Sample Sources

Blood and bone marrow are normally sterile sites. The presence of bacteria in these locations indicates a significant, often life-threatening infection (bacteremia, sepsis, or bone infection)

Effective collection and handling are critical for accurate diagnosis and patient management

Blood

Peripheral Blood

Indications
- Suspicion of bacteremia or sepsis (e.g., fever, chills, hypotension, altered mental status)
- Fever of unknown origin (FUO)
- Endocarditis (infection of the heart valves)
- Osteomyelitis (bone infection)
- Follow-up of patients on antibiotic therapy
- Suspected disseminated infections
Collection Technique
- Venipuncture: The most common method
- Site Preparation: Meticulous aseptic technique is essential to avoid contamination:
  - Clean the venipuncture site (usually an arm vein) with 70% isopropyl alcohol
  - Allow the alcohol to dry completely
  - Clean the site with Chloraprep or povidone-iodine
  - Apply Chloraprep or povidone-iodine in a concentric circle, starting from the center and moving outward
  - Allow the antiseptic to dry for the appropriate contact time (usually 30-60 seconds)
- Blood Volume: The volume of blood collected is CRUCIAL. This is a key factor in sensitivity:
  - Adults: Typically, 2-3 sets of blood cultures are drawn, each set consisting of 8-10 mL of blood: per bottle (aerobic and anaerobic)
  - Children: Volumes vary depending on age and size (e.g., 1-5 mL per bottle). Pediatric bottles are often used
  - Infants: Smaller volumes (e.g., 0.5-1 mL per bottle), often collected using a needle and syringe
- Timing
  - Acute Sepsis: Draw blood cultures immediately before antibiotic administration
  - Intermittent Bacteremia: Collect blood cultures at different times (e.g., two or three sets at intervals of 30-60 minutes)
  - Endocarditis: Collect 3-5 sets over 1-2 hours before antibiotics
- Number of Sets: Generally, 2-3 sets are recommended in adults. A “set” typically means one aerobic and one anaerobic bottle (from a single venipuncture site). This helps improve the detection of both facultative and anaerobic organisms
- Order of Draw: Generally, collect blood cultures before other blood tests to avoid contamination from other tubes and additives
- Collection from Central Lines: If a central line is suspected to be the source of infection, blood can be drawn from the central line and a peripheral site. The order of draw is critical to determine the origin of the infection and differentiate between contamination and true infection
Blood Culture Bottles
- Types
  - Aerobic Bottles: Contain air, promoting the growth of aerobic and facultative anaerobic organisms
  - Anaerobic Bottles: Contain an oxygen-free environment, allowing the growth of anaerobic organisms
  - Pediatric Bottles: Smaller volumes are used
  - Specialty Bottles: Some systems have bottles designed for specific organisms (e.g., fungal bottles, mycobacterial bottles)
- Culture Media: Blood culture bottles contain a broth medium that supports bacterial growth. They also typically contain an anticoagulant (e.g., sodium polyanethol sulfonate (SPS)) to prevent clotting, and may also contain antimicrobial agents to neutralize any antibiotics in the patient’s blood
- Bottle Inoculation: Blood is injected directly into the bottles. Follow the manufacturer’s instructions regarding the amount of blood and proper inoculation technique
- Aerobic vs. Anaerobic: Inoculate aerobic bottles first. This minimizes the amount of air introduced into the anaerobic bottle
Transportation and Storage
- Prompt Transport: Blood culture bottles should be transported to the laboratory as quickly as possible. Delays can affect the recovery of bacteria
- Room Temperature: Store blood culture bottles at room temperature (20-25°C) until they can be processed. Do not refrigerate unless specified by the lab’s protocol
- Incubation: Blood culture bottles are incubated in automated blood culture systems that continuously monitor for bacterial growth

Intravenous Catheters (Central Lines, Peripheral IVs)

Indications
- Suspicion of catheter-related bloodstream infection (CRBSI). This is a significant concern in hospitalized patients
- Fever in a patient with a central venous catheter (CVC) or peripheral IV
- Signs of local infection at the catheter insertion site (e.g., redness, swelling, purulent drainage)
- Persistent bacteremia despite appropriate antibiotic therapy
Collection Techniques: There are two main methods:
- Catheter Blood Culture vs. Peripheral Blood Culture
  - Simultaneous Collection: Blood is drawn simultaneously from the catheter and a peripheral vein
  - Quantitative Analysis: Often, laboratories will do a quantitative blood culture (e.g., using a filtration method). This can help to differentiate between a catheter-related infection and bacteremia from another source
  - Differential Time to Positivity (DTP): If the catheter blood culture becomes positive significantly sooner than the peripheral blood culture (e.g., by 2 hours or more), this strongly suggests a catheter-related infection
  - Blood Culture Ratios (Bacterial Load): The ratio of colony counts from the catheter to peripheral blood can aid in determining the source of the infection. A ratio of 3:1 or greater (catheter to peripheral) is suggestive of catheter-related infection
- Catheter Removal and Tip Culture (If the catheter is being removed)
  - Tip Culture: The catheter tip is aseptically cut and sent to the laboratory for culture. This can help identify the causative organism of a catheter-related infection. The lab will roll the catheter tip on a culture plate and incubate to observe for growth
  - Semi-Quantitative Method (Modified Maki Method): This involves rolling the catheter tip over an agar plate
  - Quantitative Method: The catheter tip is homogenized, and a quantitative culture is performed
Important Considerations
- Aseptic Technique: Strict aseptic technique is critical. The same site preparation guidelines as for peripheral blood cultures apply
- Flush: After drawing blood from the catheter, flush the catheter with sterile saline to remove any blood clots
- Documentation: Clearly document the source of the blood culture (e.g., right femoral catheter, peripheral IV)
- Clinical Correlation: Results must be interpreted in conjunction with the patient’s clinical condition

