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Antibiotics Basmah almaarik

Learn about different antibiotic agents, sensitivity testing methods, and definitions relevant to antimicrobial therapy in the lab setting. Understand MIC, MBC, troubleshooting, and various testing techniques like Etest and Microscan.

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Antibiotics Basmah almaarik

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  1. Antibiotics Basmahalmaarik Lab # 1

  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. Sir Alexander Fleming(1881 –1955) “One sometimes finds what one is not looking for“Penicillin He observed inhibition of staphylococci on an agar plate contaminated by aPenicilliummold

  4. Classification According to Mode of Action: Stop cell wall synthesis eg. Penicillin DNA Inhibit cell membrane Ribosome Inhibit nucleic acid synthesis Inhibit protein synthesis

  5. 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

  6. 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.

  7. 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

  8. 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

  9. 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

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

  11. How to prepare standard inoculum size: • Standard inoculum should have turbidity equivalent to 0.5 McFarland standard. • Should be from a freshly overnight growth and then compared against Wickerham Card. McFarland standard set

  12. Wickerham Card • plastic laminated card with thick black and white lines to facilitate the preparation of bacterial and yeast suspensions when comparing the turbidity for disk diffusion and other tests that require a specified McFarland turbidity.

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

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

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

  16. 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.

  17. Broth Dilution Method • Prepare a 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.

  18. Broth Dilution Method • Add 1 drop of test organism in each tube of set 1 using a pastuer pipette. 7. Incubate 24 hr x 37C

  19. 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

  20. 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.

  21. 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.

  22. MIC

  23. MBC

  24. Epsilometer test Etest (strip test) a rectangular strip that has been impregnated with antibiotic, used to determine MIC.

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

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

  27. 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

  28. Student work in the lab • Prepare a 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 • Add 1 drop of test organism in each tube of set 1 using a pastuer pipette. • Incubate 24 hr x 37C • Next day: observe MIC and calculate antibiotic concentration.

  29. Thank You

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