Microbiological examination of urine

Rules for collection and transportation of biomaterial

For routine testing, the patient collects the material independently. The main issue of the pre-analytical phase is that urine microflora multiplies rapidly at room temperature. This leads to an artificially inflated bacterial count and false-positive results. According to regulations, the delivery of the sample to the laboratory and the start of the study must take no more than 1–2 hours.

Self-collection algorithm:

• Hygiene: thoroughly wash hands and the area of the external genitalia.
• Container: use a sterile container (remove the lid carefully, never touching the edges).
• Midstream technique: pass the first portion of urine into the toilet. Then pause urination, and fill the container with the midstream portion up to half its volume. Close it tightly afterwards.

Alternative collection methods are rarely used. Catheterization is not used for routine diagnostics due to the high risk of introducing infection. Suprapubic aspiration is indicated only in specific, clinically justified cases.

Quantitative inoculation technique (Gold's method)

Sector inoculation is used to isolate a pure culture of microorganisms and count bacteria. CLED agar (cystine-lactose-electrolyte-deficient agar) or 5% blood agar is used as a nutrient medium.

A standard 2 mm bacteriological loop is used, which captures exactly 0.005 mL of biomaterial.

Sector inoculation technique:

1. The Petri dish is conventionally divided into 4 sectors: A, I, II, and III.
2. Sector A: primary urine inoculation is performed with a dense streak (30–40 streaks).
3. The loop is flamed in a burner flame.
4. Sector I: 4 streaks are made from sector A into sector I.
5. Sector II: similarly, 4 streaks are made from sector I into sector II.
6. Sector III: 4 streaks are made from sector II into sector III.

After inoculation, the dishes are incubated in an incubator at $37^\circ\text{C}$ for 24 to 72 hours.

Interpretation of colony counts and clinical assessment

The degree of colonization is assessed by counting the grown colonies across sectors, which allows data to be extrapolated to 1 mL of urine. The further the growth extends across the sectors, the higher the degree of bacteriuria:

• Growth only in sector A: low to moderate colonization (from $<1000$ to $50\,000$ microbial bodies per 1 mL).
• Growth in sectors A and I: high colonization (from $100\,000$ to $1$ million).
• Growth in sectors A, I, and II: very high colonization ($5–10$ million).
• Growth in all sectors: critical colonization ($100$ million or more).

The main clinical objective is to differentiate infection from contamination (sample pollution by normal microflora).

Differentiation criteria:

• Contamination: bacterial count less than $10^3$ CFU/mL, an association (polyculture) of several microorganism species is detected.
• Doubtful result: borderline titer of $5 \times 10^3$ CFU/mL. Requires a repeat test.
• Infectious process: diagnostic threshold $\ge 10^4$ CFU/mL. Usually, a monoculture of the pathogen is detected.

After isolating a pure culture, its identification is performed and an antibiogram is set up. Dynamic changes in the degree of bacteriuria (decrease in titer) are used by physicians to monitor the efficacy of prescribed therapy.

Briefly: answers to topical questions

Class and exam questions

What else to study on this topic

• Features of nutrient media: CLED agar and 5% blood agar
• Algorithm for biochemical identification of the isolated pure culture
• Methodology for setting up an antibiogram
• Therapy monitoring and interpretation of changes in the bacteriuria titer

Other topics in the «Clinical Microbiology» section

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