Revival Of Freeze-Dried Cultures
Most lyophilized cultures 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 have been sealed perfectly and other ampoules may crack during storage. We therefore recommend to revive the cultures within a few weeks.
Opening of an ampoule: Make a file mark on the ampoule if there is none. Wrap up the ampoule in sterile paper cloth and crack the ampoule with your fingers. Ampoules of hazardous pathogens should be opened in an exhaust protective cabinet specially designed to protect the worker.
With a Pasteur pipette, add a few drops of broth (Brain Heart Infusion or similar) to the contents of the ampoule. Dissolve all the material and transfer all the material to suitable solid medium. Recommended medium and conditions for incubation are given on the information sheets sent with the cultures, or will be found in the CCUG catalogue. It may be a good practice to subcultivate on to two or more different medium formulations or incubation conditions.
Discard all material used in the above revival procedure.
Most Helicobacter are microaerophilic (or microaerobic) organisms, but the amounts of oxygen (5% ?) and carbon dioxide (5% ?) are not critical. Some species and some strains are quite easily cultivated, while others are extremely fastidious.
- Freshly prepared horse blood agar (5% blood, Columbia agar II, BBL) meaning that the plates should be only one or at the most 3 days old.
- Microaerobic conditions with CO2 and H2: Since 1997 we use 10% CO2, 10% H2 and 80% nitrogen (a gas mixture compatible with other applications). The anaerobic steel jar is evacuated with a vacuum pump (20 to 30% of the original atmosphere is left). The jar is then filled with the above gas mixture up to atmospheric pressure. Conditions (with hydrogen) provides normally high humidity which is essential.
- The blood agar surface may be flooded with a few drops of broth.There is no standard incubation time.
- Make subcultures from young cultures.
- Do not attempt cultivation of the most difficult organisms before you have a long experience of the easiest ones
- Check the CCUG site for information on particular strains.
- Very fastidious species: Helicobacter bizzozeronii, Helicobacter salomonis.
- Very fastidious strains: The type strain of Helicobacter felis.
- Growth representative of the species: Helicobacter pylori CCUG 15815 B
- Relatively easily grown strain: Helicobacter pylori CCUG 39500 = ATCC 43504