Sample Collection and Transport Protocols
The reliability of a CSF microbiological study directly depends on the quality of the preanalytical phase. A physician collects the material via lumbar puncture or puncture of the lateral cerebral ventricles. Strict aseptic technique is required to prevent contamination. The collected 1–2 mL of CSF is placed in a strictly sterile tube.
A critical factor for successful diagnostics is proper transportation. The primary hazard at this stage is sample cooling. Several pathogens, particularly Neisseria meningitidis (meningococcus), are highly sensitive to low temperatures and rapidly perish when the tube cools.
Consequently, the sample must be delivered to the laboratory within 15–20 minutes while the CSF is still warm. Special equipment is used for transport, such as thermos flasks, heating pads, or isothermal boxes capable of maintaining a temperature of 37 °C. If immediate delivery is impossible for any reason, the sample must be stored in an incubator at 37 °C (storage for several hours is acceptable).
Laboratory Processing and Direct Microscopy
The first phase of laboratory analysis is preparing a smear for microscopic examination.
The standard preparation algorithm includes mandatory concentration of cells and microorganisms. To achieve this, the CSF tube is centrifuged for 5 minutes at 3500 rpm. After centrifugation, the supernatant is discarded, and a smear is prepared from the resulting sediment.
However, an important exception applies: if the received cerebrospinal fluid is visually turbid or contains a characteristic pellicle, the smear must be prepared immediately without prior centrifugation.
Microscopy has high diagnostic value, particularly in purulent meningitis. This method rapidly detects tuberculosis pathogens, meningococci, and opportunistic flora, providing the clinician with crucial information even before complete bacteriological analysis is finished.
Culture Examination (CSF Inoculation)
To isolate a pure culture of the pathogen, inoculation is performed on enriched nutrient media. The standard set includes 5% blood agar, serum agar, and thioglycollate broth. The inoculation technique involves applying 2–4 drops of native CSF or resuspended sediment onto the medium surface and spreading it thoroughly with a loop or spreader.
Inoculated plates are incubated at 37 °C using different gas atmospheres to cover the maximum spectrum of potential pathogens:
- Plate 1: Incubated under normal atmospheric conditions.
- Plate 2: Incubated in an enhanced carbon dioxide ($CO_2$) concentration.
Using carbon dioxide is strictly justified: $CO_2$ is a potent growth stimulant for microorganisms such as pathogenic neisseriae, pneumococci, and listeriae.
Additional media are used when specific infections are suspected. For instance, if the clinical picture suggests Haemophilus influenzae infection, inoculation is performed on chocolate agar. Once visible colonies appear, microbiologists proceed to identify the isolated microorganism.
Algorithm Completion and Result Interpretation
The final and most crucial clinical step of the analysis is determining the antibiotic susceptibility of the isolated microbial culture. If the initial culture yields no growth (negative result), the protocol requires a repeat culture after 8–10 days.
Result interpretation relies on the fact that normal cerebrospinal fluid is sterile. Therefore, the isolation of any microorganism under proper collection conditions is considered pathological, and the microbe is recognized as the etiologic agent of the disease.
A specific approach is required for secondary meningitis. If meningeal inflammation develops as a complication of existing purulent processes (such as otitis, sinusitis, phlegmon, or abscess), the clinician must ensure a microbiological examination of the primary infection site. The primary goal is to detect and match identical pathogen strains in the primary purulent focus and the CSF.