Hemo Cult I Vos 2012

M47-A
Vol. 27 No. 17
Replaces M47-P
Vol. 26 No. 31
Principles and Procedures for Blood

Cultures; Approved Guideline
This document provides recommendations for the collection, transport, and processing of
blood cultures as well as guidance for the recovery of pathogens from blood specimens
taken from patients who are suspected of having bacteremia or fungemia.
A guideline for global application developed through the Clinical and Laboratory
Standards Institute consensus process.
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M47-A
ISBN 1-56238-641-7
Volume 27 Number 17 ISSN 0273-3099
Principles and Procedures for Blood Cultures; Approved Guideline
Michael L. Wilson, MD
Michael Mitchell, MD
Arthur J. Morris, MD, FRCPA
Patrick R. Murray, PhD
Larry G. Reimer, MD
L. Barth Reller, MD
Michael Towns, MD
Melvin P. Weinstein, MD
Sybil A. Wellstood, PhD
W. Michael Dunne, Jr, PhD
Robert C. Jerris, PhD
David F. Welch, Ph, D(ABMM)
Abstract
Clinical and Laboratory Standards Institute document M47-A—Principles and Procedures for Blood Cultures; Approved
Guideline addresses the laboratory detection of bacteremia and fungemia by use of blood cultures. Included in this guideline are
recommendations for the: 1) clinical importance of blood cultures; 2) specimen collection and transportation; 3) critical factors in
the recovery of pathogens from blood specimens; 4) special topics, including pediatric blood cultures, catheter-related
bloodstream infections, infective endocarditis, patients receiving antimicrobial therapy, rare and fastidious pathogens, and test of
cure; 5) reporting results; 6) interpreting blood culture results; 7) safety issues; and 8) quality assurance.
Clinical and Laboratory Standards Institute (CLSI). Principles and Procedures for Blood Cultures; Approved Guideline. CLSI
document M47-A (ISBN 1-56238-641-7). Clinical and Laboratory Standards Institute, 940 West Valley Road, Suite 1400,
Wayne, Pennsylvania 19087-1898 USA, 2007.
The Clinical and Laboratory Standards Institute consensus process, which is the mechanism for moving a document through
two or more levels of review by the healthcare community, is an ongoing process. Users should expect revised editions of any
given document. Because rapid changes in technology may affect the procedures, methods, and protocols in a standard or
guideline, users should replace outdated editions with the current editions of CLSI/NCCLS documents. Current editions are
listed in the CLSI catalog, which is distributed to member organizations, and to nonmembers on request. If your organization
is not a member and would like to become one, and to request a copy of the catalog, contact us at: Telephone: 610.688.0100;
Fax: 610.688.0700; E-Mail: customerservice@clsi.org; Website: www.clsi.org
(Formerly NCCLS)

Number 17 M47-A
Copyright ©2007 Clinical and Laboratory Standards Institute. Except as stated below, neither this
publication nor any portion thereof may be adapted, copied or otherwise reproduced, by any means
(electronic, mechanical, photocopying, recording, or otherwise) without prior written permission from
Clinical and Laboratory Standards Institute (“CLSI”).
CLSI hereby grants permission to each individual member or purchaser to make a single reproduction of
this publication for use in its laboratory procedure manual at a single site. To request permission to use
this publication in any other manner, contact the Executive Vice President, Clinical and Laboratory
Standards Institute, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898, USA.
Suggested Citation
(CLSI. Principles and Procedures for Blood Cultures; Approved Guideline. CLSI document M47-A.
Wayne, PA: Clinical and Laboratory Standards Institute; 2007.)
Proposed Guideline
October 2006
Approved Guideline
May 2007
ISBN 1-56238-641-7
ISSN 0273-3099
ii Leal Universidad Nacional de Colombia

Licensed to: Aura
Volume 27 M47-A
Committee Membership
Area Committee on Microbiology
Mary Jane Ferraro, PhD, MPH John D. Turnidge, MD Robert P. Rennie, PhD
Chairholder Women’s and Children’s Hospital University of Alberta Hospital
Massachusetts General Hospital North Adelaide, Australia Edmonton, Alberta, Canada
Boston, Massachusetts
Michael L. Wilson, MD Thomas R. Shryock, PhD
James H. Jorgensen, PhD Denver Health Medical Center Elanco Animal Health
Vice-Chairholder Denver, Colorado Greenfield, Indiana
University of Texas Health
Science Center Advisors Jana M. Swenson, MMSc
San Antonio, Texas Centers for Disease Control and
Ellen Jo Baron, PhD Prevention
Donald R. Callihan, PhD Stanford Univ. Hospital & Medical Atlanta, Georgia
BD Diagnostic Systems School
Sparks, Maryland Stanford, California Melvin P. Weinstein, MD
Robert Wood Johnson Medical
Freddie Mae Poole Lynne S. Garcia, MS School
FDA Center for Devices and LSG & Associates New Brunswick, New Jersey
Radiological Health Santa Monica, California
Rockville, Maryland Matthew A. Wikler, MD, MBA,
Richard L. Hodinka, PhD FIDSA
John H. Rex, MD, FACP Children’s Hospital of Philadelphia Pacific Beach BioSciences
AstraZeneca Philadelphia, Pennsylvania San Diego, California
Cheshire, United Kingdom
Michael A. Pfaller, MD Gail L. Woods, MD
Daniel F. Sahm, PhD University of Iowa College of University of Arkansas for Medical
Eurofins Medinet Medicine Sciences
Herndon, Virginia Iowa City, Iowa Little Rock, Arkansas
Fred C. Tenover, PhD, ABMM

Centers for Disease Control
and Prevention
Atlanta, Georgia
Subcommittee on Principles and Procedures for Blood Cultures
Michael L. Wilson, MD Michael Towns, MD David F. Welch, PhD, D(ABMM)

Chairholder BD Diagnostic Systems University of Texas Southwestern
Denver Health Medical Center Sparks, Maryland Medical Center
Denver, Colorado Dallas, Texas
Melvin P. Weinstein, MD
Michael Mitchell, MD Robert Wood Johnson University Staff
U Mass Memorial Medical Center Hospital
Worcester, Massachusetts New Brunswick, New Jersey Clinical and Laboratory Standards
Institute
Arthur J. Morris, MD Sybil A. Wellstood, PhD Wayne, Pennsylvania
Diagnostic Medlab US Department of Agriculture
Auckland, New Zealand Riverdale, Maryland
John J. Zlockie, MBA
Vice President, Standards
Patrick R. Murray, PhD Advisors
NIH
Bethesda, Maryland W. Michael Dunne, Jr., PhD Tracy A. Dooley, BS, MLT(ASCP)
Washington University School of Staff Liaison
Larry G. Reimer, MD Medicine
University of Utah School of St. Louis, Missouri Donna M. Wilhelm
Medicine Editor
Salt Lake City, Utah Robert C. Jerris, PhD
Children’s Healthcare of Atlanta Melissa A. Lewis
L. Barth Reller, MD Atlanta, Georgia Assistant Editor
DUHS Clinical Laboratories
Durham, North Carolina
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iv Leal Universidad Nacional de Colombia

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Volume 27 M47-A
Contents
Abstract ....................................................................................................................................................i
Committee Membership........................................................................................................................ iii
Foreword .............................................................................................................................................. vii
1 Scope..........................................................................................................................................1
2 Standard Precautions..................................................................................................................1
3 Definitions .................................................................................................................................1
3.1 Acronyms......................................................................................................................3
4 Clinical Importance of Blood Cultures ......................................................................................4
4.1 Diagnostic Importance ..................................................................................................4
4.2 Prognostic Importance ..................................................................................................4
5 Specimen Collection and Transportation...................................................................................4
5.1 Timing of Blood Cultures .............................................................................................4
5.2 Number of Blood Cultures............................................................................................4
5.3 Volume of Blood for Culture........................................................................................5
5.4 Distribution of Blood Between Aerobic and Anaerobic Blood Culture Bottles ...........5
5.5 Disinfection of Skin and Prevention of Blood Culture Contamination ........................6
5.6 Blood Culture Collection ..............................................................................................6
5.7 Specimen Rejection Criteria for Blood Culture Specimens..........................................7
6 Methods and Procedures ............................................................................................................7
6.1 Critical Factors in the Recovery of Pathogens From Blood Specimens .......................7
6.2 Blood Culture Methods...............................................................................................10
6.3 Fungal Blood Cultures ................................................................................................13
6.4 Mycobacterial Blood Cultures ....................................................................................14
6.5 Special Topics.............................................................................................................15
7 Reporting Results.....................................................................................................................23
7.1 Written and Electronic Reports...................................................................................23
8 Contaminants ...........................................................................................................................26
9 Safety Issues ............................................................................................................................26

9.1 Agents Associated With Laboratory-Acquired Infections..........................................27
9.2 Protective Measures ....................................................................................................27
10 Quality Assurance....................................................................................................................31
10.1 Preexamination Process ..............................................................................................31
10.2 Examination Process...................................................................................................34
10.3 Postexamination Process.............................................................................................35
References.............................................................................................................................................36
Additional References...........................................................................................................................45
Summary of Delegate Comments and Subcommittee Responses.........................................................46

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Contents (Continued)
The Quality Management System Approach ........................................................................................50
Related CLSI/NCCLS Publications ......................................................................................................51
vi Leal Universidad Nacional de Colombia

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Volume 27 M47-A
Foreword
The incidence of sepsis continues to increase in the United States: the most recent published data indicate
that as many as 660,000 cases occur annually.1 Because the morbidity and mortality attributable to sepsis
is high, the prompt and accurate detection of bacteremia and fungemia is important for improving patient
care. The laboratory test that is used to detect the presence of bacteria (bacteremia) or fungi (fungemia) in
the blood is the blood culture.
During the past 30 years, a number of studies have been conducted to: 1) define the clinical significance
of blood cultures; 2) define the critical factors in the recovery of pathogens from the blood; 3) establish
the best medium formulations and other laboratory practices; 4) evaluate and compare commercial blood
culture systems; and 5) develop interpretive criteria. Because of the clinical importance of bacteremia and
fungemia, and therefore the importance of blood cultures, guidelines are needed so that laboratories and
providers use optimal laboratory methods and interpret the results correctly. To date there has not been a
single document that incorporates these data into consensus guidelines. Such guidelines are also needed to
help control healthcare costs, as the costs attributable to the recovery of contaminants from blood cultures
are high.
Key Words
Bacteremia, bacteria, blood culture, bloodstream infection, fungemia, fungi, mycobacteria, sepsis
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Volume 27 M47-A
Principles and Procedures for Blood Cultures; Approved Guideline
1 Scope
The laboratory detection of bacteremia and fungemia remains one of the most important functions of
clinical microbiology laboratories. During the past 30 years, a number of studies have defined the critical
factors in the recovery of pathogens from blood and the optimal laboratory methods for recovering
specific pathogens, and have established the performance characteristics of blood culture systems. Despite
this information, there remains a need for guidelines for the collection, processing, and interpretation of
blood cultures.
Several in vitro blood culture devices are cleared by the United States Food and Drug Administration
(FDA) for use in the United States. These devices typically are available for use in other countries.
This guideline is intended to provide guidance to clinical microbiologists and other laboratorians (e.g.,
pathologists, laboratory supervisors, laboratory managers) for the recovery of pathogens from blood
specimens taken from patients who are suspected of having bacteremia or fungemia. Specific
recommendations will be offered for the collection, transport, and processing of blood cultures. The
existing blood culture technology will be reviewed and the relative benefits of these technologies will be
compared. Procedures for the identification of pathogens will not be addressed. Antimicrobial
susceptibility testing of bacteria is addressed in CLSI documents M2—Performance Standards for
Antimicrobial Disk Susceptibility Tests,2 M7—Methods for Dilution Antimicrobial Susceptibility Tests for
Bacteria That Grow Aerobically,3 and M11—Methods for Antimicrobial Susceptibility Testing of
Anaerobic Bacteria.4 Antimicrobial susceptibility testing of fungi is covered in CLSI/NCCLS documents
M27—Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts5 and M38—
Reference Method for Broth Dilution Antifungal Susceptibility Testing of Filamentous Fungi.6
2 Standard Precautions
Because it is often impossible to know what isolates or specimens might be infectious, all patient and
laboratory specimens are treated as infectious and handled according to “standard precautions.” Standard
precautions are guidelines that combine the major features of “universal precautions and body substance
isolation” practices. Standard precautions cover the transmission of all infectious agents and thus are
more comprehensive than universal precautions which are intended to apply only to transmission of
blood-borne pathogens. Standard and universal precaution guidelines are available from the US Centers
for Disease Control and Prevention.7 For specific precautions for preventing the laboratory transmission
of all infectious agents from laboratory instruments and materials and for recommendations for the
management of exposure to all infectious disease, refer to the most current edition of CLSI document
M29—Protection of Laboratory Workers From Occupationally Acquired Infections.8
3 Definitions
antiseptic - a substance that inhibits the growth and development of microorganisms without necessarily
killing them.9
automated blood culture system - a blood culture system that uses mechanical systems to incubate,
agitate, and/or monitor blood culture bottles for microbial growth.
bacteremia – the presence of bacteria in the bloodstream; NOTE: Bacteria isolated from blood may be
the cause of sepsis, indeterminate as a cause of sepsis, or contaminants.10
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Institute. All rights reserved.
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biphasic blood culture system - a blood culture system in which a single container (vial) has separate
chambers for solid- and liquid-based media; NOTE: Most biphasic systems are designed so that the solid
medium can be irrigated with the liquid medium.
blind subcultures – subcultures performed as a routine laboratory procedure irrespective of any objective
evidence of microbial growth.10
blood culture – a specimen of blood that is submitted for bacterial or fungal culture; NOTE: This is
irrespective of the number of bottles or tubes into which the specimen is divided or distributed.10
blood culture series – a group of temporally related blood cultures that are collected to determine
whether a patient has bacteremia or fungemia.10
blood culture set – the combination of blood culture bottles or tubes into which a single blood specimen
is inoculated.10
bloodstream infection – an infection associated with bacteremia or fungemia.
breakthrough bacteremia – bacteremia that persists while a patient is receiving antimicrobial therapy
for an episode of bacteremia; NOTE 1: Breakthrough bacteremia that occurs early usually is the result of
inappropriate or inadequate antimicrobial chemotherapy; NOTE 2: Breakthrough bacteremia that occurs
late usually is the result of a focus of infection (e.g., an abscess) that has not been drained adequately.10
chlorhexidine gluconate – the digluconate salt of chlorhexidine9; NOTE: It is used as a topical agent for
cleansing and disinfecting the skin.
contaminant – a microorganism isolated from a blood culture that was introduced into the culture during
specimen collection or processing and that was not pathogenic for the patient from whom blood was
collected10 (i.e., the isolates were not present in the patient’s blood when the blood was sampled for
culture).
conventional (manual) blood culture system – a blood culture system that processes bottles without the
use of mechanical systems (i.e., manually).
culture – 1) the intentional growing of microorganisms, such as bacteria or fungi, in a controlled

environment, for purposes of identification or other scientific study, or for commercial and/or medicinal
use; 2) the product resulting from the intentional growth of microorganisms.
culture medium – a substance or preparation used for the cultivation and growth of microorganisms.
disinfectant – a substance used to reduce the concentration of bacteria, fungi, or viruses on a surface.
false positive – a positive test result for a disease or condition when the disease or condition is not
present; NOTE: For blood cultures, 1) a culture that yields a microbial isolate(s) that is determined not
to be the cause of sepsis, or 2) a culture with objective evidence of microbial growth (i.e., an instrument
signal that indicates microbial growth) but for which subcultures and stains are negative.
fungemia – the presence of fungi (yeasts or molds) in the bloodstream.10
inadequate blood volume – a blood culture bottle containing less than 80% of the recommended
minimum volume indicated on the bottle label.
indeterminate isolates – a microorganism of undetermined clinical importance.10

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Volume 27 M47-A
lysis-centrifugation blood culture systems - blood culture systems that utilize chemicals to lyse the
blood cells, thereby releasing microorganisms into the fluid, followed by centrifugation to concentrate the
microorganisms in the pellet; NOTE: These systems are most often used for fungal and mycobacterial
blood cultures.
manual blood culture - see conventional blood culture system.
povidone iodine – a water-soluble complex of iodine with polyvinylpyrrolidone11; NOTE 1: Applied as

an antiseptic in the form of solutions or ointments, it releases iodine; NOTE 2: Used as a topical agent for
disinfecting the skin.
sepsis – systemic inflammatory response syndrome (SIRS) plus infection.12,13
septic episode – an episode of sepsis, severe sepsis, or septic shock for which a blood culture or blood
culture series is drawn; NOTE: The blood cultures may or may not yield microorganisms that are
subsequently determined to be the cause of sepsis, severe sepsis, or septic shock.10
septic shock – sepsis with arterial hypotension despite adequate fluid resuscitation.12,13
severe sepsis – sepsis associated with organ dysfunction, hypoperfusion, or hypotension.12,13
subculture – a bacterial or fungal culture made from material derived from another culture, such as the
blood-broth mixture in blood culture bottles or a culture made by transferring microorganisms to a fresh
medium from a previous culture; a method used to prolong the life of a particular strain where there is a
tendency to degeneration in older cultures.11,14
systemic inflammatory response syndrome (SIRS) – a physiologic state believed to be triggered by

systemic activation of the innate immune response; NOTE: Under the current definition, SIRS is
considered to be present when patients have more than one of the following clinical and/or laboratory
findings: body temperature >38 °C or <36 °C; heart rate >90/minute; hyperventilation evidence by
respiratory rate >20/minute or PaCO2 <32 mm Hg; and white blood cell count >12,000 cells/µL or <4000
cells/µL.12,13
terminal subculture – a subculture taken from the blood-broth mixture in blood culture bottles at the end
of the routine incubation and testing period; NOTE: Because subcultures are done on blood culture when
there is objective evidence of microbial growth, terminal subcultures may be done as a matter of routine
on blood culture bottles lacking evidence of microbial growth (i.e., “negative” blood culture bottles).
tincture of iodine – an alcoholic solution of iodine and potassium iodide9; NOTE: It is used as a topical
agent for disinfecting the skin.
true positive – a positive test result for a disease or condition when the disease or condition is present;
NOTE: For blood cultures, a culture that yields a microbial isolate(s) that is determined to be the cause
of sepsis.
venipuncture – puncture of a vein 9; NOTE: A method used to collect blood specimens for culture.
3.1 Acronyms
CMBCSs continuous monitoring blood culture systems

