BLOOD CULTURE

Indications
Precautions
Technique
Types
Reading and interpretation

Indications:

Bacteremia :typhoid , brucellosis,

infective endocarditis, puerperal
sepsis
Septicemia: septic shock, enteric
fever, endocarditis, gram negative
septicemia , anaerobic septicemia

Timing of Blood Cultures

Optimal time: just before anticipated onset

of the chill or fever,
Typically collected as soon as possible after
the
onset of fever or chills or whenever serious
infection is suspected
Collection, whenever possible, prior to the
administration of antimicrobial agents.
Simultaneous collection of two to three sets
in the initial evaluation or at different times

Sites for obtaining Blood

Cultures

Venipuncture is the method most

commonly used
Whenever possible, blood for culture
should not be drawn through an
indwelling intravenous or intra-arterial
catheter
Reason: Potential risk of higher
contamination!

Skin preparation
Poor skin preparation prior to drawing
blood
cultures is the most common cause of culture
contamination.
False-positive
blood cultures may be
associated
with increased length of hospital stay and
increased
pharmacy
and
laboratory
charges.

70% alcohol for 30 sec from centre to periphery

2% iodine tincture for 60 sec or 10% povidone
for 120 sec
70% alcohol for 30 sec from centre to periphery
Allow to dry completely
Sterile gloves
Repalpitation of vein only with disinfected finger
Change needle if unsuccessful
Cleanse rubber stopper of the blood culture
bottle with 70% alcohol before blood inoculation

Volume of Blood for

Culture

The volume of blood drawn per culture is

the single most important variable in
recovering microorganisms from the blood
of bacteremic or fungemic patients.
Laboratories should routinely monitor the
volume of blood cultured as a quality
assurance activity
Each ml of blood,, up to 10 ml, can increase
the sensitivity of the blood culture by 3 5%..

Optimal Volume of Blood

for Culture

Adults:
20 ml per set (two
bottles)
Pediatric
- Neonates 1-2 ml per bottle
- Infants and children 2-5 ml
per bottle
- Adolescents 10-20 ml per
set

Number of Blood
Cultures Sets

With adequate volume of blood, 2 3 blood culture sets are sufficient to

detect nearly all episodes of
bacteremia and fungemia

Duration of Blood
Cultures

7-10 days
14 days for anaerobic
4 weeks at least for brucella
The duration is shortened with the
use of automated techniques.

Transport from Bed Side to

the Lab

Prior to transport, vials should be properly

identified
Transport time should be as fast as possible
Transport temperatures should not be extreme
preferably at RT
never frozen nor refrigerated
not higher than normal body temperature
Vial leakage should be considered all the time
use adequate transport containers
wear gloves

Blood culturing methods

AUTOMATED

MANUAL

Blood Culture Media

No one medium or system is capable of

detecting all microorganisms.
Automated systems offers special media
for:
Aerobes
Anaerobes
Yeasts/Fungi
Mycobacteria
Pediatric patients

Automated method
Various types of bottles
available
to isolate a range of
organisms
i.e. aerobic, anaerobic,
Mycobacterium

The system detects Carbon

Dioxide production this
indicates presence
of respiring organisms
False positives can arise
from blood samples with
high WBC count

colorimetric sensor and

reflected light in Bact
Alert
microorganisms are present in the

If
sample, CO2 is produced as the organisms
metabolize the substrate in the culture
medium.
When
the
growth
of
the
microorganisms produces CO2, the gaspermeable, liquid emulsion sensor installed
in the bottom of each culture bottle
changes from a blue-green to yellow color.
Positive bottles are determined by one of
three software algorithms and are indicated
as a yellow sensor and high reflectance
units. Negative bottles result from darker

Bactalert

 Some media contain resins

in order to absorb
antibiotics
Resins are non-ionic and cationic

exchangers that neutralize many different

antibiotics which might be present due to
the pretreatment of the patient
help to lyse blood cells so that intracellular organisms are set free
provide the organisms with growthcentres to
enhance speed and recovery rate

Manual method

Biomerieux : Hemoline Performance DUO (HEMOLINE DUO

Diphasic bottle

Combination of:

