Technical Data #3000a / 2005.09.01
The culturing of blood for the detection of microorganisms is an important function of the clinical microbiologist since it enables the diagnosis of bacteremia. Blood Culture Bottles are used for organism detection in blood samples. Blood specimens may be injected into partially evacuated Prepared Culture Bottles using a syringe or directly from the patient using the QUELAB closed-system Blood Taking Unit.
FORMULA
Many different types of bacteria and fungi have
been identified as causative agents of septicemia. For this reason, many diverse
culture media formulations are available in Prepared Blood Culture Bottles. The majority
of these media contain 0,03% SPS (Sodium Polyanetholesulfonate), a polyanionic anticoagulant,
which additionally inhibits complement and lysozyme activity, interferes with phagocytosis
and inactivates aminoglycosides. Carbon dioxide in Prepared Blood Culture Bottles
is stimulatory for many microorganisms. The following media formulations are available
as specified:
PRECAUTIONS, STORAGE AND SIGN OF DETERIORATION
Prepared Blood Culture Bottles are for IN VITRO diagnostic use only. Store Prepared
Blood Culture Bottles at room temperature, protected from direct light. Media should
not be used if the expiry date has passed. Prepared Blood Culture Bottles should
not be used if there are signs of contamination.
PROCEDURE
Rigourous disinfection of the skin using bactericidal agents,
such as 70% isopropyl alcohol followed by 1% to 2% tincture of iodine or by an iodophor,
such as 10% Providonme-Iodine Solution, should be performed after palpation of the
site. The use of a closed collection system, consisting of a sterile Blood Taking
Unit (tubing with needles on both ends) and an evacuated bottle containing culture
medium, is preferable to collection using a sterile syringe.
Each bottle contains a rubber stopper held in place by a topped screw cap surrounded
by a plastic band. The rubber stopper must not be taken off the bottle. Blood is
added by puncturing the rubber stopper with the needdle attached to the syringe containing
the blood specimen or with the needle of the Blood Taking Unit.
Blood specimens of 2, 5 or 10ml generally are added to bottles containing 20 (pediatric),
50 or 100ml of medium to achieve a 1:10 blood medium ratio and in a two- or three-bottle
set (aerobic, anaerobic, hypertonic medium).
Biphasic bottles containing 25ml of broth medium require 5ml of blood, since there
is a total volume of 50ml including the agar slant. For increased recovery of yeast
and other fungi, 5ml are added to each of two BHI with BHIA Surface biphasic bottles.
Pediatric Blood Culture Bottles (20ml) are used for isolation of aerobic and microaerophilic
organisms when the volume of the blood sample is reduced.
The approximate amount of blood added is determined by noting the level of the
liquid in the bottle relative to the graduation marks before and after addition of
the blood. Bottles and blood-taking units are designed so that blood collection
is through a closed system. Each specimen should be cultured aerobically and anaerobically.
For aerobic growth, filtered air must be allowed to enter the bottle through a
Venting Unit after blood collection.
Following blood collection, the bottles are shaken to thoroughly mix the blood and
medium and are then labelled.
All bottles should be transported to the laboratory as soon as possible and immediately
incubated at 35 +/- 2ºC in an upright position. Biphasic bottles should be incubated
either in an upright position or on their side with the agar surface up. In this
way, colonies of bacteria may be observed on the surface of the agar after incubation.
The purpose of the biphasic bottle is to shorten the time necessary to obtain isolated
organisms for identification procedures.
Lower incubation temperatures may be preferred for the isolation of specific organisms.
Culture for the detection of fungi may be incubated at 22 to 30ºC, whereas certain
bacterial species, such as Listeria, grow well at 20 to 25ºC.
A total incubation period of 7 days is generally sufficient for routine isolation
procedures. Alternatively, the bottles can be incubated for at least 14 days before
discarding those that do not show evidence of growth. A 14-day incubation period
has been reported to be adequate for the recovery of yeast; if dimorphic fungi are
suspected to be present, blood cultures may require incubation for an additional
2 weeks.
Following inoculation and incubation of the bottles, microorganisms recovered from
the medium may be Gram stained and identified by conventional plate and identification
procedure. The first important subculture test to be performed on an isolated from
a blood culture is to investigate susceptibility of the organism to antimicrobial
agents. If anaerobes are suspected, subculture should be inoculated anaerobically.
CULTURE AND ISOLATION
The test bottles should be visually examined within 24 h and at daily intervals thereafter.
The bottles should be carefully removed from the incubator to avoid disturbing the
sedimented blood and examined for any visual evidence of microbial growth, such as
turbidity, hemolysis, gas production, or formation of discrete colonies.
Growth in biphasic bottles may be first seen on the agar surface. If no growth is
visible in the broth or on the agar surface, tilt the bottle to wash broth over the
agar surface and reincubate.
Since autolysis of some microorganisms may occur after prolonged incubation of inoculated
blood culture bottles, subcultures should be taken at various incubation intervals.
