Bacteriological urine analysis

Quantitative inoculation technique (Gold's method)

To isolate a pure culture of microorganisms and perform an accurate quantitative bacterial count in the test material, microbiological practice employs sector inoculation, also known as Gold's method. Specialized media are traditionally chosen as the nutrient base for growing bacteria: cystine-lactose-electrolyte-deficient agar (abbreviated as CLED agar) or 5% blood agar.

The main working tool when performing this analysis is a standard bacteriological loop. Its diameter is exactly 2 mm, which allows the laboratory technician to capture a strictly defined volume of liquid — exactly 0.005 mL of urine.

The inoculation procedure is strictly regulated and includes the following consecutive steps:

1. The Petri dish is mentally divided into four separate zones: starting sector A and subsequent sectors designated by Roman numerals I, II, and III.
2. In sector A, primary inoculation of the delivered urine sample is carried out by making thirty to forty dense streaks across the agar surface.
3. After this, the bacteriological loop is thoroughly flamed in a burner flame to completely destroy any microbes remaining on it.
4. From primary sector A, exactly four streaks are made into the adjacent zone I.
5. Similarly, four streaks are made from zone I to zone II.
6. The process of material transfer is completed with four streaks from zone II to the final zone III.

Petri dishes inoculated in this manner are placed in an incubator where the temperature is constantly maintained at 37 °C. The incubation time for the cultures ranges from one to three days (24 to 72 hours).

The role of bacteria of the genus Proteus in urinary tract pathology

Bacteria belonging to the genus Proteus often act as culprits in severe infectious processes of the urinary tract. Normally, these microorganisms are natural representatives of the facultative microflora of the vagina and human large intestine. Furthermore, their detection in wastewater serves as an important sanitary indicator of fecal environmental contamination.

The key virulence factor of these bacteria in urinary system infections is their ability to produce a specific enzyme — urease. The mechanism of pathogenic action and subsequent stone formation is as follows:

• Urease secreted by bacteria actively breaks down urea, which is normally present in urine.
• As a result of this biochemical reaction, a large amount of ammonia is released.
• The accumulation of ammonia leads to a sharp alkalization of the medium, that is, a significant increase in the urine pH level.
• In an alkaline environment, the solubility of magnesium and calcium salts drops sharply.
• The end result of this pathological cascade is salt crystallization and the formation of kidney stones.

It is important to note that after a past infection caused by Proteus, protective immunity is not formed in the patient, which makes repeated episodes of the disease quite possible.

Identification and properties of Proteus

For an accurate diagnosis, the bacteriological method requires careful identification of the pathogen. Primary inoculation is carried out on lactose-containing differential nutrient media. Since bacteria of the genus Proteus exhibit extremely pronounced biochemical activity, they are distinguished from other members of the family Enterobacteriaceae by a number of specific features.

The most important differential properties of Proteus include:

• The ability to produce the enzymes phenylalanine deaminase and urease.
• Active hydrogen sulfide production during metabolic processes.
• Ability to liquefy gelatin.
• Lack of lactose fermentation (this is a key negative sign).

Of particular diagnostic significance is Schukevich's method: inoculation is performed directly into the condensation water accumulating on slanted agar. This allows for the visual detection of the characteristic "swarming" growth inherent specifically to these bacteria.

The antigenic structure of the microorganism is represented by the somatic O-antigen and flagellar H-antigen. The bacteria themselves are characterized by high stability in the external environment: they are able to survive in weak solutions of disinfectants and phenol, and also easily withstand thermal exposure up to 60 °C for a whole hour.

To treat Proteus infections, a specific coli-Proteus bacteriophage or antibiotics are used, although the latter are prescribed strictly after receiving antibiogram results. Specific prophylaxis (vaccination) against these pathogens has not been developed to date.

Bacteriological confirmation in other infections

In addition to urinalysis, bacteriological methods are critical for the diagnosis and treatment monitoring of other infectious conditions. For example, in the development of reactive arthritis following an intestinal infection, etiotropic therapy is prescribed exclusively when laboratory confirmation is available. Such confirmation is provided by a positive stool culture or the detection of specific antibodies to intestinal group pathogens in diagnostic titers. As first-line drugs in such cases, aminoglycoside antibiotics are used (amikacin is administered intravenously, gentamicin is also used). Fluoroquinolones serve as an alternative group, but an important restriction applies in pediatric practice: their use is strictly authorized only for children over 12 years of age.

In the case of respiratory diseases (for example, when evaluating the outcomes of chemotherapy), the result is also formulated based on laboratory data. If a positive microscopy was recorded in the patient before the start of treatment, the outcome is registered as "effective course of chemotherapy confirmed by sputum microscopy." If bacterial growth is present on solid or liquid nutrient media, the wording changes to "confirmed by sputum culture." If both diagnostic tests were initially positive, the medical documentation records an outcome confirmed by both sputum microscopy and culture.

Class and exam questions

What else to study on this topic

• Features of microorganism growth on CLED agar and 5% blood agar.
• Antigenic structure of Enterobacteriaceae: the role of O- and H-antigens.
• Principles of performing an antibiogram for the selection of etiotropic therapy.
• Application of the coli-Proteus bacteriophage in clinical practice.
• Interpretation of sputum culture results on liquid and solid nutrient media.

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