Enumeration of protozoan ciliates in activated sludge: determination of replicate number using probability

A new approach to the enumeration of ciliate protozoa in activated sludge is described. A 25 microL sub-sample volume is optimal for routine analysis using a standard slide and 24 x 24 mm cover slip requiring between 20 and 40 min per sub-sample for full enumeration and identification of species. However, to achieve high probability (>or=95%) of recovering all species large numbers of replicates are required (i.e. 23-47). To achieve high probabilities of recovery using less replicates it is necessary to neglect rare species with low densities (<0.5%); based on the assumption that they do not play a significant role in plant performance. The precise number of replicates required for different probabilities of recovering species is determined by conducting an initial pilot survey analysing a minimum of 8 replicates and using a probability equation to determine the optimum replicate number for that particular plant. Six replicate 25 microL sub-samples provided excellent species recovery (90-95% excluding up to 3 rare species), while analysing just two or three replicates, as commonly used in previous wastewater studies, only gave probabilities of 25 and 50% respectively for the same recovery. Ciliate analysis should be completed within 8h of collection with significant changes in community structure occurring beyond this period.