Test Catageory
فئة التحليل
Microbiology
Refrence Books
كتب مراجع
1. Bailey & Scott’s Diagnostic Microbiology
2. Laboratory Manual and Workbook in Microbiology
Skills Required
المهارات المطلوبة
1. Specimen collection
2. Inoculation technique
3. Identification of organisms
4. Antibiotic susceptibility testing
5. Interpretation of results
Video Resources
مصادر فيديو
Principle
Urine specimen is inoculated on culture media plates allowing growth of bacteria present. Colonies are then identified by morphology, staining, and biochemical tests. Sensitivity profiling determines efficacy of different antibiotics against the isolate, guiding optimal antibiotic choice.
Solutions & Equipments
1. Sterile Urine collection container
2. Culture plates (blood agar/MacConkey agar)
3. Identification media
4. Antibiotic sensitivity discs
5. Incubator
6. Autoclave
7. Microscope
8. Biochemical tests
Sample Type
Mid-stream clean catch urine
Procedure
Sample Collection
1. Have patient wash hands prior to collection to prevent contamination
2. Instruct patient to spread labia and retract foreskin if uncircumcised while collecting midstream urine
3. Have patient void a small amount of urine first, then collect 10-20mL of midstream urine without stopping the flow of urine
4. Transfer collected urine into sterile, leak-proof container
Inoculation
1. Mix urine specimen gently to homogenize
2. Using a calibrated loop, inoculate 1μL of urine onto 5% sheep blood agar plate in a zig-zag pattern over 1 cm2 area
3. Also inoculate 0.001mL of urine onto MacConkey agar plate
4. Streak inoculum over a third of the plate to obtain isolated colonies
5. Allow plates to dry 3-5 minutes before incubating
Incubation
1. Tape blood agar and MacConkey agar plates together for the same specimen
2. Invert inoculated plates and incubate aerobically at 35-37°C for 18-24 hours
Reading Plates
1. After overnight incubation, remove plates and examine
2. Note presence or absence of bacteria growth on plates
If growth present:
1. Quantify growth in terms of CFU/mL
2. Note colony morphology (size, shape, color, hemolytic pattern if on blood agar)
Identification
1. Choose isolated colonies from both plates representing predominant organism
2. Gram stain isolates to determine Gram reaction
3. Perform biochemical testing such as catalase, coagulase, indole
4. Use results to identify organism using diagnostic algorithms
Antibiotic Sensitivity Testing
1. Prepare inoculum suspension equivalent to 0.5 McFarland standard
2. Dip antibiotic susceptibility discs into suspension to impregnate
3. Place discs onto Mueller Hinton agar plate inoculated with test organism
4. Incubate plate for 18 hours at 35°C
5. Measure zone of inhibition around each antibiotic disc
6. Interpret sensitive/resistant based on established breakpoints
Results
Quantitative and qualitative culture results; isolated organism identification and antibiotic susceptibility profile
Interpretation Presence of pathogenic bacteria at ≥105 CFU/mL considered diagnostic of UTI. Antibiotic sensitivity guides best therapeutic option.
Notes:
1. Use quality control organisms to ensure growth media, biochemical, and antibiotic discs potency
2. Ensure proper aerobic incubation temperatures and duration
3. Accurately measure zones of inhibition and interpret antibiotic sensitivity as per CLSI guidelines
4. Carefully identify pathogenic organisms using morphological, gram staining, and biochemical properties
M. Sulieman
mohammad@mlsgaate.com
The acid fast bacilli (AFB) stain is a differential stain used to detect Mycobacterium species, including Mycobacterium tuberculosis.
