Laboratory strategy and medium selection
Since different microorganisms can present with a similar clinical picture, microbiology employs the "medium battery" principle. This involves the simultaneous use of a whole set of selective and differential diagnostic media for inoculating a single sample. This significantly increases the chances of successful detection and primary identification of the causative agent.
Each medium in the "battery" is designed for a specific group of microorganisms:
- Endo agar — used for the isolation of Enterobacteriaceae.
- Egg-yolk salt agar — optimal for the growth of staphylococci.
- CPC agar (with cetylpyridinium chloride) — used for the selective isolation of Pseudomonas aeruginosa.
- Sabouraud agar — designed for the cultivation of fungi.
- Thioglycolate broth — necessary for detecting anaerobes.
Inoculation tactics depending on the biotope
The choice of culture medium directly depends on whether the sampling site is normally sterile.
- Sterile loci (blood, cerebrospinal fluid). Normally, these fluids contain no microorganisms. Any detected microbe is a potential pathogen. Therefore, the material is inoculated onto enriched culture media to provide the most favorable growth conditions for the widest possible range of microorganisms.
- Non-sterile loci (biotopes with normal microflora). Such samples always contain concomitant flora. To prevent it from overgrowing the target pathogen, selective (elective) media are used. Their main task is to suppress the growth of normal flora and allow the specific pathogen to multiply.
Quantitative analysis in purulent-inflammatory infections
When diagnosing infections caused by opportunistic microorganisms, qualitative detection (a "yes/no" answer) is insufficient. Quantitative determination (assessment of bacterial load) is mandatory. This is necessary to differentiate the true etiological role of the microbe from simple carriage.
To determine concentration, the serial dilution method or Gold's streak inoculation (sectoral plating) is used. The interpretation of results is based on the following criteria:
- High concentration: isolation of one or two species of microorganisms in a large quantity (CFU). This directly indicates their role as the primary etiological factor.
- Low concentration: detection of an association of multiple species in a small quantity. As a rule, this indicates sample contamination with the patient's normal microflora or environmental flora.
Cultivation and antibiotic susceptibility
At the second stage of the study, each portion of the material is pre-inoculated into an enrichment medium (e.g., selenite broth at a 1:10 ratio), and parallel inoculations are made 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 an anaerobic infection is suspected, cultivation is carried out in special devices called anaerostats.
There are specific protocols, for example, for the isolation of salmonellae. In this case, material from the enrichment medium and native bile is plated onto bismuth sulfite agar for the next 3 days.
After counting colonies and determining the species, antibiotic susceptibility is assessed. Important rule: the analysis is performed only for etiologically significant pathogens. If a microbial association (several significant pathogens) is detected, drug susceptibility is determined for each member of this association separately.
Briefly: answers to topical questions
What specific enriched culture media are used in the laboratory for blood cultures for sterility?
Liquid and biphasic enriched culture media are used for blood cultures for sterility. Laboratory practice utilizes:
- Bile broth — used for the isolation of typhoid pathogens.
- Sugar broth — used as an enrichment medium.
- Biphasic medium — includes sloped nutrient agar and semi-solid glucose broth.
- Thioglycolate broth — designed for detecting anaerobes and microaerophiles.
- Sabouraud agar — used for detecting fungi.
How is Gold's sectoral streak inoculation performed technically?
Sectoral inoculation according to Gold's method is performed using a standard 2 mm bacteriological loop. The technique includes the following sequential steps:
- The Petri dish is conventionally divided into four sectors (A, I, II, III).
- Primary inoculation of the material is performed in sector A by making 30–40 streaks.
- The bacteriological loop must be flamed.
- Four streaks are made from sector A into sector I.
- Four streaks are made from sector I into sector II.
- Four streaks are made from sector II into sector III.
Incubation of the cultures is carried out in an incubator at 37 °C for 24–72 hours.
What enrichment media, besides selenite broth, are used for the preliminary inoculation of clinical material?
For preliminary inoculation and enrichment of clinical material, besides selenite broth, the following are used:
- Sugar broth — an enrichment medium, particularly for streptococci.
- Salt broth — an accumulation medium for staphylococci.
- Alkaline peptone water — an enrichment medium when inoculating feces and wound discharge to isolate cholera vibrios and Aeromonas spp.
To memorize the profile of media, use associations based on initial letters or meaning: Endo — Enterobacteriaceae (matching syllables), Egg-yolk salt — Staphylococcus aureus (yellow color of colonies).
Class and exam questions
Why are multiple nutrient media used simultaneously in cultures?
Because different pathogens can cause diseases with similar clinical presentations, the "battery of media" principle is applied. Using several differential-diagnostic and selective media simultaneously ensures the target pathogen is not missed.
Why determine the number of microorganisms in purulent infections?
Purulent-inflammatory infections are often caused by opportunistic flora. Quantitative analysis is necessary to distinguish the true pathogen (present in high concentration) from normal carriage or sample contamination.
What media are used for blood and CSF inoculation?
Blood and cerebrospinal fluid are normally sterile loci. Enriched culture media are used for them to ensure the unimpeded growth of any microorganisms present in the sample.
How is antibiotic susceptibility determined in mixed infections?
If an association of several pathogens is detected in the material, drug susceptibility must be determined individually for each associate, provided they are etiologically significant.
What else to study on this topic
- Mechanisms of normal flora suppression in selective media
- Technique of Gold's sectoral streak inoculation
- Algorithm for setting up the serial dilution method
- Design and operating principles of the anaerostat
- Growth characteristics of salmonellae on bismuth sulfite agar
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