Preanalytical Phase: Specimen Collection Guidelines
Proper blood collection is a critical success factor for the entire procedure. For suspected acute sepsis, it is recommended to perform 2–3 consecutive blood cultures at 30–60 minute intervals.
Key Rules:
- Timing: Blood must be drawn strictly during the temperature rise (when the patient experiences chills), rather than at the peak of fever.
- Asepsis: Thorough skin disinfection over the venipuncture site is required to prevent contamination with normal skin flora. The skin is wiped first with 70% isopropyl alcohol, then treated with a 5% iodine tincture (or chlorhexidine). Use a sterile syringe exclusively.
- Volume: At least 10 mL of blood is required for an adult patient, while 5 mL is sufficient for children.
Inoculation Technique and Selection of Culture Media
Inoculation must be performed promptly: immediately at the bedside or with the fastest possible transport of the syringe to the laboratory. Before piercing the rubber stopper of the bottle, the needle must be replaced with a new sterile one, and the bottle cap itself disinfected with 70% alcohol for one minute. All media must be pre-warmed to room temperature.
A crucial requirement is blood dilution. The sample is added to the medium at a ratio of 1:10 to 1:60. This is done intentionally to overcome the potent natural bactericidal properties of blood (to maximally dilute phagocytes, antibodies, and the complement system), which could otherwise inhibit the growth of the pathogen.
To cover the full spectrum of potential pathogens, a set of different media is used:
- Liquid nutrient media (the basis for classical cultivation).
- Diphasic (dual) medium. Includes a solid phase (slanted nutrient agar) and a liquid phase (semi-solid glucose broth). Blood is inoculated into the broth, after which the agar surface is gently washed with it.
- Thioglycollate broth (specific for detecting anaerobes and microaerophiles).
- Sabouraud medium (liquid formulation used when fungal sepsis is suspected).
Incubation, Reading, and Interpretation of Results
Culture bottles are placed in an incubator at 37 °C and inspected daily. When using a diphasic medium, the laboratory technician simply tilts the bottle to wet the agar with broth: if a pathogen is present, colonies become visible on the slanted agar. This eliminates the need to open the container for subculturing and significantly reduces the risk of accidental airborne contamination.
Result Reporting Algorithm:
- In the case of a positive result (growth detected), pathogen identification and antimicrobial susceptibility testing (antibioticogram) are performed immediately.
- A preliminary negative report can be issued on days 9–10.
- A definitive negative result is issued only after 4–6 weeks of incubation. This prolonged duration is necessary to rule out slow-growing bacteria in the bloodstream.
Reliability Assessment: Any growth is regarded as pathology (bacteremia). However, the risk of errors always exists. False-positive results occur due to contamination with the patient's skin flora or environmental microbes during collection. False-negative results are often associated with the pathogen being present in the bloodstream but failing to grow due to insufficient dilution and preserved blood bactericidal activity. True bacteremia is established when identical microbial strains are isolated from multiple separate blood draws.