Project Description

Bioanalytical laboratory needed to conduct 3 studies as part of food clinical trials including:

1- Study of the changes in faecal bacteria populations using 16SrRNA probes labelled with Cy3 for specific bacterial groups or the nucleic acid stain DAPI for total bacterial counts. The bacterial groups selected for enumeration will be Bifidobacterium spp., Bacteroides/Prevotella spp., Lactobacillus/Enterococcus spp., Clostridiumcoccoides- Eubacterium rectale group, Clostridium histolyticum group, and Atopobium cluster including most Coriobacteriaceae species, using the specific probes Bif164, Bac303, Lab158, Erec482, His150, and Ato291, respectively

2. Fluorescence insitu Hybridisation (FISH), as described by Rycroft et al. (2001) and Daims et al. (2005). Briefly, faecal samples (375?l) fixed in 4% paraformaldehyde (pH7.2) overnight at 4?C were then centrifuged at 1500 ×g for 5 min, washed twice with phosphate buffer saline (PBS0.1M,pH7.0), re-suspended in a mixture of PBS/99% ethanol (1:1v/v) and stored at ?20?C for up to 3 months. For the hybridisations, 20?l of each sample was pipetted onto Teflon-and poly-l-lysine-coated, six-well (10mm diameter each) slides. Slides were dried at 46C for 15 min and then submerged in a series of ethanol solutions (50, 80, and 96%, 3 min each). This process was used for all samples, except those where the Lab 158 probe was used. Sample slides probed with Lab 158 were subjected to an additional step with 50 ?l of lysozyme (1mg/mL in 100mM Tris-HCl, pH8.0) at 37?C for 15 min prior to being submerged in the ethanol solutions. A probe/hybridization buffer mixture (5 ?l of a 50ngstock of probe plus 45 l of hybridization buffer) was applied to the surface of each well. Hybridisations were performed for 4 hour in an ISO20 oven (Grant Boekel). Slides were stored in the dark at 4?C (for a maximum of 3 days) until cells were counted. Slides were enumerated using a Nikon E400 Eclipse microscope fitted with an EPI-fluorescence attachment, 15 randomized views were counted for each sample.

3. Plasma propionate