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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. 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

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
  • Luminescent bacteria = Luminous bacteria
  • Characterization
    • Visible Light →

Aerobic

Large cell density

    • Morphology →

G (-), short rod, flagella

light production
Light production
  • Luciferase

FMNH2 + O2 + RCHO

FMN + RCOOH + H2O + LIGHT

Luciferase

luminescent bacteria1
Luminescent bacteria
  • Ecology
    • Saprophyte, parasite, symbiosis, free-living
  • Genus:
    • Marine: Vibrio, Photobacterium, Aeromonas
    • Land: Xenorhabdus
slide5

表一、 Vibrio, Photobacterium, Aeromonas 之特性差異

+ 正反應或有生長。

- 負反應或無生長。

mesophilic bacteria
Mesophilic bacteria
  • Growth temperature range: 15 – 45 ℃
  • Optimum growth temperature: 25 ℃
materials and method
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

slide9

Sample 2

Sample 1

Sample 3

Sample 4

medium
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

medium1
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

medium2
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

results
Results
  • 10 isolates
    • 2EL
    • 3EL1
    • 3EL3D
    • 3EL3S
    • 3EM1D
    • 3EM2D
    • 3EM2S
    • 3EM3
    • 4EL
    • 4SL
slide24

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。

slide25

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。

discussion
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
references
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
references1
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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