One of the most important functions of the clinical microbiology laboratory is the detection and characterization of organisms causing bloodstream infections The laboratory detection of bacteremia and fungemia using blood cultures is one of the most simple and commonly used investigations to establish the etiology of bloodstream infections. Rapid, accurate identification of the bacteria or fungi causing bloodstream infections provides vital clinical information required to diagnose and treat sepsis.

1. BLOOD CULTURE
AS AN IMPORTANT DIAGNOSTIC
TOOL IN MEDICAL MICROBIOLOGY
BY
OSAYANDE CHELSEA IMUETINYANOSA
A SEMINAR PRESENTATION IN PARTIAL FULFILMENT FOR INTERNSHIP
IN MEDICAL MICROBIOLOGY, NATIONAL HOSPITAL ABUJA.
SEMINAR SUPERVISOR: SCT. NDIKE U.P. (PMLS)
CO-ORDINATOR: SCT. AKHIGBE ALEX (CMLS)
13TH JANUARY, 2023

2. OUTLINE
INTRODUCTION
DEFINITION OF TERMS
WHAT IS BLOOD CULTURE
HISTORY
SAMPLE COLLECTION
LABORATORY ANALYSIS
INSTRUMENTATION
QUALITY CONTROL
CONCLUSION
RECOMMENDATION
REFERENCES

3. INTRODUCTION
One of the most important functions of the clinical microbiology laboratory is the
detection and characterization of organisms causing bloodstream infections
(Gonzalez et al., 2020).
The laboratory detection of bacteremia and fungemia using blood cultures is one of
the most
simple and commonly used investigations to establish the etiology of bloodstream
infections.
Rapid, accurate identification of the bacteria or fungi causing bloodstream infections
provides vital clinical information required to diagnose and treat sepsis (CLSI
document M47-A, 2007). `

4. DEFINITION OF TERMS
Bacteremia
• The presence of bacteria in the blood. It may be transient,
intermittent or continuous. he presence of bacteria in the
blood. It may be transient, intermittent or continuous
Fungemia:
• The presence of fungi in the blood
Septicemia:
• Clinical syndrome characterized by fever, chills, malaise,
tachycardia, etc. when circulating bacteria multiply at a rate
that exceeds removal by phagocytosis
(Singer et al., 2016).

5. WHAT IS A BLOOD CULTURE?
A blood culture is a laboratory test in which blood, taken from the patient, is inoculated
into bottles containing culture media to determine whether infection-causing
microorganisms (bacteria or fungi) are present in the patient’s bloodstream (Singer et
al., 2016).
Blood cultures are intended to:
Confirm the presence of microorganisms in the bloodstream
 Identify the microbial etiology of the bloodstream infection
Help determine the source of infection (e.g., endocarditis)
Provide an organism for susceptibility testing and optimization of antimicrobial therapy
(Singer et al., 2016).

6. HISTORY
Manualithic
(pre-1970)
• Dr. Jean Antoine
Villemin déveloped
biological blood
culture
• recognition of
“Liquoid” as a
potent anticoagulant
Bactecene
(1970 to 1990
• Automated blood
culture systems first
became available in
the 1970s
• 1984 a new
generation of
BACTEC
instruments was
released that
used spectrophoto
metry to detect
CO2.[
Continuous
Monitorassic
(1990 to 2000)
• BacT/Alert
colorimetric
microbial detection
system in 1990
• Bactec 9000 series
• ESP
Ampliaissance
(post-2000) ages
• Molecular based
assays or high-
resolution
spectrometry
• Increase speed of
detection
(Ombelet et al., 2019)

7. WHEN SHOULD A BLOOD CULTURE BE PERFORMED?
Blood cultures should always be requested when a bloodstream infection or sepsis is
suspected.
Clinical symptoms in a patient which may lead to a suspicion of a bloodstream
infection are:
undetermined fever (≥38°C) or hypothermia (≤36°C)
shock, chills, rigors
severe local infections (meningitis, endocarditis, pneumonia, pyelonephritis, intra-
abdominal suppuration).
abnormally raised heart rate
low or raised blood pressure
raised respiratory rate
Baron (et al., 2005).

8. SAMPLE COLLECTION

9. BLOOD CULTURE MEDIUM
FIGURE 8 | Dissection of blood culture bottle (Ombelet et al., 2019).

10. MEDIA FORMULATIONS
 Base :
soybean casein digest (trypticase soy broth)
supplemented peptone broth
brain heart infusion broth
Anticoagulant :
sodium polyanethol sulfonate (SPS)
Headspace Atmoshere:
CO2 and N2 for anaerobic bottles and ambient
air supplemented with CO2 for aerobic bottles
Additives
Reducing agent
Polymeric resin beads
Yeast extract
(Gross et al., 2018).

