Pathogenic Microorganisms

Bacteria  Fungi  Parasites

Culture and Sensitivity

Identification of Bacteria Bacteria Gram’s Stain Gram’s +ve CocciBacilli Gram’s -ve CocciRods

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

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

Standardized Disc-Agar Diffusion Method by Kirby-Bauer Method and Clinical Laboratory Standards Institutes (CLSI) Antibiotic Sensitivity Testing

Variables Affecting the Results of Sensitivity Testing

Components of the medium: Examples: * PABA antagonizes Sulfonamides * Ca 2+ 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

Should be standardized to be 10 5 – 10 6 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 be 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 35 o C. Should be adjusted at 35 o C. N.B: Several antimicrobial agents may loose their activity at this temperature. eg: Chlortetracycline eg: Chlortetracycline

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

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

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

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

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

Procedure: Apply the antibiotic discs by means of sterile forceps or disc dispenser Apply the antibiotic discs by means of sterile forceps or disc dispenser Incubate at 35 o C for 16 – 18 hrs. Incubate at 35 o C for 16 – 18 hrs. IP10 GM10 Am CT10 SXT

Results: Measure the diameter of each inhibition zone 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. IP10 GM10 Am CT10 SXT

Results: DiscAntibiotic Zone diameter (mm) Susceptibility Am30S10F10AmikacinStreptomycinFucidine91425RIS Antimicrobial agent Disk content ( µ g) Resistant Intermediat e SusceptibleAmikacinStreptomycinFucidine ≤ 14 ≤ 11 ≤ – ≥ 17 ≥ 15 ≥ 22 Zone diameter (mm)