CRBSI catheter-related bloodstream infection
HACEK group Haemophilus, Actinobacillus, Cardiobacterium, Eikenella, Kingella
HBV hepatitis B virus
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HCV hepatitis C virus

HIV human immunodeficiency virus
LIS laboratory information system
VAD venous access device
4 Clinical Importance of Blood Cultures
4.1 Diagnostic Importance
The presence of living microorganisms circulating in the bloodstream of a patient has substantial
diagnostic and prognostic importance.15 The positive blood culture either establishes or confirms that
there is an infectious etiology for the patient’s illness. Moreover, it also provides the etiologic agent for
antimicrobial susceptibility testing which, in turn, allows optimization of antibiotic therapy.
4.2 Prognostic Importance
From a prognostic standpoint, a blood culture that grows a clinically important pathogen indicates failure
of the host’s defenses to contain the infection at its primary location or failure of the physician to remove,
drain, or otherwise eradicate that focus of infection. The type of pathogen recovered from blood also
provides important prognostic information.15
5 Specimen Collection and Transportation
5.1 Timing of Blood Cultures
Only a few studies have tried to establish the optimal timing of blood cultures to maximize the recovery
of pathogens from blood. Experimental data show that an influx of bacteria into the bloodstream is
followed by a lag of approximately one hour before chills and fever develop.16 Although it has been
common practice to obtain blood cultures at arbitrary intervals of 30 to 60 minutes,17 Li et al18 showed no
difference in microbial recovery when blood specimens were drawn for culture simultaneously or at
spaced intervals for up to 24 hours. Thompson et al observed no significant differences in positivity rates
of blood cultures obtained in relation to the fever spikes of patients.19
As a practical matter, blood cultures should be obtained simultaneously (or over a short timeframe).
Drawing blood at intervals is only indicated when it is necessary to document continuous bacteremia in
patients with suspected infective endocarditis or other endovascular (e.g., catheter-related) infections.
5.2 Number of Blood Cultures
Several studies have been published addressing the optimal number of blood cultures that are needed to
detect bacteremia or fungemia. This variable is reviewed in detail by Aronson and Bor,20 who present the
theoretical basis for this issue.
The first publication was in 1975, when Washington reported results from 80 patients using 20-mL blood
specimens.21 In that study, the cumulative yield of pathogens from three blood cultures was 80% for the
first culture, 88% from two cultures, and 99% from three cultures.21 In 1983, Weinstein et al reported
results from 282 patients using 15-mL blood specimens. The results from that study were similar to those
reported by Washington; the cumulative yield of pathogens from these cultures was 91% from the first
culture and >99% (281/282) from two cultures.22 In both of these studies, blood cultures were performed
using manual blood culture systems; in 2004 Cockerill reported the results of a similar study from 163
patients when blood cultures were performed using a continuous-monitoring blood culture system
(CMBCS). In that study, the cumulative yield of pathogens from three blood cultures, with a blood
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volume of 20 mL each (excluding patients with infective endocarditis), was 65% from the first culture,
80% from two cultures, and 96% with three cultures.23 For patients with infective endocarditis, the yield
was 90% from first blood culture.23 The lower cumulative yield from the cultures performed on a
CMBCS, as compared with the yields from manual blood culture systems, may be the result of a number
of variables: differences in the broth media that were used, the detection mechanisms, use of different
antimicrobial agents over the 20 years that these studies were performed, the volume of blood cultured,
and, most importantly, different definitions of true-positive and contaminated blood cultures.21-23 The
present guideline is to collect two to three sets per episode. Single blood cultures should never be drawn
from adult patients; this practice results in an inadequate volume of blood cultured, and the results of
single blood cultures are more difficult to interpret. Blood cultures should not be repeated for two to five
days, because the blood does not become sterile immediately following the start of antimicrobial therapy.
The use of so-called surveillance blood cultures has been advocated as a means to allow earlier detection
of sepsis in certain patient populations—such as those in intensive care, undergoing transplantation, or
with vascular catheters—or as a "test of cure." Blood cultures obtained for the prediction of septic
episodes are of limited value and should not be performed routinely, as these cultures do not improve
patient management but add substantial costs.24-26
Similarly, most patients with bacteremia or fungemia can be followed clinically and do not need follow-
up blood cultures to document that the bacteremia or fungemia has cleared. There are, however, two
exceptions to this: the first is for patients with infective endocarditis, where documenting that bacteremia
or fungemia has cleared may be used to assess and guide therapy; the second is for patients with
Staphylococcus aureus bacteremia not related to infective endocarditis, where positive follow-up blood
cultures drawn at 48 to 96 hours were the strongest predictors of complicated S. aureus bacteremia.27
5.3 Volume of Blood for Culture
The volume of blood drawn for culture is the most important variable in detecting bacteremia or
fungemia.18,23,28-35 This observation is based on data published from many studies of adult patients with
bacteremia and fungemia; only limited data have been published from studies of infants and young
children. For adult patients, the yield of pathogens increases in direct proportion to the volume of blood
that is cultured from 2 to 30 mL.18,23,28,29,33 The yield still increases when 40-mL (or even higher) volumes
of blood are cultured, although the increase may no longer be in direct proportion to the volume of blood
cultured.18 For pediatric patients, the limited data that have been published also indicate that the yield of
pathogens increases in direct proportion to the volume of blood that is cultured.34,36 Some of these data are
based on observations of the numbers of bacteria that are present in blood, with the implication that
culturing higher volumes of blood containing small numbers of bacteria will improve recovery of bacteria
by culture.34,36-45 For adults, the recommended volumes for blood cultures are 20 to 30 mL per culture
(i.e., per venipuncture). For infants and younger children, the volume of blood drawn should be no more
than 1% of the patient’s total blood volume. There are no published data for determining when volumes
considered to be appropriate for adults can be used for older children.
5.4 Distribution of Blood Between Aerobic and Anaerobic Blood Culture Bottles
The historic practice has been to divide the blood drawn for culture equally between aerobic and
anaerobic blood culture bottles. This practice was questioned in the late 1980s and early 1990s after
several studies reported that the incidence of anaerobic bacteremia began to decrease, starting in the
1970s.42-47 Several studies performed at about the same time reported data to support the concept of
routinely inoculating blood drawn for culture into aerobic bottles only, using anaerobic bottles only for
select patients. Based on these data, a number of authors have recommended use of aerobic bottles only
for routine cultures.39,45-50 A recent study reported data that raise doubts about those recommendations.49
In this study, use of paired aerobic/anaerobic blood culture bottles yielded more staphylococci, members
of the family Enterobacteriaceae, and anaerobes when compared to paired aerobic blood culture bottles.50
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Because the data are conflicting and inconclusive, and because the recommendation that anaerobic blood
culture bottles be limited to use in select patient populations has never been validated by controlled
clinical studies to define these patient populations, it is recommended that routine blood cultures include
paired aerobic/anaerobic blood culture bottles.50-52 When less than the recommended volume of blood is
drawn for culture, the blood should be inoculated into the aerobic vial first; any remaining blood should
then be inoculated into the anaerobic vial. Inoculating blood culture bottles in this manner is important
because most bacteremias are caused by aerobic and facultative bacteria, which will be recovered better
from aerobic bottles. In addition, pathogenic yeasts are recovered almost exclusively from aerobic bottles,
as are strict aerobes, such as Pseudomonas and Stenotrophomonas. For those laboratories opting to use
aerobic bottles only, it is important that two bottles be used for each blood culture to help ensure that
adequate volumes of blood are cultured.
5.5 Disinfection of Skin and Prevention of Blood Culture Contamination
Most blood cultures are drawn by venipuncture. In order to minimize the risk of contamination with skin
flora, the venipuncture site requires disinfection. A number of disinfectants have been used clinically
during the past 50 years, including rubbing alcohol (70% isopropyl), tincture of iodine, povidone-iodine,
iodophors, chlorine-peroxide, and chlorhexidine gluconate.39,53-59 Several studies comparing these
disinfectants have been published, from which the following conclusions can be made:
• Tincture of iodine, chloride peroxide, and chlorhexidine gluconate are superior to povidone-iodine
preparations.
• Tincture of iodine and chlorhexidine gluconate are probably equivalent.
Iodine-containing preparations require sufficient time to disinfect surfaces (30 seconds for tincture of
iodine and 1.5 to 2 minutes for iodophors). Chlorhexidine gluconate requires the same amount of time as
tincture of iodine, but is not associated with allergic reactions and does not need to be cleaned off the skin
after the venipuncture is completed. The primary disadvantage to chlorhexidine gluconate is that it cannot
be used to disinfect skin of infants less than two months of age; however, it is the recommended skin
disinfectant for older infants, children, and adults.
5.6 Blood Culture Collection
Blood cultures should be collected using standard precautions. Strict aseptic technique should be used
throughout the procedure. Blood for culture should be drawn from veins, not arteries.20,40,53 Arterial blood
cultures are not associated with higher diagnostic yields than venous blood cultures and are not
recommended.53 Blood cultures obtained from indwelling intravascular access devices, such as
intravenous catheters and ports, are associated with greater contamination rates than are blood cultures
obtained by venipuncture.60-62 Although blood occasionally may need to be obtained from intravenous
lines and similar access devices, a culture of blood from such a device should be paired with another
culture of blood obtained by venipuncture to assist in interpretation in the event of a positive result.
If blood cultures for bacteria or fungi are collected through an intravenous line, it is not necessary to
discard the initial volume of blood or flush the line with saline to eliminate residual heparin or other
anticoagulants.63 Moreover, the antimicrobial activity of heparin is effectively eliminated in protein-rich
culture media.64,65
After the venipuncture site is identified, the rubber septum on the blood culture bottle(s) or tube(s) should
be disinfected with 70% isopropyl alcohol and allowed to dry. The site of the venipuncture should then be
disinfected; typically this means cleansing the site first with 70% isopropyl alcohol, allowing it to air dry,
followed by application of the main disinfectant, then allowing that substance to sit for the recommended
amount of time. The person drawing the culture should not palpate the vein after skin disinfection unless a
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sterile glove is worn. It is recommended that blood be drawn into a sterile syringe and then transferred to
the blood culture tube or bottle. Blood can be drawn directly into collection tubes containing sodium
polyanetholsulfonate (SPS), but should never be drawn into tubes containing other anticoagulants. The
blood from an SPS tube can then be transferred to blood culture medium. Drawing blood directly into
blood culture vials (e.g., using a needle holder designed for collecting blood into tubes) is not
recommended because of the risk of reflux of the broth media back into the vein and also because the
volume of blood draw into the bottle or tube cannot be controlled. Collection devices are available from
some manufacturers; these should be used according to the manufacturers’ recommendations. Blood
culture bottles should be kept upright when these devices are used. Blood culture bottles/tubes should be
inverted gently several times to prevent clotting.
For many years it was standard practice to change needles before inoculating blood culture bottles/tubes.
Although several studies showed that using the same needle to both draw blood as well as to inoculate
blood culture bottles did not significantly increase contamination rates,66-68 a meta-analysis did show
slightly higher contamination rates when needles were not changed.69 Because of the risk of sustaining an
accidental needlestick, many facilities activate the safety feature of the needle, remove and discard the
contaminated sharp, and apply a safety transfer device to the syringe before filling the bottles.
Regardless of the method used to collect blood cultures, laboratories should validate that their process is
effective in minimizing contamination rates to an acceptable range, typically ≤ 3%.
5.6.1 Transporting Specimens to the Laboratory
Blood culture bottles/tubes should be sent to the laboratory within two hours; delays in entering blood
culture bottles into continuous-monitoring blood culture instruments (particularly if the bottles are
incubated at 35 to 37 °C) may delay or impede detection of growth. Holding bottles at room temperature
is not recommended for anything longer than a few hours. Blood culture bottles/tubes should never be
refrigerated or frozen after they have been inoculated; refrigeration or freezing can kill some
microorganisms, and freezing fluid-filled containers may cause the container to break. Blood culture
specimens collected for lysis-centrifugation also should not be held for more than eight hours; holding
cultures longer than this may decrease the yield or delay the growth of certain bacterial pathogens.70-73
5.7 Specimen Rejection Criteria for Blood Culture Specimens
Blood culture specimens that meet the following criteria should be rejected and another specimen
collected:
• incorrectly labeled or unlabeled vials;

