The RAPD fingerprints obtained from colonies processed in this way were identical to those produced from conventionally extracted high molecular weight DNA (Fig. 4). However, it was found that consistent profiles were only obtained if the RAPD HSP activation PCR was set up immediately after the boiling and chilling cycles of the colony extraction procedure. The amplified PCR fingerprints deteriorated after subsequent frozen storage of the Chelex® resin extracted DNA. To overcome this potential problem, we examined if prolonged frozen storage (-20°C) of the resuspended colony in Chelex® resin prior to full extraction by boiling was possible. This procedure did

not affect the quality of the RAPD profiles (Fig. 4). The ability to fingerprint from frozen stored colony material

provided a high throughput strategy that could be used to systematically screen the multiple colony types isolated from human faeces as part of a Lactobacillus strain feeding study (see below). Figure 4 Reproducibility of single colony RAPD fingerprints. The polymorphismsamplified by primer 272 from conventionally extracted DNA compared to single colony Chelex® extracted DNA are shown for two LAB strains as follows: lane 1, L. rhamnosus strain MW standard DNA extraction; lanes 2 to 4, single colonies of strain MW that were picked into Chelex® resin, stored frozen and then extracted immediately prior to PCR; lane 5, L. acidophilus strain LMG 8151 standard DNA extraction; lanes 6 to Selonsertib ic50 8, single colonies of strain LMG 8151 that were processed with Chelex® as described. The size of relevant molecular size markers (lane M) are shown

in bp. Lactobacillus species feeding study design A small scale proof-of-principle human feeding study was performed to evaluate if the colony-fingerprint strategy could be used to track specific LAB strains from ingestion as capsule recovery from faeces. A capsule for oral administration was formulated to commercial Flavopiridol (Alvocidib) standards which contained two Lactobacillus species isolates: L. salivarius strain NCIMB 30211 (1.8 × 1010 colony forming units [cfu] per capsule) and L. acidophilus strain NCIMB 30156 (5.6 × 109 mean cfu per capsule). Twelve volunteers participated in a feeding study where the capsule was taken daily for 14 days; faecal samples were provided on days before, during and after consumption as described in the Methods. The volunteers were not advised to change their diets in any way other than to take the capsule once a day with some food on each of the trial days. At each faecal sampling point, LAB were plated as described below, enumerated and multiple colonies genotyped by RAPD.