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Blood Culture Essentials


Blood Culture Essentials

This educational webpage is based on the Blood Culture Educational Booklet that you can access HERE

What Is a Blood Culture?

A blood culture is a laboratory test in which blood, taken from the patient, is inoculated into bottles containing culture media to determine whether infection-causing microorganisms (bacteria or fungi) are present in the patient’s bloodstream. 

Blood cultures are intended to:

  • confirm the presence of microorganisms in the bloodstream    
  • identify the microbial etiology of the bloodstream infection  
  • help determine the source of infection  
  • provide an organism for antimicrobial susceptibility testing  and optimization of antimicrobial therapy

*  Adapted from ESCMID (European Society of Clinical Microbiology and Infectious Diseases) guidelines, 2012. 6

Why Are Blood Cultures Important?

Blood culture is the most widely used diagnostic tool for the detection of bacteremia and fungemia. It is the most important way to diagnose the etiology of bloodstream infections and sepsis and has major implications for the treatment of those patients.

A positive blood culture either establishes or confirms that there is an infectious etiology for the patient’s illness.1 A positive blood culture also provides the etiologic agent for antimicrobial susceptibility testing, enabling optimization of antibiotic therapy.1 Sepsis is one of the most significant challenges in critical care, and early diagnosis is one of the most decisive factors in determining patient outcome. Early identification of pathogens in the blood can be a crucial step in assuring appropriate therapy, and beginning effective antibiotic therapy as early as possible can have a significant impact on the outcome of the disease.7,8

Providing adequate antibiotic therapy within the first 24-48 hours leads to: 9-13

  • decreased infection-related mortality (20-30%)  
  • earlier recovery and shorter length of hospital stay  
  • reduced risk of adverse effects  
  • reduced risk of antimicrobial resistance  
  • cost reduction (length of stay, therapy, diagnostic testing)

When Should a Blood Culture Be Performed? 

Blood cultures should always be requested when a bloodstream infection or sepsis is suspected.15

Clinical symptoms in a patient which may lead to a suspicion of a bloodstream infection are:16

  • undetermined fever (≥38.3°C) or hypothermia (≤36°C)  
  • shock, chills, rigors
  • vomiting  
  • severe local infections (meningitis, endocarditis, pneumonia, pyelonephritis, intra-abdominal suppuration…)  
  • abnormally raised heart rate (>90 beats/min)  
  • low blood pressure (<90 mm Hg)  
  • raised respiratory rate (>20 breaths/min)

Blood cultures should be collected: 

  • as soon as possible after the onset of clinical symptoms;  
  • ideally, prior to the administration of antimicrobial therapy.17

If the patient is already on antimicrobial therapy, recovery of micro-organisms may be increased by collecting the blood sample immediately before administering the next dose and by inoculating the blood into bottles containing specialized antimicrobial neutralization media.17,18

What Volume of Blood Should Be Collected?

The optimal recovery of bacteria and fungi from blood depends on culturing an adequate volume of blood. The collection of a sufficient quantity of blood improves the detection of pathogenic bacteria or fungi present in low quantities.

The volume of blood that is obtained for each blood culture set is the most significant variable in recovering micro-organisms from patients with bloodstream infections. 19,20

Blood culture bottles are designed to accommodate the recommended blood-to-broth ratio (1:5 to 1:10) with optimal blood volume.21 Some commercial blood culture systems use a blood-to-broth ratio that is less than 1:5. This ratio is acceptable because proprietary substances that bind to and inactivate the inhibitory substances are added to blood specimens.1


For an adult, the recommended volume of blood to be obtained per culture is 20 to 30 mL.1,17 

Since each set includes an aerobic and an anaerobic bottle, each bottle should be inoculated with approximately 10 mL of blood. This volume is recommended to optimize pathogen recovery when the bacterial/fungal burden is less than 1 Colony Forming Unit (CFU) per mL of blood, which is a common finding. It is also generally recommended that two or three bottle sets (2 bottles per set) are used per septic episode, meaning, for adults, 40 to 60 mL of blood collected from the patient for the 4 to 6 bottles, with 10 mL per bottle. For each additional milliliter of blood cultured, the yield of microorganisms recovered from adult blood increases in direct proportion up to 30 mL.22 This correlation is related to the relatively low number of CFU in a milliliter of adult blood.1


The optimal volume of blood to be obtained from infants and children is less well prescribed, however, available data indicate that the yield of pathogens also increases in direct proportion to the volume of blood cultured.17, 23  The recommended volume of blood to collect should be based on the weight of the patient (see Table 1)24, and an aerobic bottle should be used, unless an anaerobic infection is suspected.25

Recent studies have shown that facultative anaerobes are detected earlier or solely in the anaerobic blood culture bottle in certain pediatric populations suggesting that they should continue to be used. For newborns, infants, and children, the volume of blood drawn should be no more than 1% of the patient’s total blood volume.1

Specially formulated blood culture bottles are commercially available for use in children <2 years of age. They are specifically designed to maintain the usual blood-to-broth ratio (1:5 to 1:10) with smaller blood volumes, and have been shown to improve microbial recovery.1 

aMinimum and maximum recommended total blood volume to be drawn for paediatric blood culture.