• broken, damaged, or leaking bottles/tubes;
• clotted tubes; or
• tubes containing anticoagulants other than SPS (refer to Section 6.1, Critical Factors in the Recovery
of Pathogens From Blood Specimens).
6 Methods and Procedures
6.1 Critical Factors in the Recovery of Pathogens From Blood Specimens
VOLUME
The optimal recovery of bacteria and fungi from blood depends on culturing an adequate volume of
blood. The direct correlation between the volume of blood cultured and yield relates to the low number of
colony forming units (CFU) in a milliliter of adult blood. For each additional milliliter of blood cultured,
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the yield of microorganisms recovered from adult blood increases.30,33 Pediatric patients often have higher
numbers of microorganisms in their blood, and satisfactory results are achieved with smaller blood
culture volumes. However, low-level bacteremia may also occur in children, and the recommended
volume of blood to collect is based on the patient’s total blood volume and age.38 Section 5, Specimen
Collection and Transportation, discusses the recommended blood volumes to collect safely from adult and
pediatric patients in order to maximize yield. Laboratories must also follow manufacturers’ instructions
and collect the recommended blood volumes for the system.
Surveys74 indicate that laboratories often receive less than the recommended culture amounts; laboratories
should process these cultures and note on the report that the volume collected was less than optimal to
achieve adequate sensitivity. Monitoring blood volume and providing feedback to staff should be part of
an institution’s quality assurance program to improve patient care and resource utilization.75
BLOOD-TO-BROTH RATIO
Normal human blood contains substances that inhibit microbial growth. Among these are complement,
lysozyme, phagocytes, antibodies, and antimicrobial agents (if the patient is receiving treatment with
antimicrobial agents prior to collection of blood for culture). To reduce the concentration of these
inhibitory factors, and thereby minimize their inhibitory activity, blood should be diluted in broth media
at a blood-to-broth ratio of 1:5 to 1:10.76 Failure to maintain this ratio may result in false-negative cultures
from underfilling bottles. Some commercial blood culture systems use a blood-to-broth ratio that is less
than 1:5—a ratio that is acceptable because of the addition of proprietary substances that bind to and
inactivate the inhibitory substances present in blood. Pediatric blood specimens can be inoculated into
pediatric vials designed to maintain the blood-to-broth ratio with smaller blood volumes, but there is
minimal data to indicate that use of these bottles improves microbial recovery.
MEDIA (TYPES, INDICATIONS/FORMULATIONS)
Numerous broth medium formulations are available for conventional and automated blood culture
methods. Soybean-casein digest broth is the most widely used basal medium, and brain-heart infusion
(BHI), Columbia, Brucella, thiol, thioglycolate, and supplemented peptone broths have also been used to
recover aerobic and anaerobic microorganisms. Several Middlebrook formulations are available for
automated and commercial blood culture systems to enhance recovery of mycobacteria.
Each automated blood culture system has its own specific medium formulations to culture aerobic and
anaerobic microorganisms. Several well-controlled, large clinical studies comparing the systems and
media indicate that most perform acceptably.39,77-79 However, comparisons are complicated by the
variations in the same basal medium formulations marketed by different manufacturers. Manufacturers
enrich their media with a variety of proprietary supplements, varying concentrations of SPS, and different
headspace atmospheres to enhance microbial growth.79 Regardless of the method used, a combination of
complementary medium formulations may be necessary to optimize recovery rates of all potential
pathogens, because no single medium or system recovers all microorganisms optimally.
Recommendations for quality control requirements for media, including procedures for receiving media
from the manufacturer (e.g., visual inspection and contamination checks) and quality assurance guidance,
are outlined in CLSI document M22—Quality Control for Commercially Prepared Microbiological
Culture Media.80
Other blood culture methods include biphasic systems containing both broth medium and agar or a solid
surface, and lysis-centrifugation.39,77,78 Details about these systems and how to select a system are
discussed in Section 6.2, Blood Culture Methods.
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ADDITIVES (ANTICOAGULANTS, RESINS, CHARCOAL)
All broth blood culture media contain anticoagulants to inhibit blood clot formation. The most effective
anticoagulant, SPS, neutralizes lysozyme, inhibits phagocytosis, inactivates some aminoglycosides, and
inhibits part of the complement cascade.79 The typical concentration of SPS ranges from 0.025 to 0.05%,
although some commercial systems have concentrations as low as 0.006%. Despite inhibiting the growth
of several bacteria, including Neisseria species, Peptostreptococcus anaerobius, Moraxella catarrhalis,
and Gardnerella vaginalis,81-83 SPS remains the most common anticoagulant and has been shown to
increase the rate and speed of recovery of both gram-positive and gram-negative microorganisms.
Heparin, ethylenediamine tetraacetic acid (EDTA), and citrate are toxic to microorganisms; blood should
not be inoculated into blood collection tubes containing these anticoagulants.
In addition to diluting blood in broth, and adding SPS to reduce the inhibitory effects of antimicrobials in
blood cultures, some manufacturers have added antimicrobial-binding or adsorbing agents to their
systems to enhance the recovery of microorganisms from the blood of patients receiving antimicrobial
therapy. Compared to media without additives, more microorganisms, particularly staphylococci and
yeasts, are recovered from formulations with additives.21,73,84,85 However, these media are more expensive,
and there are increased costs to the laboratory to work up the increased numbers of contaminants that also
are recovered.
INCUBATION CONDITIONS
Temperature
Bacterial and fungal blood cultures should be incubated at 35 °C after collection and delivery to the
laboratory. Although delays in incubating cultures after collection do not affect yield, delays should be
minimized to avoid prolonging the time to detection of microbial recovery. Because there are conflicting
reports about the effects of delays on microbial recovery in lysis-centrifugation tubes, these tubes should
be processed within eight hours after blood collection.73
Atmosphere
To accommodate the volume of blood to be inoculated into a bottle, a portion of the headspace
atmosphere is evacuated to create a partial vacuum; thus, blood culture bottles contain an atmosphere in
the bottle headspace that has a lower pressure than the atmosphere. With some manual systems, one bottle
must be vented with a needle to create an aerobic atmosphere. Automated blood cultures are also
manufactured with varying amounts of carbon dioxide added to the ambient atmosphere in the headspace
of the aerobic vial. The atmosphere in the anaerobic bottle headspace contains carbon dioxide and
nitrogen.
See Section 5.4, Distribution of Blood Between Aerobic and Anaerobic Blood Culture Bottles, for
additional discussion of this issue.
Length of Incubation
For conventional manual methods, incubation for seven days is recommended; some slower-growing,
fastidious bacterial pathogens (Bartonella, Legionella, Brucella, Nocardia) and dimorphic fungi may
require longer incubation. The standard incubation period for routine blood cultures performed by
automated systems is five days,39,86 including cultures for Brucella spp., Haemophilus spp., Actinobacillus
spp., Cardiobacterium spp., Eikenella corrodens, Kingella spp., and the nutritionally variant streptococci
(e.g., Abiotrophia and Granulicatella spp.). Prolonged incubation and testing periods to recover bacteria
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from patients suspected of having infective endocarditis also appear unnecessary, as discussed in Section
6.5.3, Special Considerations: Infective Endocarditis.23,87
Published data88,89 suggest that four or even as few as three days90,91 may be adequate to recover 95 to
97% of clinically significant bacteria and yeasts from some systems. However, the current
recommendation remains five days of incubation for automated systems. Adequate data from studies
validating a shorter incubation time may satisfy accrediting bodies and permit laboratories to adapt
shorter blood culture monitoring protocols.
AGITATION
Studies indicate that bottle agitation, particularly during the first 24 hours of incubation, increases yields
and improves the speed of detecting microorganisms in aerobic bottles.92,93 This presumably is due to
increased oxygenation of the broth medium. The current CMBCSs agitate bottles continuously. Agitation
of anaerobic bottles does not adversely affect growth of bacteria.
MONITORING FREQUENCY/SUBCULTURES
Conventional blood culture methods require daily or more frequent visual examination for evidence of
macroscopic growth. Cultures should be examined with bright fluorescent bulbs or with incandescent,
transmitted light for turbidity, hemolysis, gas production, surface colony formation, or change in blood
color. Subcultures to blood and chocolate agar and/or Gram or acridine orange stains of aerobic vials after
24 to 48 hours of incubation facilitate early detection of microorganisms. Blind/terminal subcultures of
negative cultures are considered unnecessary by many and may increase contamination rates and
needlestick injuries.39 Repeat subcultures of previously positive blood cultures are ineffective for
detecting polymicrobial bacteremias.
Some manual blood culture systems, particularly those with agar media that are an integral part of the
bottle, also require daily visual inspection to detect evidence of microbial growth.
CMBCSs monitor aerobic and anaerobic vials at regular 10- to 24-minute intervals for evidence of
growth. Subcultures are performed when a positive signal indicates growth. Routine, blind, or terminal
subcultures from negative blood cultures performed on automated systems are unnecessary when cultures
have been monitored for at least five days.94 Subcultures should be held for a period determined by the
type of organism(s) being considered.
6.2 Blood Culture Methods
Blood cultures can be processed in the laboratory either by manual or by automated methods. For the sake
of discussion, the term manual blood culture techniques will refer to methods that do not employ
instrumentation to monitor the growth of microorganisms. Manual blood cultures can be further
subdivided into visually monitored (‘conventional’) broth-based cultures, cultures using biphasic media,
and the lysis-centrifugation technique. Automated CMBCSs use instrumentation to detect microbial
growth in broth media by monitoring byproducts of bacterial or fungal metabolism.
6.2.1 Manual Blood Cultures
6.2.1.1 Conventional Broth Cultures
Conventional blood cultures typically consist of paired aerobic and anaerobic bottles. Standard medium
choices include brain-heart infusion, Columbia, or trypticase soy broths for aerobic to microaerophilic
microorganisms, and thiol or thioglycolate formulations for microaerophilic to anaerobic bacteria. Each
medium may be supplemented with additional factors (e.g., hemin, vitamin K1, L-cysteine) to support the
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growth of fastidious microorganisms. The volume of broth selected for conventional cultures should
reflect the anticipated draw volume so that a blood-to-broth ratio ranging from 1:5 to 1:10 is achieved.53
Commercially prepared blood culture bottles have broth volumes ranging from 18 to 100 mL.
The atmosphere in the headspace of commercially prepared blood culture bottles is evacuated to remove
O2 and replaced with CO2 and N2 at pressures lower than atmosphere pressure. Following inoculation, the
bottle designated for aerobic microorganisms may require venting to reintroduce O2 for aerobic
metabolism (refer to the manufacturer’s recommendations).
After inoculation with blood, bottles are incubated at 35 °C and examined visually for evidence of
bacterial growth. Changes that suggest microbial growth include turbidity of the blood-broth mixture,
growth of microcolonies, hemolysis, color changes, and gas production. Initial inspection occurs after 12
to 24 hours of incubation with twice daily inspection for two days followed by daily inspection for days 3
to 7.95 Gram staining of the broth along with subculture during the initial examination can hasten early
detection of growth in conventional cultures.96,97 However, this practice does little to improve detection of
growth thereafter, and terminal subcultures of negative bottles should be discouraged.98,99 Bottles should
be incubated for seven days.
6.2.1.2 Biphasic Cultures
The use of a blood culture bottle containing both agar and broth medium was initially designed for the
recovery of Brucella species from blood.100 These “biphasic” blood culture systems have also been shown
to be effective for the recovery of fungi and bacteria from blood. The popularity of this method has been
hindered by the complexity of bottle preparation and the relative lack of commercial sources for the
bottles. Currently, a commercially prepared system is available that consists of a broth culture bottle and
an attachable cylinder housing a paddle coated with an agar-based medium on either side. Bottles are
available with a variety of broth and volume combinations to support use for pediatric or adult patients
and a range of aerobic to anaerobic microorganisms. Formulations are also available for the recovery of
mycobacteria. To initiate a culture, blood is introduced into the broth bottle, and the agar-containing
cylinder is attached to the top of the bottle. Subcultures are accomplished simply by inverting the bottle
and allowing the blood/broth mixture to wash over the agar surface. The assembly is then incubated in the
upright position and observed for the growth of microorganisms either in the broth (as described for
conventional blood culture systems) or on the agar surface.
Compared to conventional manual blood culture bottles, biphasic blood culture systems provide improved
recovery of aerobic and facultative anaerobic bacteria and fungi; biphasic systems also have decreased
time to detection and allow for recovery of isolated colonies.101-110 However, the commercial biphasic
systems yield higher contamination rates and do not perform as well for the recovery of anaerobic
bacteria.105,106,108,110 Similar to manual blood cultures, biphasic media should be monitored visually for a
total of seven days. Biphasic bottles should be incubated for seven days.
6.2.1.3 Lysis-Centrifugation
Lysis-centrifugation refers to a blood culture method in which microorganisms are released from lysed
blood and are then separated from blood components by centrifugation into a density layer at the bottom
of the collection tube. Microorganisms concentrated into this layer are then transferred from the lysis-
centrifugation tube to solid media. The performance of the lysis-centrifugation method is equivalent to
that of biphasic blood culture systems in terms of time to recover isolated colonies and the overall
recovery rate of filamentous fungi and yeasts.
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6.2.2 Continuous-Monitoring Blood Culture Systems79
Two of the three commercially available CMBCSs rely on detection of increased CO2 production by
microorganisms. Both of these systems are modular in design to support low or high bottle capacity,
depending on the number of blood cultures that need to be processed. A variety of media formulations are
available for use with each system, including aerobic and anaerobic formulations with or without
additives designed to enhance recovery of microorganisms from patients receiving antimicrobial therapy.
Media for the recovery of mycobacteria from blood have also been developed for both systems. Aerobic
bottles are mechanically aerated during the course of incubation to increase the pO2 in the broth medium.
The growth detection apparatus of both systems is similar. A sensor located on the bottom of the blood
culture bottle is separated from the liquid medium by a membrane that is selectively permeable to CO2.
As CO2 is produced by actively metabolizing microorganisms, it diffuses across the membrane and
displaces hydrogen ions, causing acidification of the sensor with a concomitant shift in the emission
spectrum of the detection dye (either colorimetric or fluorometric, depending on the system). A light-
emitting diode illuminates the sensor every ten minutes, and reflected light is captured by a photo
detector. The change in signal intensity is proportionate to the emission shift of the sensor dye, which is
related to the amount of dissolved CO2 in the culture medium. Data is collected and transferred to a
computer, where software algorithms analyze the data and recognize either increasing rates or sustained
production of CO2. Bottles matching criteria for significant CO2 increases are signaled for removal and
subculture.
The third CMBCS detects microbial growth by measuring the headspace gas pressure within the blood
culture bottle. This pressure change is attributable to microbial O2 consumption and/or N2, H2, or CO2
production. Aerobic and anaerobic bottles are monitored every 12 and 24 minutes, respectively, and
pressure readings are translated into growth curves over time after correction for atmospheric pressure
changes. A software algorithm translates pressure changes into likely microbial growth patterns and
signals a positive bottle for removal and subculture. At present, two medium formulations in two bottle
sizes are approved for general use with this system.
The advantages to CMBCSs include decreased staff needs for laboratories processing large numbers of
blood cultures, a reduction in contamination rates because monitoring is noninvasive, and decreased time
to detection of positive cultures secondary to the increased rate of monitoring. However, the latter is
dependent on the frequency of instrument inspection and subculture of flagged bottles. On shifts when
microbiologists are not available, central laboratory personnel should be scheduled to inspect the
instrument(s), remove positive bottles for subculture and prepare Gram stains for evaluation at a later
time, or be trained to examine and report Gram-stained smears. The ability to prepare and interpret Gram
stains and smears on all shifts can affect information delivery time and changes in therapeutic options.111
6.2.3 Preservation of Isolates for Further Testing
Blood culture isolates are of unique value in patients with infectious processes. Microorganisms may be
used for research, clinical, epidemiological, educational, microbiological, therapeutic, and commercial
reasons. During the period of active testing, preserving isolates is most conveniently accomplished by
serial subculture to appropriate media on a periodic basis. The frequency of subculture varies for different
organisms because of intrinsic viability, as well as phenotypic and genotypic stability. Subculture every
two to three days is likely to preserve the organism, at least through several passages.
Laboratories should develop procedures for short- and long-term storage of blood culture isolates to
ensure access to viable organisms in case additional testing is needed. All blood culture isolates, except
known contaminants, should be maintained for seven to ten days. This is most important for
microorganisms whose clinical significance cannot be determined by initial blood culture results. The
simplest approach to the storage of bacteria and yeasts for short periods of time is to keep them on agar
media. These cultures may be stored at room temperature, but storage at 5 to 8 °C may enhance the
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duration of viability. Cultures maintained in this fashion are subject to drying, but most clinically
important bacteria will survive for days to weeks. Sealing the cultures to prevent water loss from the
medium may allow preservation of viability during intermediate-term storage. There are no detailed
descriptions of the adequacy of these approaches, and individual laboratories will have variable success,
depending on internal environmental conditions.
If possible, laboratories should have procedures for both short- and long-term storage of pathogenic blood
culture isolates in case additional testing is needed. This is particularly important for isolates that may be
associated with repeated/relapsing infections in an individual, changing resistance patterns caused by
antimicrobial therapy, and confusing interpretation (e.g., patients who may also have endogenous
infection with the same species). Ultra-low temperature (e.g., -70 oC) storage or lyophilization is
recommended for long-term storage. Sterile cryoprotective agents, such as 20% skim milk, 10% glycerol,
and 5% DMSO, improve the viability of organisms preserved at ultra-low temperatures, but the optimal
agent varies by organism group. To prepare isolates for long-term storage, an aliquot of late log-phase
growth of a fresh broth culture is centrifuged and the pellet resuspended in several milliliters of fresh
broth medium to which the cryoprotective agent has been added at the appropriate concentration.
Aliquots, typically ~0.5 mL, of this suspension are transferred into cryopreservation vials. Such vials
must be able to withstand ultra-low temperatures, be securely sealed, and provide a suitable surface for
labeling the contents of the vial. A record of the location of each preserved isolate should be prepared to
facilitate retrieval of isolates as needed. When needed, cryopreserved cultures should be rapidly thawed
by use of a 35 °C waterbath, followed by subculture appropriate for the organism.
Lyophilization may provide the most dependable method for long-term storage of many bacteria, but is
not appropriate for microorganisms damaged by removal of intracellular water, such as molds and some
bacteria. Specialized equipment, storage vials, and procedures are required for lyophilization.
Additional detailed methods for microorganism storage are available elsewhere.112-114
6.3 Fungal Blood Cultures
6.3.1 Population at Risk
The increased incidence of infections with documented fungemia corresponds to improvements in blood
culture techniques and changes in the medical management of patients.15,45 Fungemia with yeasts is well-
documented in patients with traumatic injuries or ulcerations of the gastrointestinal mucosa, recipients of
broad-spectrum antibacterial antimicrobial agents or hyperalimentation, patients with intravenous access
devices, and those with immunosuppressive diseases. In recent years there has been an increase in the
recovery of dimorphic fungi (e.g., Histoplasma, Blastomyces, Coccidioides) and filamentous fungi (e.g.,
Fusarium, Scedosporium) in blood cultures of patients with AIDS, hematologic malignancies, bone
marrow and solid organ transplantations, and other causes of severe immune compromise. Although
infections with Aspergillus and Zygomycetes are common in severely immunocompromised patients,
documentation of fungemia in these patients is infrequent.
The yeasts most commonly isolated in blood cultures include Candida albicans, Candida glabrata,
Candida tropicalis, Candida parapsilosis, and Cryptococcus neoformans. Other Candida species (e.g.,
Candida krusei, Candida lusitaniae, Candida guilliermondii), Malassezia furfur, Rhodotorula species,
and Trichosporon species are recovered less frequently.115,116
Histoplasma capsulatum is the most commonly isolated dimorphic fungus; Blastomyces dermatitidis and
Coccidioides immitis are recovered less frequently.101,117,118 Fusarium and Scedosporium are the most
commonly isolated filamentous fungi, with Exophiala, Rhinocladiella, and Aspergillus recovered less
frequently.119-121
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6.3.2 Critical Factors
Numerous studies have documented better recovery of yeasts in aerobic broths compared with anaerobic
broths.45,46,115 Although improved recovery of yeasts was obtained with biphasic blood culture systems
using both agar and broth media, this is generally unnecessary with current commercial blood culture
systems. Agitation of broth-based systems increases aeration and improves recovery of yeasts.122 This can
be accomplished either through the use of automated commercial blood culture systems or mechanical
agitation of manual broth systems for the first 24 hours of incubation. Most yeasts and yeast-like fungi
will be detected within two to five days of incubation, although some yeasts (e.g., C. glabrata, C.
neoformans) may require extended incubation. Optimal growth of M. furfur requires supplementation of
media with lipids (e.g., olive oil). Although dimorphic fungi can grow in most commercially prepared
aerobic blood culture broth media, detection may require up to two to four weeks of incubation. For this
reason, use of broth-based automated blood culture systems is unreliable. If infection with a dimorphic or
filamentous fungus is suspected, blood should be processed by lysis-centrifugation and plated onto agar
media.70,118 Although some fungi (e.g., Fusarium and Scedosporium) may grow within three to five days,
cultures should be incubated for a minimum of four weeks.
6.3.3 Methods—Manual
Three types of manual blood culture systems are used for the detection of fungemia: 1) bottles of nutrient
broth, 2) biphasic systems, and 3) lysis-centrifugation systems. Yeasts can be satisfactorily recovered in
any of the three systems; however, dimorphic and filamentous fungi can only be recovered reliably in
biphasic broths and in the lysis-centrifugation system. A variety of aerobic media have been used with
equivalent success for the detection of yeast infections. Anaerobic broths should not be used. Biphasic
systems are acceptable for the recovery of yeasts and dimorphic and filamentous fungi, although
incubation of the cultures for four weeks is required for reliable detection of dimorphic fungi. For
optimum performance of the biphasic systems, gentle continuous mixing of the bottles for the first 24
hours of incubation is required.122 Poor recovery of dimorphic fungi in biphasic systems can be attributed
to an insufficient incubation period. Dimorphic and filamentous fungi are detected earlier in lysis-
centrifugation systems than with other manual blood culture systems. For optimal recovery of fungi in
lysis-centrifugation systems, the concentrated blood sediment should be inoculated onto multiple agar-
based media and incubated at both 27 to 30 °C and 35 to 37 °C.123
6.3.4 Methods—Automated
With the current CMBCSs, recovery of yeasts is best in aerobic broth formulations. In some studies, the
use of factors that inactivate antimicrobial agents has improved recovery and time to detection of yeasts.85
In general, special media formulated for the recovery of yeasts are unnecessary.124,125
6.4 Mycobacterial Blood Cultures
6.4.1 Population at Risk
The incidence of positive blood cultures with mycobacteria was relatively uncommon until the advent of
the AIDS epidemic. Mycobacteremia is also documented in patients with other immunosuppressive
diseases (e.g., leukemia, severe combined immunodeficiency syndrome, multiple myeloma, and other
solid tumor malignancies), recipients of high-dose steroid therapy or cytotoxic chemotherapy, and
patients with long-term vascular access devices.
Documented mycobacteremia with Mycobacterium tuberculosis is relatively uncommon in developed

countries. However, in developing countries recovery of this organism in blood cultures from
immunocompromised patients is commonplace.124 In contrast, Mycobacterium avium complex (MAC) is
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the most common mycobacterium isolated in the blood of immunocompromised patients in developed
countries, although in recent years the incidence of mycobacteremia has decreased.126-128
Other slowly growing mycobacteria, including Mycobacterium kansasii, Mycobacterium simiae,

