The Australian Wine Research Institute > Services to Industry > Winemaking > Laboratory methods > Microbiological > Preparation of cycloheximide stock solution

Preparation of cycloheximide stock solution

Introduction
Cycloheximide is added to some media (mainly solid media), such as YPD agar, WLN agar and MRS-AJ agar, to suppress the growth of most yeast. Some yeasts, including Debaromyces housenii, Kluyveromyces marxianus and Dekkera/Brettanomyces species are cycloheximide resistant, but growth of most other yeast that can grow in grape juice and wine can be inhibited by 10 mg/L cycloheximide in the medium. To select specifically for the growth of Brettanomyces/Dekkera it is necessary to increase the cycloheximide concentration to 100 mg/L. Stock solution of cycloheximide for use in media preparation is made up in 96% ethanol.

**Safety issues**

  • Cycloheximide is extremely toxic! Use extreme care when handling: use gloves and appropriate protective clothing; avoid inhalation of dust by weighing out cycloheximide in a fume hood. For further safety and related toxicity information (MSDS information), refer to the Sigma-Aldrich website.
  • 96% ethanol is flammable. Do not use cycloheximide stock solution near a naked flame (bunsen).
  • Place warning label on stock solution bottle.

Time frame of preparation
Allow 20 minutes to prepare cycloheximide stock solution(s).

Equipment

  1. Analytical balance

Materials

  1. Weighing tray (optional) or other appropriate receptacle for weighing. Note: the solid powder may be adversely affected by static. Under these circumstances, it is not recommended to use plastic weighing trays.
  2. Spatula
  3. Measuring cylinder
  4. Appropriate chemical storage bottle with screw cap seal
  5. Aluminium foil

Reagents
Depending upon the application, cycloheximide stock solutions of different concentration can be prepared (see table below):

  • 0.5 mg/mL in 96% ethanol (general yeast suppression)
  • 5.0 mg/mL in 96% ethanol (isolation of Brettanomyces/Dekkera)
Application/Component Stock solution volume
200 ml
(for 400-600 plates)		 500 ml
(for 100-1500 plates)		 1 L
(for 2000-3000 plates)
0.5 mg/mL stock solution (for general yeast suppression)
Cycloheximide 100 mg 250 mg 0.5 g
96% Ethanol 200 mL 500 mL 1 L
5.0 mg/mL stock solution (for isolation of Brett./Dekk.)
Cycloheximide 1.0 g 2.5 g 5.0 g
96% Ethanol 200 mL 500 mL 1 L

Procedure

  1. Remove the cycloheximide from cold storage (4°C) and allow to equilibrate to room temperature for a few minutes to minimize moisture uptake.
  2. Weigh the cycloheximide in the fume hood.
  3. Add the ethanol and mix to dissolve.
  4. Store in a foil covered, sealed bottle in cold storage (4°C).

Addition of cycloheximide stock solution to microbiological media

  1. After autoclaving agar media (eg. YPD agar, WLN agar or MRS-AJ agar), place in a water bath (55°C) and allow to cool for 30-60 minutes.
  2. When agar medium is cool, aseptically dispense appropriate volume of cycloheximide stock solution at a rate of 20 mL per litre of medium (see below).*Do not under any circumstances pipette by mouth*
  3. Replace bottle cap/closure of agar medium and swirl thoroughly to mix.
  4. The final concentration of cycloheximide required in microbiological media is dependent upon the intended application (see table below):
    • 10 mg/L: general yeast suppresion – use 0.5 mg/mL cycloheximide stock solution
    • 100 mg/L: isolation of Brettanomyces/Dekkera; use 5.0 mg/mL cycloheximide stock solution

Addition rates of cycloheximide stock solutions to microbiological media.
 

Application Volume of stock solution required for mL agar
250 mL agar
(10-15 plates)		 500 mL agar
(20-30 plates)		 1 L agar
(40-60 plates)
General yeast suppression (10 mg/L cycloheximide final concentration)
Addition rate of cycloheximide
stock solution (0.5 mg/mL) required		 5 mL 10 mL 20 mL
Isolation of Brett./ Dekk. (100 mg/L cycloheximide final concentration)
Addition rate of cycloheximide
stock solution (5.0 mg/mL) required		 5 mL 10 mL 20 mL

Other notes

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. cold storage (4°C) or dark cupboard.

Interpretation of results

Any colonies from wine origin found on YPD- or WLN-cycloheximide agar are likely to be Dekkera/Brettanomyces sp. while colonies from wine origin found on MRS-cycloheximide agar are likely to be lactic or acetic acid bacteria. However, microscopic examination should be used to determine whether the isolates are yeast or bacteria. Yeast colonies which grow slowly and produce an acid reaction (yellow halo) on WLN-cycloheximide agar are more likely to be Brett./ Dekk.
Note that cycloheximide resistant yeast can grow on MRS – cycloheximide agar as well as YPD- and WLN-cycloheximide agar.

References

Fleet, G. H. (1993) The microorganisms of winemaking – isolation, enumeration and identification. In: ‘Wine microbiology and biotechnology’. Ed G. H. Fleet Harwood Academic Publishers: Switzerland, pp. 1-25.

Barnett, J.A., Payne, R.W., Yarrow, D. (1990) Yeasts: characteristics and identification. Second Edition. Cambridge University Press, p. 31.

Appendix 1 Quality assurance

Cultures of control microorganisms can be tested for growth on the media or plates for quality assurance.
Positive control : Dekkera bruxellensis AWRI 952
Negative control : Saccharomyces cerevisiae AWRI 796