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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. 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)

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Isolation and characterization of bacteria producing substances against gram positive bacteria

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  1. Isolation and characterization of bacteria producing substances against gram positive bacteria 報告學生:吳佩珊 楊于萱

  2. 目標 • Actinomycete~Micromonospora spp. Streptomycete spp. • Bacillus spp.

  3. 表一. 目前已知可以產生抗生素之菌株

  4. 材料 • 潮濕的中性土壤 • 選擇性培養基Starch-casein agar (SCA) + cycloheximide • TSA + cycloheximide

  5. 材料 • MacConkey agar • Phenylethanol agar • Mannitol salt agar • SIM agar • MR-VP broth

  6. 試劑 • Kovact’s reagent • Methyl red • 3%H2O2

  7. 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

  8. 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

  9. 測試菌 Staphylococcus aureus CCRC 10781 Staphylococcus epidermidis CCRC 10783 Staphylococcus saprophyticus CCRC 10786 Streptococcus bovis CCRC 14729 Streptococcus pyogenes CCRC 10797 Bacillus cereus CCRC 10603 Bacillus subtilis CCRC 10255 Micrococcus luteus CCRC 10449 Micrococcus varians CCRC 12152

  10. 測試純化的細菌是否對G(+)細菌 有抑制效果

  11. 生化測試 • Catalase • Methyl red test • SIM test

  12. 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

  13. 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.圓形乳黃色菌落,直徑約1.5mm,於SCA培養基上孢子呈白色

  14. 放線菌2.經革蘭氏染色後之顯微鏡圖

  15. 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.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

  16. 放線菌3.經革蘭氏染色後之顯微鏡圖

  17. 放線菌4.黃色菌落,直徑約1mm,形態與菌3相似

  18. 放線菌5.乳黃色菌落,直徑約2.5mm,表面突起邊緣呈放現狀放線菌5.乳黃色菌落,直徑約2.5mm,表面突起邊緣呈放現狀

  19. 表一、放線菌對G（＋）測試菌之抑制現象

  20. 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

  21. 表二、細菌對G（＋）測試菌之抑制現象

  22. 分離株測試抗生素產生的情形

  23. 胞子染色鏡檢圖 1 2 4 3

  24. Catalase test Indole test

  25. 分離株在基紅甲平板上之測試 1 2 4 3

  26. G（＋）產胞細菌

  27. 細菌編號2

  28. 細菌編號1.3.4

  29. 參考文獻 • David Hendricks Bergey (1989) Actinoplanetes. Bergey’s manual of systematic 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.

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