The Australian Wine Research Institute > Services to Industry > Winemaking > Laboratory methods > Microbiological > Preparation of MRS + apple juice medium

Preparation of MRS + apple juice medium

Aim or introduction
MRS + Apple Juice Medium used for isolating and enumerating lactic acid bacteria, especially Oenococcus species from wine. Cycloheximide can be added to suppress growth of most yeasts at a concentration of 10 mg/L.

Safety issues
Avoid breathing dust. Cycloheximide is very toxic and flammable. Wear gloves when handling it.

Preparation required
You can prepare the cycloheximide solution if required. Refer to cycloheximide method. Stock solution can be stored in the fridge.

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. MRS nutrient medium (e.g. Oxoid® or Amyl®)
  2. Agar
  3. MRS supplement
  4. Apple juice
  5. Purified water (e.g. distilled)
  6. Cycloheximide solution (0.5 mg/mL)

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)
MRS Broth* 11.5 g 23 g 46 g
MRS supplement (Amyl®) 10 mL 20 mL 40 mL
Apple Juice
(to give final concn. 20% v/v)		 50 mL 100 mL 200 mL
Agar* 5 g 10 g 20 g
Purified water 190 mL 380 mL 760 mL
**After autoclaving**
Cycloheximide solution
(0.5 mg/mL)		 5 mL 10 mL 20 mL

*Note: MRS agar medium contianing both these ingredients can also be obtained.

  • The apple juice is clear juice containing no preservatives or other additives, available from supermarkets.
  • Cycloheximide stock solution: 0.5 mg/ml in 96% ethanol.

Procedure

  1. Weigh the nutrient media using the top pan balance.
  2. Measure the apple juice and MRS supplement and mix with the media.
  3. Add the water and mix thoroughly.
  4. Adjust the pH to 5.5 if necessary.
  5. Pour solution into bottles leaving sufficient headspace and loosely screw on lids
  6. Sterilise by autoclaving at 121C for 15 minutes (allow the autoclave pressure to return to normal before opening)
  7. Carefully remove bottles from autoclave and place in the water bath to cool to 55C (approximately 60 minutes).
  8. When cool, add the cycloheximide if required and swirl to mix.
  9. Pour sufficient volumes of the molten medium at a minimum temperature of 45C into Petri dishes to give a depth of approximately 5 mm. As soon as the medium is set, dry in laminar flow cabinet.
  10. 10. Store made up plates inverted in the fridge, sealed in plastic bags

    11. NB. Cycloheximide is rapidly inactivated by direct light (especially ultra violet light) – never expose cycloheximide solutions, mixtures or plates to direct sun light – short exposure to laboratory lighting is acceptable. Protect plates from light during storage, ie dark cupboard or refrigerator.

    References

    1. Oxoid manual
    2. Amyl manual