Pathogenic Microorganisms
Pathogenic Microorganisms
Bacteria Fungi Parasites
Culture and Sensitivity
Identification of Bacteria
Bacteria Gram’s Stain
Gram’s +ve Gram’s -ve
Cocci Bacilli Cocci Rods
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.
Biochemical Tests:•Primary tests.•Secondary tests.
Identification of Bacteria
Addit ional Tests:Addit ional Tests:• such as seriological testssuch as seriological tests
Standardized Disc-Agar
Diffusion Method
by
Variables Affecting the Results
of Sensitivity Testing
• 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. MediumMueller – Hinton Agar
• 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
3. Incubation condition
• IncubationPeriod:
The usual incubation time for sensitivty testing should be16 -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 35oC.N.B: Several antimicrobial agents may loose their activity at this
temperature. eg: Chlortetracycline
4. Selection of Antimicrobials
Microorganism .Spectrum of the antibioticPatient condition.
Materials:
Test microorganism:
S.aureus , E.coli or Pseudomonas.
Mueller-Hinton agar plate.Mueller-Hinton agar plate.Sterile cotton swab.Sterile cotton swab.
Ps
Set of standardized antibiotic discs.Set of standardized antibiotic discs.
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.
Incubate at 35oC for 16 – 18 hrs.
F10S10
AmG10
SXT
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.
F10S10
AmG10
SXT
Results:Disc Antibiotic Zone diameter
(mm(Susceptibility
Am30S10F10
AmikacinStreptomycin
Fucidine
91425
RIS
Antimicrobial agent Disk content(µg( Resistant Intermediate Susceptible
AmikacinStreptomycin
Fucidine
301010
≥14 ≥11 ≥14
15- 1612 – 1415 - 21
≤17 ≤15 ≤22
Zone diameter (mm(
كل عام وأنتم بخير
With my Best Wishes,,,Manal Abu El-Khair