A universally applicable and rapid method for measuring the growth of streptomyces and other filamentous microorganisms by methylene blue adsorption-desorption

Abstract

Quantitative assessment of growth of filamentous microorganisms, such as streptomycetes, is generally restricted to determination of dry weight. Here, we describe a straightforward methylene blue-based sorption assay to monitor microbial growth quantitatively, simply, and rapidly. The assay is equally applicable to unicellular and filamentous bacterial and eukaryotic microorganisms.

Fig 1

Characterization of methylene blue binding by different microorganisms. (A and B) Linear correlations between adsorbed (filled symbols) or desorbed (open symbols) methylene blue (measured at 660 nm) and dry weight (mg) (A) or optical density value (measured at 600 nm or 750 nm) (B) for a variety of microorganisms are shown. (A) The organisms analyzed were Streptomyces coelicolor M145 (full line; circles), Penicillium chrysogenum Ib (dashed line; squares), and Anabaena sp. PCC7120 (dotted line; diamonds). (B) The organisms analyzed were Escherichia coli K-12 (full line; circles), Synechococcus elongatus PCC7942 (dashed line; squares), and Saccharomyces cerevisiae (dotted line; diamonds). The optical density of S. elongatus PCC7942 was measured at 750 nm, and that of the other species was measured at 600 nm. (C) Methylene blue adsorption and desorption curves for S. coelicolor at 25°C. Methylene blue adsorption (filled circles) and desorption (open symbols) curves (measured at 660 nm) are shown in relation to the cell amount equivalents (CAE) determined by dry weight. Desorption curves with either 250 mM HCl (open triangles) or 500 mM potassium phosphate buffer, pH 7 (open circles), as the solvent are shown. All assays were performed with a staining solution of 1,500 nmol ml^-1 methylene blue solution, and experiments were repeated at least three times.

Fig 2

Application of the methylene blue adsorption assay for quantitative growth determination. (A and B) Absolute CAE of individual filamentous aggregates/pellets of S. coelicolor (A) and Penicillium chrysogenum (B) with various sizes (open circles) were determined by methylene blue adsorption and compared with the dry weights (full circles) determined for the individual aggregates. The latter was calculated as the average of several aggregates dried together to a minimal measureable level of 5 mg. (C) Aerobic growth of S. coelicolor at 30°C and in 1 ml (circles) or 2 ml (triangles) of TSB (30 g/liter, containing 100 mM MOPS-NaOH, pH 7.0) is shown. Cell amount equivalents (CAE) were measured by methylene blue adsorption (MBA). Results with standard deviations of four independent experiments are shown.