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