Blood Culture

(Manual System)

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 Definition
• Collection and inoculation of blood into culture
medium with the aim of growing pathogenic
microorganism for diagnostic purposes specially in
case of bacteremia, fungemia or septicemia.

• An etiological diagnosis of bacteremia by aerobic

and anaerobic cultivation of the blood, with
identification and susceptibility test of the isolated
organism(s).

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 Types of Bacteremia
• Primary Bacteremia – blood stream bacterial invasion
with no preceding or simultaneous site of infection with
the same microorganism.
It may be transient (e.g. following dental procedures,
vigorous chewing,instrumentation of genitourinary tract),
Intermittent (e.g. undrained abscesses), or
Continuous (e.g. endovascular infection).

• Secondary Bacteremia – isolation of a microorganism

from blood as well as other site(s)

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 Bacteremia Complication

• Warm shock – fever, increased pulse, hyperventilation, and

warm, dry flushed skin
• Cold shock – decreased blood pressure, increased pulse, and
rapid, shallow respirations
• Septic chock
• Hemodynamic changes, decreased tissue perfusion and
compromised organ & tissue function
• Mortality 40% to 50%

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 Sources of Bacteremia Spread

• Pericarditis and Peritonitis

• Pneumonias
• Pressure sores
• Prosthetic medical devices
• Total hip replacement
• Skeletal system
• Skin and soft tissue

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Organisms commonly isolated
from blood cultures
• S. aureus
• E. coli
• CONS
• Enterococcus spp.
• C. albicans
• P. aeruginosa
• Viridians streptococci
• S. pneumoniae
• • Proteus spp.

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 Indication of blood culture
• Where the possibility of septicemia or bacteremia is
suggested by the presence of fever, shock or other signs and
symptoms occurring in association with a known or
suspected local infection such as sepsis in a surgical wound,
Puerperal sepsis, Pneumonia, Bacterial Meningitis,
Endocarditis, Osteomyelitis, Enteric fever
• Pyrexia of unknown origin (temperatures of >38.3°C
(>101°F) on several occasions with fever of >3 weeks and
failure to reach a diagnosis despite 1 week of inpatient
investigation. )
• Unexplained leukocytosis or leucopenia
• Suspected fungemia especially in Immunocompromised
patients, HIV patients

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 Types of Blood Culture
• There are basically two blood culture systems:
manual and automated systems.
• In either system, certain factors are taken into
consideration if the blood culture, in the presence of
infection, will yield positive result:

Volume of blood in the medium

Dilution of blood in the medium

Duration of incubation.

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 Blood Volume
• For most systems, 10 – 20ml of blood from adults is taken and
distributed equally into the aerobic and anaerobic blood culture
bottles containing 100ml of broth.
However each system indicates the volume of blood required.
• Amount of blood for infant and children: 1-5 ml
• 1-2 ml= neonate
• 2-3ml= 1 month-2 year age
• 3-5ml= Older children

• To eliminate or minimize the effects of antibody, complement and

other antibacterial substances present in the blood, an optimal
dilution of blood in the liquid blood culture media is necessary.
• Usually 1:10 to 1:20 dilution is adequate.

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 Conventional Broth Systems
• One aerobic bottle and one anaerobic bottle per blood
collection
• Aerobic broth contains soybean casein digest broth, Tryptic
or trypticase soy broth, Brucella agar or Columbia broth base
• Anaerobic broth is usually the same as aerobic with
addition of 0.5% cysteine in an aerobic environment
• Must be sub-cultured and gram stained manually

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 Specimen Collection
• During blood culture collection all precautions should be taken
to minimize the percentage of contaminated blood culture. To
reduce the chance of contaminating organisms from the skin
the vein puncture site should ideally be prepared as follows;
Wash with soap, rinse with sterile water or saline.
Apply 1-2 % tincture of iodine or povidone –iodine and allow
drying for 1-2 minutes.
Remove the iodine with 70 % alcohol wash, if the site again
be palpated after the iodine – alcohol preparation the finger
must be disinfected or sterile gloves worn.
A tourniquet is applied to the upper arm above the vein
puncture site to distend the antecubital veins.

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Remove
Remove Flip
Flip Caps
Caps from
from the
the tops
tops of
of the
the selected
selected
culture
culture bottles.
bottles. Disinfect
Disinfect the
the septa
septa of
of the
the bottles
bottles
with
with alcohol
alcohol or
or iodine
iodine preparation
preparation and
and allow
allow to
to dry.
dry.

Perform venipuncture with syringe and collect the

desired amount of blood. If the vein is missed a
new needle should be used.

