Gram characteristics determined on single cells and at the microcolony level of bacteria immobilized on polycarbonate membrane filters

Abstract

A new protocol was developed for rapid Gram characterization of mixed bacterial populations in environmental samples. Bacteria appearing as single cells or microcolonies after incubation on polycarbonate membrane filters are stained by a Gram staining procedure combined with acridine orange staining to obtain total cell numbers. Gram-positive cells can thus be observed by light microscopy and acridine orange stained cells by epifluorescence microscopy. The mixed population of cells was immobilized on polycarbonate membrane filters by mounting them on silicone-coated coverslips, which ensured that the microcolonies that developed after incubation on a growth medium remained intact during the subsequent staining procedures. Microcolonies that developed after incubation were all scored correctly for both the Gram-negative (ten species) and Gram-positive (eleven species) test strains. When analyzing bulk soil and barley rhizosphere samples, respectively, 40 and 22% of the microcolonies formed after incubation of the filters on nutrient-rich substrate were scored as being Gram-positive. These results show that a higher number of culturable, Gram-negative bacteria were present in the rhizosphere soil than in the bulk soil. A great advantage of the protocol presented is the rapid and concurrent characterization of Gram features and the culturability of bacteria from environmental samples.