11. PREVENTING CONTAMINATION OF BLOOD CULTURES
Disinfect Skin sites for collection with an
alcohol containing disinfectant.
blood should not be collected from an
intravascular device
use of commercial diversion devices
(Doern et al., 2019).
Figure: steripath gen2 initial specimen
diversion device

12. ORDER OF DRAW
If using a winged blood collection set, then the
aerobic bottle should be filled first to prevent transfer
of air in the device into the anaerobic bottle.
If using a needle and syringe, inoculate the
anaerobic bottle first to avoid entry of air.
If the amount of blood drawn is less than the
recommended volume*, then approximately 10 ml of
blood should be inoculated into the aerobic bottle
first, since most cases of bacteremia are caused by
aerobic and facultative bacteria (Ombelet et al.,
2019).

13. WHAT VOLUME OF BLOOD SHOULD BE COLLECTED?
The American Society for Microbiology (ASM) and the Infectious Diseases Society of
America (IDSA) jointly recommend 2 to 4 collections per septic episode.
 Blood culture bottles are designed to accommodate the recommended blood- to-
broth ratio (1:5 to 1:10) with optimal blood volume.
ADULTS
for adults, 40 to 60 ml of blood collected from the patient for the 4 to 6 bottles, with 10
ml per bottle.
PEDIATRIC
The optimal volume of blood to be obtained from infants and children is less well
prescribed. The recommended volume of blood to collect should be based on the
weight of the patient (Mosta et al., 2017).

14. Weight of
patient
Patient’s total
blood volume
(ml)
Recommended
volume of blood
for culture (ml)
Total volume
for culture (ml
)
% of patient’s
total blood
volume
kg Ib Culture
no.1
Culture
no.2
≤1 ≤2.2 50-99 2 2 4
1.1-2 2.2-4.4 100-200 2 2 4 4
2.1-12.
7
4.5-27 >200 4 2 6 3
12.8-3
6.3
28-80 >800 10 10 20 2.5
>36.3 >80 >2,200 20-30 20-30 40-60 1.8-2.7
Table 1: Blood volumes suggested for cultures from infants and children
(Kellog el al., 2000).

15. LABORATORY ANALYSIS
BLOOD CULTURE SYSTEMS
INCUBATION
PROCESSING POSITIVE BLOOD CULTURE
INSTRUMENTATION
CONTAMINANT OR TRUE PATHOGEN
QUALITY CONTROL

16. BLOOD CULTURE SYSTEMS
MANUAL SYSTEMS
The bottles are visually examined for indicators of microbial growth, which might
include cloudiness, the production of gas, the presence of visible microbial
colonies, or a change in colour from the digestion of blood, which is
called hemolysis.
Conventional broth based system
Biphasic systems such as hemoline and septi-check system
Displacement (signal) system
 lysis centrifugation (isolator) system to culture blood for molds and mycobacteria.
(Mosta et al., 2017).

17. Figure: Signs of growth in blood culture bottles. (A) pellicle formation on surface; (B) gas
production; (C) turbidity (left bottle: no growth; right bottle: turbidity); (D) puff balls (Ombelet
et al., 2019).

18. BLOOD CULTURE SYSTEMS contd
CONTINUOUS-MONITORING BLOOD CULTURE SYSTEM (CMBCS).
BacT/ALERT
Colorimetric change (at the bottom of the bottle) caused by drop in pH from
increased CO2 levels
BACTEC
Change in fluorescence caused by a drop in pH from increased CO2 levels
VersaTREK
Measures pressure changes caused by gas consumption or production (Doern et
al., 2019).

19. INSTRUMENTATION
Figure: BacT/ALERT, BACTEC, VersaTREK (Singer et al., 2016).

20. Figure : Clinically significant isolates per day demonstrating recommended days of incubati
on (Bourbeau and Foltzer , 2005).

21. PROCESSING POSITIVE BLOOD CULTURES
STAINING METHODS
• Gram stain
• Acridien orange
IDENTIFICATION
• subculture
• Molecular testing
• Mass spectrometry
MALDI-TOF MS),
DIRECT
IDENTIFICATION
• Accelerate phenotest
BC kit
• BD phoenix and
biome ́rieux VITEK2
systems
• Rapid AST (RAST)
method based on
direct disk diffusion
(ddd) testing
• Enzyme-based
targeted AST
(Singer et al., 2016).

