The distribution of hybridized cells was subsequently visualised by means of a Leica TCS SP2 UV confocal laser scanning microscope (CLSM) equipped with Leica DMRXA microscope. Images were captured and processed using LCS V2.5.1040-1 software. For observation x 60 Na 1.32 lenses were applied.

The CLSM was run in the following mode: single channel for Fluorescene and double channel for Carbocyanine-5. Fluorescene was detected using excitation at 488 nm and a long pass emission filter in the range of 500-530 nm. Cy5 was detected using excitation at 633 nm and a long pass emission filter of 650-680 nm. The artificial colours green and red were assigned to the monochrome images acquired in the fluorescene and Cy5 channels respectively. The LCS software actively mixed colours so that a cell emitting red and green (the AOB) would appear yellow. For each sample, only 5 fields of view were randomly recorded in view of the time and budget available for the process.

1.1.2 Enumeration technique

An Excel spreadsheet constructed by Coskunur (2000) was used to carry out the calculation based on Equation 1 below:

where

K = average number of microcolonies in one ml of sample

A1 = area of sample spot (the area can be calculated from the diameter of the sample spot, [n(D/2)2])

A2 = area of one field view

N = average number of ammonia oxidizer microcolonies/field of view V = volume of sample applied Vo = original volume of sample

ODF = other dilution factors not considered above may be required (e. g. volume of sample spun down). Where no ODF, default value = 1 The spreadsheet was designed for the quantification of AOB population in wastewater treatment plants following FISH and quantification typically using CLSM produced images. It requires that the user inputs data concerning the number of AOB microcolonies, the shortest and longest diameter of the microcolonies, area measurements of the fields of view and sample spots and dilution factors used in FISH. The spreadsheet returns the average number of microcolonies and geometric mean diameter. This data sheet can also be used to calculate the concentration of AOB in mg/l, the % AOB in terms of total bacterial population (measured by volatile suspended solids, VSS), following an empirically determined conversion factor, in terms of total cell numbers.