Common dyes used to determine bacterial polysaccharides on agar are affected by medium acidification

In this work, we highlight effects of pH on bacterial phenotypes when using the bacteriological dyes Aniline blue, Congo red, and Calcofluor white to analyze polysaccharide production. A study of galactose catabolism in Sinorhizobium meliloti led to the isolation of a mutation in dgoK1, which was observed to overproduce exopolysaccharides when grown in the presence of galactose. When this mutant strain was spotted onto plates containing Aniline blue, Congo red, or Calcofluor white, the intensity of the associated staining was strikingly different from that of the wild type. Additionally, a Calcofluor dull phenotype was observed, suggesting production of a polysaccharide other than succinoglycan. Further investigation of this phenotype revealed that these results were dependent on medium acidification, as buffering at pH 6 had no effect on these phenotypes, while medium buffered at pH 6.5 resulted in a reversal of the phenotypes. Screening for mutants of the dgoK1 strain that were negative for the Aniline blue phenotype yielded a strain carrying a mutation in tkt2, which is annotated as a putative transketolase. Consistent with the plate phenotypes, when this mutant was grown in broth cultures, it did not acidify its growth medium. Overall, this work shows that caution should be exercised in evaluating polysaccharide phenotypes based strictly on the use of dyes.