1. Pathogenic Microorganisms

2. Pathogenic Microorganisms
 Bacteria
 Fungi
 Parasites

3. Culture and Sensitivity

4. Identification of Bacteria
Bacteria Gram’s Stain
Gram’s +ve Gram’s -ve
Cocci Bacilli Cocci Rods

5. Microscopical Examination:
•Examination of wet mount preparation.
•Examination of stained preparation.
Identification of Bacteria
Macroscopical Examination:Macroscopical Examination:
• Characters of colonies.Characters of colonies.
• Hemolysis on blood agar.Hemolysis on blood agar.
• Pigment production.Pigment production.

6. Biochemical Tests:
•Primary tests.
•Secondary tests.
Identification of Bacteria
Additional Tests:Additional Tests:
• such as seriological testssuch as seriological tests

7. Standardized Disc-Agar
Diffusion Method
by

8. Variables Affecting the
Results
of Sensitivity Testing

9. • Components of the medium:
Examples:
* PABA antagonizes Sulfonamides
* Ca2+
antagonizes Tetracyclines
• pH of the medium:
It should be adjusted in the range 7.2 – 7.4
Acidity
Activity of tetracycline and methicillin
Activity of Aminoglycosides and Erythromycin
1.Medium
Mueller – Hinton Agar

10. • Should be standardized to be 105
– 106
cfu / ml.
• This can be achieved by visual matching the
turbidity of the broth culture with a 0.5 McFerland
Standard Suspension.
• The apparent sensitivity of the organism is
inversely proportional to the inoculum size.
• A resistant mutant is much more likely to
emerge in large population.
2.Inoculum Size

11. 3.Incubation condition
• IncubationPeriod:
The usual incubation time for sensitivty testing should be
16 -18 hrs
* Sometimes, the microorganism is not killed but only inhibited
upon short exposure to antimicrobial agents
* The longer the incubation period, the greater chance for
resistant mutants to emerge.
• IncubationTemperature:
Should be adjusted at 35o
C.
N.B: Several antimicrobial agents may loose their activity at this
temperature.
eg: Chlortetracycline

12. 4.Selection of
Antimicrobials
Microorganism.
Spectrum of the antibiotic
Patient condition.

13. s
Test microorganism:
S.aureus , E.coli or Pseudomonas.
Mueller-Hinton agar plate.Mueller-Hinton agar plate.
Sterile cotton swab.Sterile cotton swab.
P
s
Set of standardized antibiotic discs.Set of standardized antibiotic discs.

14. Procedure:
Adjust turbidity of the culture to be equal to 0.5
McFerland Standard Suspension.
Inoculate Mueller-Hinton agar plate by streaking
the test organism in three different dimensions.

15. Procedure:
Adjust turbidity of the culture to be equal to 0.5
McFerland Standard Suspension.
Inoculate Mueller-Hinton agar plate by streaking
the test organism in three different dimensions.

16. Procedure:
Apply the antibiotic discs by means of sterile
forceps.

17. Procedure:
Apply the antibiotic discs by means of sterile
forceps.
Incubate at 35o
C for 16 – 18 hrs.
F10
S10
AmG10
SXT

18. Results:
Measure the diameter of each inhibition zone
*The diameter of the inhibition zones are directly
proportional to the susceptibility of the
microorganism to the antibiotics.
F10
S10
AmG10
SXT

19. Results:
Disc Antibiotic Zone diameter
(mm(
Susceptibility
Am30
S10
F10
Amikacin
Streptomycin
Fucidine
9
14
25
R
I
S
Antimicrobial agent Disk content
(µg( Resistant Intermediate Susceptible
Amikacin
Streptomycin
Fucidine
30
10
10
≥14
≥11
≥14
15-16
12–14
15-21
≤17
≤15
≤22
Zone diameter (mm(

20. ‫بخير‬ ‫وأنتم‬ ‫عام‬ ‫كل‬
With my Best Wishes,,,
Manal Abu El-Khair