Bone Marrow

Indications
- Diagnosis of bacteremia or sepsis, particularly in patients who are neutropenic or immunocompromised
- Diagnosis of specific bone marrow infections (e.g., Mycobacterium, Brucella, Salmonella)
- Evaluation of fever of unknown origin (FUO)
- Diagnosis of endocarditis when blood cultures are negative
- Detection of disseminated fungal infections
Collection Technique
- Physician Procedure: Bone marrow aspiration and biopsy are typically performed by a physician (hematologist, oncologist, or infectious disease specialist)
- Site: The iliac crest (posterior or anterior) is the most common site, but the sternum may also be used
- Aseptic Technique: Meticulous aseptic technique is crucial to prevent contamination
- Sample Volume: A small amount of bone marrow aspirate (usually 1-2 mL) is collected
- Anticoagulant: The specimen should be collected in a tube containing an anticoagulant (e.g., heparin)
- Additional Samples: A bone marrow biopsy is often performed in addition to the aspirate to obtain a solid tissue sample
- Processing: The aspirate is sent to the microbiology lab and the hematology lab
- Transport: Transport to the laboratory as quickly as possible at room temperature
Laboratory Processing
- Gram Stain: A Gram stain is performed on the bone marrow aspirate to visualize bacteria
- Culture: The aspirate is inoculated onto appropriate culture media, including blood agar, chocolate agar, and broth
- Specific Media: If specific pathogens are suspected (e.g., mycobacteria, fungi), special culture media are used
- Incubation: Cultures are incubated at 35-37°C for up to 14 days or longer if slow-growing organisms are suspected
- Microscopic Examination: Cultures are examined daily for growth
- Identification: Any bacterial isolates are identified using standard methods
- Susceptibility Testing: Antimicrobial susceptibility testing is performed to determine the antibiotic sensitivities of bacterial isolates
- Other Tests: Additional tests such as fungal stains, acid-fast stains, and molecular tests can be performed as needed

Potential Contaminants and Considerations

Skin Flora: The skin is a major source of potential contaminants
Common Contaminants
- Coagulase-negative staphylococci (CoNS) - Staphylococcus epidermidis is the most common
- Bacillus species
- Diphtheroids (Corynebacterium spp.)
- Propionibacterium acnes (from skin)
Interpreting Results
- Colony Counts: Significant growth (i.e., growth of a pathogen) usually indicates a true infection, while isolation of a small number of contaminants may be clinically insignificant
- Multiple Sets: The isolation of the same organism from multiple blood culture sets increases the likelihood of a true infection
- Clinical Correlation: ALWAYS correlate the laboratory findings with the patient’s clinical presentation and other laboratory results
- Contaminant vs. Pathogen: The lab needs to differentiate the “true” pathogen from a contaminant. If a common skin contaminant grows in one blood culture, this might be contamination. If the same organism grows in multiple blood culture sets, then it is more likely a true infection

Key Terms

Bacteremia: The presence of viable bacteria in the bloodstream. This can range from transient and clinically insignificant to severe, life-threatening sepsis
Sepsis: A life-threatening organ dysfunction caused by a dysregulated host response to infection. It is often associated with bacteremia and can lead to septic shock
Blood Culture: A laboratory test used to detect the presence of bacteria or fungi in a blood sample. This is the primary method for diagnosing bacteremia and sepsis
Aseptic Technique: A set of procedures used to prevent contamination of a specimen with microorganisms from the environment. This is crucial when collecting blood and bone marrow samples to ensure accurate results
Anticoagulant: A substance (e.g., sodium polyanethol sulfonate (SPS), heparin) added to blood culture bottles or bone marrow samples to prevent clotting. Clotting would make it difficult or impossible for the lab to grow and identify organisms
Colony Morphology: The visual characteristics of bacterial colonies growing on culture media (e.g., size, shape, color, texture, hemolysis). These characteristics can help with the preliminary identification of bacteria
Differential Time to Positivity (DTP): The time it takes for a blood culture to become positive. It is sometimes used to differentiate catheter-related bloodstream infections (CRBSI) from other sources of bacteremia; the sooner a culture from a catheter is positive relative to a peripheral draw, the more likely the catheter is the source
Catheter-Related Bloodstream Infection (CRBSI): A bloodstream infection that originates from an intravascular catheter (e.g., central venous catheter, peripheral IV)
Bone Marrow Aspirate: A sample of the liquid portion of bone marrow, obtained by aspiration. This sample can be cultured to detect microorganisms or examined microscopically
Antibiotic Susceptibility Testing: Laboratory tests performed to determine the susceptibility of a bacterial isolate to various antibiotics. The results guide antibiotic therapy

Blood & Bone Marrow

Continuous Monitoring