TOPIC: BLOOD CULTURE BATCH 2024

BLOOD CULTURE
Why do we need to do blood culture?
1.to detect the presence of organisms in the blood
2.to establish the phase of the infection
3.to determine the effectiveness of antibiotic
treatment
4.to assess the prognosis of the patient
How many blood samples should be obtained?
1. Adult patients with severe life-threatening clinical
septicemia: four (4) blood cultures, two from each
arm, collected immediately before treatment
2. Patients with suspected sub-acute bacterial
endocarditis or low-grade intravascular infection:
four blood cultures over a 24-hour period and spread
at intervals spaced no closer than one hour
• 2 collected at earliest sign of febrile episode
3. Patients with suspected bacteremia of unknown
origin and already on therapy: four to six blood
samples collected within the first forty-eight hours
• If patient's therapy cannot be suspended for
a few days, samples are taken immediately
before the next dose of the antimicrobial
agent being used
In general, blood should be collected for culture
before beginning antimicrobial therapy when any
one or a combination of the following are present in
the patient, namely;
1. Fever 38°C
2. Hypothermia < 36°C
3. Leukocytosis with shift to the left
4. Granulocytopenia
5. Hypotension
What culture media are used?
• Tryptic soy broth for aerobic organisms;
thioglycolate medium for anaerobic organisms
• Vacutainer culture tube with supplemented
peptone broth - minimize contamination;
grows both aerobes & anaerobes
• Usually, five mL of blood is added to forty-five
mL of the bottled medium
• Addition of adequate amounts of CO₂
essential to virtually all bacteria.

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 TOPIC: BLOOD CULTURE BATCH 2024

How much blood should be obtained? 4. Incubate the cultures aerobically at 35°C for 24
• In pediatric patients, the volume of blood hours. Observe, record, and submit the results.
drawn from small children is determined by
the physician
• In infants, one to two mL of blood and two
blood cultures are usually sufficient to
diagnose neonatal sepsis.
• In general: 1 to 5 mL for infants and children
• Adults: 20-30 mL per culture
Things to Remember:
1. Use 2 sterile needles
• one for blood collection
• replaced by another for delivery of the blood
into the rubber capped blood culture medium
2. Proper skin preparation: wash with soap and water,
dry, swab with 70% isopropyl alcohol followed by
concentric application of 3.5% tincture of iodine
solution
3. Aseptic collection of blood
4. Addition of anticoagulant to prevent fibrin clot
(sodium polyanetholsulfonate/SPS or sodium
amylosulfate/SAS)
• Fibrin clot may trap the organisms → delay or
prevent growth of the organism
5. Giving of antibiotics prior to collection can result to
a negative blood culture.
Factors Influencing Recovery of Organisms from
Blood: Evidence of Growth
1. Number of bacteria present 1. “Cotton ball” formation
2. Phagocytosis • The microbe binds immunoglobulins and
→ May continue for 30 minutes after collection agglutinates with or coagulates blood and
3. Complement moderated antibody plasma
4. Antibiotic therapy 2. Hemolysis
5. Media requirements • Lysis of RBC in the blood due to the
6. Number of times specimens are collected extracellular enzymes produced by certain
→ If multiple blood cultures are collected as a series, bacterial species
contaminants are usually recovered from one • Hemolysins - Extracellular enzymes produced
blood culture only by the bacteria; radially diffuse outwards from
Procedure the colonies, causing complete or partial lysis
1. Obtain a sample of blood from the counter and of the RBC
record the number of the sample. The sample has • Due to β-hemolytic Streptococci
been “seeded” with a single organism. 3. Bubble formation
2. With sterile 10-mL pipettes, obtain 8 mL of citrated • Due to Catalase-positive bacteria (e.g., strict
blood. aerobes, facultative anaerobes) which have
3. Deliver 5 mL of blood to a flask of tryptic soy broth the ability to respire using oxygen as a
and 2 mL to a tube of thioglycolate medium. terminal electron acceptor.

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 TOPIC: BLOOD CULTURE BATCH 2024

• Catalase-negative bacteria may be anaerobes, • Gram negative rods: gas bubbles, cloudy,
or they may be facultative anaerobes that only hemolysis
ferment and do not respire using oxygen as a • Bacillus and Fungi: hemolysis of RBC's with a
terminal electron acceptor (e.g., thick pellicle on top
Streptococci). • Haemophilus: may not exhibit any visual signs
4. Uniform turbidity at all
• Continuous agitation impacts ease of visual
growth detection → turbidity will increase
due to mixture with the red blood cells
5. Pellicle formation
• Certain aerobic bacteria and those containing
fimbriae (Vibrio & Bacillus) are known to grow
as a thin film called ‘surface pellicle’ on the
surface of undisturbed broth
• Consists of at least three distinctive steps:
➔ Initial attachment of bacteria to the solid
surface (wall of culture device) at the
Signs of growth in blood culture bottles. (A) pellicle
interface between air and liquid
formation on surface; (B) gas production; (C)
➔ Development of the monolayer pellicle
turbidity (left bottle: no growth; right bottle:
initiated from the attached cells
turbidity); (D) puff balls
➔ Maturation of pellicles with characteristic
three dimensional architecture
• In addition to cells, a variety of components,
mainly extracellular polymeric substances
(EPS), are needed for developing and
maintaining the pellicle matrix.

A) Strain negative for pellicle formation (turbid

culture); B) Strain forming a pellicle on the top of the
liquid media (culture broth transparent); C) Inverted
tube to examine pellicle strength.
Appearance of (+) Blood Culture Bottles:
• Streptococcus pneumoniae: may have a green
tint to the media.
• Beta hemolytic Strep: (+) hemolysis
• Staphylococci: "cotton balls" on the bottom
of the bottle or a jelly-like consistency in the
media due to coagulase.

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