Comparison of Gram-staining and alternate methods, KOH test and aminopeptidase activity in aquatic bacteria: their application to numerical taxonomy

Abstract

A miniaturized method for Gram-staining using a microtiter system introduced. Compared with other methods this procedure is more rapid and precise. A total of 466 aerobic bacterial isolates (82.6% Gram-negative, 12.7% Gram-positive and 4.7% Gram-variable) from a eutrophic lake were examined with the KOH test and for aminopeptidase (AP) activity. A complete correlation between the KOH test and Gram-reaction was not observed; this was also the case between Gram-reaction and AP activity. Almost all (91.4%) of the Gram-negative isolates were disintegrated with a 3% KOH solution, while the correlation between Gram-positive staining and a negative KOH reaction was somewhat less (88%). All Gram-variables were KOH negative. Additionally, 76.2% of the Gram-negative showed AP activity, whereas 44.1% of the Gram-positive, and 54.5% of the Gram-variable were AP negative when grown on casein-peptone-starch agar (CPS). Of the naturally occurring Gram-negative strains, 73% were both AP positive and KOH positive; only 42.4% of the Gram-positive strains were AP and KOH negative. Therefore, it is proposed to employ treatment with KOH and hydrolysis of l-alanine-4-nitroanalide as an additional taxonomic criterion for classifying naturally occurring aquatic populations. These methods should not replace the Gram-staining