How Many Blood Culture Sets Should Be Collected?

Since bacteria and fungi may not be constantly present in the bloodstream, the sensitivity of a single blood culture set is limited.

Using continuous-monitoring blood culture systems, a study investigated the cumulative sensitivity of blood cultures obtained sequentially over a 24-hour time period. It was observed that the cumulative yield of pathogens from three blood culture sets (2 bottles per set), with a blood volume of 20 mL in each set (10 mL per bottle), was 73.1% with the first set, 89.7% with the first two sets and 98.3% with the first three sets. However, to achieve a detection rate of >99% of bloodstream infections, as many as four blood culture sets may be needed.26

A contaminant will usually be present in only one bottle of a set of blood culture bottles, in contrast to a true bloodstream infection, in which multiple blood culture bottles/sets will be positive. 

If two to three sets are taken and cultures are still negative after 24-48 hours incubation, and the patient is still potentially septic, two to three additional cultures may be collected, as indicated in the following diagram.17

Which Media to Use?

Microorganisms causing bloodstream infections are highly varied (aerobes, anaerobes, fungi, fastidious microorganisms…) and, in addition to nutrient elements, may require specific growth factors and/or a special atmosphere.

In cases where the patient is receiving antimicrobial therapy, specialized media with antibiotic neutralization capabilities should be used. Antibiotic neutralization media have been shown to increase recovery and provide faster time to detection versus standard media.27-30

A blood culture medium must be:

  •  sensitive enough to recover:
    • a broad range of clinically relevant microorganisms, even the most  fastidious (Neisseria, Haemophilus…)
    • microorganisms releasing small amounts of CO₂ (Brucella, Acinetobacter…)  
  • versatile: able to provide a result for all types of sample collection (adults, infants, patients receiving antibiotic therapy, sterile body fluids…)

Which bottle should be inoculated first?

If using a winged blood collection set, then the aerobic bottle should be filled first to prevent transfer of air in the device into the anaerobic bottle.

If using a needle and syringe, inoculate the anaerobic bottle first to avoid entry of air. If the amount of blood drawn is less than the recommended volume*, then approximately 10 mL of blood should be inoculated into the aerobic bottle first, since most cases of bacteremia are caused by aerobic and facultative bacteria. In addition, pathogenic yeasts and strict aerobes (e.g. Pseudomonas) are recovered almost exclusively from aerobic bottles. Any remaining blood should then be inoculated into the anaerobic bottle.1

* For recommended volumes, see chapter 6 “What volume of blood should be collected?“

Timing of Blood Cultures

Studies have shown that the time interval between collecting two blood cultures is not considered to be a critical factor as the diagnostic yield remains the same.21

Guidelines recommend that the first two to three sets (2 bottles/set) of blood culture be obtained either over a brief time period (e.g. within 1 hour) or as a single sample taken at one time.1, 17, 21 The possible impact that the blood culture collection method used (e.g. single or multiple venipunctures, winged collection set or needle and syringe) may have on contamination rates should be considered.21

Drawing blood at spaced intervals, such as 1 to 2 hours apart, is only recommended to monitor continuous bacteremia/fungemia in patients with suspected infective endocarditis or other endovascular (i.e. catheter- related) infections.17

Two to three additional blood culture sets can be performed if the first two to three blood cultures are negative after 24-48 hours incubation in cases of severe infection or in order to increase detection sensitivity. This also depends on the microorganisms involved: while sensitivity is relatively good for organisms like Escherichia coli or Staphylococcus aureus, it is lower for Pseudomonas aeruginosa, streptococci or fungi.32

How to Collect Blood Cultures

Sample collection is a crucial step in the blood culture process. Standard precautions must be taken, and strict aseptic conditions observed throughout the procedure. Compliance with blood culture collection recommendations can significantly improve the quality and clinical value of blood culture investigations and reduce the incidence of sample contamination and “false-positive” readings. 

