IVD

 Blood Culture Medium (Biphasic Culture Medium)

Principles

Some culture systems comprise of both liquid and solid phases in the same bottle. These are known as biphasic media (Castaneda system for blood culture). The inoculum is added to the liquid medium, and when subcultures are to be made, the bottle is simply tilted to allow the liquid to flow over the solid phase. This obviates the need for frequent opening of the culture bottle to subculture.

 Packaging Contents

The biphasic media containing BHI Broth and BHI Agar. Surface is recommended for improved recovery and earlier detection of Bacteria and Fungi in blood culture

          •        Each vial containing 30 ml of BHI Broth.

          •        Each vial containing 20 ml of BHI Agar.

Storage and Stability

•        Store at room temperature. Do not refrigerate the specimen.

•        Biphasic culture medium is stable until the expiry date stated on the label (12 months).

Procedure

•        Each bottle contains a rubber stopper held in place by a topped screw cap surrounded by a plastic band. The rubber stopper must not be taken off the bottle. Blood is added by puncturing the rubber stopper with the needle attached to the syringe containing the blood specimen or with the needle of the Blood Taking Unit.

•        Each specimen should be cultured aerobically and anaerobically. For aerobic growth, filtered air must be allowed to enter the bottle through a Venting Unit after blood collection.

•        Following blood collection, the bottles are shaken to thoroughly mix the blood and medium and are then labeled.

•        All bottles should be transported to the laboratory as soon as possible and immediately incubated at 35 +/-    2°C in an upright position. Biphasic bottles should be incubated either in an upright position or on their side with the agar surface up. In this way, colonies of bacteria may be observed on the surface of the agar after incubation. The purpose of the biphasic bottle is to shorten the time necessary to obtain isolated organisms for identification procedures.

•        Lower incubation temperatures may be preferred for the isolation of specific organisms. Culture for the detection of fungi may be incubated at 22 to 30°C, whereas certain bacterial species, such asListeria, grow well at 20 to 25°C.

•        A total incubation period of 7 days is generally sufficient for routine isolation procedures. Alternatively, the bottles can be incubated for at least 14 days before discarding those that do not show evidence of growth. A 14-day incubation period has been reported to be adequate for the recovery of yeast; if dimorphic fungi

are suspected to be present, blood cultures may require incubation for an additional 2 weeks.

          •        Following inoculation and incubation of the bottles, microorganisms recovered from the medium may be Gram stained and identified by conventional plate and identification procedure. The first important subculture test to be performed on an isolated microorganism from a blood culture is to investigate the susceptibility of the organism to antimicrobial agents. If anaerobes are suspected, the subculture should be inoculated anaerobically.

Culture and Isolation

The test bottles should be visually examined within 24 h and at daily intervals thereafter. The bottles should be carefully removed from the incubator to avoid disturbing the sedimented blood and examined for any visual evidence of microbial growth, such as turbidity, hemolysis, gas production, or formation of discrete colonies. Growth in biphasic bottles may be first seen on the agar surface. If no growth is visible in the broth or on the agar surface, tilt the bottle to wash the broth over the agar surface and reincubate. Since autolysis of some microorganisms may occur after prolonged incubation of inoculated blood culture bottles, subcultures should be taken at various incubation intervals. After 24- to 48-h incubation, a small quantity (0.1 to 0.5 ml) of the blood-broth mixture should be removed using a sterile syringe and needle and subcultured to plates of enriched and selective media. This procedure should be repeated after an incubation period of 7 days if the culture appears negative, or earlier as growth appears.

Although growth in blood culture bottles may be manifested in a variety of appearances, useful information can be obtained by observing the cultures for typical appearances. If visible evidence of growth appears, the broth should be examined by the Gram stain and subcultured onto appropriate media for isolation and identification.

Precautions, Storage and Sign of Deterioration

Prepared Blood Culture Bottles are for in-vitro diagnostic use only.

Store Prepared Blood Culture Bottles at room temperature, protect them from direct light. Media should not be used if the expiry date has passed.

Prepared Blood Culture Bottles should not be used if there are signs of contamination. Biphasic culture is not recommended for the culture of anaerobic microorganisms

Disposal

Infectious waste must be disposed of in a carefully controlled manner in accordance to the guidelines.