manual blood culture techniques

MANUAL BLOOD CULTURE METHODOLOGY STILL IN USE.

Dr Mostafa Mahmoud Ahmed, Ph DConsultant Microbiologist, GDHA, Riyadh, KSA.Associate Prof. of Microbiology & Immunology.

Faculty of Medicine – Ain Shams University

BLOOD CULTURE METHODS Many methods available; the choice of which

depends mainly upon resources. The cheapest one is the manual (conventional). The expensive and sophisticated automated

systems are now in hands. Note that 90% of blood cultures (BC) are

normally negative making comparison between different systems so difficult.

There is no blood system as absolute reference one so, sensitivities and specificities cannot be accurately calculated.

A . Manual System:1. Conventional broth-based system

Simple Labor-intensive. Each bottle contains 50-100 ml of broth

supplemented with 0.025-0.05% sodium polyanethol sulfonate (SPS) to prevent clotting.

Two bottles aerobic (vented) and anaerobic ones.

To be inspected daily for signs of growth.

Signs of growth: turbidity, hemolysis, surface pellicle, or froth (gas production).

Another alternative is blind subculture daily.

The main disadvantage of the conventional system is the highest rate of contamination during subculture techniques.

Prolonged incubation and subculture is needed to detect Brucella species.

2 -Biphasic systems- Conventional broth that can flood solid agar in a closed

system. Different devices available:.

i- Hemoline system Castaneda medium is the principle of it. Was developed in 1947 in France (Biomerieux). Contains 40 ml broth and solid agar. Originally described for aerobic culture, however,

anaerobic culture bottles available. Inoculation of agar is by flooding it by inversion of

bottles. Colonies easily detected on surface of agar by

inspection daily for 7 days.

Hemoline system

Hemoline system

ii- Septi-Check system-was developed by Hofhann-La Roche,

Switzerland in 1980s.- Now marketed by BD systems in USA.-It was based on the dip slide bacteriologic urine-culture kit.- modified to fit onto 100-ml blood-culture bottles, the paddles being coated withappropriate media (chocolate, MacConkey agar).- The bottles contain 70 ml of broth medium. -The paddle is inoculated by the blood in broth by simple inversion of the bottle.

- The agar surfaces and the broth should be visually inspected for microbial growth twice daily for the first 2 days of incubation, and thereafter once a day up to the seventh day of incubation. - For anaerobic blood cultures, conventional broth bottles that should not be vented are available for this system.- advantages: less contamination on subcultures of positive.- disadvantage: daily inspection. - other biphasic systems available but not popular as the previously described ones.

Septi-check system

3 -Gas capture or broth displacement (Signal) system:

- Was developed in 1986 by Oxoid. - Based upon the principle that microbial metabolism produces positive pressure in a sealed bottle, which can be visualized in a secondary plastic cylinder (Signal device) attached to the broth tube via a needle. - Aerobic and anaerobic microorganisms causing sepsis can be isolated from one Signal bottle.- However the use of both aerobic and anaerobic bottles in other systems was superior to single bottle use.

Oxoid signal devices

4- Lysis-centrifugation system (Isolator)

-A single-tube blood-culture system based on lysis, centrifugation and subsequent direct plating on appropriate media was described in 1978.- it is now distributed by Oxoid Isolator System.- The Isolator 10 tubes contain the following reagents in 0.7 mL of aqueous solution: saponin (cell-lysing agent), propylene glycol (blocks the foaming tendency of saponin), and SPS (anticoagulant).

- At least 6 ml of blood should be inoculated into these tubes, which must be processed as soon as possible, or at most 8 h after the inoculation of blood.

- Tubes are centrifuged in the laboratory, preferably at 3000 g for 30 min. The supernatant is discarded, and the pellet is inoculated on solid media.

- The Isolator 1.5 used for pediatric patients inoculated with a maximum of 1.5 ml of

blood, and is processed without centrifugation.- Easy manipulation and detect more pathogens.

Isolator Blood Culture System

Some characters of commercial BC systems:

الله بسمالرحيم الرحمن

Thank you Alsharjah UAE 2014