title: Distinction of Gram-positive and -negative bacteria using a colorimetric microbial viability assay based on the reduction of water-soluble tetrazolium salts with a selection medium.
abstract: Bacteria are fundamentally divided into two groups: Gram-positive and Gram-negative. Although the Gram stain and other techniques can be used to differentiate these groups, some issues exist with traditional approaches. In this study, we developed a method for differentiating Gram-positive and -negative bacteria using a colorimetric microbial viability assay based on the reduction of the tetrazolium salt {2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt} (WST-8) via 2-methyl-1,4-napthoquinone with a selection medium. We optimized the composition of the selection medium to allow the growth of Gram-negative bacteria while inhibiting the growth of Gram-positive bacteria. When the colorimetric viability assay was carried out in a selection medium containing 0.5µg/ml crystal violet, 5.0 µg/ml daptomycin, and 5.0µg/ml vancomycin, the reduction in WST-8 by Gram-positive bacteria was inhibited. On the other hand, Gram-negative bacteria produced WST-8-formazan in the selection medium. The proposed method was also applied to determine the Gram staining characteristics of bacteria isolated from various foodstuffs. There was good agreement between the results obtained using the present method and those obtained using a conventional staining method. These results suggest that the WST-8 colorimetric assay with selection medium is a useful technique for accurately differentiating Gram-positive and -negative bacteria.
authors.affiliation: Biotechnology and Food Research Institute, Fukuoka Industrial Technology Center, 1465-5 Aikawamachi, Kurume, Japan. tukatani@fitc.pref.fukuoka.jp
authors.forename: Tadayuki
authors.initials: T
authors.lastname: Tsukatani
authors.forename: Hikaru
authors.initials: H
authors.lastname: Suenaga
authors.forename: Tomoko
authors.initials: T
authors.lastname: Higuchi
authors.forename: Masanobu
authors.initials: M
authors.lastname: Shiga
authors.forename: Katsuya
authors.initials: K
authors.lastname: Noguchi
authors.forename: Kiyoshi
authors.initials: K
authors.lastname: Matsumoto
mesh_terms: D000900:Anti-Bacterial Agents / Q000378:metabolism; D001431:Bacteriological Techniques / Q000379:methods*; D003124:Colorimetry / Q000379:methods*; D003470:Culture Media / Q000737:chemistry*; D005840:Gentian Violet / Q000378:metabolism; D006090:Gram-Negative Bacteria / Q000254:growth & development / Q000302:isolation & purification*; D006094:Gram-Positive Bacteria / Q000254:growth & development / Q000302:isolation & purification*; D050296:Microbial Viability; D010084:Oxidation-Reduction; D012641:Selection, Genetic; D013194:Staining and Labeling / Q000379:methods; D013778:Tetrazolium Salts / Q000378:metabolism
chemical_list: D000900:Anti-Bacterial Agents; D003470:Culture Media; D013778:Tetrazolium Salts; D005840:Gentian Violet