Pre-analytical phase: sample collection rules
Proper blood collection is a critical factor for the success of the entire test. In suspected acute sepsis, it is recommended to perform 2–3 consecutive tests at 30–60 minute intervals.
Key rules:
- Timing: Blood must be collected strictly during the temperature rise (when the patient experiences chills), rather than at the peak of fever.
- Asepsis: Thorough disinfection of the skin over the vein is required to prevent contamination with normal microflora. The skin is first wiped with 70% ethanol, then treated with 5% iodine tincture. Use an exclusively sterile syringe.
- Volume: For an adult patient, at least 10 mL of blood must be obtained; 5 mL is sufficient for children.
Inoculation technique and choice of culture media
Inoculation must be performed urgently: immediately at the bedside or with the fastest possible delivery 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 must be disinfected with 70% alcohol for one minute. All media must be pre-warmed to room temperature.
The most crucial condition is blood dilution. The sample is added to the medium in a ratio from 1:10 to 1:60. This is done intentionally to overcome the powerful natural bactericidal properties of blood (to maximally dilute phagocytes, antibodies, and the complement system), which could inhibit the growth of the pathogen.
To cover the entire spectrum of potential pathogens, a set of different media is used:
- Liquid nutrient media (the basis for classical culture).
- Biphasic (dual) medium. Includes a solid phase (slanted nutrient agar) and a liquid phase (semi-liquid glucose broth). Blood is added to the broth, after which the agar surface is gently washed with it.
- Thioglycollate broth (specific for detecting anaerobes and microaerophiles).
- Sabouraud medium (liquid variant used when fungal sepsis is suspected).
Incubation, evaluation, and interpretation of results
The culture bottles are placed in an incubator at 37 °C and inspected daily. When using a biphasic medium, the laboratory technician simply needs to tilt the bottle to wet the agar with broth: if the 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.
Reporting algorithm:
- In case of a positive result (growth appearance), the microorganism is immediately identified and an antibiogram is performed.
- A preliminary negative response can be issued on days 9–10.
- The definitive negative result is issued only after 4–6 weeks of incubation. Such a long period is necessary to rule out the presence of slow-growing bacteria in the blood.
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 from the external environment during collection. False-negative results are often associated with the presence of the pathogen in the bloodstream that fails to grow due to insufficient dilution and preserved bactericidal properties of the blood. True bacteremia is defined as a situation where absolutely identical strains of microorganisms are isolated when comparing results from different sites of the same patient.
Briefly: answers to topical questions
Which specific types of microorganisms most frequently cause false-positive blood culture results due to skin contamination?
False-positive blood culture results due to skin microflora contamination may be associated with:
- Coagulase-negative staphylococci (predominantly Staphylococcus epidermidis).
- Coryneform bacteria (Corynebacterium spp.).
Which types of slow-growing bacteria require prolonged blood culture incubation of up to 4–6 weeks?
Prolonged incubation of blood cultures (up to 4–6 weeks) is necessary to detect slow-growing microorganisms. Sources indicate the following types of bacteria that require prolonged cultivation:
- Mycobacterium tuberculosis (M. tuberculosis) — cultivation time is 3–4 weeks.
- Brucella (Brucella spp.) — primary culture is incubated for up to 30 days due to slow growth rate.
By what visual signs does the laboratory technician determine bacterial growth in a flask with classical liquid medium?
The laboratory technician determines bacterial growth in a classical liquid nutrient medium based on several visual changes. The main signs include:
- Turbidity of the medium — can be uniform (diffuse) or accompanied by sediment formation.
- Pellicle formation — surface growth, for example, in the form of a grayish-silvery pellicle.
- Appearance of sediment — formation of wall-adherent, bottom, crumbly, or fine-grained sediment.
- Gas production — active or intensive release of gas bubbles.
What criteria are used to prove true bacteremia and distinguish it from contamination?
True bacteremia is distinguished from contamination based on the following criteria:
- Comparison with cultures from other sites in the same patient: isolation of identical strains confirms the diagnosis.
- When isolating coagulase-negative staphylococci or Corynebacterium spp., confirmation by two positive blood cultures is required; repeat blood collection is performed as close in time to the first test as possible.
- A false-positive result may be associated with sample contamination by skin microflora or the culture medium.
Remember the key pre-analytical rule: blood collection is performed during the chills stage, not at the peak of temperature. At the height of fever, bacteria may be intensively destroyed, leading to a false-negative result.
Class and exam questions
Why is blood diluted with nutrient medium in a 1:10 ratio and higher?
This is necessary to overcome the natural bactericidal properties of blood. Dilution sharply reduces the concentration of complement, specific antibodies, and phagocytes, allowing the pathogen to multiply in vitro.
What is the main advantage of a biphasic nutrient medium?
It allows visual detection of colony growth on the slanted agar without the need to open the bottle for routine subculturing. This significantly reduces the risk of accidental sample contamination.
How to prove that the isolated microbe is the cause of sepsis rather than accidental contamination?
The main criterion for truth is comparison with cultures from other sites of the same patient. If an identical strain is isolated, bacteremia is confirmed.
What else to study on this topic
- Mechanisms of natural blood bactericidal activity (role of complement and antibodies)
- Specifics of working with thioglycollate broth in anaerobic infection
- Specifics of fungal cultivation on Sabouraud medium
- Tactics of serial blood collections in acute sepsis
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