Isolation and characterization of bacteria producing substances against gram positive bacteria...
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Transcript of Isolation and characterization of bacteria producing substances against gram positive bacteria...
Isolation and characterization of bacteria producing
substances against gram positive bacteria
報告學生 :吳佩珊 楊于萱
目標
• Actinomycete~Micromonospora spp.
Streptomycete spp.
• Bacillus spp.
Microorganism Number of antibiotics Total
Eubacteria Pseudomonas 82 274
Enterobacteria 36
Bacillus 171
Actinomycetes Streptomyces 1922 2078
Micromonospora 21
Nocardia 45
Actinoplanes 5
Actinomadura 4
Streptosporangium 7
Streptoverticillium 8
Thermoactinomyces 9
Chainia 2
表一 . 目前已知可以產生抗生素之菌株
材料
• 潮濕的中性土壤
• 選擇性培養基 Starch-casein agar (SCA)
+ cycloheximide
• TSA + cycloheximide
材料
• MacConkey agar
• Phenylethanol agar
• Mannitol salt agar
• SIM agar
• MR-VP broth
試劑
• Kovact’s reagent
• Methyl red
• 3%H2O2
實驗架構• 將土壤篩濾
• 1g 土+ 9mL 0.85% 生理食鹽水,作十倍連續稀釋
• 取 0.1mL 於 SCA 平板與 TSA 平板 ( 皆加入 Cycloheximide)
• 挑 single colony 作 pure culture
測試
•將 pure culture 於 TSA 平板直徑處劃一條線
•放線菌培養四天
•一般細菌培養一天
•接測試菌
測試菌Staphylococcus aureus CCRC 10781Staphylococcus epidermidis CCRC 10783Staphylococcus saprophyticus CCRC 10786Streptococcus bovis CCRC 14729 Streptococcus pyogenes CCRC 10797Bacillus cereus CCRC 10603Bacillus subtilis CCRC 10255Micrococcus luteus CCRC 10449Micrococcus varians CCRC 12152
•測試純化的細菌是否對 G(+) 細菌
有抑制效果
生化測試
• Catalase
• Methyl red test
• SIM test
放線菌 1.乳黃色菌落 ,直徑約 3mm, 表面突起呈放線狀 ,孢子呈白色 ,培養一週會使 SCA 培養基
變黃
放線菌 2.圓形乳黃色菌落 ,直徑約 1.5mm, 於 SCA 培養基上孢子呈白色
放線菌 2. 經革蘭氏染色後之顯微鏡圖
放線菌 3.黃褐色菌落 ,直徑約 2mm, 菌落中央突起呈星形放射狀 ,具粉色孢子
放線菌 3. 經革蘭氏染色後之顯微鏡圖
放線菌 4.黃色菌落 ,直徑約 1mm, 形態與菌 3相似
放線菌 5.乳黃色菌落 ,直徑約 2.5mm, 表面突起邊緣呈放現狀
編號 測試菌
S. aureus
S. epidermidis
S. saprophyticus
S. bovis
S. pyogenes
B. cereu
s
B. subtilis
M. luteus
M. varians
1 - - - - 1 - - 2 2
2 3 5 1 - 5 3 5 5 10
3 - - - - - - 1 - -4 - - - - - - - - 11
5 - 3 - - - - - - 5
*數字代表抑制範圍 (mm)
*- 表示無抑制現象
* S. aure,Staphylococcus aureus;S. epidermidis,Staphylococcus epidermidis;S. saprophyticus,Staphylococcus saprophyticusus ;S. bovis,Streptococcus bovis ;S. pyogenes,Streptococcus pyogenes;B. cereus,Bacillus cereus ;B. subtilis,Bacillus subtilis;M. luteus,Micrococcus luteus ;M. varians,Micrococcus varians
表一、放線菌對 G (+)測試菌之抑制現象
結果
• 對 G(+) 細菌有抑制效果的一般細菌型態觀察
1. 表面皺褶 ,凸起 ,灰白色菌落 ,邊緣圓滑
2. 表面明顯突起 ,有透明液泡 ,灰白色菌落 ,邊緣不規則
3. 表面皺褶 (較 1密 ), 凸起 ,灰白色菌落 ,邊緣圓滑
