Transcript Antibiotic sensitivity testing

Pathogenic Microorganisms
Pathogenic Microorganisms



Bacteria
Fungi
Parasites
Culture and Sensitivity
Identification of Bacteria
Bacteria Gram’s Stain
Gram’s +ve
Cocci
Bacilli
Gram’s -ve
Cocci
Rods
Identification of Bacteria
Microscopical Examination:
• Examination of wet mount preparation.
• Examination of stained preparation.
Macroscopical Examination:
• Characters of colonies.
• Hemolysis on blood agar.
• Pigment production.
Identification of Bacteria
Biochemical
• Primary tests.
• Secondary tests.
Tests:
Additional Tests:
• such as seriological tests
by
Standardized Disc-Agar
Diffusion Method
Variables Affecting the
Results
of Sensitivity Testing
1. Medium
Mueller – Hinton Agar
•
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
2. Inoculum Size
• 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.
3. Incubation condition
•
IncubationTemperature:
Should be adjusted at 35oC.
N.B: Several antimicrobial agents may loose their activity at
this temperature.
eg: Chlortetracycline

•
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.
4. Selection of Antimicrobials
Microorganism .
 Spectrum of the antibiotic
 Patient condition.

Materials:
Test microorganism:
S.aureus , E.coli or Pseudomonas.

S

Set of standardized antibiotic discs.

Mueller-Hinton agar plate.

Sterile cotton swab.
E Ps
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.

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.

Procedure:

Apply the antibiotic discs by means of sterile
forceps.
Procedure:

Apply the antibiotic discs by means of sterile
forceps.
G10
Am
F10
S10
SXT

Incubate at 35oC for 16 – 18 hrs.
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.

G10
Am
F10
S10
SXT
Results:
Disc
Antibiotic
Am30 Amikacin
S10 Streptomycin
F10 Fucidine
Zone diameter Susceptibility
(mm)
9
14
25
R
I
S
Zone diameter (mm)
Antimicrobial agent Disk content
(µg)
Resistant
Intermediat Susceptible
e
Amikacin
Streptomycin
Fucidine
30
10
10
≤ 14
≤ 11
≤ 14
15 -16
12 – 14
15 - 21
≥ 17
≥ 15
≥ 22
‫كل عام وأنتم بخير‬
With my Best Wishes,,,
Manal Abu El-Khair