Mycobacterium xenopi, and Mycobacterium genavense, have been recovered from immunocompromised
patients with disseminated disease.127,128 Bacteremia with rapidly growing mycobacteria (e.g., M.
fortuitum, M. chelonae, M. abscessus, and M. mucogenicum) is more commonly associated with
contamination of long-term intravascular catheters and prosthetic devices.129
6.4.2 Critical Factors
Mycobacteria are not particularly fastidious microorganisms and can grow in conventional blood culture
broths if the incubation period is extended.130 Despite this observation, optimal recovery of mycobacteria
from blood specimens requires supplementation of broth cultures with fatty acids (e.g., oleic acid),
albumin, and carbon dioxide. Some species (e.g., M. genavense, M. haemophilum) require supplements
such as mycobactin and hemin, hemoglobin, or ferric ammonium citrate. Whereas some mycobacterial
species have a temperature preference of 25 to 30 °C, strains associated with mycobacteremia can be
recovered at 35 to 37 °C. Because mycobacteria have a slow generation time, all cultures should be
incubated for a minimum of four weeks.
6.4.3 Methods—Manual
Blood specimens must be processed with a lytic agent to release intracellular bacteria before culture
media are inoculated. Blood specimens should be collected in either a sterile tube with an anticoagulant
(e.g., SPS, heparin, or citrate but not EDTA) or a lysis-centrifugation tube. Whole blood should not be
directly inoculated onto solid media or into broths without a lytic agent. Blood specimens collected in a
lysis-centrifugation tube can be inoculated onto a variety of solid, broth, or biphasic media; however,
recovery of mycobacteria in specimens processed on solid media is inferior to recovery in broth and
biphasic systems.131,132 Detection of positive cultures in the biphasic systems is slower than in broth-based
automated systems.133,134
6.4.4 Methods—Automated
A variety of automated commercial systems have been developed for the recovery of mycobacteria. Each
manufacturer has developed broth-based media supplemented with various growth factors and
antimicrobial agents that are used specifically with their culture systems. Some systems require the initial
lysis of blood before inoculation into the broth cultures; whereas in other systems anticoagulated, whole
blood can be processed. Some differences have been reported in the overall detection and time to
detection in these systems135-137; however, extensive comparisons have not been performed.
6.5 Special Topics
6.5.1 Pediatric Blood Cultures
As for adult patients, two to three blood cultures should be collected within a 24-hour period. Because
anaerobic bacteria are rarely recovered in pediatric patients, some investigators have recommended the
use of aerobic bottles only.138,139 Anaerobic bottles may be considered in high-risk groups that include
neonates from mothers that have had prolonged rupture of membranes during childbirth or maternal
chorioamnionitis; chronic oral or sinus infection; cellulitis (especially perianal and sacral); abdominal
signs and symptoms; bite wounds; septic phlebitis; and neutropenic patients receiving steroids.139
The same methods of skin antisepsis for adults apply to pediatrics, with the exception in neonates with the
potential to develop subclinical hypothyroidism due to iodine.140 For all patients, topical iodine
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compounds must be completely removed after phlebotomy. Chlorhexidine gluconate as a skin antiseptic
is approved for use in pediatric patients two months of age and older. For patients who are younger than
two months of age, use of 70% isopropyl alcohol is an acceptable alternative for skin disinfection. As
with recommendations in adults, blood drawn from an intravenous catheter should be accompanied by a
peripheral draw.
Pediatric blood cultures differ from adult blood cultures primarily in the amount of blood obtained for
culture. Many of the studies in adults indicating blood volume, ratio of blood to broth, number and timing
of cultures, and the need to use blood culture bottles specifically designed for adult blood cultures are
lacking for pediatrics. Caution must be used in extrapolating these data to pediatric patients. It has been
common practice to draw the minimum amounts of blood because of the: 1) smaller volumes of blood in
pediatric patients; 2) difficulty in phlebotomy; 3) potential for increased transfusions due to the amounts
of blood drawn for all laboratory procedures; and 4) presumed high levels of bacteria in the blood in
pediatric patients with bacteremia.141 While many pediatric infections are characterized by high numbers
of microorganisms in the blood, a small but significant number of infections have relatively low numbers
of microorganisms.36-38,142-145 Processing larger blood volumes increases the pathogen recovery
yield,138,142,146-148 and decreases the time to detection.36,146,149 As stated previously, for infants and younger
children, the volume of blood to be drawn for culture should not exceed 1% of the patient's blood volume.
On an individual basis, suggested blood culture volumes to be drawn have been based on other clinical
parameters, such as patient weight and hematocrit.38,147,150
6.5.2 Catheter-Related Bloodstream Infections
Catheter-related bloodstream infections (CRBSIs) are common causes of healthcare-associated infections.

