Microbiology - Blood Cultures

The culture of microorganisms from blood is required for the laboratory diagnosis of bacteraemia, infective endocarditis and several infective conditions associated with pyrexia of unknown origin (PUO).

Blood is normally sterile. However bacteria can enter the bloodstream through different routes:

  • a focus of infection within the body
  • through broken skin or mucous membranes
  • by direct introduction of contaminated material into the bloodstream

Bacteria are normally removed from the bloodstream within minutes. Systemic infection only occurs when the host defences are overwhelmed or evaded.

Blood cultures should only be taken when there is a clinical need and not as routine. Bacteraemia can be suspected in the presence of a large number of patient symptoms and signs and clinical judgement is therefore required.

The following indicators should be considered:

  • Core temperature outside normal limits
  • Chills or rigors
  • Raised or very low WBC count
  • Focal signs of infection
  • Abnormal heart and respiratory rate (raised) and blood pressure (low or raised)

It must also be remembered that signs of sepsis or bacteraemia may minimal or absent in immunocompromised patients, the very young and the elderly.

The Microbiology department examines blood cultures using the Biomerieux BacT/ALERT 3D. Once loaded on this system the bottles are continuously monitored and the positives are flagged as soon as they appear. The bottles are routinely incubated for 5 days before being discarded as negative.

The multiplication of microorganisms in positive cultures generates CO2. As this increases the sensor in the bottle turns to a lighter colour. By measuring reflected light the BacT/ALERT 3D monitors and detects colour changes in the sensor.


Bottle type Specimen type Specimen volume
BacT/ALERT SA Standard Aerobic (blue top). Blood or normally sterile body fluid Up to 10 ml
BacT/ALERT SN Standard Anaerobic (purple top) Blood or normally sterile body fluid >Up to 10 ml
BacT/ALERT PF Pediatric FAN (yellow top) Blood Up to 4 ml
BacT/ALERT MB Mycobacteria Blood  (black top) Whole blood 3-5 ml


Number and Timing of Samples

With the exception of endocarditis, bacteraemia is intermittent. In order to maximise the chances of recovering viable organisms blood cultures should not be taken during a temperature spike but should be taken after the spike or in the troughs between spikes.

For most patients two sets of blood cultures are normally recommended. The two sets should be taken at separate times and from separate sites.


Clinical condition  Procedure
Adults and adolescents
Septicaemia, meningitis, pneumonia, therapy osteomyelitis,  arthritis.
Comment: One 10 - 20 ml sample from each arm. Therapy should not be delayed pending blood cultures.
2 cultures before therapy

Subacute bacterial endocarditis
Comment: Collections should be spaced over 24 hours. Cultures should be taken at the start of the fever spikes. Collect 3 more if the first 3 are negative after 24 hours.

3 cultures in 24 hours

Acute bacterial endocarditis
Comment: Therapy should not be delayed pending blood cultures.

3 cultures within 1- 2 hours  before  therapy   
 Low grade intra-vascular infection
Comment: Space collections at least 1 hour apart. Collect 2 at the first sign of pyrexia.
 3 cultures in 24 hours
Bacteraemia of unknown origin {patient is on therapy)
Comment: Collect just before antibiotic is administered
 4 - 6 cultures in 48 hours
Younger children/neonates 
Comment: Two cultures are usually sufficient to diagnose bacteraemia in newborns. 1 - 2 ml samples


Blood Cultures Bottle Sets

In order to maximise the recovery of organisms the following combinations of bottles should be used under normal circumstances:

Standard set: 1 BacT/ALERT SA Standard Aerobic (blue top) and BacT/ALERT SN Standard Anaerobic (purple top).

The paediatric bottle (BacT/ALERT PF Pediatric FAN (yellow top)) should be used for small children or patients from whom only a small volume of blood can be obtained.

Important Considerations Relating to the Collection of Blood

When taking a blood culture together with blood for Biochemistry or Haematology, it is essential that the Microbiology sample is collected first.

The correct sequence is:

  1. Blood Culture
  2. Biochemistry Tube
  3. Coagulation Tube
  4. Full Blood Count Tube

Do not use existing peripheral lines/cannulae or sites immediately above peripheral lines. If a central line is present blood may be taken from this and a separate peripheral site when investigating infections related to the central line.

A significant problem encountered with blood cultures collection is contamination with skin organisms. The following procedure is recommended in order to minimise the chances of contamination.

  1. Wash your hands with soap and water. Dry hands.
  2. Clean any soiled skin on the patient with soap and water and dry the skin.
  3. Apply a disposable tourniquet (if applicable) and palpate to identify a vein.
  4. When blood cultures are collected from a central venous catheter, the access port must be disinfected with a suitable antiseptic (e.g. 2% clorhexidine in 70% isopropyl alcohol steret).
  5. Clean the venepuncture site with the antiseptic starting from the centre of the site and scrubbing outward with a circular motion to cover approximately 10 cm for 30 seconds. Do not go back over the parts of the site that have been already scrubbed. You MUST allow the alcohol to dry as this is an essential step to kill the organisms.
  6. Before using the blood culture bottles check the expiry date and discard if out of date.
  7. Label each bottle with the patient's details. The following information should be on each bottle: (A) Patient's full name (B) Date of birth (C) Date and time of collection.
  8. Fill the request form correctly. The minimum information required should be the same as on the blood culture bottle. Clinical data should also be provided together with any antibiotic therapy.
  9. Do not affix labels on the barcodes on the bottles as these are required for loading the bottles on the BacT/ALERT system.
  10. Make sure the high risk samples are appropriately labelled and packaged for transport.
  11. Remove the plastic caps from the bottles.
  12. Decontaminate the rubber stopper with a suitable antiseptic. Allow the alcohol to evaporate before inoculating the bottle.
  13. Collect between 10 and 20 ml of blood from the patient. Inoculate the two blood culture bottles with equal amounts. The aerobic bottle (blue top) must always be inoculated first to avoid the release of air from the syringe into the anaerobic bottle. If only a paediatric bottle is used collect up to 4 ml and add to this bottle.
  14. Dispose of the needle and syringe appropriately (eg in a sharps bin).
  15. Please both bottles in a specimen bag.
  16. Arrange for the bottles to be transported to the laboratory as soon as possible.
  17. When multiple sets of blood cultures are collected the sets should be collected from different venepuncture sites and at different times.

Clinical Data

It is important that all relevant clinical data are provided on the request form. This should include the type of sample (eg venous, arterial, line blood). History of recent foreign travel is also important as it may be necessary to extend the incubation time for certain organisms (eg when Brucella is suspected).


When bottles become positive the BacT/ALERT systems flags them automatically. A gram stain is carried out from each positive bottle to indicate the type of organism that may be growing (eg gram positive or gram negative rods and cocci). This information is then telephoned to the clinician or relevant ward and to the Consultant Microbiologist .