The Australian Wine Research Institute > Services to Industry > Winemaking > Laboratory methods > Microbiological > Preparation of WL nutrient agar medium

Preparation of WL nutrient agar medium

Aim or introduction
WL nutrient medium is used for the culturing of yeast species. WL nutrient medium can be prepared as liquid media, or most commonly, as agar plates and slopes.
WL nutrient medium contains a colouring agent which can be used to differentiate between colony types.

Safety issues
Avoid breathing dust.

Time frame of preparation
Allow 20 minutes to make media, 45 minutes to autoclave and 60 minutes to cool before pouring plates.

Equipment

  1. Steriliser or autoclave
  2. Top pan balance
  3. Waterbath (set to 55°C)
  4. Laminar flow cabinet

Materials

  1. Bottles with screw on caps capable of withstanding sterilisation (e.g. Schott)
  2. Weigh tray
  3. Measuring cylinder

Reagents

  1. WL nutrient agar medium (e.g. Oxoid® or Amyl®)
  2. Purified water (e.g. distilled)

Medium can be made up in the volumes required using the proportions indicated below:

for 250 mL(10-15 plates) for 500 mL(20-30 plates) for 1 L(for 40-60 plates)
WL Nutrient Medium 18.75 g 37.5 g 75 g
Purified Water 250 mL 500 mL 1000 mL

Procedure

  1. Weigh the nutrient media using the top pan balance.
  2. Add the water and mix thoroughly.
  3. Pour solution into bottles leaving sufficient headspace and loosely screw on lids
  4. Sterilise by autoclaving at 121°C for 15 minutes (allow the autoclave pressure to return to normal before opening)
  5. Carefully remove bottles from autoclave and place in the water bath to cool to 55°C (approximately 60 minutes).
  6. When cool, swirl to mix and use.
  7. Pour sufficient volumes of the molten medium at a minimum temperature of 45°C into Petri dishes to give a depth of approximately 5 mm. As soon as the medium is set, dry in laminar flow cabinet.
  8. When set, the plates are to be inverted, stacked back in the plastic bag that they were packed in. Ensure the bag is labeled with the date, media type and appropriate expiry date (generally 3 months from preparation).
  9. Store made up plates inverted in the fridge, sealed in plastic bags.

Trouble Shooting
If the plates don’t set after 20 minutes, there is either a problem with that batch of WL agar, or the agar has been overheated destroying the gelling properties.

References

  1. Oxoid manual
  2. Amyl manual