Standard Operating Procedure: Urine Culture

1. Purpose and Scope

To perform quantitative culture of urine specimens to detect and enumerate bacterial pathogens causing urinary tract infections (UTI).

2. Specimen Requirements

• Type: Mid-stream clean-catch urine, catheterized specimen, or suprapubic aspirate.
• Volume: Minimum 5-10 mL.
• Storage/Transport: Process within 2 hours of collection or refrigerate at 2-8°C for up to 24 hours.
• Rejection Criteria: Improperly labeled, contaminated, or delayed (>24 hours refrigerated) specimens.

3. Safety and Precautions

• Wear appropriate PPE (lab coat, gloves).
• Handle all specimens as potentially infectious.
• Work in a biological safety cabinet if available.

4. Equipment and Reagents

• Calibrated inoculating loop (1 µL or 10 µL) or calibrated pipette.
• Culture media: Blood agar, MacConkey agar, CLED (Cystine Lactose Electrolyte Deficient) agar.
• Incubator (35-37°C), Bunsen burner or incinerator.
• Colony counter, microscope.

5. Step-by-Step Procedure

1. Specimen Preparation: Mix urine specimen gently by inversion.
2. Inoculation:

• Using a calibrated 1 µL loop: Touch loop to well-mixed urine and streak onto blood agar and MacConkey/CLED agar.
• Using a calibrated 10 µL loop: Use for specimens with suspected low colony counts.
• Alternatively, use calibrated pipette to deliver exact volume onto agar surface.

1. Streaking Pattern: Use quadrant streak method to isolate individual colonies.
2. Incubation: Incubate plates at 35-37°C for 18-24 hours in ambient air (or 5% CO₂ if required).
3. Colony Count: After incubation, count colonies on the plate.

• Calculate CFU/mL: Number of colonies × dilution factor (1000 for 1 µL loop; 100 for 10 µL loop).

1. Interpretation:

• ≥10⁵ CFU/mL: Significant bacteriuria (UTI likely).
• 10⁴-10 CFU/mL: Equivocal (repeat culture if symptomatic).
• <10⁴ CFU/mL: Usually contamination (unless symptomatic or catheterized specimen).

1. Identification: Identify organisms based on colony morphology, Gram stain, and biochemical tests.
2. Antimicrobial Susceptibility Testing: Perform on significant isolates.

6. Quality Control

• Verify calibrated loop volume monthly by weighing water delivered.
• Check media sterility and performance with known organisms.
• Include QC strains (E. coli ATCC 25922) for culture and AST.

7. Reporting Results

• Report organism(s) identified and colony count in CFU/mL.
• Report mixed growth if ≥2 organisms present with no predominant organism.
• Include susceptibility results for significant isolates.