Dipicolinic acid (DPA) assay revisited and appraised for spore detection

Abstract

Delayed gate fluorescence detection of dipicolinic acid (DPA), a universal and specific component of bacterial spores, has been appraised for use in a rapid analytical method for the detection of low concentrations of bacterial spores. DPA was assayed by fluorimetric detection of its chelates with lanthanide metals. The influence of the choice and concentration of lanthanide and buffer ions on the fluorescence assay was studied as well as the effects of pH and temperature. The optimal system quantified the fluorescence of terbium monodipicolinate in a solution of 10 µM terbium chloride buffered with 1 M sodium acetate, pH 5.6 and had a detection limit of 2 nM DPA. This assay allowed the first real-time monitoring of the germination of bacterial spores by continuously quantifying exuded DPA. A detection limit of 10⁴Bacillus subtilis spores ml^–1 was reached, representing a substantial improvement over previous rapid tests.

References

1. J. M. Hausenbauer, W. M. Waites and P. Setlow, J. Bacteriol., 1997, 129, 1148.
2. P. J. Setlow, Appl. Bact. Symp. Supp., 1994, 76, 49S.
3. M. R. Blake and B. C. Weimer, Appl. Environ. Microbiol., 1997, 63, 1643.
4. M. Te Giffel PhD Thesis, Wageningen Agricultural University, Wageningen, The Netherlands, 1997.
5. D. D. Slaten, R. I. Oropeza and S. B. Werner, Public Health Rep., 1992, 107, 477.
6. P. McClure, Methods to study germination of bacterial spores, presentation to Fundamental and Applied Aspects of Bacterial Spores, a FEMS International Conference, Cambridge, July 1997.
7. L. D. S. Smith, Botulism, The Organism, Its Toxins, The Disease, Charles C. Thomas, Springfield, IL, 1977.
8. B. M. Lund, J. Ind. Microbiol., 1993, 12, 144.
9. K. L. Dodds, J. Ind. Microbiol., 1993, 12, 139.
10. D. L. Gatto-Menking, H. Yu, J. G. Bruno, M. T. Goode, M. Miller and A. Zulich, Biosens. Bioelectron., 1995, 10, 501.
11. J. J. Quinlan and P. M. Foegeding, J. Rapid Methods Automation Microbiol., 1998, 6, 1.
12. S. Udo, J. Agric. Chem. Soc. Jpn., 1936, 12, 386.
13. J. F. Powell, Biochem. J., 1953, 54, 210.
14. W. G. Murell, Bact. Spore, 1969, 1, 216.
15. G. P. Kalle and P. S. Khandekar, J. Biosci., 1983, 5, 43.
16. M. W. Tabor, J. MacGee and J. W. Holland, Appl. Environ. Microbiol., 1976, 31, 25.
17. A. D. Warth, Appl. Environ. Microbiol., 1979, 38, 1029.
18. H. Paulus, Anal. Biochem., 1981, 114, 407.
19. E. Ghiamati, R. Manoharan, W. H. Nelson and J. F. Sperry, Appl. Spectrosc., 1992, 46, 357.
20. D. Helm and D. Naumann, FEMS Microbiol. Lett., 1995, 126, 75.
21. J. Volke and V. Volkova, Chem. Listy, 1954, 48, 1031.
22. G. S. Porter, M. W. Brown and M. R. W. Brown, Biochem. J., 1967, 102, 19c.
23. O. R. Brown, J. A. Harrison and K. S. Sastry, Electroanal. Chem. Interfacial Electrochem., 1975, 58, 387.
24. S. Ferraro, P. Passamonti, V. Bartoccia and F. Pucciarelli, J. Chem. Soc., Faraday Trans., 1997, 93, 289.
25. Y. Matano, Y. Yasuda and K. Tochikubo, FEMS Microbiol Lett., 1993, 109, 189.
26. N. Grecz and T. Tang, J. Gen. Microbiol., 1970, 63, 303.
27. I. R. Scott and D. J. Ellar, Biochem. J., 1978, 174, 635.
28. A. D. Warth, Anal. Biochem., 1983, 130, 502.
29. T. D. Barela and A. D. Sherry, Anal. Biochem., 1976, 76, 351.
30. L. E. Sacks, Appl. Environ. Microbiol., 1990, 56, 1185.
31. D. L. Rosen, C. Sharpless and L. B. McGown, Anal. Chem., 1997, 69, 1082.
32. P. M. Pellegrino, N. F. Fell Jr., D. L. Rosen and J. B. Gillespie, Anal. Chem., 1998, 70, 1755.
33. S. Lis, Chem. Anal. (Warsaw), 1993, 38, 443.
34. B. R. Judd, Phys. Rev., 1962, 127, 750.
35. G. S. Ofelt, J. Chem. Phys., 1962, 37, 511.
36. E. D. Moorhead, V. E. Dauyotis and M. M. Stephen, Anal. Chim. Acta., 1984, 162, 161.
37. S. H. Jung, S. K. Yoon, J. G. Kim and J. G. Kang, Bull. Kor. Chem. Soc., 1992, 13, 650.
38. J. G. Kim, S. K. Yoon and J. G. Kang, Bull. Kor. Chem. Soc., 1992, 13, 54.
39. J. G. Kim, S. K. Yoon and J. G. Kang, Bull. Kor. Chem. Soc., 1996, 17, 854.
40. S. I. Weissman, J. Chem. Phys., 1942, 10, 214.
41. F. R. G. E. Silva and O. L. Malta, J. Alloys Compounds, 1997, 250, 427.
42. E. Soini and T. Lövgren, CRC Crit. Rev. Anal. Chem., 1987, 18, 104.
43. R. D. Bautista, A. I. Jiménez, F. Jiménez and J. J. Arias, Talanta, 1996, 43, 421.
44. F. S. Richardson, Chem. Rev., 1982, 82, 541.
45. P. Guerriero, U. Casellato, S. Sitran, P. A. Vigato and R. Graziani, Inorg. Chim. Acta., 1987, 133, 337.
46. B. M. Alsaadi, F. J. C. Rossotti and R. J. P. Williams, J. Chem. Soc., Dalton Trans., 1980, 597.
47. P. Guerriero, U. Casellato, S. Sitran, P. A. Vigato and R. Graziani, Inorg. Chim. Acta, 1987, 139, 67.
48. J. Albertsson, Acta Chem. Scand., 1972, 26, 958.
49. T. K. Trout, J. M. Bellama, R. A. Faltynek and F. E. Brinckman, Inorg. Chim. Acta, 1989, 155, 13.
50. I. Grenthe, J. Am. Chem. Soc., 1961, 83, 360.
51. D. J. Ellar, 1997, personal communication.