Conjugate (second addition)

• Using a clean (or rinsed) measuring tube, pour 10
  mL of Buffer (E) into dish to cover chip.

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• Add 6 drops of Conjugate (D) and mix by hand
  periodically (2 or 3 times) over 30 minutes,
  discard liquid.

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STEP 5 Wash

• Using a clean (or rinsed) measuring tube, pour 10
  mL of Buffer (E) into dish to cover chip. Mix
  gently by hand for about 1-2 minutes, discard
  liquid. Repeat two more times for a total of 3
  washes.

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STEP 6 Develop Color

• Prepare Developer Mixture by combining Developer
  Diluent (G1) and Active Developer (G2).

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• Prepare 10mL of Developer Mixture by combining 10
  ml of Developer Diluent (G1) and 0.1 ml of Active
  Developer (G2) in a clean measuring tube. (Or,
  simply add G10.6 ml into G2 60mL and use
  immediately.)

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• Pour 10 mL Developer Mixture into dish to cover
  the chip wait 20 minutes.

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• Discard liquid and rinse chip with cold tap water
  for a few seconds.

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• Place chip upright at an angle and air dry.
  Compare sample intensity with calibration column
  on the chip.

wet
dry