Isolation and Characterization Isolation and Characterization of Mesophilic Luminescent Bacof Mesophilic Luminescent Bac

teriateria

作者：作者：柯明喬柯明喬、、賴文彬賴文彬指導老師：趙維良老師指導老師：趙維良老師

Luminescent bacteria = Luminous Luminescent bacteria = Luminous bacteriabacteria

CharacterizationCharacterizationVisible Light → Visible Light →

AerobicAerobicLarge cell densityLarge cell density

Morphology →Morphology →G (-), short rod, flagella G (-), short rod, flagella

Luminescent bacteria Luminescent bacteria

LuciferaseLuciferase

FMNHFMNH22 + O + O22 + RCHO + RCHO

FMN + RCOOH + HFMN + RCOOH + H22O + O + LIGHT LIGHT

Light production Light production

Luciferase

EcologyEcologySaprophyte, parasite, symbiosis, free-livingSaprophyte, parasite, symbiosis, free-living

Genus: Genus: Marine: Marine: VibrioVibrio, , PhotobacteriumPhotobacterium,, Aeromonas Aeromonas Land: Land: XenorhabdusXenorhabdus

Luminescent bacteria Luminescent bacteria

GenusGenusCharacteristicsCharacteristics

Accumulation of β-hydroxybutyrateAccumulation of β-hydroxybutyrate Utilization of mannitolUtilization of mannitol

VibrioVibrio －－ ＋＋

PhotobacteriumPhotobacterium ＋＋ －－

AeromonasAeromonas －－ －－

＋＋ 正反應或有生長。 正反應或有生長。－－ 負反應或無生長。 負反應或無生長。

表一、 表一、 VibrioVibrio, , PhotobacteriumPhotobacterium,, Aeromonas Aeromonas 之特性差異之特性差異

Accumulation of β-hydroxybutyrat

+ －－

Growth temperature range: 15 – 45 ℃Growth temperature range: 15 – 45 ℃

Optimum growth temperature: 25 ℃Optimum growth temperature: 25 ℃

Mesophilic bacteria Mesophilic bacteria

procedure procedure

Materials and method Materials and method

Collection

Isolation

Identification

Sampling: sea-fish skin (S) & enteron (E)

Culture Medium: modified MSWYE, luminous medium

Condition: 25 , aerobic, ℃

Streak plate method

Morphology observation

Use of mannitol

Accumulation of β-hydroxbutyrate

Sample 1 Sample 2

Sample 3 Sample 4

SamplingSampling

Luminous medium (per Liter)Luminous medium (per Liter)NaClNaCl 30 g30 g

NHNH44ClCl 5 g5 g

Yeast extractYeast extract 5 g 5 g

CaCOCaCO33 1 g1 g

GlycerolGlycerol 3 ml3 ml

Pancreatic digest of caseinPancreatic digest of casein 5 g5 g

KK22HPOHPO44 3.9 g3.9 g

KHKH22POPO44 2.1 g2.1 g

MgSOMgSO44 7H‧7H‧ 22OO 1 g1 g

KClKCl 0.75 g0.75 g

1 M Tris buffer (pH 7.5)1 M Tris buffer (pH 7.5) 50 ml50 ml

AgarAgar 20 g20 g

MediumMedium

Modified MSWYE (per Liter)Modified MSWYE (per Liter)NaClNaCl 23.4 g23.4 g

MgSOMgSO44 7H‧7H‧ 22OO 6.98 g 6.98 g

KClKCl 0.75 g 0.75 g

Protease peptoneProtease peptone 1 g1 g

Yeast extractYeast extract 1 g 1 g

AgarAgar 20 g20 g

Use NaOH (1N) adjust to pH 7.6Use NaOH (1N) adjust to pH 7.6

MediumMedium

Basal medium (per Liter)Basal medium (per Liter)NaClNaCl 23.4 g 23.4 g

MgSOMgSO44 7H‧7H‧ 22OO 24.6 g 24.6 g

KClKCl 1.5 g1.5 g

CaClCaCl22 2H‧2H‧ 22OO 2.9 g2.9 g

Artificial sea water (per Liter)Artificial sea water (per Liter) 500 ml500 mlNaClNaCl 23.4 g 23.4 g MgSO4 7H2O‧MgSO4 7H2O‧ 24.6 g 24.6 g KClKCl 1.5 g1.5 gCaCl2 2H2O‧CaCl2 2H2O‧ 2.9 g2.9 g

