Laboratory Strategy and Media Selection
Because different microorganisms can cause similar clinical presentations, microbiology utilizes the "media battery" principle. This involves the simultaneous inoculation of a single sample onto a panel of selective and differential diagnostic media. This significantly increases the likelihood of successfully detecting and initially identifying the causative agent.
Each medium in the battery serves a specific group of microorganisms:
- Endo agar — used for the isolation of Enterobacteriaceae.
- Mannitol salt agar (MSA) — optimal for staphylococcal growth.
- Cetrimide agar — applied for the selective isolation of Pseudomonas aeruginosa.
- Sabouraud dextrose agar — designed for fungal cultivation.
- Thioglycolate broth — required for the detection of anaerobes.
Inoculation Tactics Based on Biotope
The choice of culture medium directly depends on whether the collection site is normally sterile.
- Sterile sites (blood, cerebrospinal fluid). Normally, these fluids contain no microorganisms. Any detected microbe is potentially pathogenic. Therefore, specimens are inoculated onto enriched culture media to provide optimal conditions for the growth of the widest possible spectrum of microorganisms.
- Non-sterile sites (biotopes with normal microbiota). These samples always contain resident flora. To prevent them from overgrowing and masking the target pathogen, selective (enrichment) media are used. Their primary function is to suppress normal flora while permitting the specific pathogen to multiply.
Quantitative Analysis in Suppurative Infections
When diagnosing infections caused by opportunistic pathogens, a qualitative result (a simple "yes/no" answer) is insufficient. Quantitative determination (assessment of microbial load) is mandatory. This is necessary to differentiate the true etiologic role of the microbe from mere colonization or carriage.
To determine concentration, investigators use serial dilutions or Gold's streak plating (sectoral inoculation). Results are interpreted based on the following criteria:
- High concentration: Isolation of one or two microbial species in high colony-forming units (CFU). This directly points to their role as the primary etiologic factor.
- Low concentration: Detection of a polymicrobial association in low numbers. As a rule, this indicates contamination of the sample by the patient's normal microbiota or environmental flora.
Cultivation and Antimicrobial Susceptibility
In the second stage of investigation, each portion of the specimen is preliminarily inoculated into an enrichment broth (e.g., selenite broth at a 1:10 ratio), with parallel inoculations onto solid and liquid media (blood agar, Endo agar, thioglycolate broth).
Incubation conditions are standard: cultures are placed in an incubator at 37 °C. If anaerobic infection is suspected, cultivation is performed in specialized devices called anaerobic jars.
Specific protocols exist, such as for the isolation of Salmonella species. In this case, material from the enrichment broth and native bile is streaked onto bismuth sulfite agar over the following 3 days.
After counting colonies and identifying the species, antimicrobial susceptibility testing is performed. An important rule: the test is conducted only for etiologically significant pathogens. If a microbial association (multiple significant pathogens) is identified, susceptibility must be determined for each member of the association individually.