It is estimated that there are more than 250,000 cases of CRBSI annually in the United States,151 with an
attributable mortality of 12 to 35%.152 Important risk factors for CRBSI include: type of catheter (non-
tunneled central venous catheters long-term, tunneled central venous catheters short-term, peripheral
catheters), duration of catheter placement, and site of insertion.153 Despite their frequent occurrence,
CRBSIs are difficult to diagnose. Clinically, there may be absence of inflammation around the catheter
exit site and the presence of only nonspecific clinical signs suggestive of sepsis. Documentation of these
infections in the clinical microbiology laboratory is also problematic due to the lack of a well-established
gold standard for laboratory diagnosis. Diagnosis of catheter exit site and tunnel tract infections will not
be covered in these guidelines.
There have been a number of proposed methods for diagnosing these infections in the clinical
microbiology laboratory. These methods include: semiquantitative and quantitative cultures of catheter
segments; paired peripheral and catheter blood cultures; quantitative peripheral and catheter blood
cultures; differential time-to-positivity for peripheral versus catheter blood cultures; and endoluminal
brush staining with acridine orange, among others. A meta-analysis of the various methods used to
diagnose CRBSI was published in 1997.154 Although that review failed to show conclusively which of
these methods was best for diagnosis of CRBSI, it did conclude that a quantitative culture was the most
accurate method for catheter-segment culture, while unpaired quantitative catheter blood culture was the
single most cost-effective test, especially for long-term catheters. Diagnostic methods that rely on
catheter-segment cultures, however, require removal or exchange of the catheter, and should not be
performed in the absence of simultaneous peripheral (i.e., obtained by venipuncture) blood cultures. It has
been estimated that 75 to 85% of catheters are removed unnecessarily during evaluation of new fever.155
In order to avoid the unnecessary removal of a central venous catheter, methods that permit the diagnosis
with the catheter in place may be attempted. Furthermore, because removal of a surgically implanted
catheter is frequently a management challenge, it is important to distinguish CRBSI from skin
contamination, catheter colonization, or infection from a source other than the catheter.
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6.5.2.1 Recommendations for Short-term Peripheral Catheters
Obtain two sets of peripheral blood cultures from the patient via venipuncture. Remove the catheter
aseptically and culture by the semiquantitative method of Maki156 (with these catheters it is usually the
external surface of the catheter that is colonized, leading to infection).
• Interpretation of culture results:
— If one or more blood culture sets are positive AND the catheter-segment culture is positive (>15
colonies for semiquantitative) for the same organism: suggestive of CRBSI.
— If one or more blood culture sets are positive AND the catheter-segment culture is negative:
inconclusive; however, suggestive of CRBSI if positive for Staphylococcus aureus or Candida
spp. AND in the absence of any other identifiable source of infection.
— If both blood culture sets are negative AND the catheter-segment culture is positive, despite the
colony count: suggestive of catheter colonization, not CRBSI.
— If both blood culture sets are negative AND the catheter-segment culture is negative: CRBSI is
unlikely.
6.5.2.2 Recommendations for Nontunneled and Tunneled Central Venous Catheters and Venous Access
Ports (VAP)
Obtain at least two sets of blood cultures from the patient suspected of having a CRBSI, with at least one
set drawn from a peripheral venipuncture and labeled as such. The other set should be drawn aseptically
from the catheter hub or through the VAP septum, close to the same time of collection of the peripheral
set and labeled as such.
• Interpretation of culture results (Method 1):
— If both sets recover the same organism (as determined by identification and antimicrobial
susceptibility profiles): suggestive of CRBSI, in absence of any other identifiable source of
infection.
— If both sets are positive for the same organism AND the set drawn through the catheter becomes
positive >120 minutes earlier: suggestive of CRBSI, in absence of any other identifiable source of
infection. (If the differential time to positivity is <120 minutes, a CRBSI is still possible, if both
sets are positive for an organism with the same identification and antibiotic susceptibility profile.)
— If both sets are positive AND the set drawn through the catheter has at least a fivefold greater
number of CFUs/mL: suggestive of CRBSI, in absence of any other identifiable source of
infection. This method requires use of a manual quantitative blood culture system, such as
lysis/centrifugation method.
— If only the blood culture set drawn from the catheter becomes positive: inconclusive for CRSBI,
and suggests either colonization of the catheter or contamination during the collection of the
culture.
— If only the blood culture set drawn peripherally becomes positive: inconclusive; however,
suggestive of CRBSI if positive for Staphylococcus aureus or Candida spp. AND in the absence
of any other identifiable source of infection. Documentation of CRSBI would require a positive
semiquantitative or quantitative culture of the catheter segment with the same organism, or
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additional positive catheter or peripheral blood cultures with the same organism, in the absence of
any other identifiable source of infection.
— If both sets are negative: CRBSI is unlikely.
Or,
• Obtain two sets of blood cultures aseptically through independent peripheral venipunctures.
• Remove the suspected catheter and aseptically cut the distal 5-cm segment of the catheter and submit
it to the laboratory for culture by either Maki’s semiquantitative roll-plate method, or quantitative
culture following vortexing or sonication.
• Interpretation of culture results (Method 2):
— If one or more of the blood culture sets AND the catheter-segment culture are positive with the
same organism, as determined by identification phenotype and antimicrobial susceptibility
profiles, then a CRSBI is likely.
— If one or more of the blood culture sets are positive AND the catheter-segment culture is
negative, this may represent a CRSBI if positive for Staphylococcus aureus or Candida spp. AND
in the absence of any other identifiable source of infection. Documentation of CRSBI would
require additional positive peripheral cultures with the same organism, in the absence of any other
identifiable source of infection.
— If the blood culture sets are negative AND the catheter-segment culture is positive: suggests
colonization of the catheter, as opposed to a CRBSI.
— If both blood culture sets and the catheter-segment culture are negative: a CRBSI is unlikely.
Method 1 might be appropriate in those instances in which an attempt is being made to save the catheter
from having to be removed from the patient. If the decision has been made to remove the suspected
catheter from the patient, then Method 2 might be more appropriate.
6.5.3 Special Considerations: Infective Endocarditis
Blood culture results are critical to the diagnosis and management of patients with infective endocarditis.
It therefore is imperative that optimal procedures are used, such as those stated in the earlier sections of
this guideline. If optimal culture techniques are used, positive blood cultures will be obtained in over 90%
of infective endocarditis cases.87 The following recommendations are those that specifically apply to the
diagnosis of infective endocarditis.
When to Obtain Cultures
The first issue when evaluating a patient with suspected infective endocarditis is to determine when to
obtain the blood cultures. The continuous nature of most bacteremias associated with infective
endocarditis, however, renders timing less important.
• Acute Infective Endocarditis
In cases of suspected or known infective endocarditis that may be caused by highly virulent pathogens
such as Staphylococcus aureus, blood cultures should be drawn immediately to avoid unnecessary delays
in treatment.
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Recommendation: Obtain blood culture sets within a 30-minute period before administration of empiric
antimicrobial agents.
• Subacute Infective Endocarditis
If the patient presents with complaints that are suggestive of subacute disease, there is no urgent need to
obtain the cultures before starting initiation of empiric therapy. For these infections, it is far more
important to attempt to establish the microbiological diagnosis.
Recommendation: Obtain blood culture sets with the sets spaced 30 minutes to one hour apart. This may
help document a continuous bacteremia, which may provide additional clinical value, especially if the
echocardiogram is negative or equivocal.
Skin Antisepsis
Adequate skin disinfection is particularly important when evaluating patients with suspected infective
endocarditis. In patients who present with subacute infective endocarditis and have a prosthetic heart
valve(s), the principal pathogens are skin flora such as coagulase-negative staphylococci. Therefore, it is
especially important that blood cultures should be obtained by venipuncture and not from indwelling
intravascular devices. Catheter-drawn blood cultures are associated with an increased risk of
contamination, which might lead to a false conclusion of infective endocarditis.
Recommendation: Following adequate skin antisepsis, obtain blood for culture from separate, peripheral
venipuncture sites. Do not obtain blood for culture from indwelling catheters.
Number of Blood Cultures
The optimal number of blood cultures to be drawn varies, but a single blood culture set clearly is
inadequate. It has been demonstrated that positive blood cultures due to skin contaminants usually result
in a single-positive blood culture set when multiple sets were obtained.15 Therefore, multiple sets would
help a clinician to distinguish a “false-positive” blood culture, due to skin contaminants, from “true-
positive” blood cultures. The other reason that multiple blood culture sets should be obtained is due to the
increased volume of blood cultured, which is the single most important factor in the recovery of
microorganisms from blood. A single blood culture, therefore, would not enable determination of possible
continuous bacteremia, would not enable distinction between contamination and true bacteremia, and
would not provide the appropriate volume. Because subacute infective endocarditis has the highest pretest
probability of a positive blood culture, a total of three to five blood culture sets should suffice. If initial
blood cultures are negative, alternative diagnostic strategies should be considered.
Recommendation: Initially obtain three blood culture sets from patients presenting with possible infective
endocarditis. If those sets are negative at 24 hours, obtain two more sets of cultures, for a total of five sets
overall.
Blood Culture Media
No single blood culture system, nor any single culture medium, can detect all microorganisms that might
be present in the bloodstream of patients with suspected infective endocarditis. Use of an anaerobic
culture complements aerobic media in the recovery of facultative anaerobes, such as streptococci,
especially the nutritionally variant streptococci, and as such should still be included with every blood
culture set.
Frequently patients who are undergoing investigation for possible infective endocarditis have already
been placed on antimicrobial agents; this is the single most common reason for “culture-negative”
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infective endocarditis. In order to overcome the potential inhibitory activity of those antimicrobial agents
on bacterial growth, it is important to utilize a blood culture medium that has been designed to counteract
or neutralize this inhibitory effect.
In earlier studies of infective endocarditis, it was reported that another potential cause of “culture-
negative” endocarditis is the presence of the so-called “nutritionally variant streptococci” (NVS). These
streptococcal species, now called Abiotrophia defectiva, Granulicatella adiacens, and Abiotrophia
elegans, require supplemental vitamin B6 or cysteine for growth. Commercially available blood culture
media today either already contain these supplements, or the human blood added to the culture medium
provides them. Subcultures of blood culture bottles showing chains of gram-positive cocci consistent with
streptococci but without growth on sheep blood agar with a tryptic soy base are clues to the presence of
NVS. It is best practice to employ techniques to recover these microorganisms, which account for an
estimated 5 to 6% of viridans streptococcal endocarditis cases.157
Duration of Incubation of Blood Culture
Commercial blood culture systems recover most clinically important pathogens from blood within five
days, which is now the recommended duration of incubation for routine blood cultures. Previously it was
held that at least two weeks of incubation were required158 for optimal recovery of the more fastidious
microorganisms that may cause infective endocarditis. The data to support this recommendation were
based on older culture systems and media, and do not reflect the situation today. Recently reported data23
from the Mayo Clinic documented that all episodes of infective endocarditis were detected within a five-
day period of incubation of blood cultures using a CMBCS. In addition, a recent study in which an
extensive blood culture protocol was used with extended incubation showed only three clinically relevant
results after five days of incubation in 215 patients with suspected infective endocarditis159; these were
two Mycobacterium avium complexes and one Legionella pneumophila. All HACEK microorganisms (24
isolates) were recovered within five days.159,160
Recommendation: Incubate blood culture bottles for five days. If all blood culture sets are negative at five
days and the diagnosis of infectious endocarditis is still under consideration, subculture all bottles from
the blood culture sets to chocolate agar.
6.5.3.1 Special Considerations: Diagnosis of Fungal Infective Endocarditis
Fungal infective endocarditis includes cases caused by both yeasts and molds. This disease entity, once
considered rare, is now being reported more frequently.161 It has been stated that the increase in this
disease can be attributed to “medical progress,” because many of the risk factors for this disease are
associated with technologic advances: long-term intravenous catheterization; hyperalimentation;
immunosuppression; implantation of prosthetic devices, including heart valves and pacemakers;
prolonged use of antimicrobial agents; and cardiac surgery.161 Use of drugs of addiction is another
important risk factor for the development of fungal endocarditis.162
Typically a fungal etiology for infective endocarditis is not under initial consideration until the routine
blood culture results are reported as negative. In a recent worldwide review of 270 cases of fungal
endocarditis, this diagnosis was included in the differential diagnosis in only 48 (18%) of the cases at the
time of presentation.162 The most common fungal pathogens implicated in infective endocarditis are
Candida albicans and non-albicans Candida spp.161,162
Many of the same technical factors for diagnosis of bacterial endocarditis apply to the diagnosis of fungal
endocarditis. Most routine manual and automated blood culture system media are able to support the
growth of yeasts such as Candida spp. If routine blood cultures are negative with these media, then it may
be warranted to specifically request a “fungal blood culture,” in which media are used that optimally
support the growth of most yeasts. This is rarely indicated, however, and only if the routine methods used
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are known to be suboptimal for yeasts. Overall, if optimal methods are utilized, the percentage of positive
blood cultures in fungal endocarditis for Candida spp., is 83 to 95%.163
6.5.4 Patients Receiving Antimicrobial Therapy
Patients are frequently receiving antimicrobial therapy at the time of blood culture collection. The
possibility that these antimicrobials inhibit recovery of bacterial pathogens has long been a concern.
Recent research corroborates this concept by showing that when blood cultures are obtained before and
after initiation of antimicrobial agents, the postantibiotic cultures are less likely to yield
microorganisms.164
Medium formulations have been developed for continuous-monitoring blood culture systems that add
resins or proprietary activated charcoal substances intended to remove antimicrobial agents. In laboratory
experiments, the effectiveness of these substances in adsorbing or neutralizing antimicrobials varies from
one drug class to another, and the interval required for removal may be hours to days.165 Comparisons of
each of these products against each other show that they perform similarly in overall recovery rates of
microorganisms.166,167 In multiple trials, these media perform better than standard media.85,168-172 In some
studies, recovery is enhanced exclusively for samples obtained from patients who are receiving
antimicrobials at the time of blood culture collection, but in others these supplemented media perform
better for overall recovery of microorganisms, even in patients who have not been treated. Hence, it is not
clear that the advantage these formulations have is specifically the result of antimicrobial removal.
Among individual microorganisms, staphylococci consistently grow more often from one of these media,
and in selected studies, streptococci, Enterobacteriaceae, nonfermenters, and yeasts are also recovered
more often.
Microorganisms judged to be contaminants may also grow more frequently in these media.173 Moreover,
resin- and charcoal-containing products are more expensive than standard blood culture media. In one
study, these media only enhanced recovery of microorganisms that were already being effectively treated
with antimicrobials, and their recovery had little impact on patient management.174 In contrast, another
study showed important clinical benefits from the improved recovery in a tertiary care setting.174
6.5.5 Rare and Fastidious Pathogens
These microorganisms are infrequently recovered from blood, but when encountered may represent
serious infection. The recovery of a member of the HACEK group of bacteria in blood is among the
major criteria for diagnosis of infective endocarditis.175 In the past, the recovery of these microorganisms
required special procedures,98,176 but the current automated technology provides generally reliable
detection of fastidious isolates (e.g., Haemophilus spp., Abiotrophia spp., Cardiobacterium spp.,
Actinobacillus spp.) within the same timeframe of routine blood cultures.159,160 In contrast to the majority
of blood cultures yielding nonfastidious pathogens, which become signal-positive in the first 24 to 36
hours of incubation, bacteremia caused by fastidious microorganisms may require up to five days of
incubation and testing before becoming signal-positive. Microorganisms such as Legionella, Bartonella,
or Mycoplasma are more optimally diagnosed by means of immunodiagnostic or molecular techniques, as
are the uniformly uncultivable (by usual bacterial culture systems) agents such as Coxiella,
Chlamydophila, Rickettsia, and Tropheryma.177,178 With the exception of Bartonella spp., most rare or
fastidious bacteria are still cultivable by usual bacterial culture systems and can be recovered using blood
culture protocols currently in use by most clinical laboratories. The usual five-day cycle practiced in
many laboratories with continuous-monitoring instruments will permit detection of even
Abiotrophia/Granulicatella and Francisella.
A phenomenon sometimes associated with highly fastidious bacteria in blood cultures is that of signal-
positive/Gram stain-negative cultures. The bacterium may not be revealed by Gram stain of the contents
of a positive bottle for a number of reasons. Failure to observe the bacterium usually derives from an
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atypical morphology or unusually small size (e.g., Abiotrophia spp. may produce bizarre shapes or sizes,
while Francisella may be so small and indistinct that they blend in with background on the slide). In
some cases, as with the mycoplasmas, they may be unable to be Gram stained. An acridine orange stain
may, in these cases, reveal the bacterium.179 For example, use of either acridine orange or a Gram's stain
using carbol fuchsin as the counterstain may be necessary to see Campylobacter, Helicobacter, or
Brucella on smears.
Detection of fastidious gram-negative bacteria in blood cultures should always elicit safety precautions
because of the highly infectious nature of some of these bacteria (e.g., Francisella or Brucella). These can
be safely handled and presumptively identified in microbiology laboratories that use Biological Safety
Level-2 (BSL-2) practices; use of the biological safety cabinet is recommended at the earliest possible
stage of blood culture examination.
Abiotrophia and Granulicatella: These bacteria can be detected in the automated blood culture systems,
usually after the second day of incubation.180 Isolation in subculture depends on the presence of pyridoxal
or cysteine in the medium. This requirement can be met by supplemented media, by cocultivation with
staphylococci around which satellite colonies will grow, or by use of enriched chocolate and some
anaerobe agars. Consequently, these bacteria may be isolated on chocolate or anaerobe subcultures but
not on blood agar if a base such as TSA is used.
Bartonella: Bartonella spp. can be recovered from blood by culture but the yield is low; in most cases
special handling is necessary. The persistent and recurring nature of Bartonella bacteremia elicits strong
antibody responses, making serology a diagnostically useful approach.181 Numerous PCR assays have
been developed for direct detection of Bartonella spp. in clinical specimens, including those from patients
with endocarditis.182 If blood cultures are performed, lysis-centrifugation in combination with plating on
freshly prepared, enriched, blood or chocolate media is optimal.183 Plates should be incubated at 35 °C in
a humid atmosphere containing elevated CO2 for 14 to 21 days. Alternatively, a lytic broth system may be
used, followed by subculture to solid media upon termination of a seven-day incubation period.
Brucella: In the past, isolation of Brucella from blood was done using biphasic media and prolonged
incubation. With the advent of CMBCSs, recovery of Brucella spp. differs little from that of other
bacteria.184,185 While a commercially available biphasic system would still be suitable, the sensitivity and
efficiency of detection is best (and also when compared to lysis-centrifugation) with the CMBCSs.186
Studies from geographic areas endemic for brucellosis suggest that most isolates can be recovered within
three days. The rate of detection by the seventh day is at least 95%, and rarely an isolate may require
longer than seven days for detection.187,188 When one of the CMBCSs is used, most isolates are recovered
from aerobic bottles only.
Campylobacter: Systemic disease due to various species of Campylobacter can occur.189 Though usually
associated with acute gastroenteritis, C. jejuni may also be recovered from blood. Infrequently
encountered Campylobacter species, such as C. fetus, C. lari, or C. upsaliensis, can be recovered using
the CMBCS typically after two to three days of incubation. Growth of C. jejuni is slower at 35 to 36 °C
than at 42 °C, so subcultures may appear negative after the first 24 hours. Colonies should be visible after
48 hours of incubation, provided the plates are incubated in a microaerobic atmosphere.
Francisella: F. tularensis grows in standard blood culture media. Although these bacteria typically
require cysteine supplementation for optimal recovery, this is usually not necessary. F. tularensis varies
in its growth rate; hence, the length of incubation required before detection is unpredictable.190,191 There
are relatively rapidly growing strains that behave as practically nonfastidious, while other strains require
more than ten days of incubation. Due to its tiny, pleomorphic characteristics, F. tularensis is easily
missed upon examination of Gram-stained contents of blood culture bottles. Subcultures of F. tularensis
may at first grow on standard sheep blood agar (SBA), but upon subsequent passage will fail to grow on
SBA. Some isolates may initially grow only on chocolate agar plates.
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HACEK Group: Identification of a bacterium from the group of HACEK microorganisms is highly
suggestive of infective endocarditis, even in the absence of an obvious focus of infection and of typical
physical findings. The group includes Haemophilus spp. (other than H. influenzae), Actinobacillus
actinomycetemcomitans, Cardiobacterium spp., Eikenella corrodens, and Kingella spp. They cause a
variety of infections, in addition to endocarditis, which also may be accompanied by bacteremia.
Regardless of the focus of infection, most blood cultures that yield these microorganisms are positive
within the first week.192 In a study focused on HACEK endocarditis employing biphasic and lysis-
centrifugation methods, blood cultures became positive in a mean of 3.4 days.193 If a high index of
suspicion for HACEK endocarditis exists, and cultures are negative after five days, a longer incubation
period or terminal subculture may be necessary.
Helicobacter: There are several newly described or recently reclassified species of Helicobacter. They
can occur as bacteremic pathogens, usually in immunocompromised patients, with H. cinaedi as the most
frequently isolated species. These bacteria can be detected as early as the third day of incubation by
automated blood culture systems, but they often require more than five days to signal positive.194-196
Optimal recovery of these bacteria requires extending the incubation period to seven days and adding a
terminal subculture onto enriched blood agar followed by incubation in a hydrogen-enriched microaerobic
atmosphere.
Legionella: Legionella spp. are common causes of community-acquired pneumonia in many geographic
regions, but Legionella bacteremia secondary to pneumonia is unusual.197 Legionella bacteremia can also
occur following renal transplantation, and cases of prosthetic valve endocarditis caused by Legionella
spp. have been reported.198 Culture of Legionella requires use of buffered charcoal yeast extract (BCYE),
and subculture onto BCYE after a five-day incubation period of routine blood cultures is acceptable, as is
use of BCYE agar in conjunction with the lysis-centrifugation system.
Leptospira: Although methods for recovering leptospires from blood have been described, these bacteria
are unlikely to be recovered from blood cultures.199 Other laboratory methods for diagnosis should be
used in cases of suspected leptospirosis.
Mycoplasma: Current automated blood culture systems designed to recover common pathogenic bacteria
are not reliable for recovery of Mycoplasma spp. from blood. M. hominis may be fortuitously isolated
from conventional blood cultures, and addition of supplements such as gelatin or arginine may enhance
recovery, but slow growth and specialized medium requirements and mycoplasma-specific cultivation
techniques should be employed when this organism is suspected on clinical grounds.200 For optimal
recovery, blood should be inoculated onto specific media (e.g., SP4 glucose at pH 4.5) that can be used
for both Mycoplasma pneumoniae and Mycoplasma hominis (provided that arginine is added for the
latter).
7 Reporting Results
The results of blood cultures, whether positive or negative, are critical to patient care. Therefore, the
reporting of blood culture results must be effective and consistent throughout the culture process. The
report must allow the healthcare provider to quickly and accurately identify the status and any test results
for an ordered blood culture. Though a fairly wide range of reporting mechanisms may exist for different
types of laboratories and healthcare systems, several generic guidelines pertain.
7.1 Written and Electronic Reports
7.1.1 Specimen Status
It is important that the healthcare provider be able to determine the status of a specimen quickly and
efficiently. Specific information includes the answers to the following questions:
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• Has the specimen been ordered?

• Has the specimen been collected?
• What testing was ordered on the specimen?
• Has the specimen been received by the laboratory?
• Is the specimen acceptable for the testing requested?
• Is the requested testing in progress?
A record should be created in the laboratory information system (LIS) as soon as possible for each blood
culture ordered. Data concerning change in the specimen status, as described above, should be entered
into the record as soon as possible.
7.1.2 Culture Data
Each record should include a result, as described below. Culture data and other information should be
verified as quickly as possible. Critical value results, as described below, must be communicated as soon
as possible (within 60 minutes). Noncritical culture information should be entered into the culture record
within four hours of verification.
7.1.3 Preliminary Written Results
The following may serve as examples of specimen status reports:
• blood culture ordered, specimen not received;

• testing in progress, no results to date;
• no growth at 24 hours;
• no growth at 48 hours; and
• positive blood culture.
At the first detection of a positive blood culture, written and verbal reports, including the Gram stain
results, should be issued to the healthcare provider responsible for the patient as described in Section
7.1.5, Critical Value (Verbal) Reports. Preliminary written reports include, as available:
— final Gram stain result;
— preliminary identification (based on colonial and microscopic morphology, preliminary test

results, etc., according to laboratory protocol); and
— final antimicrobial susceptibility data.
7.1.4 Final Written Results
The final blood culture result should include one of the following:
• Test Canceled
• No Growth – The total incubation time, according the laboratory protocol, should be indicated in
the final report.
• Positive Blood Culture – A verbal report, as described in Section 7.1.5, Critical Value (Verbal)
Reports, should be issued for any final positive blood cultures if one was not issued during the
preliminary status of the testing.
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The final written report includes, as available:
— final Gram stain result;

