Pathogenic Pathogenic MicroorganismsMicroorganisms

Pathogenic MicroorganismsPathogenic Microorganisms

Bacteria Fungi Parasites

CultureCulture and and SensitivitySensitivity

Identification of BacteriaIdentification of Bacteria

Bacteria Gram’s Stain

Gram’s +ve Gram’s -ve

Cocci Bacilli Cocci Rods

Microscopical Examination:Microscopical Examination:• Examination of wet mount preparation.Examination of wet mount preparation.• Examination of stained preparation.Examination of stained preparation.

Identification of BacteriaIdentification of Bacteria

Macroscopical Macroscopical Examination:Examination:

• Characters of colonies.Characters of colonies.• Hemolysis on blood agar.Hemolysis on blood agar.• Pigment production.Pigment production.

Biochemical Tests:Biochemical Tests:• Primary tests.Primary tests.• Secondary tests.Secondary tests.

Identification of BacteriaIdentification of Bacteria

Additional Tests:Additional Tests:• such as seriological testssuch as seriological tests

Standardized Disc-Standardized Disc-AgarAgar

Diffusion MethodDiffusion 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. Medium1. 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 Size2. Inoculum Size

3. Incubation condition3. 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 Should be adjusted at 3535ooCC..N.B: N.B: Several antimicrobial agents may loose their Several antimicrobial agents may loose their

activity at this temperature.activity at this temperature. eg: Chlortetracyclineeg: Chlortetracycline

4. Selection of 4. Selection of AntimicrobialsAntimicrobials

Microorganism .Microorganism .Spectrum of the antibioticSpectrum of the antibioticPatient condition.Patient condition.

Materials:Materials:

Test microorganism:Test microorganism:

S.aureus , E.coli S.aureus , E.coli oror Pseudomonas. Pseudomonas.

Mueller-Hinton agar Mueller-Hinton agar plate.plate.Sterile cotton swab.Sterile cotton swab.

S E Ps

Set of standardized antibiotic Set of standardized antibiotic discs.discs.

Procedure:Procedure: Adjust turbidity of the culture to be equal to Adjust turbidity of the culture to be equal to

0.5 McFerland Standard Suspension.0.5 McFerland Standard Suspension. Inoculate Mueller-Hinton agar plate by Inoculate Mueller-Hinton agar plate by

streaking the test organism in three different streaking the test organism in three different dimensions.dimensions.

Procedure:Procedure: Adjust turbidity of the culture to be equal to Adjust turbidity of the culture to be equal to

0.5 McFerland Standard Suspension.0.5 McFerland Standard Suspension. Inoculate Mueller-Hinton agar plate by Inoculate Mueller-Hinton agar plate by

streaking the test organism in three different streaking the test organism in three different dimensions.dimensions.

Procedure:Procedure: Apply the antibiotic discs by means of Apply the antibiotic discs by means of

sterile forceps.sterile forceps.

Procedure:Procedure: Apply the antibiotic discs by means of Apply the antibiotic discs by means of

sterile forceps.sterile forceps.

Incubate at 35Incubate at 35ooC for 16 – 18 hrs.C for 16 – 18 hrs.

F10S10

AmG10

SXT

Results:Results: Measure the diameter of each inhibition zoneMeasure the diameter of each inhibition zone

* The diameter of the inhibition zones are * The diameter of the inhibition zones are directly proportional to the susceptibility of directly proportional to the susceptibility of the microorganism to the antibiotics.the microorganism to the antibiotics.

F10S10

AmG10

SXT

Results:Results:DiscDisc AntibioticAntibiotic Zone Zone

diameter diameter (mm)(mm)

SusceptibilitSusceptibilityy

Am30Am30

S10S10

F10F10

AmikacinAmikacin

StreptomyciStreptomycinn

FucidineFucidine

99

1414

2525

RR

II

SSAntimicrobial Antimicrobial agentagent

Disk Disk contentcontent

(µg)(µg)ResistantResistant IntermediaIntermedia

teteSusceptiblSusceptiblee

AmikacinAmikacin

StreptomyciStreptomycinn

FucidineFucidine

3030

1010

1010

≤ ≤ 1414

≤ ≤ 1111

≤ ≤ 1414

15 -1615 -16

12 – 1412 – 14

15 - 2115 - 21

≥ ≥ 1717

≥ ≥ 1515

≥ ≥ 2222

Zone diameter (mm)

بخير وأنتم عام بخير كل وأنتم عام كل

With my Best Wishes,,,Manal Abu El-Khair