4. 表面皺褶 ,凸起 ,灰白色菌落 ,邊緣不規則
編號 測試菌
S. aureus
S. epidermidis
S. saprophyticus
S. bovis
S. pyogenes
B. cereu
s
B. subtilis
M. luteus
M. varians
1 - 10 3 - 4 5 - 5 6
2 - 4 - 5 14 - - 6 6
3 - 18 3 11 12 6 - 5 3
4 - - - 10 9 4 - 3 -
*數字代表抑制範圍 (mm)
*- 表示無抑制現象
* S. aure,Staphylococcus aureus;S. epidermidis,Staphylococcus epidermidis;S. saprophyticus,Staphylococcus saprophyticusus ;S. bovis,Streptococcus bovis ;S. pyogenes,Streptococcus pyogenes;B. cereus,Bacillus cereus ;B. subtilis,Bacillus subtilis;M. luteus,Micrococcus luteus ;M. varians,Micrococcus varians
表二、細菌對 G (+)測試菌之抑制現象
分離株測試抗生素產生的情形
表三、細菌之形態觀察及生化測試結果測試 編號
1 2 3 4
Gram stain G (+) G (+) G (+) G (+)spore (+) (+) (+) (+)rod (+) (+) (+) (+)
catalase (+) (+) (+) (+)indole (-) (-) (-) (-)
H2S (-) (-) (-) (-)motility (-) (-) (-) (-)MR test (-) (-) (-) (-)manitol (+) (-) (+) (+)
7.5% NaCl (-) (-) (-) (-)anaerobic
growth(-) (+) (-) (-)
*(+)表示有反應 ; (-)表示無反應
胞子染色鏡檢圖
1 2
3 4
Catalase test Indole test
分離株在基紅甲平板上之測試
1
3
2
4
G (+)產胞細菌
Rod Cocci
Strict anaerobic (+) Strict anaerobic (-)Bacillus sp., Sporolactobacillus sp.
Catalase (+) Catalase (-) Bacillus sp.
Bacillus sp.
VP (+) VP (-)
Indole (+) Indole (-)
7%NaCl (+) 7%NaCl (-)
Anaerobic growth(+)
Anaerobic growth (-)
Mannitol (+) Mannitol (-)B. polymyxa B. cereus B. mycoides
B. coagulans
55℃(+)
55℃ (-)
B. coagulans B. cereus, B. mycoides細菌編號 2
Bacillus sp.
Indole (+) Indole (-)
7%NaCl (+) 7%NaCl (-)
Anaerobic growth(+)
Anaerobic growth(-)
Mannitol(+)
Mannitol(-)
Spore round (+) Spore round (-)B. stearothermophilyB. megaterium
B. brevis, B. alcalophilusB. megaterium, B. lenteus B. fastidiosus
細菌編號 1.3.4
參考文獻– David Hendricks Bergey (1989) Actinoplanetes. Bergey’s manual of s
ystematic bacteriology. Volume 2:P.1104-1139.
– David Hendricks Bergey (1989) Actinoplanetes. Bergey’s manual of systematic bacteriology. Volume 4:P.2442-2450.
– David Hendricks Bergey (1974) Actinomycetes and Related Organisms. Bergey’s manual of determinative bacteriology.847~852.
– James J. Champou, Lawrence Corey Frederick C. Neidbardt, James J. Plorde C. George Ray, Kennetb J. Ryan (1990) Actimicrobics and Chemotherapy of Bacterial and Viral Infections. Medical Microbiology An Introduction to Infectious Disease.209,231.
– Juan Carlos Oscariz, Inigo Lasa, and Antonio G. P. (1999)Detection and characterization of cerein 7, a new bacteriocin produced by Bacillus cereus with a broad spectrum of activity. FEMS Microbiology Letter. 178, 337-341.