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  • Induction of viable but non-culturable (VBNC) state in Salmonella cultured in M9 minimal medium containing high glucose.

Induction of viable but non-culturable (VBNC) state in Salmonella cultured in M9 minimal medium containing high glucose.

Biological & pharmaceutical bulletin (2014-08-12)
Yuta Morishige, Masaaki Tanda, Ko Fujimori, Yoshiki Mino, Fumio Amano
ABSTRACT

An environmental isolate of Salmonella enterica serovar Enteritidis (SE) clone, SE Cl#15-1, loses its culturability during 72-h culture in M9 minimal medium containing 0.8% glucose, a concentration twice higher than that in normal M9 medium, whereas the bacterium retains its culturability in normal M9 medium. Live/dead analysis using the 5-cyano-2,3-di(p-tolyl) tetrazolium chloride (CTC)-reduction assay revealed that SE cells cultured in M9 medium containing 0.8% glucose died with time when in the "viable but non-culturable" (VBNC) state. Assay of the culturability of SE cells in the used supernatant (0.4 spent M9 or 0.8 spent M9) also indicated that 0.8 spent M9 soon showed a lethal effect on intact SE cells. These results suggest that large amounts of glucose metabolites might have been responsible for the toxicity. Analysis of the 0.8 spent M9 revealed that formate rapidly accumulated in the medium. The pH of the medium rapidly dropped to 4.7, leading to conversion of formate to formic acid, which might have damaged the bacterial cell membrane. These results suggest that the excessive amount of glucose in the M9 medium might have injured SE cells in the VBNC state by being metabolized to formic acid and other acidic compounds.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Pyruvic acid, natural, ≥95%, FG
Sigma-Aldrich
Pyruvic acid, 95%
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Pyruvic acid, 98%
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5-Cyano-2,3-di-(p-tolyl)tetrazolium chloride, ≥85% (HPLC)
Sigma-Aldrich
Pyruvic acid, ≥97%, FG
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Formic acid, reagent grade, ≥95%
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DAPI, for nucleic acid staining
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Formic acid, ACS reagent, ≥88%
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Formic acid, ≥95%, FCC, FG
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Formic acid solution, BioUltra, 1.0 M in H2O
Sigma-Aldrich
Formic acid, puriss. p.a., ACS reagent, reag. Ph. Eur., ≥98%
Sigma-Aldrich
Formic acid, ACS reagent, ≥96%
Sigma-Aldrich
Formic acid, puriss., meets analytical specifications of DAC, FCC, 98.0-100%