It is recommended that blood cultures should be collected only by members of staff (medical, nursing, phlebotomist or technician) who have been fully trained and whose competence in blood culture collection has been assessed.33

10 Key Steps to Good Sample Collection:

  1. Prior to use, examine the bottles for evidence of damage, deterioration or contamination. Do not use a bottle containing media which exhibits turbidity or excess gas pressure, as these are signs of possible contamination.
  2. Check the expiry date printed on each bottle. Discard bottles that have expired.
  3. Strictly follow the collection protocol in use in the healthcare setting, including standard precautions for handling blood at the bedside.
  4. Blood culture bottles should be clearly and correctly labelled, including patient identification, date and collection time, puncture site (venipuncture or intravascular device). 
  5. For adults, each blood culture set should include an aerobic and an anaerobic bottle.
  6. For adults and older children, blood should be drawn from veins in the antecubital fossae.1, 21
  7. It is recommended to avoid drawing blood from a venous or arterial catheter, since these devices can be colonized with either bacteria or fungi.1, 21
  8. Carefully disinfect the skin prior to collection of the sample using an appropriate disinfectant, including chlorhexidine in 70% isopropyl alcohol, tincture of iodine, povidone-iodine in swab or applicator form.1, 21
  9. Transport the inoculated bottles at room temperature and the completed blood culture request to the clinical microbiology laboratory as quickly as possible, preferably within 2 hours per CLSI.1 Delays beyond 2 hours could result in a delay in positive bottle detection or could cause a false negative result. If delays are expected, it is important to refer to the manufacturer’s Instructions For Use (IFU) for guidance.
  10. All blood cultures should be documented in the patient’s notes, including date, time, collection site and indications.

How Many Days of Incubation Are Recommended?

A study by Bourbeau, et al. (JCM, 2005) demonstrated that 97.4% of clinically significant isolates were recovered within the first 3 days of incubation and 93.8% within 2 days of incubation.35

Incubation of fastidious microorganisms

Another study by Cockerill, et al. (CID, 2004) demonstrated that, when using a continuous-monitoring blood culture system, 99.5% of non-endocarditis bloodstream infections and 100% of endocarditis episodes were detected within 5 days of incubation.22 This data suggests that extended incubation periods previously recommended for detection of the fastidious micro-organisms* that sometimes cause endocarditis, are no longer necessary when using continuous-monitoring blood culture systems.17

With the exception of Bartonella spp., most rare or fastidious bacteria are still cultivable by traditional bacterial culture systems and can be recovered using blood culture protocols currently in use by most medical laboratories.1

* including Brucella, Capnocytophaga and Campylobacter spp., and the HACEK group (Haemophilus species, Aggregatibacter species, Cardiobacterium hominis, Eikenella corrodens and Kingella species) 36

Is It a Contaminant or a True Pathogen?

Contamination of blood cultures during the collection process can produce a significant level of false-positive results, which can have a negative impact on patient outcome.

A false positive is defined as growth of bacteria in the blood culture bottle that were not present in the patient’s bloodstream, and were most likely introduced during sample collection.

Contamination can come from a number of sources: the patient’s skin, the equipment used to take the sample, the hands of the person taking  the blood sample, or the environment.

Certain microorganisms such as coagulase-negative staphylococci, Bacillus spp, Cutibacterium spp., diphtheroids, Micrococcus spp. rarely cause severe bacterial infections or bloodstream infections. These are common skin contaminants, and a though they are capable of causing serious infection in the appropriate setting, their detection in a single blood culture set can reasonably be identified as a possible contaminant without clinical significance. However, it is important to consider that coagulase-negative staphylococci are the primary cause of both catheter- and prosthetic device-associated infections and may be clinically significant in up to 20% of cases.37

The most difficult interpretation problem for the physician is  whether the organism recovered from a blood culture is a true pathogen causing bloodstream infection, or a contaminant. If it is a contaminant, the patient may be treated unnecessarily with antibiotics, leading to additional patient risks. Interpretation of true pathogen versus contaminant should be  based on whether the blood has been collected with a venipuncture or an intra-vascular device, and whether the same organism was recovered from multiple blood culture sets. This illustrates the crucial nature of having collection site information included with the blood culture request sent to the laboratory. 

In contrast to patients with infective endocarditis or other true positive bloodstream infections, patients whose blood cultures grow contaminants usually have only a single blood culture that is positive. This information is of great practical value for physicians, and underlines the importance of taking two to three blood culture sets from different anatomical sites.17

The American Society for Microbiology and CLSI recommend targeting contamination rates of 1% of the total of collected sets.1,17

Impact of Contamination Rates

A contaminated blood culture can result in unnecessary antibiotic therapy, increased length of hospitalization and higher costs.

It has been found that each false positive result can lead to:  

  • increased length of stay - on average 1 day37    
  • 39% increase in intravenous antibiotic charges37    
  • $5,000 to $8,720 additional charges38, 39
  • 20% increase in laboratory charges37  
  • 3 days longer on antibiotics37

The content on this website and associated materials do not constitute medical advice and should not be considered a substitute for the individual professional judgement of any physician or other health care practitioner regarding the appropriate course of action for a particular patient. All recommendations should be independently reviewed with appropriate medical staff in light of the needs of any particular institution and its patients. bioMérieux makes no guarantee or representation regarding the accuracy, completeness, or usefulness of this information for any particular purpose, including but not limited to any cost savings.