— final identification; and
— final antimicrobial susceptibility data.
• Other Information—Any additional information that may impact the interpretation of the final test
result should be included in the written report. Examples of such information include inadequate
blood volume collected, prolonged transport time, or other factors as described in Section 10,
Quality Assurance.
7.1.5 Critical Value (Verbal) Reports
Any blood culture test result that may have an important impact on patient care (i.e., critical values)
should be reported according to laboratory policy. Such policies must be designed, in consultation with
the healthcare provider served by the laboratory, to ensure compliance with standards of medical care.
Critical values should be communicated verbally, as described below, unless alternative communication
strategies have been established and validated to ensure timely and accurate communication of results;
also, document that the critical value results were received by the healthcare provider.
The policy regarding communication of critical values should ensure that information is communicated
quickly and accurately to the healthcare provider. A mechanism for identifying an alternative licensed
caregiver must be provided for cases in which the requesting healthcare provider is unavailable.
The first laboratory result (whether stain or culture) documenting a positive blood culture should be
communicated as a critical value for every positive blood culture. Critical value reports should be issued
as soon as possible (within 60 minutes) after laboratory verification of the abnormal result.
Communication of information, generated by subsequent testing, may not be required unless the
information is significantly different from the information communicated in the original critical value
communication.
Several other specimen reports should be communicated as critical values. A critical value report should
be issued when unacceptable specimens are received for blood culture testing and a new blood culture is
needed (e.g., a broken blood culture bottle).
A critical value result should be issued for any correction to previously issued preliminary or final results,
whether the changed results were issued as critical values or by routine reporting. The written report
should clearly indicate that the results represent a corrected report, and provide details regarding the
change to the previously issued report.
Each critical value report should include the following components, which must be documented in the
laboratory record:
• full name of the person issuing the report;
• date and time of unsuccessful and successful attempts to contact a healthcare provider responsible
for the patient;
• full name of the person to whom the report is issued;
• the abnormal value reported, with emphasis that the result is a critical value; and
• documentation that the person receiving the report “read-back” the result.
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Written and/or electronic reporting, according to the laboratory protocol, must follow all critical value
verbal reports.
8 Contaminants
In order to minimize blood culture contamination, each laboratory should have policies describing: 1)
blood culture collection techniques that minimize contamination; and 2) a standardized process for the
evaluation of blood culture isolates to determine the contamination rate. Even when the best blood
collection protocols are used it may not be possible to reduce the contamination rate below 2%.39
Microorganisms commonly associated with contaminated blood cultures (e.g., Bacillus spp.,
Corynebacterium spp., Propionibacterium spp., coagulase-negative staphylococci, Aerococcus spp.,
Micrococcus spp.) are also capable of causing systemic, blood-borne infection in the appropriate setting.
In many instances, a potential contaminant is recovered from one or both bottles of a single blood culture
set. Without a second blood culture for comparison, it is virtually impossible to assign significance to a
questionable isolate. Therefore, the evaluation of an isolate with low virulence potential recovered from a
single blood culture set (one or both bottles) should be limited to the extent at which medically important
microorganisms can be excluded from the identification. Susceptibility testing should not be done on
suspected contaminants. All isolates should be saved for a few days so that additional studies can be
performed if an identical organism is recovered from subsequent blood cultures of the same patient.
Recovery of multiple isolates of the same organism(s) from independent blood cultures warrants full
identification and susceptibility testing of the initial isolate(s). The publication of Richter et al. provides a
detailed description of a laboratory-based algorithm for minimizing the extent of evaluation of blood
culture contamination.201
9 Safety Issues
The risk of injury or laboratory-acquired infection must be minimized for laboratory personnel.
Publications such as CLSI/NCCLS document GP17—Clinical Laboratory Safety202 and ISO 15190,
Medical laboratories—Requirements for safety203 provide information on the implementation of an
effective safety program for laboratory activities. These documents address important issues, including
program organization, facility design, maintenance and inspection of equipment, personal safety, signage,
and labeling. Specific areas addressed include fire prevention; chemical, electrical, microbiological, and
radiation safety; waste disposal; evacuation and emergency response; program evaluation; and other
laboratory safety issues.
CLSI/NCCLS documents M298 and H3—Procedures for the Collection of Diagnostic Blood Specimens
by Venipuncture204 provide additional information regarding safety issues directly related to blood culture
examination.
Pathogens present in samples collected for blood culture examination have the potential for causing
infection in laboratory workers through percutaneous injury or by direct contact with the worker’s skin,
eyes, or mucus membranes. In addition, indirect exposure of personnel, by contaminated surfaces or to
pathogens isolated in culture, provides another potential mechanism for laboratory-acquired infection.
The risk of infection is decreased by implementation of procedures that minimize the chance of exposure
of personnel to infectious materials. Such procedures include use of standard precautions, use of personal
protective equipment and barriers, use of safety devices and equipment, and proper handling of specimens
and biohazardous waste. For certain pathogens, like hepatitis B virus and Neisseria meningitidis,
immunization of laboratory personnel provides an effective adjunct to engineering controls. Institution
policies must comply with federal and local regulations.
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9.1 Agents Associated With Laboratory-Acquired Infections
9.1.1 Transmission of Hepatitis Viruses and Retroviruses
The importance of preventing percutaneous injury cannot be overemphasized, especially during the
collection of samples by venipuncture. The average risk of infection after needlestick injury is ~18% for
hepatitis B virus (HBV), 1.8% for hepatitis C virus (HCV), and ~0.3% for human immunodeficiency
virus (HIV). See the most current edition of CLSI document M29.8 Though transmission of HBV, HCV,
and HIV through direct cutaneous and mucus membrane exposure have been well documented, precise
incidence studies defining the risk of transmission after such exposures have not been well defined. Safety
strategies to prevent transmission of HBV are considered sufficient for prevention of HCV and HIV
transmission.
9.1.2 Transmission of Agents Other Than Hepatitis Viruses and Retroviruses
While the hepatitis viruses and retroviruses present the greatest risk of transmitting infection from blood
samples collected for blood culture examination, the handling and manipulation of patient samples and
cultures in the laboratory present an increased risk of infection due to other pathogens. Though
percutaneous exposure represents the most common mode of transmission of the hepatitis viruses and
retroviruses, transmission of other infectious agents within the laboratory is most commonly due to
aerosol or small-droplet formation. Therefore, engineering and work practice controls that minimize the
formation and dissemination of infectious droplets and aerosols must be employed for activities that
might produce them.
Some naturally occurring agents that are associated with high risk for laboratory-acquired infection may
be isolated by blood culture procedures. Because the isolation of such isolates is not predictable, all blood
culture isolates should be handled according to protocols based on Biosafety in Microbiological and
Biomedical Laboratories (BMBL).205
9.2 Protective Measures
Prevention of infection transmitted by samples submitted for blood culture examination, or pathogens
isolated from these samples, depends on compliance with an effective laboratory-wide exposure control
plan. Protocols for activities related to blood culture examination should provide detailed instructions for
the following protective measures:
Hand Washing
Frequent hand washing is a critical component for preventing transmission of laboratory-acquired

infection. Hands, or other skin surfaces, must be washed immediately after direct contact with blood or
any potentially infectious material. Hands should also be washed before donning gloves and after removal
of gloves, after completion of work, and when leaving the laboratory or moving into a clean area within
the laboratory. MMWR Guideline for Hand Hygiene in Health-Care Settings206 provides information for
establishing a hand hygiene program for a variety of healthcare settings.
Barrier Protection
OSHA regulations mandate that institutions provide employees with appropriate personal protective
equipment. Gloves must be worn during the collection of samples for blood culture examination and
changed between each patient contact. Gloves must be changed immediately if they become contaminated
by blood or show any sign of breakage or loss of barrier function. Gloves should also be worn in the
laboratory specimen receiving and processing areas, in the mycobacteriology laboratory, and other areas
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of the laboratory where hands may come into contact with infectious materials or contaminated surfaces.
Gloves should be worn when handling and disposing of biohazardous waste.
Facial protection should be used whenever there is a possibility of splashing of blood or other infectious
material. A face shield, fluid-resistant mask with eye protection, or splashguard can provide this
protection.
Fluid-resistant, long-sleeved, closed-front protective clothing should be worn for activities related to
blood culture processing and examination. Protective clothing must be removed immediately if visibly
contaminated. Contaminated protective clothing should be discarded as biohazardous waste or laundered
according to the institution’s policy. Protective clothing must be removed before leaving the laboratory or
moving into a clean area of the laboratory.
Biological Safety Cabinets
Class IIA or IIB biological safety cabinets should be used for laboratory activities that have a significant
risk for formation of small droplets or aerosols.
Sterilization, Disinfection, and Decontamination
The collection and manipulation of samples for blood culture examination presents risks for
contamination of equipment and environmental surfaces with infectious material. Detailed instructions for
the prevention and management of infectious material spills must be provided by general laboratory
procedures. Decontamination of laboratory equipment should follow instructions provided by the
manufacturer as well as standard laboratory practice. Individuals responsible for transporting diagnostic
specimens must be trained in packaging that is compliant with applicable local and federal regulations.
Transport procedures must provide containment for potential spills and provide instructions for response
to spills recognized during transport.
Samples for blood culture examination may be transported through a pneumatic tube system if care is
taken to prevent breakage. In the event of breakage during transport, detailed instructions must be
provided for response to different types of potential contamination. If potentially infectious material may
have leaked out of the transport carrier, the entire system must be shut down while potentially
contaminated routes and carriers are identified from the system’s traffic records. Detailed instructions for
decontamination of the stations, tube system, and carrier components (carrier, liner, containment pouches,
etc.) must be prepared according to the manufacturer’s instructions.
If a spill involves agents that are transmissible via inhalation, the room should be closed for a minimum
of 30 minutes to allow droplets to settle. Appropriate personal protective equipment must be worn during
clean-up procedures; gown, gloves, and facial protection are a minimum. Use of puncture-resistant gloves
is required if broken glass or other sharp object is involved in the spill. Use of respirators is required if an
infectious aerosol may have been formed, or for agents with high risk for respiratory transmission, such as
M. tuberculosis. Water impermeable shoe covers must be worn for large spills on the floor.
The exact procedures employed by laboratory protocol should take into account the volume and type of
spilled material, infectious agent potentially present and its concentration, and type of contaminated
surface. In general, the response to spills includes the following components:
• containment and absorption;
• removal of residual material using an aqueous detergent;
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• decontamination by flooding the surface using an intermediate hospital disinfectant, such as diluted
household bleach. Concentration and time of exposure will depend on the type of surface
contaminated;
• removal of disinfectant and rinse with water;
• drying surface to prevent slipping; and
• disposal of materials used for decontamination and all contaminated materials that could not be
effectively decontaminated.
Standard Precautions
Standard precautions provide engineering and work practice controls that will minimize the risk of
contact with infectious materials or, in the case of accidental contact, minimize the duration of exposure.
All employees must receive initial training in the basic principles and practices related to standard
precautions. In addition, retraining related to the concepts of standard precautions should be incorporated
into the laboratory’s continuing education program.
A critical component of standard precautions is the prevention of percutaneous exposure. CLSI/NCCLS

document X3—Implementing a Needlestick and Sharps Injury Prevention Program in the Clinical
Laboratory207 provides guidelines for implementing an appropriate program for activities related to blood
culture examination. Several strategies should be considered:
• Minimize the use of needles and other sharp instruments (e.g., use safety devices for venting bottles
and subculturing).
• When needles must be used, as in the collection of samples for blood culture examination, use
safety devices and practices to minimize the chance of needlestick injury. Recapping needles, when
necessary, and other manipulation of exposed needles, like those that may be required to vent
aerobic blood culture bottles, should be performed using techniques that prevent directing the point
of the needle to any part of the body.
• If blood is collected by syringe, the needle used for collection should not be replaced before
inoculation of blood culture bottles. The use of intermediate collection tubes or bottles increases the
risk of blood culture contamination, as well as the risk of injury during subsequent transfer; their
use, therefore, is discouraged.
• Needles should be equipped with a mechanism to minimize the risk of injury. The safety
mechanism should be used, according to the manufacturer’s instructions.
• Needles must be placed in an approved sharps container after use. The container should be close to
the site of use with the opening clearly visible. Sharp containers should not be overfilled.
• A sharps container must be used if exposed needles or other sharps must be transported to or within
the laboratory.
• The use of glass should be avoided for laboratory supplies that come in contact with patient
samples or infected materials.
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Management of Laboratory Accidents
Laboratory personnel must be trained and maintain competence in risks, prevention, and management of
accidents related to job performance. The components of postexposure management should include acute
exposure management, exposure reporting, evaluation of risk associated with exposure, and postexposure
prophylaxis. Detailed protocols, in compliance with OSHA and other federal regulations as well as the
institution’s infection control program, should be established to address all aspects of personnel
management after exposure to potentially infectious materials.
Engineering controls and processes that will minimize exposure of laboratory personnel to infectious
material should be provided for activities performed in dirty areas. Specifically, activities that increase the
risk of exposure of laboratory personnel to infectious material in dirty areas should be clearly prohibited
by protocol. Such high-risk activities include: mouth pipetting; nail biting; smoking; eating; contact lens
manipulation; contact of eye, nose, or mouth with hands or other environmental surfaces; etc. Laboratory
personnel should avoid use of contact lenses in dirty areas. If absolutely required, goggles or other eye
protection should be used.
Procedures associated with a high risk for splashing should be performed in a biological safety cabinet or
behind a splashguard.
Centrifuges must be operated according to the manufacturer’s instructions. Centrifuges should be

equipped with sealed rotors or safety cups. Plastic tubes with sealing screw-top tubes should be used
whenever possible. Before use, tubes must be examined carefully for signs of damage. The contents
should be examined for signs of breakage before opening the sealed rotor or safety cups. Rotors or safety
cups and centrifuged samples should be opened within a biological safety cabinet for samples likely to
contain agents spread by airborne transmission.
Respiratory Protection
Respiratory protection must be provided when laboratory personnel may be exposed to agents associated
with a high risk for airborne transmission, through contact with infected patients, specimens obtained
from infected patients, or laboratory isolates. Laboratory personnel performing activities related to blood
culture examination for mycobacteria may be exposed to such risks. Performance of personal respirators
must comply with current CDC, NIOSH, and OSHA recommendations with regard to filter efficiency for
1-µm particles; fit testing to document face-seal integrity; ability to fit various facial sizes and
characteristics; the ability of healthcare workers to evaluate face piece fit each time they put on a
respirator; and other applicable criteria.
Laboratory Instruments and Equipment
Instruments and equipment should only be operated according to the manufacturer’s instructions and with
sufficient engineering and work practice controls to minimize any type of injury to laboratory personnel.
Parts of any device that contact patient samples or infectious material as part of normal use should be
decontaminated regularly during routine maintenance and before disposal or repair. Equipment that has
been contaminated, or operated in a “dirty” area of the laboratory, must be decontaminated immediately
according to manufacturer’s instructions. NOTE: Dried blood must be considered as infectious as liquid
blood. Uncontaminated supplies, reagents, and materials must be stored separately from patient samples
and stored cultures. Subcultures of patient or other isolates must be stored in an organized and secure
manner that ensures easy access and identification at the time of retrieval or disposal. Containers (ideally
plastic) designed for low-temperature storage must be used for samples or isolates that require freezer
storage.
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Safety Training
Initial safety training and evaluation of ongoing competence must be provided to laboratory personnel, in
an effective format, for all activities they perform in the laboratory. The training, monitoring activities,
incidence response, and documentation related to safety must comply with federal and local regulations
and should be a core component of the laboratory’s quality assurance program.
Special Precautions Related to Blood Culture Examination
Unless there is increased suspicion for the isolation of a highly infectious agent requiring higher-level
biosafety practices, BSL2 practices are adequate for routine activities related to blood culture
examination.
CLSI/NCCLS document H3204 provides guidance for preparing protocols for blood sample collection and
blood culture examination. The phlebotomist must follow procedures to minimize the risk of transmitting
infection from contact with the patient or specimens taken from the patient. An important aspect of
patient safety related to blood culture examination is the procedure for identification of the patient. Two
reliable identifiers must be obtained and compared to the requisition form. The protocol must be reliable
for patients who are conscious and cooperative as well as for those patients who are not. Examples of
reliable information include: full name, current address, hospital identification number, social security
number, or birth date. The patient’s location is not reliable for patient identification.
Samples collected for blood culture examination should be placed in a leakproof primary container with a
secure closure. The requisition slip should not be placed in the primary container because of the
possibility of contamination. Primary containers with separate pockets, like plastic bags, provide a
convenient system to minimize contamination of requisition forms. The samples should be transported to
the laboratory in secondary containers that are able to contain the specimen in the case of a break with
leakage from the primary container.
For blood culture specimens transported by pneumatic tube, plastic bags that have been shown to be
leakproof within the system should be used for transport. Place bottles in a primary plastic transport bag
and close securely. Secure the bottles together using tape or a rubber band around both bottles. Place the
secured bottles into a secondary plastic transport bag and close securely. The sample and test requisition
slip are placed in the transport carrier that is then closed, locked, and transported to the laboratory by
standard protocol. No other specimens should be transported with a specimen for blood culture
examination.
Waste generated by activities related to blood culture examination must be managed according to
biohazardous waste disposal procedures of the institution, in compliance with relevant federal and local
regulations. CLSI/NCCLS document GP5—Clinical Laboratory Waste Management208 provides
information concerning key elements for waste management and guidance for establishing an effective
waste management system within the laboratory.
10 Quality Assurance
10.1 Preexamination Process
The preexamination process for blood culture examination includes the following components: patient
evaluation; test selection and ordering; sample collection; sample transport; sample receipt; and sample
processing. Each of these components includes multiple procedures or processes for successful
completion.
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10.1.1 Patient Evaluation
Blood culture examination may provide critical information that impacts therapy and/or prognosis for
selected patient groups; therefore, it is imperative that every organization develops guidelines to identify
appropriate patients for blood culture analysis. Guidelines should also identify clinical scenarios with a
low prior probability of bacteremia or fungemia for which blood culture examination is not
recommended. False-positive blood culture examination for such low-risk patients is likely to have a
negative impact on the patient’s outcome and/or the institution’s cost of caring for the patient.
Example QA Indicator: Proportion of patients admitted with presumed bacterial pneumonia, from whom
blood cultures were collected as part of the initial diagnostic work-up.
10.1.2 Test Selection and Ordering
The laboratory should work with the organization’s healthcare practitioners to ensure that clinical practice
standards result in selection of the most appropriate use of blood cultures.
Protocols for when to draw or not to draw blood cultures should be available for all practitioners who use
a laboratory. Requests for blood culture examination must be submitted using a standardized process
(paper or electronic) established by the organization in compliance with relevant local and federal
regulations. The laboratory must ensure that healthcare providers have been trained in the accurate
completion of relevant paper or electronic requisition forms. All critical information must be included in
the requisition in a legible form, including, but not limited to: unequivocal patient identifiers; unequivocal
identification of the authorized healthcare provider requesting the examination; specimen type and detail;
specific examination requested; patient diagnosis (ICD-9-CM code); and pertinent clinical information.
Example QA Indicator 1: Proportion of patients with blood cultures who have the recommended number
of blood culture sets submitted. Collection of two or three blood culture sets is recommended per episode.
Example QA Indicator 2: Proportion of patients with more than the recommended number of blood
cultures submitted. Collection of two or three blood culture sets is recommended per episode for the
initial patient evaluation. Collection of another two or three blood culture sets may be indicated after 48 to
72 hours if the initial culture sets were noninformative. “Surveillance” cultures are not recommended.
10.1.3 Sample Collection
Upper extremity venipuncture is recommended for the collection of samples for blood culture
examination under most circumstances. CLSI/NCCLS document H3204 provides guidance to laboratories
for the preparation of procedures related to sample collection for blood culture. Collection of samples by
arterial puncture or lower extremity venipuncture may increase the risk of patient injury and of culture
contamination. Specimens collected through lines also have a greater risk for contamination.
Detailed protocols for collection of samples will minimize the medical errors that can occur with
venipuncture (including misidentification of samples or patients, incorrect collection vessel, incorrect
timing of collection, formation of hematomas, nosocomial anemia, and hemoconcentration). A training
program with documentation of trainee competency is essential for healthcare providers who will draw
blood cultures. Such protocols should be designed with the goals of minimizing the risks for both the
patient and phlebotomist, as well as ensuring the collection of a sample that will produce informative
results from the blood culture examination. The use of a dedicated team for collecting blood culture
samples should be considered.
The following information must be provided for all blood culture specimens:
• patient’s first and last name;
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• a unique identification number;

