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Citation Information : Polish Journal of Microbiology. VOLUME 66 , ISSUE 3 , ISSN (Online) 2544-4646, DOI: 10.5604/01.3001.0010.4867, September 2017
License : (CC BY-NC-ND 4.0)
Received Date : 24-January-2017 / Accepted: 12-April-2017 / Published Online: 27-September-2017
Bacteria play a fundamental role in the cycling of nutrients in aquatic environments. A precise distinction between active and inactive bacteria is crucial for the description of this process. We have evaluated the usefulness of Coomassie Blue G250 for fluorescent staining of protein containing potentially highly active bacteria. We found that the G250 solution has excitation and emission properties appropriate for direct epifluorescence microscopy observations. It enables fast and effective fluorescent visualization of living, protein-rich bacteria, both in freshwater environment and culture. Our results revealed that the number of G250-stained bacteria from eutrophic lake was positively correlated with other standard bacterial activity markers, like number of bacteria containing 16S rRNA, bacterial secondary productionor maximal potential leucine-aminopeptidase activity. In case of the E. coli culture, the percentage of bacteria visualized with G250 was similar to that of bacteria which accumulated tetracycline. Compared to other common methods utilizing fluorogenic substances forbacteria staining, the approach we evaluated is inexpensive and less hazardous (for example mutagenic) to the environment and researchers. It can be regarded as an additional or alternative method for protein rich, active bacteria staining.
Amann R., F.O. Glöckner and A. Neef. 1997. Modern methods in subsurface microbiology: In situ identification of microorganisms with nucleic acid probes. FEMS Microbiology Reviews. 20: 191–200.
Ammor M.S., A.B. Flórez, A. Margolles and B. Mayo. 2006. Fluorescence spectroscopy: a rapid tool for assessing tetracycline resistance in Bifidobacterium longum. Can. J. Microbiol. 52: 740–746.
Bastviken D. and L. Tranvik. 2001. The Leucine Incorporation method estimates bacterial growth equally well in both oxic and anoxic lake waters. Appl. Environ. Microbiol. 67: 2916–2921.
Bengtsson M.M., K. Sjøtun, A. Lanzén and L. Øvreås. 2012. Bacterial diversity in relation to secondary production and succession on surfaces of the kelp Laminaria hyperborea. ISME J. 6: 2188–2198.
Böllmann J., K. Rathsack and M. Martienssen. 2016. The precision of bacterial quantification techniques on different kinds of environmental samples and the effect of ultrasonic treatment. J. Microbiol. Methods 126: 42–47.
Bradford M.M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248–254.
Carlsson N., C.C. Kitts and B. Åkerman. 2012. Spectroscopic characterization of Coomassie blue and its binding to amyloid fibrils. Anal. Biochem. 420: 33–40.
Chróst R.J. 1990. Microbial ectoenzymes in aquatic environments, pp. 47–78. In: Overbeck J. and J.R. Chróst (eds.). Aquatic Microbial Ecology: Biochemical and Molecular Approaches. Springer, New York.
Chróst R.J. and H. Rai. 1993. Bacterial secondary production, pp. 92–117. In: Overbeck J. and R.J. Chróst (eds.). Microbial Ecology of Lake Pluβsee. Springer-Verlag, New York.
Franklin T.J. and G.A. Snow. 2005. Facilitated uptake of antimicrobial drugs, pp. 129–135. In: Franklin T.J. and G.A. Snow (eds.). Biochemistry and molecular biology of antimicrobial drug action, bio-chemistry and molecular biology of antimicrobial. Springer Science-l-Business, Media, Inc., NY
Freese H.M., U. Karsten and R. Schumann. 2006. Bacterial abundance, activity, and viability in the eutrophic River Warnow, northeast Germany. Microb. Ecol. 51: 117–127.
Fuhrman J.A. and F. Azam. 1982. Thymidine incorporation as a measure of heterotrophic bacterioplankton production in marine surface waters: Evaluation and field results. Mar. Biol. 66: 109–120.
Georgiou C.D., K. Grintzalis, G. Zervoudakis and I. Papapostolou. 2008. Mechanism of Coomassie brilliant blue G-250 binding to proteins: A hydrophobic assay for nanogram quantities of proteins. Anal. Bioanal. Chem. 391: 391–403.
Glazier S.A. and J.J. Horvath. 1995. Feasibility of fluorescence detection of tetracycline in media mixtures employing a fiber optic probe. Anal. Lett. 28: 2607–2624.
Gottesman S. and M.R. Maurizi. 1992. Regulation by proteolysis: energy-dependent proteases and their targets. Microbiol. Rev. 56: 592–621.
