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Isolation and Characterization of Mesophilic Luminescent Bacteria

Isolation and Characterization of Mesophilic Luminescent Bacteria. 作者： 柯明喬 、 賴文彬 指導老師：趙維良老師. Luminescent bacteria . Luminescent bacteria = Luminous bacteria Characterization Visible Light → Aerobic Large cell density Morphology → G (-), short rod, flagella .

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Isolation and Characterization of Mesophilic Luminescent Bacteria

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  1. Isolation and Characterization of Mesophilic Luminescent Bacteria 作者：柯明喬、賴文彬 指導老師：趙維良老師

  2. Luminescent bacteria • Luminescent bacteria = Luminous bacteria • Characterization • Visible Light → Aerobic Large cell density • Morphology → G (-), short rod, flagella

  3. Light production • Luciferase FMNH2 + O2 + RCHO FMN + RCOOH + H2O + LIGHT Luciferase

  4. Luminescent bacteria • Ecology • Saprophyte, parasite, symbiosis, free-living • Genus: • Marine: Vibrio, Photobacterium, Aeromonas • Land: Xenorhabdus

  5. 表一、 Vibrio, Photobacterium, Aeromonas 之特性差異 ＋ 正反應或有生長。 － 負反應或無生長。

  6. Accumulation of β-hydroxybutyrat + －

  7. Mesophilic bacteria • Growth temperature range: 15 – 45 ℃ • Optimum growth temperature: 25 ℃

  8. Materials and method • procedure Collection Sampling: sea-fish skin (S) & enteron (E) Culture Medium: modified MSWYE, luminous medium Condition: 25℃, aerobic, Isolation Streak plate method Identification Morphology observation Use of mannitol Accumulation of β-hydroxbutyrate

  9. Sample 2 Sample 1 Sample 3 Sample 4

  10. Sampling

  11. Medium • Luminous medium (per Liter) NaCl 30 g NH4Cl 5 g Yeast extract 5 g CaCO3 1 g Glycerol 3 ml Pancreatic digest of casein 5 g K2HPO4 3.9 g KH2PO4 2.1 g MgSO4‧7H2O 1 g KCl 0.75 g 1 M Tris buffer (pH 7.5) 50 ml Agar 20 g

  12. Medium • Modified MSWYE (per Liter) NaCl 23.4 g MgSO4‧7H2O 6.98 g KCl 0.75 g Protease peptone 1 g Yeast extract 1 g Agar 20 g Use NaOH (1N) adjust to pH 7.6

  13. Medium • Basal medium (per Liter) NaCl 23.4 g MgSO4‧7H2O 24.6 g KCl 1.5 g CaCl2‧2H2O2.9 g Artificial sea water (per Liter) 500 ml NaCl 23.4 g MgSO4‧7H2O 24.6 g KCl 1.5 g CaCl2‧2H2O 2.9 g

  14. Results • 10 isolates • 2EL • 3EL1 • 3EL3D • 3EL3S • 3EM1D • 3EM2D • 3EM2S • 3EM3 • 4EL • 4SL

  15. 2EL

  16. 3EL1

  17. 3EL3D

  18. 3EL3S

  19. 3EM1D

  20. 3EM2D

  21. 3EM2S

  22. 4EL

  23. 4SL

  24. 表二、分離株生化測試結果表 。 ＋ 正反應或有生長。 － 負反應或無生長。 ND無法判斷。 生長溫度測試以 luminous medium 平板培養 2 天。 Mannitol 利用以 basal medium 加入 0.2% mannitol 培養。 β-hydroxybutyrate 累積實驗以 basal medium 加入 0.2% glucose 培養。 除溫度測試外，所有測試皆以 25℃ 培養 2 天後觀察。

  25. 表三、分離株在不同培養基上亮度差異 ＋ 發亮。 － 不發亮。 ND無法判斷。

  26. Discussion • 不同樣本分離得發光菌多樣性不同 • 樣本生活環境 • 樣本表皮與腸道分離得發光菌多樣性不同 • 採樣時間距離樣本上岸時間太長 • 表皮與腸道溫度差異 • 同一菌株在不同培養基上發亮情形不同 • 培養基成分

  27. References • Hendrie, M. S., W. Hodgkiss, and J. M. Shewan. 1970. The indentification, taxonomy and classification of luminous bacteria. J. Gen. Microbiol. 64: 151-169. • Schwarz, J. R., and R. R. Colwell. 1974. Effect of hydrostatic pressure on growth and viablity of Vubrio parahaemolyticus. Appl. Microbiol. 26: 977-981 • Nealson, K. H. 1978. Isolation, indentification and manipulation of luminous bacteria. Methods Enzymol. 57: 153-166

  28. References • Orndorff, S. A., and R. R. Colwell. 1980. Distribution and identification of luminous bacteria from the Sargasso. Appl. Environ. Microbiol. 39: 983-987 • PE-BEE: WORLD: http://soils1.cses.vt.edu/ch/biol_4684/Microbes/Photo.html • 發光菌簡介：http://science.scu.edu.tw/micro/1024/learn/02micro_bio/chao000/chao016.htm

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