• date and time of collection;
• other information or label required by institution policy; and
• identification of the person collecting the specimen.
Compared to blood samples collected for most other types of laboratory examination, samples for blood
culture examination require additional procedures to maximize the detection of microbial pathogens and
to minimize the risk of contaminating the blood culture. Protocols for blood culture examination must
follow the manufacturer’s instructions for specific blood sample requirements. When possible, samples
should be collected before administering antimicrobial agents.
Example QA Indicator 1: Blood culture contamination rate. The goal for blood culture contamination
rate, whether analyzed overall or stratified by location, phlebotomist, etc., should be less than 3%.
Example QA Indicator 2: Proportion of blood culture bottles inoculated with more or less than the
recommended volume of blood. For adults, each blood culture bottle should be inoculated with 10 mL of
blood.
Example QA Indicator 3: Proportion of blood cultures submitted that include only a single inoculated
bottle.
Example QA Indicator 4: Proportion of blood cultures submitted that must be rejected for any cause.
10.1.4 Sample Transport
Samples for blood culture should be transported to the laboratory as quickly as possible in a manner to
ensure maintenance of sample integrity and compliance with applicable safety regulations. It is
recommended that samples for blood culture examination be delivered to the laboratory within two hours
of collection and transport.
Example QA Indicator: Proportion of blood cultures submitted with prolonged (>2 hour) transport time.
10.1.5 Sample Receipt and Processing
Samples for blood culture examination must be promptly received after delivery to the laboratory,
assessed for acceptability (with respect to collection, specimen volume, transport time and condition,
paperwork, labeling, etc.), accessioned into laboratory records, processed with media inoculation, as
required (refer to CLSI document M2280 for general QC requirements for media), and transferred to the
site of blood culture examination. Special instructions for the handling or incubation of samples should be
provided for times when processing within the laboratory will be delayed.
The status of samples for blood culture examination that are determined to be unacceptable at arrival in
the laboratory must be communicated immediately to the ordering physician or patient location according
to the laboratory’s policy for reporting critical test results.
Blood culture specimens that meet the following criteria should be processed but the provider notified
that the specimen is not optimal:
• inadequately filled bottles/tubes;

• insufficient numbers of cultures;
• single blood cultures; and
• blood inoculated into only aerobic or anaerobic bottles.
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When more than the recommended number of blood cultures is submitted, the specimens should be
processed but the provider notified that this will increase the risk of phlebotomy-induced anemia and is
more likely to result in the recovery of contaminants.
There are two common approaches to determine the adequacy of fill of bottles: visual comparison with a
volume standard and weighing bottles. Weighing bottles is more accurate, whereas visual comparison can
be done more quickly.
Example QA Indicator: Documentation of communication for rejected blood culture specimens.
10.2 Examination Process
The examination process for blood cultures includes the following components:
• procedures for the detection of microorganisms;

• identification of isolates;
• susceptibility testing of relevant isolates;
• verifying the reliability of test results; and
• interpretation of test results.
Each of these components includes multiple procedures or processes for successful completion. Detailed
laboratory protocols must be prepared for each process. These protocols must comply with relevant
regulations of the institution, government, and accreditation agencies. The specific procedures must also
comply with manufacturers’ instructions for equipment or kits, as described in package inserts or
operator’s manuals. CLSI document GP2—Laboratory Documents: Development and Control209 provides
guidelines for the preparation of effective procedures.
Rules for submitting isolates from blood culture samples for additional identification and susceptibility
testing must be included in the procedure for blood culture examination. The extent of testing may be
limited for likely contaminants or subsequent isolates of previously worked-up pathogens; however, the
procedure for limiting work-up of blood culture isolates must be validated and periodically reevaluated to
ensure that clinically relevant information is not being missed.
Activities performed during the examination process may produce information that could be important for
patient care. Such information, like results of staining or “preliminary” identification procedures, must be
communicated to the healthcare providers, but in a manner that ensures that the preliminary nature of the
report is clearly indicated. Verification of the final results should include a review of preliminary results.
Significant inconsistencies (e.g., preliminary report shows gram-negative diplococci, but final report
shows Streptococcus pneumoniae) should be reviewed by the laboratory director (or designee) and
communicated immediately to the healthcare practitioner according to the laboratory’s policy for critical
result reporting. Preliminary results should be included in the final blood culture examination report.
Objective guidelines for the interpretation of blood culture examinations should be available to ordering
clinicians (e.g., interpretive criteria and clinical significance for both positive and negative results). The
procedure manual for blood culture examination should include guidelines for the identification of
potential false-negative and false-positive results, as well as causes for such. Documentation should
identify other clinical or laboratory information that might be necessary to accurately interpret a blood
culture examination and/or avoid false-positive or false-negative results (e.g., number of samples
collected for blood culture examination; antimicrobial therapy before blood culture collection; other
laboratory signs, such as increased WBC; positive cultures from other infected sites; type and severity of
immune compromise; pretest probability of infection caused by a fastidious organism not reliably
detected by the routine blood culture examination; etc.).
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Example QA Indicator 1: Effectiveness of critical value results and documentation of communication.

Critical results from blood cultures should be communicated to an appropriate healthcare provider within
one hour of validation, and details of the communication should be clearly recorded in the culture record.
Example QA Indicator 2: Proportion of blood cultures that require correction to transmitted results.
10.3 Postexamination Process
The postexamination process for blood culture examination includes the following components:
• result reporting and archiving; and

• sample management.
Each of these components includes multiple procedures or processes for successful completion.
10.3.1 Reporting
Blood culture results must be verified as accurate before being reported. Methods and guidelines for
manual validation must be validated before use. After verification, the results must be communicated to
clinicians in accordance with well-documented, verified protocols. The laboratory reports must be legible
and organized so that results are clearly and unequivocally accessible to the healthcare providers
reviewing the report. Clear, standardized terminology should be used and abbreviations should be
avoided. When necessary, use of only institutionally defined abbreviations will minimize the chance of
misinterpreting the results. The use of “standard report” result comments may also minimize transcription
errors in preparation of examination reports.
The laboratory director or designee should review corrected reports on a daily basis. Cumulative
summaries of examinations with corrected reports should be reviewed periodically as part of the
laboratory QA process.
Example QA Indicator: The results of QA activities should be communicated with healthcare providers
in order to identify opportunities for improvement. The effectiveness of interventions designed to improve
quality related to blood cultures should be documented by continued monitoring of the relevant QA
indicator.
10.3.2 Record Management

Laboratory records for activities related to blood culture examination, including final reports, must be
archived in accordance with federal, local, and accreditation or certification service standards. Protocols
for record storage must clearly define the records to be stored, storage medium, methods for efficient
retrieval, duration of storage, and destruction of records after the end of storage. CLSI/NCCLS document
GP26—Application of a Quality Management System Model for Laboratory Services,210 Appendix D,
provides guidance concerning the retention schedule for various laboratory records.
10.3.3 Consultation
The laboratory medical director should be available to healthcare providers to discuss questions related to
the results or interpretation of blood culture examinations in the context of the patient’s specific clinical
condition. The laboratory director should recommend any appropriate additional clinical information or
follow-up laboratory examinations that could contribute to the interpretation of blood culture examination
reports.
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standard (5 mL) NR BACTEC system. Diagn Microbiol Infect Dis. 1991;14:111-118.
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Krisher KK, Gibb P, Corbett S, Church D. Comparison of the BacT/Alert PF pediatric FAN blood culture bottle with the standard pediatric
blood culture bottle, the Pedi-BacT. J Clin Microbiol. 2001;39:2880-2883.
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Wilson ML, Mirrett S, Meredith FT, et al. Controlled clinical comparison of BACTEC Plus Anaerobic/F to standard Anaerobic/F as the
anaerobic companion bottle to Plus Aerobic/F medium for culturing blood from adults. J Clin Microbiol. 2001;39:983-989.
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Jessamine PG, Hoban DJ, Forward KR. Positive BACTEC resin cultures do not influence antimicrobial selection. Diagn Microbiol Infect
Dis. 1990;13:281-284.
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McDonald LC, Fune J, Gaido LB, et al. Clinical importance of increased sensitivity of BacT/Alert FAN aerobic and anaerobic blood culture
bottles. J Clin Microbiol. 1996;34:2180-2184.
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Durack DT, Lukes AS, Bright DK, Duke Endocarditis Service. New criteria for diagnosis of infective endocarditis: utilization of specific
echocardiographic findings. Am J Med. 1994;96:200-209.
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Geraci JE, Greip PR, Wilkowske CJ, Wilson WR, Washington JA II. Cardiobacterium hominis endocarditis: four cases with clinical and
laboratory observations. Mayo Clin Proc. 1978;53:49-53.
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Larson AM, Dougherty MJ, Nowowiejski DJ, et al. Detection of Bartonella (Rochalimaea) quintana by routine acridine orange staining of
broth blood cultures. J Clin Microbiol. 1994;32:1492-1496.
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Murray CK, Walter EA, Crawford S, McElmeel ML, Jorgensen JH. Abiotrophia bacteremia in a patient with neutropenic fever and
antimicrobial susceptibility testing of Abiotrophia isolates. Clin Infect Dis. 2001;32:e140-e142.
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Bannatyne RM, Jackson MC, Memish Z. Rapid detection of Brucella bacteremia by using the BACTEC 9240 system. J Clin Microbiol.
1997;35:2673-2674.
185
Ruiz J, Lorente I, Perez J, Simarro E, Martinez-Campos L. Diagnosis of brucellosis by using blood cultures. J Clin Microbiol. 1997;
35:2417-2418.
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Yagupsky P, Peled N, Press J, Abramson O, Abu-Rashid M. Comparison of BACTEC 9240 Peds Plus medium and isolator 1.5 microbial
tube for detection of Brucella melitensis from blood cultures. J Clin Microbiol. 1997;35:1382-1384.
187
Yagupsky P. Detection of Brucellae in blood cultures. J Clin Microbiol. 1999;37:3437-3442.
188
Ozturk R, Mert A, Kocak F, et al. The diagnosis of brucellosis by use of BACTEC 9240 blood culture system. Diagn Microbiol Infect Dis.
2002;44:133-135.
189
Krause R, Ramschak-Schwarzer S, Gorkiewicz G, et al. Recurrent septicemia due to Campylobacter fetus and Campylobacter lari in an
immunocompetent patient. Infection. 2002;30:171-174.
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Haristoy X, Lozniewski A, Tram C, Simeon D, Bevanger L, Lion C. Francisella tularensis bacteremia. J Clin Microbiol. 2003;41:2774-
2776.
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1996;34:1995-2000.
192
Doern GV, Davaro R, George M, Campognone P. Lack of requirement for prolonged incubation of Septi-Chek blood culture bottles in
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Trivett-Moore NL, Rawlinson WD, Yuen M, Gilbert GL. Helicobacter westmeadii sp. nov., a new species isolated from blood cultures of
two AIDS patients. J Clin Microbiol. 1997;35:1144-1150.
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Kiehlbauch JA, Tauxe RV, Baker CN, Wachsmuth IK. Helicobacter cinaedi-associated bacteremia and cellulitis in immunocompromised
patients. Ann Intern Med. 1994;121:90-93.
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Rihs JD, Yu VL, Zuravleff JL, Goetz A, Muder RR. Isolation of Legionella pneumophila from blood with the BACTEC system: a
prospective study yielding positive results. J Clin Microbiol. 1985;22:422-424.
198
Chen TT, Schapiro JM, Loutit J. Prosthetic valve endocarditis due to Legionella pneumophila. J Cardiovasc Surg. 1996;37;631-633.
199
Palmer MF, Zochowski WJ. Survival of leptospires in commercial blood culture systems revisited. J Clin Pathol. 2000;53:713-714.
200
Waites KB, Canupp KC. Evaluation of BacT/ALERT system for detection of Mycoplasma hominis in simulated blood cultures. J Clin
Microbiol. 2001;39:4328-4331.
201
Richter SS, Beekmann SE, Croco JL, et al. Minimizing the workup of blood culture contaminants: implementation and evaluation of a
laboratory-based algorithm. J Clin Microbiol. 2002;40:2437-2444.
202
CLSI/NCCLS. Clinical Laboratory Safety; Approved Guideline—Second Edition. CLSI/NCCLS document GP17-A2. Wayne, PA:
NCCLS; 2004.
203
ISO. Medical laboratories—Requirements for safety. ISO 15190. Geneva: International Organization for Standardization; 2003.
204
CLSI/NCCLS. Procedures for the Collection of Diagnostic Blood Specimens by Venipuncture; Approved Standard—Fifth Edition.
CLSI/NCCLS document H3-A5. Wayne, PA: NCCLS; 2003.
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205
Biosafety in Microbiological and Biomedical Laboratories (BMBL). 5th edition. US Department of Health and Human Services; Public
Health Service, Centers for Disease Control and Prevention; and National Institutes of Health. February 2007.
206
Guideline for hand hygiene in health-care settings. MMWR. Vol. 51/No. RR-16; October 25, 2002.
207
CLSI/NCCLS. Implementing a Needlestick and Sharps Injury Prevention Program in the Clinical Laboratory; A Report. CLSI/NCCLS
document X3-R. Wayne, PA: NCCLS; 2002.
208
CLSI/NCCLS. Clinical Laboratory Waste Management; Approved Guideline—Second Edition. CLSI/NCCLS document GP5-A2. Wayne,
PA: NCCLS; 2002.
209
CLSI/NCCLS. Clinical Laboratory Technical Procedure Manuals; Approved Guideline—Fourth Edition. CLSI/NCCLS document GP2-
A4. Wayne, PA: NCCLS; 2002.
210
CLSI/NCCLS. Application of a Quality Management System Model for Laboratory Services; Approved Guideline—Third Edition.
CLSI/NCCLS document GP26-A3. Wayne, PA: NCCLS; 2004.
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Additional References
McDonald LC, Weinstein MP, Fune J, Mirrett S, Reimer LG, Reller LB. Controlled comparison of
BacT/Alert FAN aerobic medium and BACTEC fungal blood culture medium for detection of fungemia.
J Clin Microbiol. 2001;39:622-624.
Mirrett S, Joyce M, Reller LB. Validation of performance of plastic versus glass bottles for culturing
anaerobes from blood in BacT/ALERT SN medium. J Clin Microbiol. 2005;12:6150-6151.
Reimer LG. Laboratory detection of mycobacteremia. Clin Lab Med. 1994;14:99-105.
Salfinger M, Stool EW, Piot D, Heifets L. Comparison of three methods for recovery of Mycobacterium
avium complex from blood specimens. J Clin Microbiol. 1988;26:1225-1226.
Weinstein MP. Emerging data indicating that extended incubation of blood cultures has little clinical
value. Clin Infect Dis. 2005;41:1681-1682.
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Licensed to: Aura Leal and Laboratory
Universidad Standards
Nacional Institute. All rights reserved.
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Clinical and Laboratory Standards Institute consensus procedures include an appeals process that
is described in detail in Section 8 of the Administrative Procedures. For further information,
contact CLSI or visit our website at www.clsi.org.
Summary of Delegate Comments and Subcommittee Responses

M47-P, Principles and Procedures for Blood Cultures; Proposed Guideline
General
1. I suggest the following sections and text be reviewed to make the document less USA-centric and more global:
- Section 6.2.2—delete “cleared by US FDA” in two locations

