Culture media and bacteria
The media used were 7H9 liquid medium with 10% albumin, dextrose, catalase supplement and 0.05% Tween 80, and 7H11 agar medium with 10% oleic acid, albumin, dextrose, catalase supplement (Becton Dickinson, Oxford, UK). They were made selective by the addition of 100 μg carbenicillin, 200 U polymyxin B, 20 μg trimethoprim and 10 μg amphotericin B per ml (Mast, Bootle, UK) [4]. The two experiments both used 6-week, female Balbc mice, which were infected, with a mouse passaged H37Rv strain of M. tuberculosis suspended in 0.1% gelatin. The spleens and lungs were obtained at sterile autopsy and were homogenised as described elsewhere [5] in 5 ml water. From this suspension 100 μl amountsfrom the neat suspension and from serial 10-fold dilutions in 1 ml, were inoculated onto duplicate thirds of selective 7H11 medium plates. The number of colonies was counted after 3–4 weeks incubation at 37°C to give the plate count. A negative plate therefore had <25 cfu / organ. For the broth counts, serial 10-fold dilutions of the organ homogenate were made, in triplicate, in 1 ml amounts to 9 ml amounts of selective 7H9 broth with 0.05% Tween 80 in plastic 28 ml screw-capped bottle. In the acute infection, 10 serial dilution were set up, so as to obtain 30 tubes in all, while in the chronic infection 6 serial dilutions were set up yielding 18 tubes in all. These were incubated at 37°C and examined at 3 and 6 weeks and finally at 9 weeks for the characteristic growth of M. tuberculosis, namely a clear supernatant in undisturbed cultures with an upwards swirl of white growth on shaking,. Probable numbers of bacilli (pnb) per organ were obtained from a table of densities of organisms estimated by the dilution method [6]. Samples of the positive growth from 18 broth cultures were plated out on 7H11 medium.
Acute infection experiment
Each of 6 mice was infected by the intravenous route with 200 μl of a suspension of a containing 2.6 × 106 cfu of the H37Rv strain. Plate and broth counts were carried out 7 days later.
Chronic infection experiment
Each of 88 mice was infected by the intra-peritoneal route with 200 μl of a suspension containing 3.1 × 103 cfu of the H37Rv strain. Mice in our experiments are usually housed in an isolator, connected by a tunnel port to a Class 1 safety cabinet through which air from the environment is sucked. The intra-peritoneal route was chosen so that mice could be kept throughout the experiment in the isolator to prevent cross infection with mouse pathogens during exposure to the outside air in the Class 1 cabinet. One day after infection, samples of 6 mice yielded scanty or no colonies in plate counts of spleens and lungs. After a further 4 weeks these organ counts in 6 mice had risen to 2.24 × 104 in spleens and 1.15 × 104 in lungs. The mice were then divided into 6 experimental groups, 4 of which were vaccinated with various DNA vaccines over a 4-week period and 2 were unvaccinated controls. At 12 weeks after infection only 9 of 36 lungs and 18 of 36 spleens yielded positive growth on plates. At 10 months after infection, the 36 remaining mice were sacrificed, and plate and broth counts were set up on all, using dilutions estimated from a sample of 4 mice sacrificed 3 weeks earlier.
Statistics
The results of the plate and broth counts were examined by 2-way analysis of variance using the Stata package, release 8 (Stata Corp., College Station, TX)