Legionella parisiensis Brenner et al.
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2. Using a single tube of #1099 broth (5 to 6 ml), withdraw approximately 0.5 to 1.0 ml with a Pasteur or 1.0 ml pipette. Rehydrate the entire pellet.
3. Aseptically transfer this aliquot back into the broth tube. Mix well.
4. Use several drops of the suspension to inoculate a #1099 agar slant and/or plate.
5. Incubate the tubes and plate at 37°C in an atmosphere of 5% CO2 for 48 to 72 hours. Loosen the screw caps during the incubation period.
Legionella species are nutritionally fastidious and are inhibited by media of poor caliber, so strict attention should be paid to the quality of the ingredients. The pH of the medium should be checked when cool, and is most critical. Unbuffered medium should not be used. Exposure of medium to light, particularly when hot, may also result in the accumulation of peroxides that can be inhibitory to the bacteria.
Before rehydration, vials may be stored at +2 to +8oC for extended periods with minimal loss of viability. Subculturing to maintain culture is discouraged because of the possibility of mutation, selection of variants, or contamination. To minimize changes, it is recommended that cells be harvested and stored at ‑70oC or below.
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Brenner DJ, et al. Ten new species of Legionella. Int. J. Syst. Bacteriol. 35: 50-59, 1985.