A volume of 250 pl of fixed sample was centrifuged at 13000 x g for 3 minutes and the supernatant was removed. The sample was washed once by adding 1 ml PBS and centrifuged again. The sample was then divided into four tubes: a negative control containing no probe to observe autofluorescence, a negative control to observe non-specific binding events, a positive control where a universal eubacterial probe was added (Bact 338) and a sample to be hybridised by a specific AOB detection probe. The samples were serially dehydrated in successively increasing concentrations of molecular grade ethanol (60%, 80%, 100% v/ v). After adding 1 ml of the ethanol solution, the sample was vortexed and left for 3 minutes. The sample was then centrifuged at 13000 x g for 3 minutes and the supernatant was removed.

The following step is to hybridize the samples. Hybridisation buffer (HB) was prepared according to Amann et al (1990). HB was added so that the final volume including the probe will be 40 pl. Thus, for the negative control for autofluorescence, 40 pl HB is added. For a hybridisation containing only one probe (2ul), 38ul HB is added. For a hybridisation containing two probes ( 2+2 pl) 36 pl HB is added. The samples were prehybridized for 15 minutes at the hybridisation temperature. After prehybridisation, 2 pl of probe (50 ng/ pl) was added to the samples that were then incubated at the optimal hybridisation temperature for the given probe (Table 1) for at least 4 hours (or overnight).

Following hybridisation, the samples were centrifuged at 13000 x g for 3 minutes and the supernatant was removed. A volume of 0.5 ml of wash buffer was added and the sample was mixed using a pipette before being incubated for 15 minutes at the same temperature as the hybridisation step. The washing step was again repeated.

The samples were centrifuged again at 13000 x g for 3 minutes, the supernatant was removed and 1 ml of MilliQ water was added. Finally, the samples were centrifuged, the supernatant removed and the samples resuspended in 100 ul MilliQ water.

A 10 ul aliquot of the sample was added to a gelatine-coated slide with Teflon-coated wells of a known diameter (Appendix 4.1) and allowed to dry in a hybridization oven at 30oC. The sample spot on the slide was mounted in a small drop of the antifadent-Citifluor (AFI, Canterbury, UK). A cover glass was sealed carefully on the top of the slide by applying clear nail varnish to the edges to prevent movement during microscopy. The slide was then stored at -20oC in the dark and was prepared for viewing.