Processing of Blood culture specimens

Blood culture analysis is one of the most important functions of the microbiology laboratory, as the medical staff relies on the information obtained to aid in the diagnosis of bacteraemia or septicaemia and fungaemia. This can be achieved using either automated system or the traditional non-automated or manual system.

Taking of blood culture samples

This is basically taking a blood sample into a pair of blood culture bottles, aerobic and anaerobic and a further sampling usually two hours apart or more in endocarditis. This increases the yield of positive result and may allow better recognition of contamination. This is usually done as soon as symptoms present, preferably before antibiotics are given, for example shortly after pyrexia spike when bacteria are more likely to be found in the blood stream. The procedure is different in children and infants where specific paediatric blood culture bottles are used. It is ideal that this is done by an experienced member of staff as aseptic technique is very important at this stage to avoid contamination. In most conditions other than endocarditis, bacteraemia is intermittent, being related to the fevers and rigors which occur 30-60 minutes after the entry of organisms into the bloodstream (Ford, 2010).

It is recommended that 20-30ml blood be cultured as volume of blood cultured is the most critical factor in the detection of bloodstream infection, but most modern systems only require approximately 10ml of blood to the two blood culture bottles and a few systems restrict the volume to 5ml. There is a direct relationship between blood volume and yield, with approximately a 3% increase in yield per ml of blood cultured. In neonates, 1-2ml is recommended.

Further pairs of blood culture samples are taken over three days in cases of endocarditis or three blood culture samples over one day if therapy is important. This is usually repeated at 48 hours if the result is negative. These samples are incubated on the Bactec FX Blood culture analyser for 5 days or 7 days for endocarditis patients. Samples must be clearly labelled with full patent details including name and date of birth, hospital/NHS number and attached to a patient test request form containing the clinical details. The addition of full clinical details is vital as it gives laboratory and medical staff information on the likely organisms to be isolated. These clinical details may include sub-acute bacterial endocarditis(SBE), infective endocarditis(IE), endocarditis, native valve endocarditis(NVE), prosthetic valve endocarditis(PVE), vegetative, heart murmur/new murmur, mitral valve and aortic valve.

Transportation of the Blood culture samples.

Blood culture samples should be sent to the laboratory as soon as possible after sampling. They can be stored in an appropriate incubator or kept at ambient temperature before sending. Once samples are received in the laboratory, they are processed immediately by either loading them on the automated culture system or processing them manually.

Manual Blood culture system

Blood culture bottles (aerobic and anaerobic) containing culture medium are used in manual blood culture systems and incubated at the appropriate temperature usually 37 degrees centigrade and incubated for 24 hours, 48hours and 5 days (or 10 days if it is an endocarditic sample). The blood samples are subcultured to blood agar, chocolate agar, Fastidious Anaerobic agar and Chromogenic UTI agar after incubation of 24 hours, 48hours and 5 days (or 10 days if it is an endocarditis sample). An example of manual blood culture system is Biomerieux Hemoline. Growth of organisms is identified by an increase in turbidity of the culture medium and/or the haemolysis of red blood cells. This method is labour intensive and requires the frequent checking of the blood culture bottles in the first 48 hours on receipt of the sample for macroscopic evidence of microbial growth. Blind subculture at 24-48 hours and also at the end of the incubation may pose the risk of contaminating the sample or potential infection risk to the laboratory staff whenever the blood culture sample is opened.

Automated Blood culture system

An automated blood culture system should be able to support the rapid growth of a wide range of pathogenic bacteria including fastidious organisms.

The recent improvement in the automated systems involves the detection of CO₂ which is produced due to glucose metabolism during microbial growth. In BD BACTEC FX blood culture system for example, which is used for the rapid detection of bacteria and fungi in clinical specimens, the increase in the production of CO₂ due to microbial glucose metabolism causes a change in fluorescence or reflectance in a chemical sensor located in the bottom of the bottle. The sensor is monitored by the analyser every ten minutes for an increase in fluorescence which is proportional to the amount of CO₂ present. A positive reading indicates the presumptive presence of viable organisms in the vial. Resins can be added to the medium to neutralise a wide range of antibiotics and enhance the recovery of organisms. This instrument can monitor a total of four hundred vials or blood culture bottles which are arranged in four drawers. The racks are continuously incubated at 36 degrees centigrade and agitated for maximum recovery of organisms.