- Section 6.3.4—delete “available in United States”
- Section 6.5.2—the reference to a US study is OK in the context, but information about the rest of the world
should also be provided
- Section 9—delete the last sentence that refers to specific OSHA and CFR requirements
- Section 9.1.2—delete the specific US regulation or at least cite comparable regulations from other countries.
• Sections 6.2.2 and 6.3.4: The wording has been changed as suggested.
Section 6.5.2: This is intended as a supporting citation only. No change is made.
Section 9: This sentence has been deleted as suggested.
Section 9.1.2: This section has been revised substantially in response to comment #15. The cited reference
is now available online (without cost) and thus should be considered a global resource. No other changes
are made.
2. Excellent document. Recommendations for culturing blood in order to specifically isolate brucella should focus
on optimal use of media for the CMBCSs or use Isolator Tubes with subculture to appropriate agar media. For
the CMBCS the current formulations of the anaerobic media do not support the growth of brucella as
documented in some of the studies reporting on isolation of brucella from the automated systems. Therefore, the
recommendation for brucella culture with a CMBCS should be the use of a set of aerobic media and not use the
anaerobic bottle. The number of sets to collect should probably be at least two.
• This section (6.5.5) has been revised to note that most isolates are recovered only from aerobic bottles
when a CMBCS is used.
Section 5.2, Number of Blood Cultures
3. The brief paragraph on page 5 regarding surveillance blood cultures remains unclear with regard to the value of
these cultures in certain patient populations.
• This section has been revised to address this.
Section 5.6, Blood Culture Collection
4. I am writing to submit a comment pertaining to an acceptable contamination rate for blood cultures. Last
paragraph: “...minimizing contamination rates to an acceptable range, typically < 3%.” Compare that to page 33
of the document, “The goal for blood culture contamination rate...should be below 2%.” Was it the intent that
the second statement would specify a different percentage because it is a QA Indicator? This is a bit confusing.
Our lab, as well as many others, monitors blood culture contamination rates on a monthly basis and reports that
rate to Infection Control, using the 3% rate as acceptable rate. We would like to ask the committee to consider
3% as the rate in both sections of the document.
• The second percentage found in Section 10.1.3 has been changed to 3% to make the text internally
consistent.
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5. Third paragraph, eighth sentence “....because of the risk of reflux of the broth media back into the vein...”: this
“reflux” experience was related to the old practice of directly inserting the blood culture bottle into the
venipuncture needle holder attached to the needle in the vein. Newer and safer blood culture needle devices
were introduced about five to ten years ago; these are typically butterfly needles attached to long tubing
attached to “domes” fitted over the blood culture bottles, with blood flow controlled by the vacuum in the blood
culture bottles and a septum over the protected needle contained within the “dome.” These devices have
eliminated the possibility of “reflux” of broth media into the vein. These devices are the preferred (and
predominant) method of collecting blood in the various hospitals I’ve worked. I rarely hear anymore of blood
cultures being collected by syringes or directly from a vacutainer holder attached to a needle inserted in the arm.
From my limited perspective, these devices are preferred for use in most settings because of the improved safety
(fewer needlesticks, more controlled venipuncture/blood collection). I don't know about the rest of the world,
but perhaps these devices should be mentioned in this section.
The device we use at my current lab is the Saf-TWing Blood Collection Set (Smiths Medical International Ltd.,
Hythe Kent, UK). This product is also latex-free and sterile.
• It is the policy and practice of CLSI not to use brand names or to make recommendations regarding the
use of specific commercial products. No changes are made.
6. Fourth paragraph – Changing needles before culture inoculation created a major safety hazard and numerous
needlesticks occurred, as correctly stated. But, and related to comment #5 above, the newer blood collection
devices (dome assembly set) allow the same needle to draw directly into both bottles of a single set of blood
cultures. And use of these devices has dramatically reduced needlestick injuries from this procedure.
I’d recommend an addition to this paragraph discussing the advantage of these blood collection “dome” devices,
especially relative to the safety considerations (i.e., needlestick injuries).
• It is the policy and practice of CLSI not to use brand names or to make recommendations regarding the
use of specific commercial products. No changes are made.
7. Fifth paragraph: This paragraph states that the acceptable blood culture contamination rate is < 3%. Yet, two
other sections (p. 26, Section 8, Contaminants; p. 33, Example QA Indicator 1) state “below 2%.” Please be
consistent.
• Please see the response to comment #4.
Section 6.2.1.1, Conventional Broth Cultures
8. Third paragraph – “…incubated at 35 degrees,” yet on page 9 under “Temperature” it is 35 - 37 degrees.
• The text has been changed to 35 degrees to make the document internally consistent.
Section 6.2.3, Preservation of Isolates for Further Testing
9. Third paragraph - What is the expected temperature for the ultra-low temperature storage?
• The text has been changed to include: (e.g., -70 oC).
Section 6.5.2, Catheter-Related Bloodstream Infections
10. Typo on page 16, 4th paragraph: “250 000.”
• This has been changed to "250,000."
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Section 6.5.2.1, Recommendations for Short-Term Peripheral Catheters
11. Interpretation of results should be Interpretation of Culture results (the same as in Section 6.5.2.2).
• This change has been made as suggested.
Section 6.5.3, Special Considerations: Infective Endocarditis
12. Under “Blood Culture Media,” first paragraph, second sentence—“recovery of facultative anaerobes.”
• This change has been made as suggested.
Section 8, Contaminants
13. First sentence: What does the point mean? An example would help explain the second point.
• The first part of this paragraph has been revised.
Section 9, Safety Issues
14. This section might benefit from a discussion of the blood culture collection devices (“dome” assembly, or
whatever this thing is called if it has a generic name).
• Please see response to comment #5.
Section 9.1.2, Transmission of Agents Other Than Hepatitis Viruses and Retroviruses
15. Second paragraph, last sentence: List below is missing.
• This section has been revised and the reference has been updated to the February 2007 edition. Because
this information already is available from a number of other sources, and therefore would be redundant
here, it has been deleted.
Section 10.1.4, Sample Transport
16. Sample transport has a two-hour timeframe included here. This information was not included in Section 5.6.1,
Transport of Specimens to the Laboratory.
• The text on page 7 has been changed to be consistent with the wording in Section 10.1.4.
Section 10.3.2, Specimen Management
17. This section is Record Management.
• The title of the section has been changed.
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The Quality Management System Approach

Clinical and Laboratory Standards Institute (CLSI) subscribes to a quality management system approach in the
development of standards and guidelines, which facilitates project management; defines a document structure via a
template; and provides a process to identify needed documents. The approach is based on the model presented in the
most current edition of CLSI/NCCLS document HS1—A Quality Management System Model for Health Care. The
quality management system approach applies a core set of “quality system essentials” (QSEs), basic to any
organization, to all operations in any healthcare service’s path of workflow (i.e., operational aspects that define how
a particular product or service is provided). The QSEs provide the framework for delivery of any type of product or
service, serving as a manager’s guide. The quality system essentials (QSEs) are:
Documents & Records Equipment Information Management Process Improvement

Organization Purchasing & Inventory Occurrence Management Customer Service
Personnel Process Control Assessments―External and Facilities & Safety
Internal
M47-A addresses the quality system essentials (QSEs) indicated by an “X.” For a description of the other documents
listed in the grid, please refer to the Related CLSI/NCCLS Publications section on the following page.
Purchasing &
Improvement
Organization
Management
Management
Information
Satisfaction
Assessment
Facilities &
Occurrence
Documents
Equipment
& Records
Service &
Personnel
Inventory
Control
Process
Process
Safety
X
Adapted from CLSI/NCCLS document HS1—A Quality Management System Model for Health Care.
Path of Workflow
A path of workflow is the description of the necessary steps to deliver the particular product or service that the
organization or entity provides. For example, CLSI/NCCLS document GP26⎯Application of a Quality
Management System Model for Laboratory Services defines a clinical laboratory path of workflow which consists of
three sequential processes: preexamination, examination, and postexamination. All clinical laboratories follow these
processes to deliver the laboratory’s services, namely quality laboratory information.
M47-A addresses the clinical laboratory path of workflow steps indicated by an “X.” For a description of the other
documents listed in the grid, please refer to the Related CLSI/NCCLS Publications section on the following page.
Preexamination Examination Postexamination

receipt/processing
Sample collection
Results reporting
Sample transport
Results review
and follow-up
and archiving
Interpretation
Examination
Examination
management
ordering
Sample
Sample
X X X X X X
Adapted from CLSI/NCCLS document HS1—A Quality Management System Model for Health Care.
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Volume 27 M47-A
Related CLSI/NCCLS Publications∗

GP2-A5 Laboratory Documents: Development and Control; Approved Guideline— Fifth Edition (2006). This
document provides guidance on development, review, approval, management, and use of policy, process, and
procedure documents in the medical laboratory community.
GP17-A2 Clinical Laboratory Safety; Approved Guideline—Second Edition (2004). This document contains general
guidelines for implementing a high-quality laboratory safety program. The framework is adaptable to any
laboratory. An NCCLS-CAP joint project.
H3-A5 Procedures for the Collection of Diagnostic Blood Specimens by Venipuncture; Approved Standard—
Fifth Edition (2003). This document provides procedures for the collection of diagnostic specimens by
venipuncture, including line draws, blood culture collection, and venipuncture in children. It also includes
recommendations on order of draw.
HS1-A2 A Quality Management System Model for Health Care; Approved Guideline—Second Edition (2004).
This document provides a model for healthcare service providers that will assist with implementation and
maintenance of effective quality systems.
M22-A3 Quality Control for Commercially Prepared Microbiological Culture Media; Approved Standard—
Third Edition (2004). This standard contains quality assurance procedures for manufacturers and users of
prepared, ready-to-use microbiological culture media.
M29-A3 Protection of Laboratory Workers From Occupationally Acquired Infections; Approved Guideline—
Third Edition (2005). Based on US regulations, this document provides guidance on the risk of transmission
of infectious agents by aerosols, droplets, blood, and body substances in a laboratory setting; specific
precautions for preventing the laboratory transmission of microbial infection from laboratory instruments and
materials; and recommendations for the management of exposure to infectious agents.
X3-R Implementing a Needlestick and Sharps Injury Prevention Program in the Clinical Laboratory; A
Report (2002). This report presents a step-by-step approach for implementing safer medical devices that
reduce or eliminate sharps injuries to laboratory personnel. X3-R is written in an expanded checklist format,
outlines a process that goes beyond general recommendations, and specifically addresses the needs of
professionals performing specimen collection and clinical laboratory procedures.
∗
Proposed-level documents are being advanced through the Clinical and Laboratory Standards Institute consensus process;
therefore, readers should refer to the most recent editions.
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NOTES
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Active Membership
(as of 1 April 2007)
Sustaining Members Department of Veterans Affairs Elanco Animal Health Japan Association of Clinical
FDA Center for Devices and Electa Lab s.r.l. Reagents Industries (Tokyo, Japan)
Abbott Radiological Health Enterprise Analysis Corporation
American Association for Clinical FDA Center for Veterinary Medicine Eomix, Inc. Associate Active Members
Chemistry Health Canada Eurofins Medinet
AstraZeneca Pharmaceuticals Massachusetts Department of Public FasTraQ Inc. (NV) 3rd Medical Group (AK)
Bayer Corporation Health Laboratories Future Diagnostics B.V. 48th Medical Group/MDSS (APO, AE)
BD Ministry of Health and Social Welfare - Gavron Group, Inc. 59th MDW/859th MDTS/MTL (TX)
Beckman Coulter, Inc. Tanzania Gen-Probe Aberdeen Royal Infirmary (Scotland)
bioMérieux, Inc. National Center of Infectious and Genaco Biomedical Products, Inc. Academisch Ziekenhuis -VUB
CLMA Parasitic Diseases (Bulgaria) Genomic Health, Inc. (Belgium)
College of American Pathologists National Health Laboratory Service Gentris Corporation Acibadem Labmed Clinical Laboratory
GlaxoSmithKline (South Africa) Genzyme Clinical Specialty Laboratory (Turkey)
Ortho-Clinical Diagnostics, Inc. National Institute of Standards and Genzyme Diagnostics ACL Laboratories (IL)
Pfizer Inc Technology GlaxoSmithKline ACL Laboratories (WI)
Roche Diagnostics, Inc. National Pathology Accreditation Gluco Tec, Inc. Akron’s Children’s Hospital (OH)
Advisory Council (Australia) GluMetrics, Inc. Alameda County Medical Center (CA)
Professional Members New York State Department of Greiner Bio-One Inc. Albany Medical Center Hospital (NY)
Health HistoGenex N.V. Albemarle Hospital (NC)
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ESCMID Aviir, Inc. Panaceapharma Pharmaceuticals Cadham Provincial Laboratory – MB Health
Family Health International Axis-Shield POC AS Paratek Pharmaceuticals (Canada)
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Innovation and Technology Bayer Corporation – West Haven, CT PathWork Informatics Canada)
Commission Bayer HealthCare, LLC, Diagnostics Pfizer Animal Health Canterbury Health Laboratories (New Zealand)
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Biology BD Biosciences – San Jose, CA Phadia AB (NC)
International Federation of BD Diabetes Care Powers Consulting Services Capital Health - Regional Laboratory
Biomedical Laboratory Science BD Diagnostic Systems PPD, Inc. Services (Canada)
International Federation of Clinical BD Vacutainer Systems Primera Biosystems, Inc. Capital Health System Fuld Campus (NJ)
Chemistry Beckman Coulter, Inc. QSE Consulting Capital Health System Mercer
JCCLS Beth Goldstein Consultant (PA) Radiometer America, Inc. Campus (NJ)
Joint Commission on Accreditation Bioanalyse, Ltd. Radiometer Medical A/S Catholic Health Initiatives (KY)
of Healthcare Organizations Bio-Development S.r.l. Reliance Life Sciences CDC/HIV (APO, AP)
National Society for Bio-Inova Life Sciences Replidyne CDPH (CO)
Histotechnology, Inc. International Rib-X Pharmaceuticals Central Baptist Hospital (KY)
Ontario Medical Association Quality Biomedia Laboratories SDN BHD Roche Diagnostics GmbH Central Kansas Medical Center (KS)
Management Program-Laboratory bioMérieux (NC) Roche Diagnostics, Inc. Central Texas Veterans Health Care
Service bioMérieux, Inc. (MO) Roche Laboratories System
RCPA Quality Assurance Programs Bio-Rad Laboratories, Inc. Roche Molecular Systems Centralized Laboratory Services (NY)
PTY Limited Bio-Rad Laboratories, Inc. – France Sanofi Pasteur Centura Laboratory (CO)
SDS Pathology Bio-Rad Laboratories, Inc. – Irvine, Sarstedt, Inc. Chang Gung Memorial Hospital (Taiwan)
SIMeL CA Schering Corporation Chesapeake General Hospital (VA)
Sociedad Espanola de Bioquimica Bio-Rad Laboratories, Inc. – Plano, Seneca Medical, Inc. Chester County Hospital (PA)
Clinica y Patologia Molecular TX Sequenom, Inc. Children’s Healthcare of Atlanta (GA)
Sociedade Brasileira de Analises Black Coast Corporation – Health SFBC Anapharm Childrens Hospital of Wisconsin (WI)
Clinicas Care Systems Consulting Sphere Medical Holding Christiana Care Health Services (DE)
Sociedade Brasileira de Patologia Blaine Healthcare Associates, Inc. Streck Laboratories, Inc. CHUM Hopital Saint-Luc (Canada)
Clinica Center for Measurement Standards/ITRI Sysmex America, Inc. (Long Grove, City of Hope National Medical Center (CA)
Turkish Society of Microbiology Cepheid IL) Clarian Health – Clarian Pathology
Washington G2 Reports Chen & Chen, LLC Sysmex Corporation (Japan) Laboratory (IN)
World Health Organization Comprehensive Cytometric Consulting Tethys Bioscience, Inc. Cleveland Clinic Health System Eastern Region
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M47-A

Principles and Procedures for Blood

Licensed to: Aura Leal Universidad Nacional de Colombia

Licensed to: Aura Leal Universidad Nacional de Colombia

Licensed to: Aura Leal Universidad Nacional de Colombia

ii Leal Universidad Nacional de Colombia

Area Committee on Microbiology

Fred C. Tenover, PhD, ABMM

Subcommittee on Principles and Procedures for Blood Cultures

Michael L. Wilson, MD Michael Towns, MD David F. Welch, PhD, D(ABMM)

iv Leal Universidad Nacional de Colombia

Committee Membership........................................................................................................................ iii

Foreword .............................................................................................................................................. vii

9 Safety Issues ............................................................................................................................26

Summary of Delegate Comments and Subcommittee Responses.........................................................46

Related CLSI/NCCLS Publications ......................................................................................................51

vi Leal Universidad Nacional de Colombia

Licensed to: Aura Leal Universidad Nacional de Colombia vii

viiiLeal Universidad Nacional de Colombia

Principles and Procedures for Blood Cultures; Approved Guideline

bloodstream infection – an infection associated with bacteremia or fungemia.

culture – 1) the intentional growing of microorganisms, such as bacteria or fungi, in a controlled

fungemia – the presence of fungi (yeasts or molds) in the bloodstream.10

indeterminate isolates – a microorganism of undetermined clinical importance.10

manual blood culture - see conventional blood culture system.

povidone iodine – a water-soluble complex of iodine with polyvinylpyrrolidone11; NOTE 1: Applied as

sepsis – systemic inflammatory response syndrome (SIRS) plus infection.12,13

severe sepsis – sepsis associated with organ dysfunction, hypoperfusion, or hypotension.12,13

systemic inflammatory response syndrome (SIRS) – a physiologic state believed to be triggered by

CMBCSs continuous monitoring blood culture systems

HCV hepatitis C virus

4 Clinical Importance of Blood Cultures

4.1 Diagnostic Importance

4.2 Prognostic Importance

5 Specimen Collection and Transportation

5.1 Timing of Blood Cultures

5.2 Number of Blood Cultures

5.3 Volume of Blood for Culture

5.5 Disinfection of Skin and Prevention of Blood Culture Contamination

• Tincture of iodine and chlorhexidine gluconate are probably equivalent.

5.6 Blood Culture Collection

5.6.1 Transporting Specimens to the Laboratory

5.7 Specimen Rejection Criteria for Blood Culture Specimens

• incorrectly labeled or unlabeled vials;

6 Methods and Procedures

6.1 Critical Factors in the Recovery of Pathogens From Blood Specimens

MEDIA (TYPES, INDICATIONS/FORMULATIONS)

ADDITIVES (ANTICOAGULANTS, RESINS, CHARCOAL)

6.2 Blood Culture Methods

6.2.1 Manual Blood Cultures

6.2.1.1 Conventional Broth Cultures

6.2.1.2 Biphasic Cultures

6.2.2 Continuous-Monitoring Blood Culture Systems79

6.2.3 Preservation of Isolates for Further Testing

Additional detailed methods for microorganism storage are available elsewhere.112-114

6.3 Fungal Blood Cultures

6.3.1 Population at Risk

6.3.2 Critical Factors

6.4 Mycobacterial Blood Cultures

6.4.1 Population at Risk

Documented mycobacteremia with Mycobacterium tuberculosis is relatively uncommon in developed

Other slowly growing mycobacteria, including Mycobacterium kansasii, Mycobacterium simiae,

6.4.2 Critical Factors