After 24- to 48-h incubation, a small quantity (0.1 to 0.5ml) of blood-broth mixture
should be removed by means of a sterile syringe and needle and subcultured to plates
of enriched and selective media. This procedure should be repeated after an incubation
period of 7 days if the culture appears negative, or earlier as growth appears.
Although growth in blood culture bottles may be manifested in a variety of appearances,
usefull information can be obtained by observing the cultures for typical appearances.If
visible evidence of growth appears, the broth should be examined by the Gram stain
and subcultured onto appropriate media for isolation and identification.
LIMITATIONS OF METHOD
Status of the administration of antimicrobial agents must be noted on test records
since this parameter may have a significant bearing on the microbiological test results.
Since blood culture may be negative due to the presence of antimicrobial substances
in the blood specimen, specimen should be taken before initiation of antimicrobial
therapy.
The size of the specimen theorically depends upon the numbers of organisms present.
But since this is generally unknown, ideally the quantity of blood tested should
be as large as possible.
In newborn or premature infants, it may be difficult to obtain volume of blood for
optimum ratios of blood medium. This lack is partially compensated for by the higher
numbers of bacteria seen in neonatal sepsis. However it is important to note that
the volume of blood cells is also important for Haemophilus influenzae and other
hemin-requiring species.
Obviously the quantity of medium should be as large as possible, to dilute any known
or unknown antimicrobial factors, including drugs and antibodies, that might be present.
The absence of an anticoagulant in the speciemn may cause the organisms present in
the blood to become trapped in the clot and go undetected, or the time required to
recover them may be greatly increased. In the collection of blood for culture, some
anticoagulants must not be employed. Ammonium oxalate is inhibitory to most bacteria.
Sodium citrate is inhibitory to many gram-positive organisms. The anticoagulant
SPS exhibits less toxicity and enables bacteria to survive for a much longer period
of time. In addition, SPS reduces the activity of several antibiotics. However, SPS
has been reported to inhibit some strains of Neisseria gonorrhoeae, Neisseria meningitidis,
Gardnerella vaginalis and Peptostreptococcus anaerobius.
The use of a single broth for a specimen is not recommended; exclusive use of closed
(anaerobic) bottles results in decreased recovery of aerobic organisms and fungi,
while the exclusive use of vented (aerobic) bottles results in decreased recovery
of obligately anaerobic organisms.
The use of a 10-ml inoculum (5ml into each of two BHI and BHI Agar Surface biphasic
bottles) and continuous venting is recommended for isolation of yeast and other fungi.
Cloudiness may develop in a negative culture as a result of frequent shaking.
Although QUELAB blood culture media are filtered to remove dead organisms and other
artefacts, culture media sometimes contain small numbers of dead organisms devived
from constituents of the medium, which may be visible in smears of uninoculated blood
culture media. Other sources of dead organisms visible upon Gram staining include
staining reagents, immersion oil, glass slides, splashover from a different sample
on the same slide and nonviable organisms present in the patient specimen.
Biphasic and pediatric culture bottles are not recommended for culture of anaerobic
microorganisms.
REFERENCES
CATALOGUE NUMBERS
Bottled culture media with 0,03% S.P.S. and CO2,
under vacuum. Packaging of 10 units per box.
| B.H.I. (Brain Heart Infusion) Broth, without SPS | 50 ml | 3301 |
| B.H.I. (Brain Heart Infusion) Broth | 50 ml | 3020 |
| 100 ml | 3040 | |
| Biphasic Media (B.H.I. Broth / B.H.I. Agar) | 50 ml | 3221 |
| Brucella Broth with 6% Sorbitol | 50 ml | 3031 |
| 100 ml | 3032 | |
| Brucella Broth with 10% Sucrose | 50 ml | 3181 |
| 100 ml | 3182 | |
| Columbia Broth | 20 ml | 3195 |
| 50 ml | 3491 | |
| 100 ml | 3121 | |
| Columbia Supplemented Cystein, Hemin, Menadione Broth | 50 ml | 3807 |
| Columbia Supplemented Yeast. Hemin, Menadione Broth | 50 ml | 3907 |
| 100 ml | 3600 | |
| Thioglycollate with indicator and without S.P.S. Broth | 50 ml | 3791 |
| 100 ml | 3269 | |
| T.S.B. (Tryptic Soy Broth) | 20 ml | 3011 |
| 50 ml | 3466 | |
| 100 ml | 3576 | |
| T.S.B. Supplemented Hemin, Menadione Broth | 20 ml | 3095 |
| T.S.B. without S.P.S. | 50 ml | 3371 |
| 100 ml | 3056 | |
| BLOOD TAKING SET | ||
| Blood taking set with venting unit, 100/cs, sterile, disposable | 100/cs | TPS-100 |
| Venting unit for aerobic blood culture | 50/bag | EV-50 |
| Venting unit for aerobic blood culture | 250/bag | EV-250 |
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