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MANUAL BLOOD CULTURE
METHODOLOGY STILL IN USE.
Dr Mostafa Mahmoud Ahmed, Ph D
Consultant 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
Each bottle contains 50-100 ml of broth
supplemented with 0.025-0.05% sodium
polyanethol sulfonate (SPS) to prevent
Two bottles aerobic (vented) and
To be inspected daily for signs of growth.
Signs of growth: turbidity, hemolysis,
surface pellicle, or froth (gas production).
Another alternative is blind subculture
The main disadvantage of the conventional
system is the highest rate of
contamination during subculture
Prolonged incubation and subculture is
needed to detect Brucella species.
- 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
Colonies easily detected on surface of agar by
inspection daily for 7 days.
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-
- modified to fit onto 100-ml blood-culture bottles,
the paddles being coated with
appropriate 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
- 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.
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
4- Lysis-centrifugation system
-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
- 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
- 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.