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Antibacterial tests

Antibacterial tests. Purpose: To understand chemical methods to inactivate bacteria. Theory Bactericide (bactericidal) Cide: kill; bactericide: a agent which can kill bacteria Bacteriostatic Static: not active; bacteriostatic agent: a agent which inhibit growth of bacteria.

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Antibacterial tests

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  1. Antibacterial tests • Purpose: • To understand chemical methods to inactivate bacteria. • Theory • Bactericide (bactericidal) • Cide: kill; bactericide: a agent which can kill bacteria • Bacteriostatic • Static: not active; bacteriostatic agent: a agent which inhibit growth of bacteria

  2. 窄效性藥物(narrow-specturm agents)，僅對少數微生物發揮抑制效果 適合人體醫學用 廣效性藥物(broad-spectrum agents) 可對多種微生物產生抑制或破壞的效果 是合一般消毒用

  3. 抗菌劑的作用機轉可分為下列四類：(1) 抑制細胞壁合成；(2) 抑制核酸合成或功能；(3) 抑制蛋白質合成；(4) 干擾細胞膜功能

  4. All bactericides are bacteriostatic agents depending on concentration. For example, high concentration kills bacteria but low concentration only inhibits. • Not all bacteriostatic agents are bactercides because of antibacterial mechanism. For example, tetracycline and chloramphenicol

  5. Penicillin 青黴素 Penicillium青黴菌 • The first antibiotic • 抑制細胞壁合成 • 對Gram positive 較有效 • Sir Alexander Fleming– a Scottish • LordHoward Walter Florey (Baron) - an Australian pharmacologist in Oxford • Sir Ernst Boris Chain- a German Jew

  6. Penicillium on an orange Penicillium spores

  7. Clear zone 溶菌環

  8. Ampicillin安比西林 • Ampicillin is a beta-lactam antibiotic that has been used extensively to treat bacterial infections since 1961. It is considered part of aminopenicillin in family. • Ampicillin acts as a competitive inhibitor of the enzyme transpeptidase. Transpeptidase is needed by bacteria to make their cell walls. It inhibits the third and final stage of bacterial cell wall synthesis, which ultimately leads to cell lysis.

  9. Tetracycline四環黴素 • Tetracycline antibiotics inhibit protein synthesis by inhibiting the binding of aminoacyl-tRNA to the mRNA-ribosome complex. They do so mainly by binding to the 30S ribosomal subunit in the mRNA translation complex. • Oxytetracycline is one of most commonly used antibiotics in fishery.

  10. Chloramphenicol 氯黴素 • Chloramphenicol is a bacteriostatic antimicrobial originally derived from the bacterium Streptomyces venezuelae, isolated by David Gottlieb, and introduced into clinical practice in 1949.

  11. It was the first antibiotic to be manufactured synthetically on a large scale. Chloramphenicol is effective against a wide variety of microorganisms and still widely used in low income countries because of its low cost. • This antibiotic is still used in aquatic industry(not in Taiwan). • It functions by inhibiting peptidyl transferase activity of the bacterial ribosome, binding to the 50S ribosomal subunit, preventing peptide bond formation.

  12. Streptomycin 鏈黴素 • It was the first antibiotic remedy for tuberculosis. A product purified from from the actinobacterium Streptomyces griseus.

  13. Streptomycin • Streptomycin is a bactericidal antibiotic. • It binds to the 16S rRNA of the bacterial ribosome, interfering with the binding of formyl-methionyl-tRNA to the 30S subunit. • This prevents initiation of protein synthesis and leads to death of microbial cells.

  14. 台灣對水產養殖用藥之最高殘留限量 • Ampicillin 安比西林 肌肉 魚 0.05 • Chlortetracycline、Oxytetracycline及Tetracycline 氯四環黴素、羥四環黴素及四環黴素 肌肉 魚、大明蝦、蝦 0.2 • Oxolinic acid 歐索林酸 肌肉（含皮）魚 肌肉 蝦 0.05

  15. Microbiology • Antibiotics • Tetracycline (30S ribosome)30μg 四環黴素 • Streptomycon (16S ribosom) 10μg 鏈黴素 • Chloramphenicol (protein synthesis) 30μg 氯黴素 • Ampicillin (cell wall) 10μg 安比西林 • E. coli , S. aureus • Pre-preparation • 105 CFU/mL (two tubes/table) • Spread plate of 104 CFU/plate • 玻棒 glass hockey stick • Pipet

  16. Pipet using • 一段式吸取，兩段式釋放 • 100-1000μL(0.1-1mL) • Must be in the range • Uptake slowly

  17. Spread plate • 將益體放置於plate中央 (Place the liquid suspension on the center of plate) • 玻棒放置於酒精中，取出後過火 (Use alcohol and flam to sterilize the glass rod) • 玻棒頭必須朝下 • Place the glass rod on the edge of plate to cool down • Spread the suspension • 一邊塗，一邊轉plate • 蓋好上蓋，靜置約30seconds • Leave the plates upside down

  18. 4 plates/group • Each bacterial species has two plates • Place two discs on a plate • 2 disc/plate • Incubate at 37 ℃ for 24 hours • measure the diameter (直徑)of the clear zone

  19. E. coli E. coli A T C S S. aureus S. aureus A T C S

  20. Bactericide (殺菌 ; cide: kill) vs. bacteriostatic (靜菌或抑菌:static: stay at one place or status, don’t change) • Both experiments are bacteriostatic not bactericide tests. • Bacteria do not grow yet.

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