The Early Detection of the Gram Positive Bacteria using Sonification and Fluorescent Dye in the Field

현장에서 초음파 파쇄와 형광시약을 이용한 그람 양성균의 조기 탐지

  • Published : 2006.10.30

Abstract

This study was carried out to establish the optimum condition for cell disruption with a sonificator in the detection of the gram positive bacteria, Bacillus globigii and Streptococcus epidermidis for the purpose of developing automatic fluorometer. The efficiency of sonificator on the Bacillus globigii and Streptococcus epidermidis disruption differed greatly according to the diameter of sonificator probe tip. The larger sonificator probe diameter showed greater disruption. Bacillus globigii was more disruptive than Streptococcus epidermidis. Sonificator probe of the 13 mm diameter was the most efficient one when sample was sonificated for 20 seconds. The detection limits of Bacillus globigii and Streptococcus epidermidis were $10^5CFU/m{\ell}\;and\;5{\times}10^5CFU/m{\ell}$ respectively when samples were sonificated for 20 seconds with a sonificator probe of 13 mm diameter.

본 연구에서는 sonificator를 장착하여 세포막을 파쇄하고 현장에서 형광을 이용하여 조작이 간편하고 단시간에 DNA를 측정할 수 있는 자동화된 형광기를 개발하기 위하여 그람 양성균으로 간균인 Bacillus globigii와 구균인 Streptococcus epidermidis를 대상으로 최적의 세포 파쇄조건을 확립하고자 하였다. Sonificator probe 직경에 따라 세포의 파쇄 효과가 큰 차이를 보였으며 13 mm probe로 20초동안 sonification시키는 것이 가장 효율적인 것으로 나타났으며 간균인 Bacillus globigii가 구균인 Streptococcus epidermidis보다 더 파쇄가 잘 되는 것으로 나타났다. 시료에 잠긴 Sonificator probe tip 깊이에 따라서도 세포의 파쇄 효과가 크게 나타났는데 시료에 잠긴 probe tip의 깊이가 깊을수록 큰 파쇄 효과를 발휘하였다. 선정된 최적의 파쇄 조건에서 최저 탐지농도는 Bacillus globigii, Streptococcus epidermidis 모두 $5{\times}10^5CFU/m{\ell}$의 농도를 탐지 가능한 것으로 나타났다.

Keywords

References

  1. Borthwick, K. A. J., W. T. Coakley, M. B. McDonnell, and H. Nowotny (2005), Development of a novel compact sonicator for cell disruption, J. Microbial. Methods 60, 207-216 https://doi.org/10.1016/j.mimet.2004.09.012
  2. Belgrader, P., D. Hansford, G. T. A. Kovacs, K. Vanketeswaran, R. Mariell, F. Milanovich, S. Nasarabadi, M. Okuzumi, F. Pourahmadi, and M. A. Northrup (1999), A minisonicator to rapidly disrupt bacterial spores for DNA analysis, Anal. Chem. 71, 4232-4236 https://doi.org/10.1021/ac990347o
  3. Gannon, V. P. J, P. K. King, J. Y. Kim, and E. J. Thomas (1992), Rapid and sensitive method for detection of shiga-like toxin-producing Escherichia coli in ground beef using polymerase chain reaction, Appl. Environ. Microbiol. 58, 3809-3815
  4. Fykse, E. M., J. Stand, and G. S. Olsen (2003), Application of sonification to lease DNA from Bacillus cereus for quantitative detection by real-time PCR, J. Microbiol. Methods 55, 1-10 https://doi.org/10.1016/S0167-7012(03)00091-5
  5. Bury, D., P. Jelen, and M. Kalab (2001), Disruption of Lactobacillus delbrueckii ssp. bulgaricus 11842 cells for lactose hydrolysis in dairy products: a comparison of sonication, high-pressure homogenization and bead milling, Innovative Food science & Emerging Technologies 2, 23-39 https://doi.org/10.1016/S1466-8564(00)00039-4
  6. Blechl, A. E., K. S. Thrasher, W. H. Vensel, and F. C. Greene (1992), Purification and characterization of wheat $\alpha$-gliadin synthesized in the yeast, Saccharomyces cerevisiae, Gene. 116, 119-127 https://doi.org/10.1016/0378-1119(92)90507-L
  7. James, C. J., W. T. Coakley, and D. E. Hughes (1972), Kinetics of protein release from yeast sonicated in batch and flow systems at 20kHz, Biotechnol. Bioeng. 14, 33-42 https://doi.org/10.1002/bit.260140105
  8. Calderira, J. C. G. and J. M. S. Cabral (1994), Extraction of a steroid 1,2-dehydrogenase by sonification of Arthrobacter simplex cells, Bioseparation 4, 271-278
  9. Kuboi, R., H. Umakoshi, N. Takagi, and I. Komasawa (1995), Optimal disruption methods for the selective recovery of $\beta$-galactosidase from Escherichia coli, J. Ferment. Bioeng. 79, 335-341 https://doi.org/10.1016/0922-338X(95)93991-R
  10. Feliu, J. X., R. Cubarsi, and A. Villaverde (1998), Optimized release of recombinant proteins by ultrasonication of E. coli cells, Biotechnology and Bioengineering 58(5), 536-540 https://doi.org/10.1002/(SICI)1097-0290(19980605)58:5<536::AID-BIT10>3.0.CO;2-9
  11. Ha Y. C., K. B. Choi, and J. D Choi (2006), The studies on the early detection of the gram positive bacteria using sonication and fluorescent dye in the field, Kor. J. Biotechnol. Bioeng. 100, 341-346