22. CLINICAL EVIDENCE OF BACTERIAL INFECTION IS PRESENT
features of SSTI, urosepsis, pneumonia, SIRS criteria).
Detection of the same pathogen in blood culture sets from different venipuncture
sites
Pathogens that rarely indicate contamination:
oS. aureus
oS. pneumoniae
oS. pyogenes
oEnterobacteriaceae spp. including E. coli
oH. influenzae
oP. aeruginosa
oCandida spp
oListeria monocytogenes
oNeisseria meningitidis
oAnaerobic Gram-negative rods
(Doern et al., 2019).

23. Figure: bacteria and yeast cells (Hall and Lyman, 2006).

24. CONTAMINANT OR A TRUE PATHOGEN?
A false positive is defined as growth of bacteria in the blood culture bottle that were
not present in the patient’s bloodstream, and were most likely introduced during
sample collection.
Contamination can come from a number of sources:
The patient’s skin,
The equipment used to take the sample,
The hands of the person taking the blood sample, or
The environment.
Certain microorganisms such as coagulase-negative staphylococci, viridans- group
streptococci, Bacillus spp, Propionibacterium spp., diphtheroids, Micrococcus spp.
rarely cause severe bacterial infections or bloodstream infections. These are common
skin contaminants (Hall and Lyman, 2006).

25. NEGATIVE BLOOD CULTURE
Before ruling out bacteremia, consider the following steps:
Determine whether it is likely the result could be a false-negative
Consider if:
Collection performed during current antibiotic treatment
Insufficient inoculation
 volume of each blood culture
Prolonged transportation time
Assess whether the pathogen identified is a plausible cause of the presenting
infection.If necessary, arrange for new blood culture sampling and analysis (Doern et
al., 2019).

26. CONCLUSION
Blood cultures allow the identification of pathogens, such as bacteria and fungi, in
blood and their specific resistance testing. Knowing the pathogen enables effective,
individualized antibiotic treatment, which reduces mortality, improves prognosis,
minimizes the length of hospital stays, and reduces the growing number of antibiotic-
resistant pathogens

27. REFERENCES
 Baron EJ, Weinstein MP, Dunne Jr. WM, Yagupsky P, Welch DF, Wilson DM. Cumitech 1C,
Blood Cultures IV. Coordinating ed., E.J. Baron. ASM Press, Washington D.C. 2005.
Bourbeau PP, Foltzer M. Routine incubation of BACT/ALERT* FA and FN blood culture bottles
for more than 3 days may not be necessary. J Clin Microbiol. 2005;43:2506-250
Doern GV, Carroll KC, Diekema DJ, et al. A comprehensive update on the problem of blood
culture contamination and a discussion of methods for addressing the problem. Clin Microbiol
Rev 2019;33(1). e00009-19
Doern GV, Carroll KC, Diekema DJ, et al. Practical Guidance for Clinical Microbiology
Laboratories: A Comprehensive Update on the Problem of Blood Culture Contamination and a
Discussion of Methods for Addressing the Problem. Clin Microbiol Rev. 2019; 33
(1). doi: 10.1128/cmr.00009-19
Gonzalez, M. D., Chao, T., & Pettengill, M. A. (2020). Modern Blood Culture. Clinics in
Laboratory Medicine. doi:10.1016/j.cll.2020.07.001
Hall KK, Lyman JA. Updated Review of Blood Culture Contamination. Clin Microbiol Rev.
2006,19(4):788.

28. REFERENCES
https://www.slideshare.net/MostafaMahmoud76/manual-blood-culture-techniques
Kellog JA, Manzella JP, Bankert DA. Frequency of low-level bacteremia in children from birth
to fifteen years of age.J Clin Microbiol. 2000;38:2181-2185.
Ombelet S, Barbé B, Affolabi D, Ronat J-B, Lompo P, Lunguya O, Jacobs J and Hardy L
(2019) Best Practices of Blood Cultures in Lowand Middle-Income Countries. Front. Med.
6:131. doi: 10.3389/fmed.2019.00131
Principles and procedures for Blood Cultures; Approved Guideline, CLSI document M47-A.
Clinical and Laboratory Standards Institute (CLSI); Wayne, P.A. 2007
Singer M, Deutschmann CS, Seymour CW, et al. The Third International Consensus
Definitions for Sepsis and Septic Shock (Sepsis-3). JAMA. 2016;315(8):801-810.
Singer M, Deutschmann CS, Seymour CW, et al. The Third International Consensus
Definitions for Sepsis and Septic Shock (Sepsis-3). JAMA. 2016;315(8):801-810.

29. Thank You For
Listening