MediumMedium

ResultsResults 10 isolates10 isolates

2EL2EL 3EL13EL1 3EL3D3EL3D 3EL3S3EL3S 3EM1D3EM1D 3EM2D3EM2D 3EM2S3EM2S 3EM33EM3 4EL4EL 4SL4SL

2EL2EL

3EL13EL1

3EL3D3EL3D

3EL3S3EL3S

3EM1D3EM1D

3EM2D3EM2D

3EM2S3EM2S

4EL4EL

4SL4SL

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。

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 ＋ ＋ － － ＋ Photobacterium

3EL3S － ＋ － － － Aeromonas

3EM1D ＋ ＋ － ＋ ＋ ND

3EM2D ＋ ＋ － ＋ ＋ ND

3EM2S － ＋ － － ＋ Photobacterium

3EM3 － ＋ － ＋ ＋ ND

4EL － ＋ － － － Aeromonas

4SL － ＋ － － － Aeromonas

LM mMSWYE

2EL (Vibrio sp.) + -

3EL3S (Aeromonas sp.) + +

3EM2S (Photobacterium sp.) + +

3EM3 (ND) + +

4EL (Aeromonas sp.) + -

4SL (Aeromonas sp.) + -

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。

DiscussionDiscussion

不同樣本分離得發光菌多樣性不同不同樣本分離得發光菌多樣性不同 樣本生活環境樣本生活環境

樣本表皮與腸道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 採樣時間距離樣本上岸時間太長採樣時間距離樣本上岸時間太長

表皮與腸道溫度差異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同一菌株在不同培養基上發亮情形不同 培養基成分培養基成分

Hendrie, M. S., W. Hodgkiss, and J. M. Shewan. 19Hendrie, M. S., W. Hodgkiss, and J. M. Shewan. 1970. The indentification, taxonomy and classification 70. The indentification, taxonomy and classification of luminous bacteria. J. Gen. Microbiol. 64: 151-16of luminous bacteria. J. Gen. Microbiol. 64: 151-169.9.

Schwarz, J. R., and R. R. Colwell. 1974. Effect of hSchwarz, J. R., and R. R. Colwell. 1974. Effect of hydrostatic pressure on growth and viablity of Vubrio ydrostatic pressure on growth and viablity of Vubrio parahaemolyticus. Appl. Microbiol. 26: 977-981parahaemolyticus. Appl. Microbiol. 26: 977-981

Nealson, K. H. 1978. Isolation, indentification and Nealson, K. H. 1978. Isolation, indentification and manipulation of luminous bacteria. Methods Enzymmanipulation of luminous bacteria. Methods Enzymol. 57: 153-166ol. 57: 153-166

References References

Orndorff, S. A., and R. R. Colwell. 1980. DistributOrndorff, S. A., and R. R. Colwell. 1980. Distribution and identification of luminous bacteria from tion and identification of luminous bacteria from the Sargasso. Appl. Environ. Microbiol. 39: 983-9he Sargasso. Appl. Environ. Microbiol. 39: 983-98787

PE-BEE: WORLD: http://soils1.cses.vt.edu/ch/biPE-BEE: WORLD: http://soils1.cses.vt.edu/ch/biol_4684/Microbes/Photo.htmlol_4684/Microbes/Photo.html

發光菌簡介發光菌簡介：： http://science.scu.edu.tw/micro/10http://science.scu.edu.tw/micro/1024/learn/02micro_bio/chao000/chao016.htm24/learn/02micro_bio/chao000/chao016.htm

References References