MLSM 508 Blood Culture Systems (Manual System)

Introduction Septicaemia usually portends life threatening illness and so blood cultures do receive a high priority attention in the laboratory. The recovery of microorganisms from blood is of great significance. There is no normal resident bacterial flora in the blood, so collection and handling of blood for culture should be done aseptically in order to avoid or minimize contamination from extraneous sources.

There are basically two blood culture systems: manual and automated systems. In either system, certain factors are taken into consideration if the blood culture, in the presence of infection, will yield positive result: . Volume of blood in the medium Dilution of blood in the medium . Duration of incubation.

For most systems, 10 – 20ml of blood from adults is taken from adults and distributed equally into the aerobic and anaerobic blood culture bottles containing 100ml of broth. However each system indicates the volume of blood required. To eliminate or minimize the effects of antibody, complement and other antibacterial substances present in the blood, an optimal dilution of blood in the liquid blood culture media is necessary. Usually 1:10 to 1:20 dilution is adequate.

Manual Blood Culture System Manual Blood Culture System. Blood collected for culture is transported to the laboratory without delay for immediate incubation, usually in an atmosphere enriched with CO2 . Generally, incabation is for 7 days at 370C. Extended incubation period is required for certain disease conditions e.g. endocarditis for 14 days and brucellosis for 21 days.

Examination of blood cultures Macroscopy : Blood cultures are examined daily for any physical evidence of growth. Bacterial growth : may be indicated by Turbidity Gas formation . Haemolysis In the absence of physical signs of growth, the blood culture is given a gentle shake and then returned to the incubator. If Castaneda bottle is used, the bottle should be tilted so that the broth covers the agar surface.

  Subculture : Inspection and subculture of blood cultures are carried out after 24 hours and 48 hours incubation. Some workers may prefer to further subculture on the third and fifth day of incubation. Daily inspection for signs of growth is done until the seventh day when the final subculture is done.

Technique for subculture : It is important to avoid contamination of blood culture during subculturing. To achieve this, a long, thin drawn out pasteur pipette is used to withdraw about 1 to 2 ml of broth culture into a sterile container. Alternatively, a special long wire loop may be used. All these procedures should be done in a safety cabinet.   .

Inoculate 2 blood agar plates for aerobic and anaerobic incubation, 1 chocolate agar and 1 CLED agar plates. All the plates are incubated at 370C.

Microscopy : If there is any sign of bacterial growth, a smear is made for Gram stain. The results of the Grams stain must be communicated to the clinician or the ward. Perform a direct sensitivity test based on the results of the Gram stain. Identify the isolate.   Reporting Report negative blood cultures at 48 hours and on the 7th day (Final report) All positives with full identification of the pathogen and appropriate antibiogram should be reported to the requesting physician or to the ward/clinic.