Revival Of Freeze-Dried Strains
The CCUG provides strains as freeze-dried (lyophilized) biomass in sealed glass ampoules.
Most lyophilized strains will remain viable for decades or centuries if kept in the dark at a temperature of +4°C to +8°C. However, some ampoules may not remain sealed over time and other ampoules may crack during storage. We therefore recommend to revive the strains as soon as possible or within a few weeks of receiving the ampoules.
Opening of an ampoule: using a file, make a mark on the ampoule, if there is none. Wrap the ampoule in layers of sterile paper cloth and crack the ampoule by hand. Ampoules with hazardous pathogens should be opened in an exhaust protective cabinet designed to protect the worker.
With a Pasteur pipette, add 100-200 µl of sterile broth (Brain Heart Infusion or similar) to the contents of the ampoule. Make sure the freeze-dried material is completely resuspended by pipetting. If necessary, add additional broth. Transfer all of the suspended material to a suitable solid medium.
Recommended media and conditions for incubation are given on the information sheets sent with the strains or will be found in the CCUG online catalogue. It is good practice to subcultivate on one or more different media and try different incubation conditions to be able to detect possible contaminants.
Discard safely all material used in the above revival procedure.
Always subcultivate one or more times from the primary culture before starting the work with the strains, to ensure happy bacteria!
Helicobacter
Most Helicobacter spp. strains are microaerophilic (or microaerobic) organisms. Some species and some strains are quite easily cultivated (e.g., Helicobacter pylori CCUG 39500T), while others are extremely fastidious (e.g., Helicobacter hepaticus, Helicobacter pullorum.
We use:
- Freshly prepared horse blood agar (5% blood, Columbia agar II, BBL) meaning that the plates should be only one or, at the most, two days old.
- Microaerobic conditions in a steel jar: Since 1997 we use 10% CO2, 10% H2 and 80% N2. The anaerobic steel jar is evacuated with a vacuum pump (15 of the original atmosphere is left). The jar is then filled with the above gas mixture up to atmospheric pressure. These conditions (with hydrogen) normally provide high humidity, which is essential.
- A few drops of broth may be flooded onto the blood agar surface with the biomass suspension
- No standard incubation time.
- Subcultures from young cultures, since helicobacters tend to go into coccoid, unculturable stages after time or multiple transfers.
- Check the CCUG website for information on particular strains.
- We recommend that you have experience with the easier species before attempting to culture the most difficult organisms.