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Antibiotics

Lab # 1. Antibiotics. Antimi c robi al Therapy. Natural antibiotic agents: Produced by microorganisms: Penicillium notatum – penicillin Semi-synthetic antibiotic agents: chemically modified natural agents (large group of modern antibiotics) synthetic antibiotic agents:

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Antibiotics

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  1. Lab # 1 Antibiotics

  2. Antimicrobial Therapy • Natural antibiotic agents: • Produced by microorganisms: • Penicilliumnotatum– penicillin • Semi-synthetic antibiotic agents: • chemically modified natural agents (large group of modern antibiotics) • synthetic antibiotic agents: • Chemically related to natural antibiotics but completely industrially manufactured

  3. Antibiotics are: • Natural or synthetic products that are used to kill or stop the growth of Bacteria : • bacteriostatic - stop growth (don't kill) • bactericidal – kill

  4. Why do we do sensitivity testing?? • To know which drug we use to the patient. • To Know the dose of antibiotic. It is important to use the lowest effective concentration of the antibiotic to avoid toxicity in patient.

  5. Definitions: • Control strains: • These are organisms obtained from the American Type Culture Collection (ATCC). • They should be grown in standard conditions • They have a known recorded sensitivity to antibiotics

  6. Organism UsedStandard Organism (Quality Control organism) • Staphylococcus aureus ATCC 25923 • Pseudomonas aeruginosa ATCC 27853 • Escherichia coli ATCC 25922 • Enterococcus faecalis ATCC 29212 • Klepsiella pneumoniae ATCC 700603 • Streptococcus pneumoniae ATCC 49619 • Haemophilus influenzae ATCC 49247

  7. Definitions: • Muller Hinton Medium: • It is a special media used for sensitivity testing, it dose not interfere with test results it has a: • Standard PH • Standard electrolytes

  8. Definitions: • Standard inoculum size: • A standard concentration of bacterial cells to be inoculated.

  9. How to prepare standard inoculum size: • Standard inoculum should have turbidity equivalent to 0.5 McFarland standard. • Should be from a freshly overnight growth.

  10. McFaraland: • These are made by dissolving barium sulphate in water with different concentration. • 0.5 McFaraland have a turbidity equivalent to 1x105

  11. Antibiotic Sensitivity Test • Method • 1) Broth dilution • 2) Agar diffusion (solid) -a) Kirby-Bauer test (= disc test) - b) Stock’s methods

  12. Broth Dilution Method antibiotic (dilution series) + bacterial suspension (standard amount) growth ? MIC – minimal inhibitory concentration

  13. Definitions: • Minimum Inhibitory Concentration MIC: • The lowest concentration of antimicrobial required to stop growth of bacteria. • Minimum Bactericidal Concentration MBC: • The lowest concentration of antimicrobial required to kill bacteria.

  14. Broth Dilution Method • Prepare 2 sets of 9 sterile tubes. • 1 ml  broth in each tube. • 1 ml  antibiotic of interest in tube #1. • Take 1 ml of tube #1 & add to next tube & so on tell tube #8 • Take 1 ml of tube #8 & discard.

  15. Broth Dilution Method • Add 1 drop of test organism in each tube of set 1 using a pastuer pipette. • Add one drop of control organism in each tube of set 2 • Incubate 24 hr x 37C

  16. Results: Control tube Only organism MIC  last tube showing no growth. Tube #9 has no antibiotic  has to be turbid. 1 2 3 4 5 6 7 8 9 0.5 Mg/ml 0.125 Mg/ml 0.0312 Mg/ml 0.25Mg/ml 0.0625 Mg/ml 0.0156 Mg/ml

  17. Trouble shooting • If all tubes turbid  • ?started with low antibiotic conc. • ?resistant organism • ?antibiotic not working • If all tubes clear except tube #9  • ? Started with high antibiotic conc.

  18. Results • MIC  the last dilution at which no growth is observed. The more resistant the organism is  the higher the MIC • MBC  the last dilution at which no growth is observed ; And its subculture have no growth on plate.

  19. MIC

  20. MBC

  21. Etest(strip test)

  22. Microscan • Uses standard size microtiter trays • Detection of growth: • Photometrically  24 h incubation • Fluorimetrically  short incubation • Data managed using computer-based algorithms

  23. Phoenix • Broth Microdilution test. • For growth detection it usese: • Redox indicator. • Bacterial turbidity testing.

  24. Vitek • Uses thin plastic card, comprising 30 wells  linked by capillaries • Bacterial suspension will rehydrate reagent in wells. • Growth determined turbidometrically every h  for 15 h. • Can test up to 20 antibiotics

  25. Thank You

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