Suitability of fluorescence measurements to quantify sulfate-reducing bacteria

• Published in: Journal of microbiological methods 2013-06, Vol.93 (3), p.192-197
• Main Authors: Barton, Larry L., Carpenter, Claire M.
• Format: Article
• Language: English
• Subjects: Bacterial Load - methods; bleaching; Desulfovibrio; Desulfovibrio desulfuricans - enzymology; Desulfovibrio desulfuricans - isolation & purification; Desulfovibrio gigas - enzymology; Desulfovibrio gigas - isolation & purification; desulfoviridin; Fluorescence; Light; light intensity; metal ions; Oxidation-Reduction; siroheme; sodium hydroxide; Specimen Handling - methods; sulfate-reducing bacteria; sulfates; Sulfates - metabolism; Sulfite reductase; Temperature; Time Factors
• ISSN: 0167-7012; 1872-8359
• DOI: 10.1016/j.mimet.2013.03.019

Fluorescence activity has been used to identify Desulfovibrio and has been termed the ‘desulfoviridin test’. This fluorescence is attributed to the prosthetic group of bisulfite reductase, a key enzyme in dissimilatory sulfate reduction. We have pursued the use of fluorescence measurements to quantify sulfate-reducing bacteria. Cells of D. desulfuricans and D. gigas were treated with NaOH and produced two fluorescence spectra: one with maximum fluorescence with an excitation at 395nm and an emission at 605nm and another with an excitation at 320nm and emission at 360nm. Using the fluorescence with excitation at 395nm and emission at 605nm, we explored a series of parameters to measure Desulfovibrio in pure cultures and environmental samples. Fluorescence measurements are reliable provided the cells are treated with 1.75N NaOH and the chromophore released from the cells is not exposed to strong light intensity, and is not exposed to temperatures greater than 20°C, and measurements are done within a few minutes of extraction. Bleaching of fluorescence was attributed to metal ions in solution which was not observed until metal concentrations reached 1.5mM. We propose that D. desulfuricans is appropriate as the reference organism for measurement of sulfate-reducing bacteria by fluorescence and by using fluorescence intensity, 105 cells/ml can be readily detected in environmental samples. •Optimal fluorescence is with 1.75N NaOH, 20°C, and dark environment.•Measurements at 10min use excitation at 395nm and emission at 605nm.•To quantitate unknown bacteria, Desulfovibrio desulfuricans is recommended.•Fluorescence measurements use intensity and not area under the emission peak.