Transfer the
Transfer the recommended
recommended amount amount ofof blood
blood into
into
the culture
the culture bottles
bottles using
using aseptic
aseptic technique
technique ifif
desired. First
desired. First fill
fill the
the aerobic
aerobic bottle.
bottle. Do
Do not
not overfill
overfill
the bottles!
the bottles!Any
Any remaining
remaining blood
blood may
may be
be used
used for
for
additional tests.
additional tests.

Label the
Label the bottles
bottles according
according to to the
the routine
routine
procedure.
procedure. When
When using
using aa sticker
sticker dodo not
not cover
cover the
the
tear-off
tear-off section
section of
of the
the barcode
barcode label
label ..

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 Timing of blood culture
• Before starting antimicrobial therapy
• At the time of fever peak
• Minimum 30-60 minute interval between 2 samples except
in critically ill septic patient.
• In continuous bacteremia, timing of blood culture is not
important, but in intermittent bacteremia 2 or 3 culture
should be spaced an hour apart.

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 Blood Culture Bottles

 Tryptic
Tryptic soy
soy broth
broth (TSB)
(TSB)  Fluid
Fluid thioglycolate
thioglycolate medium
medium
•• Pancreatic
Pancreatic digest
digest of
of casien.
casien. (FTM)
(FTM)
•• Enzymatic
Enzymatic soy
soy digest.
digest. •• Pancreatic
Pancreatic digest
digest of
of casien.
casien.
•• Sodium
Sodium chloride.
chloride. •• Enzymatic
Enzymatic soy
soy digest.
digest.
•• Dipotassium
Dipotassium phosphate.
phosphate. •• Sodium
Sodium chloride.
chloride.
•• Dextrose.
Dextrose. •• Dipotassium
Dipotassium phosphate.
phosphate.
•• Sodium
Sodium polyanethol
polyanethol •• Dextrose.
Dextrose.
sulphonate(SPS)
sulphonate(SPS) •• Sodium
Sodium
polyanetholsulphonate(SPS)
polyanetholsulphonate(SPS)
•• Sodium
Sodium thioglycolate
thioglycolate agar.
agar. 15
Blood Culture Bottles

AAsetset
ofof blood
blood culture:
culture:
one
one aerobic
aerobic bottle
bottle and
and
one
one anaerobic
anaerobic bottle.
bottle.

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Sodium polyanetholsulphonate (SPS)

• The anticoagulant in blood culture medium must not harm

the bacteria and must prevent clotting of the blood, which
entrap bacteria and prevent their detection.

• The most commonly used preparation in blood media is

0.025% to 0.05% SPS.

• In addition to its anticoagulant properties, SPS is also

anticomplementary, antiphagocytic, and inactivates the
certain antibiotics like gentamycin, kanamycin,
streptomycin, polymyxin B.

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 Manual Blood Culture System
• Blood collected for culture is transported to the laboratory
without delay for immediate incubation, usually in an
atmosphere enriched with CO2 .
• Generally, incubation is for 7 days at 370C.
• Extended incubation period is required for certain disease
conditions e.g. endocarditis for 14 days and brucellosis for 21
days.

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 Examination of blood cultures
• Macroscopy: Blood cultures are examined daily for any
physical evidence of growth.
Bacterial growth may be indicated by Turbidity, Gas formation
and Hemolysis

In the absence of physical signs of growth, the blood culture is

given a gentle shake and then returned to the incubator. If
Castaneda bottle is used, the bottle should be tilted so that the
broth covers the agar surface.
Castañeda Bi-phasic medium
The bi phasic system is feasible and practical method ,
it has the advantage of repeated exposure of agar
medium to actively proliferating organisms in the
liquid broth during sub culturing, which is simply by
tilting the bottle.
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 Sub-culture
• Inspection and subculture of blood cultures are carried out
after 24 hours and 48 hours incubation.

Some workers may prefer to further subculture on the third

and fifth day of incubation.

Daily inspection for signs of growth is done until the seventh

day when the final subculture is done.

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Blind Sub-culturing syringe and drip
method

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Blood Culture Bottles Incubator

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Microscopy
If there is any sign of bacterial growth, a smear is
made for Gram stain.

Perform a direct sensitivity test based on the results

of the Gram stain. Identify the isolate.
 
Reporting
Report negative blood cultures at 48 hours and on
the 7th day (Final report)
All positives with full identification of the pathogen
and appropriate antibiogram should be reported to
the requesting physician or to the ward/clinic.

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 Interpretation of positive blood
cultures
• Virtually any organism, including normal flora, can cause
bacteremia.
• A negative culture result does not necessarily rule out
bacteremia; false-negative results occur when pathogens fail to
grow.
• A positive culture result does not necessarily indicate
bacteremia; false-positive results occur when contaminants
grow.
• Gram-negative bacilli, anaerobes, and fungi should be
considered pathogens until proven otherwise.
• The most difficult interpretation problem is to determine
whether an organism that is usually considered normal skin
flora is a true pathogen.
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