Microbiological Tests

Main approaches to microbiological examination

Microbiological diagnostics rely on the study of various clinical specimens, the selection of which depends on the localization of the infectious process. Feces, sputum, urine, or blood may be sent to the laboratory.

A set of methods is used for precise pathogen identification. The bacteriological method is classic: it begins with inoculating the specimen onto selective nutrient media. Afterward, specialists isolate a pure culture of the microorganism and perform mandatory testing for its toxigenicity.

As modern adjuncts, molecular genetic methods are used, primarily PCR, which allows for the identification of the isolated culture. If it is necessary to detect a bacterial toxin directly in the test sample itself (e.g., in feces), the immunochemical approach is used—enzyme-linked immunosorbent assay (ELISA).

Diagnostic algorithm for Escherichia infections

The logic of detecting Escherichia is strictly structured from the moment of sample collection to final identification. In intestinal infections, the patient's stool is examined. If the infection is parenteral (extraintestinal), the specimens used are urine, blood, wound discharge, or tissue from a specific site of infection.

The inoculation algorithm requires that all collected material (except blood) be seeded onto differential lactose-containing media. Classic examples of such media are Endo agar and Levine's eosin-methylene blue agar. The inoculated Petri dishes are sent for incubation, which lasts 18 hours at 37 °C.

At the stage of selection and identification, laboratory technicians look for colonies capable of giving an agglutination reaction with polyvalent O-sera. Next, biochemical identification of the bacterium down to the specific species is performed. The final and most crucial stage in diagnosing Escherichia infections is the precise determination of the serological variant of the pathogen.

Specifics of mycobacteria detection

The diagnosis of mycobacterioses is largely similar to the algorithms for detecting tuberculosis, yet it has its own specific tasks. The main one is differentiation: it is necessary to clearly determine whether the isolated pure culture belongs to true tuberculosis causative agents or is a nontuberculous mycobacterium.

The primary method here is also bacteriological (culture). The process is divided into two levels:

• Primary identification takes place in basic laboratories of anti-tuberculosis dispensaries. There, the bacterial growth rate, pigment production, colony morphology, and ability to grow at various temperatures are evaluated.
• Final identification is the task of specialized laboratories. They determine the species, virulence, and Runyon group affiliation. To achieve this, complex biochemical tests are performed, and a biological assay is carried out by infecting laboratory animals (chicks, mice, or rats).

For any isolated mycobacterial culture, determining antibiotic susceptibility is a mandatory step. Bacterioscopy and serological reactions to detect antibodies are used as auxiliary tools. Skin allergy tests are of little diagnostic significance in this context.

Emergent infections: diagnosis of Aeromonas spp.

Bacteria of the genus Aeromonas inhabit aquatic ecosystems and cause infections classified as emergent (emerging or re-emerging). In humans, they provoke gastroenteritis, wound infections, and in immunocompromised individuals, severe septicemia.

The pathogenicity of these microorganisms is due to potent toxins and aggressive enzymes. Toxins include cytotoxins (aerolysin and cytolysin), hemolysins, and enterotoxins. Notably, the heat-stable enterotoxin shares antigenic similarity with cholera toxin (a heat-labile variant is also produced). Among aggressive enzymes, heat-stable serine protease and various metalloproteases, including phospholipase and chitinase, are distinguished.

The main diagnostic method for Aeromonas infections is bacteriological. If feces are examined, primary inoculation is performed into a liquid enrichment medium—alkaline peptone water. After 24 hours, the culture is subcultured onto a solid medium—blood agar supplemented with ampicillin. When examining wound discharge, inoculation is performed in parallel on both media.

For identification, colonies surrounded by a zone of hemolysis are selected. Then, tests for the presence of oxidase and catalase enzymes are performed. The process concludes with biochemical identification, the algorithm of which is similar to that for Enterobacteriaceae. Treatment of such infections is based on etiotropic antibiotic therapy.

Class and exam questions

What else to study on this topic

• Pathogenicity factors of Aeromonas spp.: toxins and enzymes
• Classification of mycobacteria by Runyon groups
• Differential lactose-containing media: Endo and Levine
• Biochemical identification of Enterobacteriaceae
• Performing a biological assay on laboratory animals

Other topics in the section "Gram-negative facultatively anaerobic rods (Enterobacteriaceae, Vibrio, Haemophilus, Pasteurella)"

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