1993 Volume 67 Issue 5 Pages 429-434
In order to facilitate the isolation of C. pneumoniae, strain similar TWAR, with high frequency, we investigated the effect of various factors on the infectivity of Chlamydia using two laboratory strains, C. pneumoniae TWAR and C. trachomatis serovar D. The factors tested were the effects of different temperatures for storage conditions, saliva from healthy person, storage media for Chlamydia, and the frequency of freezing and thawing.
Chlamydial suspension was prepared in the two media, SPG (sucrose-phosphate-glutamate buffer pH 7.5), and CT-GM (culture medium for Chlamydia which contains 1, μg/ml cycloheximide and 0.04% glucose). Chlamydial suspension was allowed to stand in each of four different thermal conditions: 37°C, room temperature (25°C), 4°C, 0°C and-75°C for 1, 3, 6, 24, 48, 72, 96, 120 and 144 hours. For storage at-75°C, one of three groups of glass vial tubes containing Chlamydia was covered with an “airmat” to prevent the rapid freezing of Chlamydia. The effect of various factors on the infectivity was assayed by inoculation of the suspension on HeLa 229 cell monolayers.
Results showed that the infectivity rapidly decreased at 37°C and room temperature, while at 4°C, 0°C and-75°C, relatively high infectivity was maintained and continued until days 4 to 6. This decreasing pattern was similar to among the media used. We were not able to find any differences in the infectivity among the samples with or without the “airmat”. No or virtually no infectivity was found after incubation of Chlamydia with saliva at 37°C. High infectivity, however, was maintained at both 4°C and 0°C.
Initial freezing and thawing resulted in about 70-80% loss of infectivity. Infectivity then gradually decreased as the number of freezings and thawings increased. Similar results were obtained for C. trachomatis serovar D.
In conclusion, our research findings indicate that samplings of throat swabs from patients without contamination by saliva is the most important factor for ensuring the successful isolation of C. pneumoniae, and that sample specimens should be transported at 4°C or 0°C and be inoculated as soon as possible. If necessary, until cell monolayers are prepared, samples should be kept at 4°C or 0°C, and should never be frozen at-75°C.