The Biomerieux BacT/Alert is another automated blood culture system which uses slightly different technology in comparison with the BD BACTEC FX. The sensor is monitored by the analyser every ten minutes for an increase CO₂ production. This increases the concentration of hydrogen ions and decreases the pH thus causing the sensor to become lighter green and eventually yellow.

The culture bottles are usually incubated for a standard five days but in some cases such as endocarditis they are incubated for seven days. This seven days incubation usually applies to fastidious organsims, and those that rarely cause human disease other than endocarditis, for example the HACEK group: Haemophilus aphrophilus, Actinobacillus actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens and Kingella kingae. Bartonella species are becoming increasingly important causes of endocarditis particularly in AIDS patients.

Blood culture bottle media

Most systems employ different media for the isolation of aerobic and anaerobic organisms, and some media are specifically designed for organisms such as fungi and Mycobacterium species.  A variety of blood culture media are commercially available and have been reviewed. They differ in the types and proportions of various supplements and anticoagulants, volumes of broth, headspace atmospheres and the presence of antimicrobial-neutralising agents.  The aerobic bottle may or may not require transient venting to increase the oxygen content in the headspace for strictly aerobic organisms such as Pseudomonas aeruginosa and Candida albicans.

	Aerobic	Anaerobic	Pediatrics
Ingredients	Quantity	Quantity	Quantity
Processed water	40ml	40ml	40ml
Soybean-Casein digest broth	2.75% w/v	2.75% w/v	2.75% w/v
Yeast extract	0.25% w/v	0.2% w/v	0.25% w/v
Animal tissue digest	0.10% w/v	0.05% w/v	0.10% w/v
Dextrose	0.06% w/v	0.2% w/v	0.06% w/v
Hemin	0.0005% w/v	0.0005% w/v	0.0005% w/v
menadione	0.00005% w/v	0.00005% w/v	0.00005% w/v
Sodium citrate	-	0.02% w/v	-
Thiols	-	0.1% w/v	-
Sodium pyruvate	0.10% w/v	0.1% w/v	0.10% w/v
Saponin	-	0.26% w/v	-
Antifoaming agent	-	0.01% w/v	-
Sodium polyanetholsulfonate (SPS)	0.020% w/v	0.035% w/v	0.020% w/v
Sucrose	0.08% w/v	-	0.08% w/v
Pyridoxal HCL (Vitamin B6)	0.001% w/v	-	0.001% w/v
Non-ionic adsorbing resin	10.0% w/v	-	10.0% w/v
Cationic exchange resin	0.6% w/v	-	0.6% w/v

The media are dispensed with added CO₂ for aerobic vials and CO₂ and N₂ for anaerobic vials. The presence of optimum blood volumes (5-10ml) is beneficial in the recovery of some organisms sensitive to Sodium polyanetholsulfonate (SPS) such as Peptostreptococcus anaerobius, because blood can neutralise the toxicity of SPS.

A blood:broth ratio

The blood:broth ratio of about 1:15 is required to remove the antibacterial effects of normal human blood, but this may be reduced to between 1:5 and 1:10 by the addition of 0.05% sodium polyanethol sulphonate (SPS).  SPS has an inhibitory effect on Neisseria species, anaerobic cocci, Streptobacillus moniliformis and Mycoplasma hominis. The inhibitory effects of SPS may be reduced by the addition of scetic to the broth. Some commercial bottles supplement the medium with materials which improve microbial recovery by adsorbing antimicrobial substances and lysing the white blood cells to release the micro-organisms into the blood-broth mixture.

Neutralisation of antimicrobials

Resins can be added to the media to neutralise a wide range of antibiotics and enhance the recovery of organisms. In addition, the addition of beta-lactamase will help to overcome the effect of beta-lactam antibiotics. 

Refereneces

Brooks G F, Butel J S and Morse S A (1998) Jawetz, Melnick and Adelberg’s Medical Microbiology. Appleton and Lange, Stamford, Cunnecticut, USA

Ford M (2010). Fundamentals of Biomedical Science; Medical Microbiology. Oxford University Press, London.