An agar slope covering one side of the bottle

40ml of broth

Both agar and broth contain growth factors, peptones, yeast

extract, hemin, NAD and vitamins
Broth contains anticoagulant (SPS: Sodium Polyanethol
Sulfonate)
Atmosphere inside bottle is enriched with Carbon Dioxide
and maintained at low pressure to create partial vacuum
Allows growth of main aerobic micro-organisms that
commonly cause sepsis
Rubber stopper and green screw cap present so easy to
directly inoculate bottle using a needle and open positive
bottles

Diphasic bottle

Using aseptic technique, the broth is inoculated

with 10-12ml of blood and mixed
The bottle is tipped to inoculate the agar slope
(do not tip blood into top of bottle)
Bottle is incubated (upright) for 7 days in total
(4 weeks if Brucellosis is suspected)
Bottle examined 2-3 times daily for colonial
growth on agar slope, broth turbidity,
haemolysis or a deposit
Colonies are usually visible where broth is in
contact with agar slope
If bottle appears negative tip the bottle to
inoculate agar slope again and re-incubate

Anaerobic bottle

80ml of broth enriched with growth factors,

peptones, reducing agents and added
anticoagulant (SPS: Sodium Polyanethol Sulfonate)
Allows growth of anaerobic micro-organisms that
commonly cause sepsis
Atmosphere inside the bottle is enriched with
Carbon Dioxide and Hydrogen and is maintained at
low pressure to create partial vacuum
Rubber stopper and red screw cap present so easy
to directly inoculate bottle using a needle and open
positive bottles

Anaerobic bottle
Using aseptic technique, the broth
is inoculated with ~5ml of blood
and mixed
Do not tip broth into lid of bottle
Bottle is incubated for 14 days in
total
Bottle examined 2-3 times daily for
turbidity, haemolysis or a deposit

Day 1
Blood culture bottles are aseptically
inoculated with blood samples and arrive in
laboratory
Diphasic bottle is tipped to inoculate agar
slope
Incubate both bottles upright at 37C
Incubate aerobic bottles for 7 days in total
(4 weeks if Brucellosis is suspected)
Incubate anaerobic bottles for 14 days in
total

Day 2
Diphasic bottle
Examine agar slope for colonies
Examine broth for turbidity, haemolysis or a deposit
Subculture on Blood agar and MacConkey agar
(even if there is no growth) and incubate at 37C in
aerobic atmosphere for 48hours in total
Subculture on Chocolate agar and incubate at 37C
in Carbon dioxide atmosphere for 48 hours in total
Perform Gram stain
Perform direct antibiotic / antifungal sensitivity
testing

Day 2
Anaerobic bottle:
Examine broth for turbidity, haemolysis or a deposit
Subculture to Blood agar and MacConkey agar and
incubate in aerobic atmosphere at 37C for 48
hours
Subculture to Chocolate agar and incubate at 37C
in Carbon Dioxide atmosphere for 48 hours
Subculture to Blood agar and incubate at 37C in
anaerobic atmosphere for at least 48 hours
Perform Gram stain / Toluidine blue
Perform direct antibiotic / antifungal sensitivity
testing

Day 3
Both bottles:
Examine subcultures for likely
pathogens
Perform appropriate identification tests
e.g. catalase, coagulase, oxidase, gram
etc
If direct sensitivities are poor, perform
antibiotic / antifungal sensitivity testing
on organism(s) grown on subcultures

Possible contaminants

Blood does NOT have a normal microbial flora

Aseptic technique MUST be used when taking
blood sample, adding it to the bottles and when
processing positive bottles in the laboratory

Common environmental organisms include:

Bacillus species
Acinetobacter species

Common skin microbial flora include:

Coagulase-negative staphylococci
Viridans streptococci
Micrococci
Corynebacterium species

Ways to prevent
contamination of blood
bottles
Sterilise patients culture
skin with alcohol wipe
or iodine solution before

taking blood sample

Use a sterile needle to take sample
The needle should not touch any other surface before sample is taken
The top of the bottles should be sterilised with alcohol wipe or iodine
solution before sample is added
The needle should not touch any other surface before adding sample
to bottles
The top of the bottle should be sterilised with alcohol wipe or iodine
solution after adding sample
When tipping the bottle to inoculate the agar slope do not tip broth
into the lid
Keep the bottle upright at ALL times
When opening a positive bottle, ALWAYS use aseptic technique i.e.
flame top of bottle when opening and closing bottle, flame loop
before and after use etc

Any Questions?