General characteristics and types of the method
Depending on the specific microorganism being targeted, the culture method is divided into several areas:
- Bacteriological — search for bacteria.
- Virological — search for viruses.
- Mycological — search for fungi.
- Parasitological — search for protozoa.
The main advantage of this approach is its high information yield. By accumulating a sufficient biomass of microorganisms, the laboratory can identify the species of the pathogen with maximum precision. However, the method has disadvantages: it is labor-intensive and requires a long waiting time for results.
Sometimes the test yields a false-negative result. This occurs due to three main reasons: the pathogen physically failed to enter the sample during collection, the microbe died during transportation to the laboratory, or specific non-culturable forms of microorganisms are present in the body.
Bacteriological method: the gold standard
Bacteriological examination is officially recognized as the "gold standard" for laboratory diagnosis of bacterial infections. Its key task is to isolate a pure culture and identify it.
Identification is carried out based on a set of properties:
- Morphological and staining properties (reaction to stains).
- Cultural properties (growth characteristics on media).
- Biochemical, toxigenic, and antigenic properties.
The clinical significance of the method is immense. It allows for an antibiogram (determining drug susceptibility) and testing the reaction of microbes to bacteriophages. Quantitative estimation of bacteria is also essential: it helps prove the etiological role of the microorganism, confirming that it is the cause of the disease rather than a random transient component.
The epidemiological significance of the method lies in epidemiological marker typing — identifying the source of infection. This is done through intraspecies identification, dividing bacteria of the same species into minor variants: phagovars, biovars, serovars, chemovars, and antibioticovars.
Virological method
This is a complex and extremely lengthy process that requires a specialized laboratory. The study takes more than 7 days and consists of three stages.
Stage 1: Inoculation (cultivation). Viruses do not grow on ordinary agar, so sensitive biological models are used: laboratory animals, chicken embryos, or living cell cultures.
Stage 2: Virus indication. Specialists need to detect the replication of the pathogen. When using animals, the clinical picture and post-mortem organ changes are assessed. In cell culture, viral replication is confirmed by the following signs:
- Cytopathic effect (CPE) — cell damage and death.
- Formation of intracellular inclusions.
- Hemadsorption — attachment of erythrocytes to infected cells.
- Plaque formation phenomenon — the appearance of "sterile" spots in a confluent cell monolayer.
- Change in the color of the nutrient medium indicator (the so-called color test).
Stage 3: Virus identification. To determine the exact species, immune reactions (HI, HAI, NT, CFT, ELISA) and molecular genetic methods (primarily PCR) are used.
Mycological and parasitological methods
For the diagnosis of mycoses, the mycological method is used. The test material is inoculated onto special media. Once a pure fungal culture is isolated, it is identified based on three groups of properties: morphological, cultural, and biochemical. An obligatory step is determining the susceptibility of the isolated fungus to antimycotics (antifungal drugs).
The parasitological method is based on cultivating protozoa on nutrient media followed by their identification. It is important to note that in routine clinical practice, this approach is rarely used.
Briefly: answers to topical questions
What specific nutrient media are used to isolate a pure fungal culture?
Special and routine nutrient media are used to isolate a pure fungal culture. Specific media include:
- Sabouraud medium — used as agar or liquid medium.
- Wort agar — used for inoculating clinical material, including in the diagnosis of candidiasis.
- Liquid wort — used for laboratory cultivation of fungi.
- Czapek medium — used for laboratory cultivation of fungi.
What groups are nutrient media classified into for bacteriological culture?
Nutrient media are classified according to their composition and purpose. They are divided into:
- Simple media.
- Complex media.
- Selective media — suppress the growth of concomitant microflora.
- Enrichment media.
- Differential-diagnostic media — designed to study the enzymatic activity of bacteria.
- Combined media — combine the properties of selective and differential-diagnostic media.
- Subculture media.
What methods are used to determine bacterial antibiotic susceptibility when compiling an antibiogram?
To determine bacterial antibiotic susceptibility and compile an antibiogram, the following are used:
- Disk diffusion method — based on the diffusion of an antibiotic from a paper disk into agar with the formation of a zone of bacterial growth inhibition.
- E-test (gradient diffusion) — uses a strip with a gradient of drug concentrations; it is used to determine the minimum inhibitory concentration (MIC).
- Serial dilution method — allows the determination of the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) in liquid media.
- Test systems — used to determine breakpoint concentrations or MICs.
What types of viral cytopathic effect (CPE) can be observed in infected cell culture?
In infected cell culture, the following can be observed:
- Cytopathic effect.
- Formation of intracellular inclusions.
- Hemadsorption — attachment of erythrocytes to infected cells.
- Plaque formation phenomenon — the formation of "sterile" spots in the cell monolayer.
- Symplast formation — particularly in mosquito cells during flavivirus cultivation.
- Change in culture medium indicator color.
To memorize the signs of virus indication in cell culture, use the acronym CVP-GC: Cytopathic effect, Viral inclusions, Plaques, Hemadsorption, Color test.
Class and exam questions
Why might the cultural method yield a false-negative result?
This occurs if the microorganism was absent at the site of biological sample collection, died during transport to the laboratory, or if the pathogen belongs to non-culturable forms of microorganisms.
Why is it necessary to determine the bacterial count during inoculation?
Quantitative evaluation helps understand the true etiological role of the microbe. This allows distinguishing a real infectious agent from bacteria that are merely present in the body.
What is intraspecies identification and what is it needed for?
This is epidemiological marking — the division of bacteria of the same species into smaller variants (serovars, phagovers, biovars). It is critically important for accurately establishing the source of infection during epidemiological investigations.
How does virus cultivation differ from bacterial cultivation?
Unlike bacteria, viruses cannot be grown on artificial nutrient media. Their cultivation strictly requires living biological models: cell cultures, chicken embryos, or laboratory animals.
What else to study on this topic
- Bacterial staining methods and tinctorial properties of microorganisms
- Rules for performing and reading the antibiogram
- Serological reactions for virus identification (IHA, CFT, ELISA)
- Cytopathic effect of viruses: types and mechanisms of development
- Characteristics of nutrient media for fungal cultivation
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