Culturing blood
By: Mazen Mohammed Qaid
Blood culture is required when bacteraemia
(septicaemia) is suspected.
Bacteraemia:
The presence of bacteria in the blood is called
bacteraemia.
Bacteraemia usually occurs when pathogens enter
the bloodstream from abscesses, infected wounds
or burns.
Septicaemia:
This is a clinical term used to describe severe life-
threatening bacteraemia in which multiplying
bacteria release toxins into the blood stream
Possible Pathogenic Bacteria isolated from
blood cultures
• Gram positive • Gram negative
1. Salmonella Typhi
1. Staphylococcus aureus 2. Other Salmonella serovars
2. Viridans streptococci 3. Brucella species
3. Streptococcus 4. Haemophilus influenzae
pneumoniae 5. Pseudomonas aeruginosa
6. Klebsiella strains
4. Streptococcus pyogenes
7. Escherichia coli
5. Enterococcus faecalis 8. Proteus species
6. Clostridium perfringens 9. Neisseria meningitidis
10. Yersinia pestis
7. Anaerobic streptococci
11. Bacteroides fragilis
-Also Mycobacterium tuberculosis (HIV-associated tuberculosis), Leptospira
species, Borrelia species, rickettsia and Candida albicans.
Commensals
• Blood is a sterile body fluid and does
not have a normal microbial flora.
• Common skin contaminants include:
① coagulase negative staphylococci
② viridans streptococci
③ micrococci
④ Corynebacterium species.
• Specimen : Whole blood
Do not refrigerate.
• Container
One aerobic and one anaerobic blood
culture bottle.
Do not vent.
Criteria of specimen rejection
① Blood collected in tubes or bottles other
than aerobic and anaerobic blood
culture bottles.
② Specimens for anaerobic blood culture
received in aerobic bottles or vice versa.
③ If the information on the label does not
match that of the request form.
Collection and transport blood specimen
• Whenever possible blood should be collected before
antimicrobial treatment has started.
Aseptic blood collection and dispensing technique
Blood must be collected and dispensed with great care to avoid
contaminating the specimen and culture medium.
1. Using a pressure cuff, locate a suitable vein in the arm. Deflate the cuff
while disinfecting the vene puncture site.
2. Wearing gloves, thoroughly disinfect the venepuncture site as follows:
– Using 70% ethanol, cleanse an area about 50 mm in diameter. Allow to air-dry.
– Using 2% tincture of iodine and a circular action, swab the area beginning at the
point where the needle will enter the vein. Allow the iodine to dry on the skin for at
least 1 minute.
3. Lift back the tape or remove the protective cover from the top of the
culture bottle(s). Wipe the top of the bottle using an ethanol-ether swab
4. Using a sterile syringe and needle, withdraw about 10-20 ml of blood
from an adult* or about 1-5 ml from children.
5. Insert the needle through the rubber liner of the bottle cap and
dispense 10–12 ml of blood into the diphasic culture medium bottle
containing 25 ml of broth When also culturing for anaerobes, dispense
about 5 ml of blood into the thioglycollate culture medium containing 50
ml of broth
Dispense the remaining approximately 2 ml of blood into a tube or bottle
containing (EDTA).
6. Using a fresh ethanol-ether swab, wipe the top of each culture bottle
and replace the tape or protective cover(s). Without delay, mix the blood
with the broth and mix the blood in the EDTA container
7. Clearly label each bottle with the name and number of the patient, and
the date and time of collection.
8. As soon as possible, incubate the inoculated media. Protect the
cultures from direct sunlight until they are incubated
Laboratory examination of blood
Day 1
1.Culture the specimen
The following media are recommended:-
● Columbia agar and Columbia broth diphasic medium with added SPS
(sodium polyanethol sulphonate), SPS prevents the blood from clotting,
neutralizes complement and other antibacterial substances in fresh blood.
Incubate at 35–37 °C for up to 7 days,(4 weeks When brucella suspcted)
● Thioglycollate broth medium
is recommended to isolate strict anaerobes. It consists of nutrient broth to
which is added thioglycollate to provide the growth of anaerobes.
No SPS in this medium b/c SPS is inhibitory to anaerobic streptococci,
therefore a sufficient volume of broth must be used to prevent the blood
from clotting . The blood should be diluted at least 1 in 10 with broth.
Incubate at 35–37 °C for up to 14 days.
2. Examine the specimen microscopically
Centrifuge a sample of EDTA venous blood or
heparinized capillary blood and make smears of
the buffy coat layers. Stain as follows:
Gram smear: To detect Gram positive and Gram
negative bacteria, particularly when the patient is
an infant or young child.
Ziehl-Neelsen smear: To detect AFB when the
patient has AIDS or suspected HIV disease.
Giemsa or rapid Field’s smear: To detect borreliae,
or parasites such as trypanosomes,malaria and
microfilariae.
Day 2
3. Examine and report the cultures.
Diphasic culture (Columbia agar and broth)
Using a hand lens, examine twice daily (up to 7
days or 4 weeks when brucellosis is suspected)
When growth is present:
Subculture on BA, Mac and Chocolate agar.
Incubate the
BA, Mac agar aerobically.
Chocolate agar in a Co2 atmosphere (candle jar)
.
Thioglycollate broth culture
Examine daily (up to 14 days) for visible signs of
bacterial growth such as turbidity above the red cell.
Subculturing a blood culture broth
A strict aseptic technique must be used to avoid
contaminating the culture.
1- Using an ethanol-ether swab, cleanse the top of the
bottle. Using a sterile needle and small syringe, insert
the needle through the rubber liner in the cap, and
withdraw about 1 ml of the broth culture.
2- Inoculate the broth on:
– BA (anaerobically , 48 hours)
– Chocolate agar (Co2, 48 hours)
– Mac (aerobically ,overnight)
Day 3
1. Staining the bacterial colonies in culture by
gram stain
2. Biochemical tests perform
3. Antimicrobial susceptibility test perform.
Using Muller Hinton agar
Day 4
1. See the biochemical tests results and record
the name of bacteria in report.
2. Measure the zone of inhibition of antibiotics
and record the report
Important:
• When a patient is seriously ill, subculture
the broth (even in the absence of visible
bacterial growth) after overnight
incubation, after 48 hours, and twice
weekly for up to 2 weeks.
