PBS was added in replacement for the withdrawal of supernatant an

PBS was added in replacement for the withdrawal of supernatant and distilled water was added 1:10 to lyse the red blood cells followed by incubation for 10 min maximum at room temperature. The tubes were spun at 2000 g for 10 min

and the supernatants removed. The pellet was resuspended in 1 mL PBS and a few glass beads were added to help disperse the pellet. The samples were then measured in the luminometer (Berthold AutoLumat Plus, Berthold Technologies, Germany) by diluting 1:10 in PBS and using 1% N-decyl aldehyde (Sigma-Aldrich, US) as the substrate. Mycobacterial luminescence was measured at baseline and at 96 h, and the growth ratio was calculated by division of the mean 96-hour luminescence value by the mean baseline value. The control samples were processed in the same way excluding the centrifugation step for removal of supernatant see more and lysis of red blood cells, as these are irrelevant for bacteria growing in growth medium alone. Growth ratios Ibrutinib were calculated and compared between different volumes of growth media, equivalent to the equation used for whole blood assays. A mean of the triplicate growth ratios

was calculated for each sample per blood/control volume. A cross sectional comparison of the median growth ratio for all data available at each volume was compared using the non-parametric Kruskal–Wallis test. Analysis including only samples with repeated measures for each volume was carried out using Friedmann ANOVA Carnitine palmitoyltransferase II test. In addition separate comparisons between each volume were analysed using Mann Whitney paired non-parametric tests. In all cases p ≤ 0.05 was termed significant. Repeated results were obtained from 9 healthy adults. Initial studies that verified the methods were performed using the original volume of blood and therefore there are more values at the 1 mL volume than at the smaller volumes. These data points were included in the initial analysis. There was no significant difference between the median

growth ratios for each of the volumes of diluted blood (Fig. 1A, p = 0.160) showing a 2-fold reduction in volume is possible in this assay system. Examining the data that included samples that had all three different volume measurements, we found that there were no significant differences between any of the volumes (Fig. 1B, p = 0.398). Additional analysis comparing each volume of blood with the original volume of 1 mL also showed no significant differences between each of the volumes tested (Fig. 1B). There were no significant differences between the different volumes of growth media for the growth of BCG (data not shown). It was also established that using polystyrene round bottom tubes with snap caps (BD Biosciences) instead of 5 mL bijous for the incubation reduced the risk of disrupting the pellet while collecting the supernatants without any changes to the resulting mycobacterial growth (data not shown).

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