5.1. Basal medium (modified from Bailey, 1957)
the following components in approximately 800 ml deionized water:
- 10 g of yeast extract
- 10 g of glucose
- 10 g of starch
- 0.25 g L-cysteine
- 20 g of agar
- Add 100 ml of 1M KH2PO4 (pH 6.7)
- Adjust the pH to 6.6 using 2.5 M KOH.
- Adjust the final volume to 1000 ml.
by autoclaving at 115˚C for 15 minutes.
Optional; in order to prevent growth of secondary bacteria, filter-sterilized nalidixic acid (dissolved in 0.1 M NaOH may be added to a final concentration of 3 µg per ml after autoclaving.
For liquid cultures, the starch may be replaced with saccharose, making the medium clear. This will facilitate when checking the turbidity or the cloudiness of the cell suspension, e.g. to see if there is any bacterial growth.
the plates for 7 days at 35°C anaerobically.
Colonies about 1 mm in diameter will appear after 4-7 days (Figs 4 a, b), and can be further confirmed by staining, LFI or PCR (see sections 6.1, 7.2 and 8). Single bacterial colonies can be screened using real-time PCR / conventional PCR by simply touching a small (10 µl) tip directly onto the colony of interest, touch the tip onto some agar to sub-culture if required, before placing the tip directly into the PCR master-mix (see section 8).
Fig. 4. a) Agar plate (basal medium) with colonies of M. plutonius. The yellow bar represents 5 mm. Photo: Lena Lundgren and Karl-Erik Johansson. b) Colony morphology of M. plutonius on basal medium. The bar represents 1 mm. Photo: Eva Forsgren.