Griebler C., B. Mindle and D. Slezak. 2001. Combining DAPI and SYBR Green II for the enumeration of total bacteria numbers in aquatic sediments. Internat. Rev. Hydrobiol. 86: 453–465.
Haglund A.L., P. Lantz, E. Törnblom and L. Tranvik. 2003. Depth distribution of active bacteria and bacterial activity in lake sediment. FEMS Microbiol. Ecol. 46: 31–38.
Karner M. and J.A. Fuhrman. 1997. Determination of active marine bacterioplankton: a comparison of universal 16S rRNA probes, autoradiography, and nucleoid staining. Appl. Environ. Microbiol. 63: 1208–1213.
Katrahalli U., S.S. Kalanur and J. Seetharamappa. 2010. Interaction of bioactive Coomassie Brilliant Blue G with protein: insights from spectroscopic methods. Sci. Pharm. 78: 869–880.
Knoll S., W. Zwisler and M. Simon. 2001. Bacterial colonization of early stages of limnetic diatom microaggregates. Aquat. Microb. Ecol. 25: 141–150.
Kiersztyn B., W. Siuda and R.J. Chróst. 2012. Persistence of bacterial proteolytic enzymes in lake ecosystems. FEMS Microbiol. Ecol. 80: 124–134.
Larimer C., E. Winder, R. Jeters, M. Prowant, I. Nettleship,R.S. Addleman and G.T. Bonheyo. 2016. A method for rapid quantitative assessment of biofilms with biomolecular staining and image analysis. Anal. Bioanal. Chem. 408: 999–1008.
Lebaron P., N. Parthuisot and P. Catala. 1998. Comparison of blue nuclei acid dyes for flow cytometric enumeration of bacteria in aquatic systems. Appl. Environ. Microbiol. 64: 1725–1730.
Long R.A. and F. Azam. 1996. Abundant protein-containing particles in the sea. Aquat. Microb. Ecol. 10: 213–221.
Luna G.M., E. Mannini and R. Donovaro. 2002. Large fraction of dead and inactive bacteria in coastal marine sediments: comparison of protocols for determination and ecological significance. Appl. Environ. Microbiol. 68: 3509–3513.
Luo S., N.B. Wehr and R.L. Levine. 2006. Quantitation of protein on gels and blots by infrared fluorescence of Coomassie Blue and Fast Green. Anal. Biochem. 350: 233–238.
Neumann U., H. Khalaf and M. Rimpler. 1994. Quantitation of electrophoretically separated proteins in the submicrogram range by dye elution. Electrophoresis 15: 916–921.
Penzkofer A. and Y. Lu. 1986. Fluorescence quenching of rhodamine 6G in methanol at high concentration. Chem. Phys. 103: 399–405.
Porter K.G. and Y.S. Feig. 1980. The use of DAPI for identifying and counting aquatic microflora. Limnol. Oceanogr. 25: 943–948.
Rodriguez G.G., D. Phipps, K. Ishiguro and H.F. Ridgway. 1992. Use of a fluorescent redox probe for direct visualization of actively respiring bacteria. Appl. Environ. Microbiol. 58: 1801–1808.
Shibata A., K. Kogure, I. Koike and K. Ohwada. 1997. Formation of submicron colloidal particles from marine bacteria by viral infection. Mar. Ecol. Prog. Ser. 155: 303–307.
Simon M. 1988. Growth Characteristics of small and large free-living and attached bacteria in Lake Constance. Microb. Ecol. 15: 151–163.
Simon M. and F. Azam. 1989. Protein content and protein synthesis rates of planktonic marine bacteria. Mar. Ecol. Prog. Ser. 51: 201–213.
Smith E.M. and P.A. del Giorgio. 2003. Low fractions of active bacteria in natural aquatic communities? Aquat. Microb. Ecol. 31: 203–208.
Suller M.T.E. and D. Lloyd. 1999. Fluorescence monitoring of antibiotic-induced bacterial damage using flow cytometry. Cytometry 35: 235–241.
Tupas L.M., B.N. Popp and D.M. Karl. 1994. Dissolved organic carbon in oligotrophic waters: experiments on sample preservation, storage and analysis. Mar. Chem. 45: 207–216.
Warkentin M., H.M. Freese, U. Karsten and R. Schumann. 2007. New and fast method to quantify respiration rates of bacterial and plankton communities in freshwater ecosystems by using optical oxygen sensor spots. Appl. Environ. Microbiol. 73: 6722–6729.
Zweifel U.L. and A. Hagstrom. 1995. Total counts of marine bacteria include a large fraction of non-nucleoid- containing bacteria (ghosts). Appl. Environ. Microbiol. 61: 2180–2185.