Isolation and Characterization of Mesophilic Luminescent Bacteria Luminescent

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

Light production u Luciferase FMNH 2 + O 2 + RCHO Luciferase FMN + RCOOH + H 2 O + LIGHT

Luminescent bacteria u Ecology l Saprophyte, parasite, symbiosis, free-living u Genus: l Marine: Vibrio, Photobacterium, Aeromonas l Land: Xenorhabdus

表一、 Vibrio, Photobacterium, Aeromonas 之特性差異 Characteristics Genus Accumulation of β-hydroxybutyrate Utilization of mannitol Vibrio － ＋ Photobacterium ＋ － Aeromonas － － ＋ 正反應或有生長。 － 負反應或無生長。

Accumulation of β-hydroxybutyrat + －

Mesophilic bacteria u Growth temperature range: 15 – 45 ℃ u Optimum growth temperature: 25 ℃

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

Sample 1 Sample 3 Sample 2 Sample 4

Sampling

Medium l Luminous medium (per Liter) Na. Cl NH 4 Cl Yeast extract Ca. CO 3 Glycerol Pancreatic digest of casein K 2 HPO 4 KH 2 PO 4 Mg. SO 4‧ 7 H 2 O KCl 1 M Tris buffer (p. H 7. 5) Agar 30 g 5 g 5 g 1 g 3 ml 5 g 3. 9 g 2. 1 g 1 g 0. 75 g 50 ml 20 g

Medium l Modified MSWYE (per Liter) Na. Cl 23. 4 g Mg. SO 4‧ 7 H 2 O 6. 98 g KCl 0. 75 g Protease peptone 1 g Yeast extract 1 g Agar 20 g Use Na. OH (1 N) adjust to p. H 7. 6

Medium l Basal medium (per Liter) Na. Cl Mg. SO 4‧ 7 H 2 O KCl Ca. Cl 2‧ 2 H 2 O Artificial sea water (per Liter) Na. Cl Mg. SO 4‧ 7 H 2 O KCl Ca. Cl 2‧ 2 H 2 O 23. 4 g 24. 6 g 1. 5 g 2. 9 g 500 ml 23. 4 g 24. 6 g 1. 5 g 2. 9 g

Results l 10 isolates l l l l l 2 EL 3 EL 1 3 EL 3 D 3 EL 3 S 3 EM 1 D 3 EM 2 S 3 EM 3 4 EL 4 SL

2 EL

3 EL 1

3 EL 3 D

3 EL 3 S

3 EM 1 D

3 EM 2 D

3 EM 2 S

4 EL

4 SL

表三、分離株在不同培養基上亮度差異 LM m. MSWYE 2 EL (Vibrio sp. ) + - 3 EL 3 S (Aeromonas sp. ) + + 3 EM 2 S (Photobacterium sp. ) + + 3 EM 3 (ND) + + 4 EL (Aeromonas sp. ) + - 4 SL (Aeromonas sp. ) + - ＋ 發亮。 － 不發亮。 ND 無法判斷。

References u Hendrie, M. S. , W. Hodgkiss, and J. M. Shewan. 1970. The indentification, taxonomy and classification of luminous bacteria. J. Gen. Microbiol. 64: 151 -169. u Schwarz, J. R. , and R. R. Colwell. 1974. Effect of hydrostatic pressure on growth and viablity of Vubrio parahaemolyticus. Appl. Microbiol. 26: 977981 u Nealson, K. H. 1978. Isolation, indentification and manipulation of luminous bacteria. Methods Enzymol. 57: 153 -166

References u Orndorff, S. A. , and R. R. Colwell. 1980. Distribution and identification of luminous bacteria from the Sargasso. Appl. Environ. Microbiol. 39: 983 -987 u PE-BEE: WORLD: http: //soils 1. cses. vt. edu/ch/biol_4684/Microbes/P hoto. html u 發光菌簡介： http: //science. scu. edu. tw/micro/1024/learn/02 mic ro_bio/chao 000/chao 016. htm