Basic steps
- Weigh the amount per litre stated on the specification sheet and use purified or distilled water. Dehydrated media absorb moisture, so weigh quickly after opening and reseal the container at once.
- Dissolve by adding the powder to about half the final volume of water with stirring, then make up to volume. Most broths dissolve at room temperature. Media containing agar must be heated with stirring to a full boil for one minute until the liquid is clear and no particles remain on the vessel wall.
- Check pH if required. Most formulations need no adjustment. If they do, adjust with 1 N NaOH or 1 N HCl measured at 25 °C, and do it before sterilisation, since autoclaving lowers pH slightly.
- Dispense into vessels filled to no more than two-thirds, with caps loosened.
- Sterilise, usually at 121 °C for 15 minutes. Formulations high in sugar or large volumes may need a shorter cycle or lower temperature as stated on the specification sheet, to avoid caramelisation.
- Pour plates once the medium has cooled to 45 to 50 °C. Too hot produces heavy condensation; too cool and it sets before pouring.
Common mistakes
- Autoclaving before the agar has fully dissolved, giving uneven gels and sediment at the bottom.
- Tipping the whole pack into the full volume of water, which forms a skin on top with dry powder underneath.
- Over-long sterilisation, darkening the medium and reducing performance (see "Why does the medium darken or turn cloudy after autoclaving?").
- Preparing with tap water; calcium, magnesium and chlorine affect some selective media.
Formulations that must not be autoclaved
Antibiotics, some dyes and other heat-sensitive supplements are added aseptically after sterilisation once the medium has cooled to about 50 °C. The specification sheet marks components to be "added after sterilisation".