A direct fluorescent antibody (FA) methodology was devised to accurately quantitate a select number of known methanogenic bacteria in the sediments of Lake Erie. The specificity of the respective fluorescent antibodies was evaluated against a limited number of hetero- and methylotrophic bacteria. The fluorescent antibodies for each of the surveyed methanogens were specific in that no cross reactions with the other methanogenic species could be shown. The autofluorescence of the methane bacteria and nonspecific absorption were masked by rhodamine isothiocyanate conjugated bovine serum albumin (RhoITC-BSA). The FA methodology devised permitted the detection of the specific methanogens in numbers as low as 10⁶ cells per gram of lake sediment. The FA method worked equally well when applied to fine silt, sand-silt, or sand sediments. The presence of large numbers of other microorganisms did not interfere with the staining process. To increase the sensitivity of this sediment analysis, organic and inorganic enrichments were pursued. These procedures permitted the selective enrichments of specific methanogens to population sizes (10⁶/ml or greater) large enough for direct FA observation. Thus, this direct FA staining methodology can specifically and accurately quantitate the known methanogenic bacteria in lake sediments. This procedure can be combined with concentration techniques to increase the sensitivity of the FA procedure. Also, this staining procedure could be adapted to other methanogenic bacteria, either known pure cultures or natural isolates, which could be obtained from lake sediments.

The greatest advantage this enumeration has over other methods of enumerating methanogens [most probable number (MPN), substrate limitations, or general cellular morphology] is that it is a direct count that is species specific.