Alternate gram staining technique using a fluorescent lectin

Abstract

Fluorescence-labeled wheat germ agglutinin binds specffically to N-acetylglucosamine in the outer peptidoglycan layer of gram-positive bacteria. The peptidoglycan layer of gram-negative bacteria is covered by a membrane and is not labeled by the lectin. By exploiting this phenomenon, an alternative Gram staining technique has been developed.The Gram stain is the most widely used taxonomic test of bacteria (5). The technique is relatively simple and, in experienced hands, gives reproducible results. The existing technique does, however, have its limitations (4). Most modifications of the Gram stain, such as that of Hucker (6), require at least four solutions and four staining steps. Furthermore, the stains used in the technique, particularly the primary stain (crystal violet), are concentrated and can be messy. The staining procedure is estimated to take approximately 3 min (2).We have developed an alternative Gram staining technique which is simpler and faster, requires fewer reagents, is easier to interpret, and, in our hands, is less susceptible to errors. This technique takes advantage of the selective binding of a lectin, wheat germ agglutinin, to N-acetylglucosamine (1,8). This molecule is a prominent component of the peptidoglycan layer found in all eubacteria except Mycoplasma spp. In gram-positive bacteria, the peptidoglycan layer is the outer portion of the cell wall. The exterior layer of gram-negative bacteria is a membrane which covers the peptidoglycan layer (9). Thus, a large molecule such as a lectin should be able to attach to the peptidoglycan layer of gram-positive bacteria but should not be able to penetrate the outer membrane and thus could not attach to the peptidoglycan of gram-negative bacteria. A total of 92 bacterial strains were tested. Gram-positive bacteria were as follows: Bacillus sp. (n = 8), Bacillus megaterium (n = 2), Corynebacterium sp. (n = 1), Lactobacillus acidophilus (n = 1), Lactobacillus lactis (n = 1), Micrococcus sp. (n = 3), Micrococcus luteus (n = 2), Mycobacterium smegmatis (n = 1), Sporosarcina ureae (n = 1), Staphylococcus aureus (n = 9), Staphylococcus epidermidis (n = 7), Staphylococcus saprophyticus (n = 1), Streptococcus faecalis (n = 4), Streptococcus mitis (n = 2), and Streptococcus pyogenes (n = 4). Gram-negative bacteria were as follows: Acinetobacter calcoaceticus (n = 2), Alcaligenes faecalis (n = 2), Cytophaga sp. (n = 1), Enterobacter aerogenes (n = 1), Enterobacter cloacae (n = 5), Escherichia coli (n = 8), Klebsiella pneumoniae (n = 4), Morganella morganii (n = 1), Proteus mirabilis (n = 2), Proteus vulgaris (n = 4), Pseudomonas sp. (n = 1), Pseudomonas aeruginosa (n = 4), Pseudomonasfluorescens (n = 1), Pseudomonas stutzeri (n = 1), Rhodospirillum rubrum (n = 1), Salmonella typhimurium (n = 1), Serratia liquefaciens (n = 1), Serratia marcescens (n = 4), and Shigella sonnei (n = 1). All strains were initially streaked to ensure purity and * Corresponding author. then were maintained at 35°C with periodic transfer. Wheat germ agglutinin labeled with flu...

A flow cytometric method for viability assessment of Staphylococcus aureus and Burkholderia cepacia in mixed culture

“…WGA is known for its specific binding to the peptidoglycan layer of Gram-positive bacteria, specifically to N-acetylglucosamine and N-acetylneuroaminic acid residues (38). In this study, WGA did not bind to B. cepacia, which is in agreement with results reported by Sizemore et al (39), who explained this finding by the presence of the outer membrane of Gram-negative bacteria, which covers the peptidoglycan layer. In contrast to this, reports from other authors showed WGA binding to Gramnegative bacteria, in particular for Escherichia coli, where the enterobacterial common antigen was labeled (40,41).…”

Generation of bioaerosols during manual mail unpacking and sorting

“…Specifically, Gram-positive bacteria are stained with fluorescent wheat germ agglutinin (WGA) whereas Gramnegative are not (Sizemore et al 1990;Fife et al 2000;Holm and Jespersen 2003). WGA binds to N-acetylglucosamine and N-acetylgalactosamine residues of the cell wall.…”

Current and future applications of flow cytometry in aquatic microbiology

“…One of the basic classical di¡erentiation criteria is Gram staining. This can be achieved by using stains impermeable to the outer membrane in the presence and absence of EDTA or through labelling with £uorescent lectins [112,113]. A £uorescent Gram stain for un¢xed organisms describes the use of hexidium iodide (HI) and SYTO 9 [113] that is also available as a mixture from Molecular Probes.…”