Materials and Methods

3.1.1 Mycobacteria! culture and culture medium

Stock cultures of M.vaccae strains Gm27 and Job5 were maintained in non-

antigenic Sauton’s media at 32®C, and on LJ slopes stored at -20°C (Appendix 1). Mycobacteria were cultured at 32°C in 50ml volumes of Sauton’s broth (Appendix 1), in 250ml Erlenemeyer flasks, in an orbital incubator (Gallenkamp) at lOOrpm. A non-ionic surfactant, tyloxapol (Sigma) 0.5%v/v, was added to the cultures to prevent adherence to the side of the flasks and to reduce clumping in the cultures. The liquid cultures were subcultured every week, and the LJ slope cultures were routinely used to start new M.vaccae cultures to prevent genetic drift. The LJ slopes were thawed for 4-8h; a 10pl loop-full of M.vaccae was used to inoculate new M.vaccae cultures.

3.1.2 Mycobacterial Growth dynamics

The growth dynamics of M.vaccae strains Gm27 and Job5 in liquid broth were unknown, and in order to standardise the experiments; these were elucidated.

3.1.2.1 Determination of Bacterial concentration using Viable Counts A viable count was performed; duplicate flasks for each strain were inoculated with 1ml of an established culture in stationary phase. The cultures were aseptically sampled, in triplicate, twice a day for 5 days. Each sample was subjected to a series of 1in10 dilutions in sterile 0.1%v/v tyloxapol, and 10pl was streaked onto individual 7H10 Middlebrook agar plates (Appendix 1), and incubated at 32°C until the bacterial colonies were clearly visible. Agar plates with greater than 100 colonies were counted, and the mean value from the triplicate data was calculated. This was done for both strains of M.vaccae, and linear regression analysis was applied to the exponential phase of the growth curves. The generation time of culture from the triplicate experiments was calculated and the mean generation time of each strain of M.vaccae determined.

3.1.2.2 Determination of Bacterial Concentration using Non-Culture Methods

The viable count gave an accurate value for the number of colony forming units in a culture, however the time taken to obtain a value was impractical for cold shock experiments. An alternative method was established.

A comparison was carried out between the viable count method and 3 methods of measuring bacterial concentration:

absolute count by haemocytometer nephelometer measurement

spectrophotometric absorbance measurement at 400nm (Aioonm)

Total count

A total count was performed using a Neubauer improved haemocytometer and a phase contrast microscope. The cultures were diluted in sterile deionised water containing 0.1%v/v tyloxapol, bacteria were disaggregated by vortexing in a sterile 1.5ml Eppendorf with four 1.5 to 2mm glass beads for 80s, lOpI was pipetted into a sealed haemocytometer. The bacteria were counted at x400 magnification. In order to ensure accuracy more than 100 bacteria were counted for each culture. Background was assessed, using sterile diluent, and subtracted from the sample counts.

Nephelometer measurements

Bacterial concentration was determined in 20ml volumes, in a turbidimeter (Merck), calibrated using 5 and 60 nephelometer unit (NTU) standards at 860nm, range 0-1000NTU. The cultures were diluted in Sauton’s media, to bring the concentrations into the above range.

Spectrophotometer measurements at 400nm

The absorbance of cultures was measured in a UVA/isible spectrophotometer (LKB Biochem, Ultrospec II), in a 500pl volume quartz cuvette with a 1cm path length, at 400nm. The blank was 500^1 of Sauton’s media. Well-mixed culture

were diluted in Sauton’s media to bring them the limits of absorbance, <3.000 absorbance units (A). The 400nm wavelength was chosen, as the orange and yellow colour of the two M.vaccae stains would interfere with the traditional eOOnm wavelength.

3.1.3. Method Comparison

Four flasks of liquid Sautons media were inoculated on 4 separate days to produce a range of bacterial concentrations. For each culture a nephelometer (turbidity meter) reading, spectrophotometer reading (A4oonm) was taken and a total count and viable count were performed. The results were compared to the viable count to determine the most efficient and accurate method of measuring the number of viable bacteria/ml.

3.1.4 Correlation of absorbance at 400nm and bacterial concentration Viable counts and absorbance measurements were carried out concurrently on a culture of M.vaccae strains Gm27 and Job5. Each strain was inoculated into separate flasks to give an initial absorbance of -0 .1. The cultures were sampled regularly and the A^oonm readings were taken, and viable count plates were set up. The A4oonm and viable count values were transformed (log2 and log 10 respectively), and plotted against each other in linear axes. A linear regression analysis was performed to calculate the linear relationship between the two methods.

3.1.5 Cold Shock Experiments

3.1.5.1 Effect of cold shock on growth of M.vaccae Gm27 and Job5

The cold shock method was adapted from published experiments with other bacteria (Jones et al., 1987; Roberts and Inniss, 1992; Goldstein et al., 1990). Flasks, containing 50ml of media, were inoculated with approximately 9x10® bacteria of M.vaccae strains JobS and Gm27, which corresponds to an initial absorbance of 0.1 A at 400nm. For each strain two flasks were inoculated, a control and cold shock flask. Both flasks were incubated at 32®C in an orbital shaker at lOOrpm to mid-exponential phase, 40h, (t=0). The cold shock flask

was transferred to a 6®C shaking waterbath (Grant OLS 200), 100 rpm. The time taken to reach 6°C was recorded for the first experiment. The flasks were sampled regularly and the A4oonm readings taken. Again the bacteria were diluted with Sauton’s media to bring the readings into the spectrophotometric range <3.000 (A). The cultures were monitored for up to 350h.