Standard Operating Procedure for Urine Microscopy, Culture, and Sensitivity (M/C/S)

Urine Microscopy, Culture, and Sensitivity are among the most common tests performed in medical microbiology laboratories worldwide.

Patient Preparation:

1. • Instruct the patient on proper collection techniques, preferably a midstream, clean-catch urine sample.
   • Provide a sterile urine container.
   • Ensure the patient washes their hands and cleans the genital area before collecting the sample.
   • Label the container with the patient’s details and date/time of collection.

Transport:

• • Transport the specimen to the laboratory within 2 hours of collection. If delayed, refrigerate at 2-8°C for up to 24 hours.

Urine Microscopy

1. Sample Preparation:
   • Mix the urine sample gently and pour approximately 10 mL into a sterile centrifuge tube.
   • Centrifuge the sample at 1500-2000 RPM for 5 minutes.
   • Discard the supernatant, leaving a small amount of urine to resuspend the sediment.
2. Microscopy Examination:
   • Place a drop of resuspended sediment on a clean glass slide, cover with a coverslip, and examine under a light microscope at 10x and 40x magnification.
   • Examine for:
     • Cells: Red blood cells (RBCs), white blood cells (WBCs), epithelial cells.
     • Casts: Hyaline, granular, cellular casts.
     • Crystals: Uric acid, calcium oxalate, etc.
     • Bacteria/Yeasts/ova: Look for the presence of bacteria, fungi, yeasts or ova of parasites.
3. Reporting:
   • Report the number of cells, casts, and crystals seen per high-power field (HPF).
   • Report the presence of bacteria or fungi (if visible).

Culture

1. Inoculation:
   • Mix the urine sample gently, then use a calibrated loop (0.001 mL) to inoculate:
     • Cystine Lactose Electrolyte Deficient (CLED) Agar: For general urinary pathogens.
     • MacConkey Agar: For identifying lactose and non-lactose fermenting organisms.
     • Blood Agar: For detecting hemolytic bacteria.
2. Incubation:
   • Incubate the plates at 35-37°C for 18-24 hours under aerobic conditions.

Sensitivity Testing

1. Isolate Identification:
   • After incubation, examine the plates for colony morphology and growth patterns.
   • Count the number of colonies:
     • <10,000 CFU/mL: No significant growth.
     • 10,000-100,000 CFU/mL: Intermediate or moderate growth.
     • >100,000 CFU/mL: Significant growth indicating infection.
   • Identify the organisms through biochemical tests or an automated system (e.g., VITEK).
2. Antibiotic Sensitivity:
   • Select a pure colony of the identified organism.
   • Perform antibiotic susceptibility testing using the  disc diffusion method on nutrient Agar.
   • Incubate at 35-37°C for 18-24 hours.
3. Measure Zones of Inhibition:
   • Measure the diameter of inhibition zones around antibiotic discs and interpret results as Susceptible/ Sensitive (S), Intermediate (I), or Resistant (R).

Reporting Results

1. Microscopy:
   • Report findings of WBCs, RBCs, epithelial cells, bacteria, and crystals (e.g., 5-10 WBCs per HPF, few bacteria).
2. Culture:
   • Report the identified organism, colony count (CFU/mL), and whether the growth is significant.
   • If no significant growth, report as “No significant growth.”
3. Sensitivity:
   • Report the antibiotic susceptibility pattern, indicating the appropriate antibiotics for treatment based on the results.