The influence of variations in the pH, NaCl concentration, temperature, and concentrations of calcium and magnesium ions on the survival of Chlamydia pneumoniae elementary bodies (EBs) outside the host cells was investigated. The survival was determined after various incubation periods by counting the inclusion-forming units after C. pneumoniae was cultured for 72 h on monolayers of HL cells. The normal physiological conditions were restored prior to infecting the HL cells with C. pneumoniae. Declines in the infectivities of C. pneumoniae EBs were observed at pH values of lower than 5 and higher than 8 or at NaCl concentrations of less than 80 mM. The viability of C. pneumoniae EBs in SPG medium decreased as the temperature and/or incubation period increased. Incubation temperatures of up to 20°C and incubation periods of up to 48 h did not affect the viability of C. pneumoniae. One hundred percent of the C. pneumnoniae EBs were infective after 1 h of incubation at 35WC, whereas 90, 50, and 40%37e survived after incubations of 8, 24, and 48 h, respectively. The viability of C. pneumoniae was unaffected within the investigated range of Ca2' and Mg2e ion concentrations in the medium. The presence of 010% fetal calf serum in the incubation medium had a stabilizing effect on the viability of C. pneumoniae. This effect became more pronounced as the incubation period increased.

SUMMARY We compared the survival of a laboratory strain of Chlamydia trachomatis serovar L-2 in different media and at different temperatures (room temperature, 4°C, and -700C). At these temperatures the best storage medium was 2SP (0 2 mol/l sucrose in 0 02 mol/l phosphate buffer supplemented with 10%o fetal calf serum). We used material obtained from patients to study the sensitivity of the culture method as a function of sample storage time and temperature.

The direct fluorescent antibody test and two culture methods were compared for accurate diagnosis of chlamydial infections. Using the same samples, 109 were found to be positive in the microtitre method with the direct confirmation test without subpassage, whereas 66 were positive in the vial method with Giemsa staining and subpassage. The direct test was evaluated for accuracy using cervical and male urethral specimens. Specimens for culture were obtained prior to sampling for the direct test. For cervical samples the sensitivity of the direct test, with the vial method taken as reference, appeared to be 72.2% with a specificity of 93.5%. With the microtitre method as standard, these values were 55.9% and 91.3%, respectively for females, and for male patients 49% and 95.6%, respectively. For cervical samples, in which sampling for the direct test was carried out prior to sampling for culture, the values were 46.3% and 93.2% respectively. Both culture method and study population influenced the sensitivity of the direct test. According to our findings, the direct test cannot replace the culture method for diagnosis of chlamydial infections.

SUMMARY Two hundred and thirty seven semen samples from 10 institutes for artifical insemination by donor (AID) in Belgium and the Netherlands were tested for the presence of Neisseria gonorrhoeae, Chlamydia trachomatis, Mycoplasma hominis, Ureaplasma urealyticum, herpes simplex virus, and cytomegalovirus. The incidence of these micro-organisms in the semen samples was 0%, 6.3%, 4.6%, 35.9%, 0%, and 0.4% respectively, and 47% of all samples were infected with one or more of the micro-organisms. As the ejaculates from which the samples had been taken had already been, or would be, used for AID, the exclusion of microbiological contamination with sexually communicable micro-organisms before insemination is indicated. Two hundred and thirty seven semen samples frozen at -1 96°C, from ten AID institutes in the Netherlands and Belgium were supplied in 0 25 ml or 0 5 ml straws. The semen had been given voluntarily by donors, who received a small sum for expenses. The samples were all diluted 2:1 or 1:1 with a cryoprotective liquid containing glycerol and sometimes also egg yolk or bovine serum albumin. Fifty samples contained ampicillin 380 mg/l and 36 contained kanamycin 5 mg/l. Forty samples came from an institution that used only fresh semen. The semen was diluted 1:1 with Eagle's modified minimum essential medium (EMEM) with 15% fetal calf serum, and was frozen to -196°C. Volumes of 120-200 ,l were diluted with 2 ml of EMEM for culture for Ngonorhoeae, C trachomatis, HSV, and CMV. Volumes of 60-200 ,l were suspended in 2 ml of U-9 medium' for culture for M hominis and U urealyticum. Materials and methods CULTURE METHODSCulture for N gonorrhoeae Semen diluted with EMEM was inoculated with a loop on to a medium consisting of GC medium base (Difco), haemoglobin (Difco), and IsoVitalex (BBL), and was incubated for 48 hours.

A total of 57 infertile women, who had been referred for in vitro fertilisation or for diagnostic laparoscopy, were tested for the presence of antibodies to Chlamydia trachomatis, Neisseria gonorrhoeae, and Mycoplasma hominis. Four were excluded from the study. Of the remaining 53, 33 had laparoscopically obvious tubal disorders, such as adhesions, distal occlusions and strictures, and 20 did not. Antibodies to C trachomatis were found in 7/33 (21.2%) v 0/20, antibodies to N gonorrhoeae in 20/38 (60.6%) v 5/20 (25%), and antibodies to M hominis in 18/24 (75%) women with tubal disorders v 13/19 (68.4%) of those with no disorder. Antibodies to C trachomatis and N gonorrhoeae were significantly (p less than 0.05) more common in women with tubal disorders. The high prevalence of antibodies to N gonorrhoeae in infertile women without tubal disorders suggests that ciliated tubal epithelium is damaged after inflammation without this being laparoscopically visible. Our results confirm the important role of N gonorrhoeae and C trachomatis in the aetiology of infertility after tubal inflammation.