Bacterial and Archaeal direct counts: A faster method of enumeration, for enrichment cultures and aqueous environmental samples

A new presence/absence method has been developed to count fluorochrome-stained bacterial and archaeal cells on membrane filters using epifluorescence microscopy. This approach was derived from the random distribution of cells on membranes that allowed the use of the Poisson distribution to estimate total cell densities. Comparison with the standard Acridine Orange Direct Count (AODC) technique shows no significant difference in the estimation of total cell populations, or any reduction in the precision of these estimations. The new method offers advantages over the standard AODC in considerably faster counting, as there is no need to discriminate between every potential cell visible on a field and fluorescent detritus, it is only necessary to confirm the presence of one cell. Additionally, the new method requires less skill, so has less reliance on expert counters, and that should reduce inter-counter variability. Although this work used the fluorochrome Acridine Orange, clearly the results are applicable to any fluorochrome used to count bacterial and archaeal cells. This method was developed using enrichment cultures for use with enrichment cultures and aqueous environmental samples where interfering detrital and mineral particles are minimal e.g., freshwater/seawater, therefore, it is not suitable for estimating total cells from sediment samples. This method has the potential for use in any situation where counts of randomly distributed items are made using a grid or quadrat system.