Mechanism and use of the commercially available viability stain, BacLight.

BACKGROUND: BacLight (Molecular Probes, Eugene, OR, USA) is a popular fluorescence-based two-component stain for determining bacterial cell viability. The main purpose of this work was to fully elucidate the mechanism and to determine why it is sometimes reported that cells stain simultaneously live and dead. METHODS: Solutions of DNA were stained with the two components, propidium iodide (PI) and SYTO9, in different combinations, and fluorescence spectra were collected. RESULTS: K(PI) and K(SYTO9) were approximately 3.7 x 10(5)/M and 1.8 x 10(5)/M. SYTO9 emissions were stronger and overlapped those of PI. Fluorescence resonance energy transfer from SYTO9 to PI was observed. It was, even under normal conditions, possible for DNA bound SYTO9 to have a component in the red region equal to that of DNA bound PI. Potentially confusing emissions were also found to occur when PI was not in sufficient excess to saturate nucleic acid (>0.4 M PI to 1 M DNA base pairs). CONCLUSIONS: The mechanism is a combination of displacement of SYTO9 by PI and quenching of SYTO9 emissions by fluorescence resonance energy transfer. Confusing results can occur if the relative intensities of the stains or the concentration of PI relative to nucleic acid are not properly accounted for.

Decreasing the hyphal branching rate of Saccharopolyspora erythraea NRRL 2338 leads to increased resistance to breakage and increased antibiotic production.

Mutation and selection for increased resistance to cell-wall synthesis inhibitors led to alterations in the hyphal branching rate of Saccharopolyspora erythraea NRRL 2338. Mutants with decreased branching frequency exhibited increased hyphal strength (estimated by in vitro micromanipulation). As the hyphal strength was increased, this led to a greater proportion of hyphal particles in liquid culture with a hyphal fragment diameter of greater than 88 microm. This, in turn, coincided with proportionately increased antibiotic production.

Viability, strength, and fragmentation of Saccharopolyspora erythraea in submerged fermentation.

Two fermentations of the commercially important erythromycin-producing filamentous bacterium Saccharopolyspora erythraea were conducted in defined media. One was glucose-limited and the other nitrate-limited. The viability of the hyphae was determined using the fluorescent stain BacLight (Molecular Probes, Eugene, OR). Also, the force required to strain hyphae to breakage was determined using micromanipulation and a sensitive force transducer. In both fermentations, fragmentation coincided with the appearance of regions in the mycelia with permeabilised membranes (considered nonviable). Under glucose-limitation, hyphal breaking force rose to 1,050 +/- 130 nN at the end of the growth phase and fell to an undetectable value as a result of glucose exhaustion. Under nitrate-limitation, hyphal breaking force fell from 900 +/- 160 nN during the growth phase to 550 +/- 40 nN in the stationary phase. In both cases image analysis showed that the dimensions of mycelia were of the same order, suggesting that the major factor influencing fragmentation was the appearance of nonviable regions (assumed to be weak). The location in which nonviable regions first appear within hyphae could not be determined because of their appearance coinciding with fragmentation.

Strength of mid-logarithmic and stationary phase Saccharopolyspora erythraea hyphae during a batch fermentation in defined nitrate-limited medium.

A method for measuring mechanical properties of Saccharopolyspora erythraea is reported with data from a batch fermentation. Briefly, hyphae were glued to the end of a tungsten filament mounted horizontally on a sensitive force transducer. Free ends of hyphae were trapped against a flat surface by a second probe. The force transducer and tungsten filament were then moved at a fixed rate, the hypha were strained, and the force resisting motion recorded. From these data the maximum force resisting motion is taken as the force at which breakage occurs. Hyphae from the mid-logarithmic phase of a simple batch fermentation on defined medium were found to have a breaking force of 890 +/- 160 nN (95% confidence), while stationary phase hyphae were weaker at 580 +/- 150 nN. Video recordings of the experiments allowed an approximation of breaking strain, which did not differ significantly between samples at 0.18 +/- 0.03. Electron microscopy was used to measure cell wall thickness, cell diameter, and hence cell wall cross-sectional area. The ultimate tensile strength was estimated to be 24 +/- 3 MPa with no difference between the two samples, the lower breaking force of the stationary phase hyphae being attributed to a thinner cell wall. Assuming a linear relationship between stress and strain, the elastic modulus was estimated to be 140 +/- 30 MPa. These values are comparable with other structural biological materials such as yeast cell walls and collagen.