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A microplate spectrofluorometric assay for bacterial biofilms

  • Original Paper
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Journal of Industrial Microbiology & Biotechnology

Abstract

A spectrofluorometric assay was developed for quantification of bacterial biofilms grown on a microtiter plate. The method involved staining biofilms formed by gram-negative and gram-positive bacteria with wheat germ agglutinin-Alexa Fluor 488 conjugate, which selectively binds to N-acetylglucosamine residues in biofilms. The fluorescence of stained biofilms was measured with a fluorescent plate reader. This method was compared with a widely used microplate colorimetric assay involving crystal violet staining of biofilms formed by both gram-negative and gram-positive bacteria. A strong linear association existed between the two methods (r 2=0.99/0.94). Being more sensitive and specific as compared to colorimetric method, the spectrofluorometric assay provides a better alternative for quantification and characterization of bacterial biofilms.

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References

  1. Costerton JW, Lewandowski Z, Caldwell DE, Korber DR, Lappin-Scott HM (1995) Microbial biofilms. Ann Rev Microbiol 49:711–745

    Article  CAS  Google Scholar 

  2. Djordjevic D, Wiedmann M, McLandsborough LA (2002) Microtiter plate assay for assessment of Listeria monocytogenes biofilm formation. Appl Environ Microbiol 68:2950–2958

    Article  CAS  Google Scholar 

  3. Itoh Y, Wang X, Hinnebusch BJ, Preston JF 3rd, Romeo T (2005) Depolymerization of beta-1,6-N-acetyl-d-glucosamine disrupts the integrity of diverse bacterial biofilms. J Bacteriol 187:382–7

    Article  CAS  Google Scholar 

  4. Jackson DW, Suzuki K, Oakford L, Simecka JW, Hart ME, Romeo T (2002) Biofilm formation and dispersal under the influence of the global regulator CsrA of Escherichia coli. J Bacteriol 184:290–301

    Article  CAS  Google Scholar 

  5. Johnson JR, Berggren T, Conway AJ (1993) Activity of a nitrofurazone matrix urinary catheter against catheter-associated uropathogens. Antimicrob Agents Chemother 37:2033–2036

    CAS  Google Scholar 

  6. Kaplan JB, Ragunath C, Velliyagounder K, Fine DH, Ramasubbu N (2004) Enzymatic detachment of Staphylococcus epidermidis biofilms. Antimicrob Agents Chemother 48:2633–2636

    Article  CAS  Google Scholar 

  7. Leriche V, Sibille P, Carpenter B (2000) Use of an enzyme-linked lectinsorbent assay to monitor the shift in polysaccharide composition in bacterial biofilms. Appl Environ Microbiol 66:1851–1856

    Article  CAS  Google Scholar 

  8. Neu TR, Marshall KC (1991) Microbial “footprints”-a new approach to adhesive polymers. Biofouling 3:101–102

    Article  Google Scholar 

  9. O’Toole G, Kaplan HB, Kolter R (2000) Biofilm formation as microbial development. Annu Rev Microbiol 54:49–79

    Article  CAS  Google Scholar 

  10. Sanford BA, De Feijter AW, Wade MH, Thomas VL (1996) A dual fluorescence technique for visualization of Staphlococcus epidermidis biofilm using scanning confocal laser microscopy. J Ind Microbiol 16:48–56

    Article  CAS  Google Scholar 

  11. Thomas VL, Sanford BA, Moreno R, Ramsay MA (1997) Enzyme-linked lectinsorbent assay measures N-acetyl-d-glucosamine in matrix of biofilm produced by Staphylococcus epidermidis. Curr Microbiol 35:249–254

    Article  CAS  Google Scholar 

  12. Wang X, Preston JF 3rd, Romeo T (2004) The pgaABCD locus of Escherichia coli promotes the synthesis of a polysaccharide adhesin required for biofilm formation. J Bacteriol 186:2724–34

    Article  CAS  Google Scholar 

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Correspondence to M. S. Madhyastha.

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Burton, E., Yakandawala, N., LoVetri, K. et al. A microplate spectrofluorometric assay for bacterial biofilms. J Ind Microbiol Biotechnol 34, 1–4 (2007). https://doi.org/10.1007/s10295-006-0086-3

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  • DOI: https://doi.org/10.1007/s10295-006-0086-3

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