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Isolation and Characterization of Mesophilic Luminescent Bacteria
作者：柯明喬、賴文彬 指導老師：趙維良老師
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Luminescent bacteria Luminescent bacteria = Luminous bacteria
Characterization Visible Light → Aerobic Large cell density Morphology → G (-), short rod, flagella
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Light production Luciferase FMNH2 + O2 + RCHO
FMN + RCOOH + H2O + LIGHT Luciferase
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Luminescent bacteria Ecology Genus:
Saprophyte, parasite, symbiosis, free-living Genus: Marine: Vibrio, Photobacterium, Aeromonas Land: Xenorhabdus
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表一、 Vibrio, Photobacterium, Aeromonas 之特性差異
Genus Characteristics Accumulation of β-hydroxybutyrate Utilization of mannitol Vibrio － ＋ Photobacterium Aeromonas ＋ 正反應或有生長。 － 負反應或無生長。
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Accumulation of β-hydroxybutyrat
+ －
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Mesophilic bacteria Growth temperature range: 15 – 45 ℃
Optimum growth temperature: 25 ℃
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Materials and method procedure Collection Culture Isolation
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
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Sample 2 Sample 1 Sample 3 Sample 4
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Sampling
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Medium Luminous medium (per Liter) NaCl 30 g NH4Cl 5 g
Yeast extract 5 g CaCO g Glycerol 3 ml Pancreatic digest of casein 5 g K2HPO g KH2PO g MgSO4‧7H2O 1 g KCl g 1 M Tris buffer (pH 7.5) 50 ml Agar g
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Modified MSWYE (per Liter)
Medium Modified MSWYE (per Liter) NaCl g MgSO4‧7H2O g KCl g Protease peptone 1 g Yeast extract 1 g Agar g Use NaOH (1N) adjust to pH 7.6
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Medium Basal medium (per Liter) NaCl 23.4 g MgSO4‧7H2O 24.6 g
KCl g CaCl2‧2H2O g Artificial sea water (per Liter) 500 ml MgSO4‧7H2O g CaCl2‧2H2O g
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Results 10 isolates 2EL 3EL1 3EL3D 3EL3S 3EM1D 3EM2D 3EM2S 3EM3 4EL
4SL
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2EL
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3EL1
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3EL3D
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3EL3S
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3EM1D
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3EM2D
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3EM2S
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4EL
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4SL
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表二、分離株生化測試結果表 。 分離株名稱 生長溫度 (℃) 利用 mannitol 聚集 β-hydroxybutyrate 可能分類 4
25 55 2EL － ＋ Vibrio 3EL1 Photobacterium 3EL3D 3EL3S Aeromonas 3EM1D ND 3EM2D 3EM2S 3EM3 4EL 4SL ＋ 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。
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3EM2S (Photobacterium sp.)
表三、分離株在不同培養基上亮度差異 LM mMSWYE 2EL (Vibrio sp.) + - 3EL3S (Aeromonas sp.) 3EM2S (Photobacterium sp.) 3EM3 (ND) 4EL (Aeromonas sp.) 4SL (Aeromonas sp.) ＋ 發亮。 － 不發亮。 ND 無法判斷。
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Discussion 不同樣本分離得發光菌多樣性不同 樣本表皮與腸道分離得發光菌多樣性不同 同一菌株在不同培養基上發亮情形不同 樣本生活環境
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
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References Hendrie, M. S., W. Hodgkiss, and J. M. Shewan The indentification, taxonomy and classification of luminous bacteria. J. Gen. Microbiol. 64: Schwarz, J. R., and R. R. Colwell Effect of hydrostatic pressure on growth and viablity of Vubrio parahaemolyticus. Appl. Microbiol. 26: Nealson, K. H Isolation, indentification and manipulation of luminous bacteria. Methods Enzymol. 57:
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References Orndorff, S. A., and R. R. Colwell Distribution and identification of luminous bacteria from the Sargasso. Appl. Environ. Microbiol. 39: PE-BEE: WORLD: 發光菌簡介：
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