Choice of broth medium
The blood-culture broth should be able to support growth of all clinically significant bacteria. It has been found that tryptic soy broth (TSB) is as good as any.
Quantity of broth
Ideally, the blood should be mixed with 10 times its volume of broth (5 ml of blood in 50 ml of broth) to dilute any antibiotic present and to reduce the bactericidal effect of human serum.
To prepare blood-culture bottles, fill a bottle with medium and then loosen the screw-cap half a turn. Cover the cap with a square piece of aluminium foil or brown paper, and autoclave the bottle for 20 minutes at 120 °C. Immediately after autoclaving, while the bottle and the medium are still hot, securely tighten the cap without removing the aluminium foil or brown paper (otherwise the cap will not be sterile). As the medium cools, a partial vacuum will be created in the bottle, which will facilitate injection of a blood specimen through the diaphragm.
The top of the cap must be carefully disinfected just before the bottle is inoculated.
Prior to distribution and before use, all blood-culture bottles should be carefully examined for clarity. Any medium showing turbidity should not be used.
If strictly aerobic bacteria (Pseudomonas, Neisseria) or yeasts are suspected, the bottle should be vented as soon as it is received in the laboratory, by inserting a sterile cotton-wool-plugged needle through the previously disinfected diaphragm. The needle can be removed once the pressure in the bottle reaches atmospheric pressure. Commercial blood-culture bottles often also contain carbon dioxide, which has a stimulating effect on growth.
In countries where brucellosis is prevalent, the use of a diphasic blood-culture bottle, with a broth phase and a solid-slant phase on one of the flat surfaces of the bottle (Castaneda bottle), is recommended for the cultivation of Brucella spp. The presence of carbon dioxide is needed for the isolation of most strains of B. abortus.