1. Antibiotics Basmah almaarik
Lab # 1

2. Antimicrobial 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:
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 a Penicillium mold

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 Used Standard Organism (Quality Control organism)
Staphylococcus aureus ATCC
Pseudomonas aeruginosa ATCC
Escherichia coli ATCC
Enterococcus faecalis ATCC
Klepsiella pneumoniae ATCC
Streptococcus pneumoniae ATCC
Haemophilus influenzae ATCC

10. 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

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

12. 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

13. 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.

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

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

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

17. 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.

18. 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.

19. 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

20. Results: MIC  last tube showing no growth.
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
Mg/ml
0.25Mg/ml
Mg/ml
Mg/ml

21. 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.

22. The more resistant the organism is  the higher the MIC
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.

23. MIC

24. MBC

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

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

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

28. 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

29. 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.

30. Thank You