Clinical

Microbiology
N E W S L E T

CMN
Vol. 36, No. 14
July 15, 2014
www.cmnewsletter.com

I N TH IS

ISS U E

105 If Specimen Collection

and Processing Guidelines
Fall, Does Anyone Hear
Them? Pre-Analytical
Conundrums in Clinical
Microbiology

Stay Current...
Stay Informed.

T E R

If Specimen Collection and Processing Guidelines

Fall, Does Anyone Hear Them? Pre-Analytical
Conundrums in Clinical Microbiology
Susan M. Harrington, Ph.D., Department of Clinical Pathology, Cleveland Clinic, Cleveland, Ohio

Abstract
Provision of accurate and meaningful test results requires that all procedures, beginning with specimen
procurement and ending with interpretation of nal results, be performed according to best practices.
Deviations may lead to under- or over-reporting of pathogens and incorrect assessment of an infectious process. Since we cannot assume that all procedures are performed according to protocols, relevant quality measures should be incorporated into routine practice. In this review, the most signicant
aspects of specimen collection, transport, and processing are discussed for the most important types
of specimens. Microbiologists need to work closely with nurses and clinicians to maintain efcient
practices and provide high-quality specimens.

Introduction
In A Treatise Concerning the Principles of Human
Knowledge, philosopher George Berkeley originally posed the idea that objects exist only if
someone is there to perceive them (1). Later, the
concept was rephrased as a question: If a tree
were to fall in the forest, and no one was around
to hear it, does it make a sound? The answer,
based on biology, is nosound occurs when
vibrations move as waves and are detected by
the earbut changes in nature would mark the
occurrence. Not only is the life of the tree diminished, but the surrounding foliage is changed by
alteration of sunlight ltered by the leaves of
the tree, the earth beneath may be enriched by
the decaying vegetable matter, and wildlife that
depends on the tree may be impacted.

Corresponding author:
Susan M. Harrington, Ph.D.,
Department of Pathology and
Laboratory Medicine, 9500
Euclid Ave./LL1-2, Cleveland,
OH 44195. Tel.: 216-4452218. Fax: 216-444-7612.
E-mail: Harrins2@ccf.org

CMN

In the world of clinical microbiology, we can

imagine that unrecognized errors represent our
trees falling with no one to hear them. Indeed,
despite our efforts to perform testing accurately,
errors and breaches in quality may go unnoticed, and patient care may be adversely affected.
Importantly, any diagnostic test starts with collection of the appropriate specimen from the patient
at the right time with the proper technique. We

may be unaware of a breakdown in specimen

collection or processing procedures, but like the
impact of the fall of the tree in the forest, the
downstream effect on the results for the patient
may be considerable.
We live in an era in which the public is increasingly aware of quality measures assessed in the
hospital or clinic setting. Rates of nosocomial
infections, patient satisfaction, and many other
outcome and quality measures are reported
publicly. Moreover, based on recent changes
to federal regulations, we may experience even
greater scrutiny of our work and possibly direct
interaction with patients. On 6 February 2014,
an amendment was made to the Federal Register. This Final Rule entitled CLIA Program
and HIPAA Privacy Rule; Patients Access to
Test Reports amends the Clinical Laboratory
Improvement Amendment (CLIA) of 1988 and
species that upon request patients have the
right to access directly laboratory reports from
laboratories that are subject to the Health Insurance Portability and Accountability Act (HIPAA)
(2). Patients may ask more questions about their
results, including deviations from best practice
that appear in the test report. We should be pre-

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105

pared to answer for all laboratory procedures, including those

conducted for specimen collection, transport, and processing.
The goal of this article is not to provide explicit details about how
to collect and process all specimen types for microbiology. These
may be found in texts and manuals, such as the Manual of Clinical
Microbiology (MCM) and the Clinical Microbiology Procedures Handbook (CMPH) (3,4). Rather, this article primarily discusses some
of the key aspects of specimen collection, transport, or processing
that signicantly impact the quality of bacterial cultures. Metrics
that may help laboratories quantify and track the quality of preanalytical procedures are also discussed.

General Principles
Documented instructions for microbiology specimen collection
and handling are essential and should provide relevant collection
guidelines for all members of the health care team and for patients
if self-collection is to occur. Placing written instructions with
photographs and supporting evidence into collection guides for
front-line staff is useful and periodic educational efforts are helpful.
For laboratories accredited by the College of American Pathologists (CAP), the CAP checklist (MIC.13250) clearly indicates the
requirement for written procedures for the pre-analytical phases
of microbiology (5) (Table 1). Laboratories bear responsibility for
ensuring that their written procedures are followed. If a provider
insists on processing a specimen that is inappropriate or inadequate, a notation indicating that recovery of pathogens may be
compromised due to improper or inadequate specimen collection
or transport should be included in the culture report.
Some general rules apply to specimen acquisition and processing:
U-iVi`LiViVi`w>iwwiV
is suspected and from body sites in which the infection is active.
U*>Viii>i>>i>>iiiiVtion requires some knowledge of the microbial species in the
differential diagnosis.
U7iii Li] iVi ` Li ViVi`
starting antimicrobial therapy.
Uw>>LiiwiViViVii`
by which the specimen was obtained helps the microbiologist
and the provider interpret the results and should be included
for all test requests in microbiology.

Table 1. Specimen collection and handling requirements

(CAP checklist 2013)
1. Method for proper collection of culture specimens from
different sources
2. Proper labeling of culture specimens
3. Use of transport media when necessary
4. Procedures for safe handling of specimens (tightly sealed
containers, no external spillage)
5. Need for prompt delivery of specimens to ensure minimum
delay in processing
6. Method for preservation of specimens if processing is delayed

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U-iVi`LiiVii`>i`ixi
U/i>iLi>i>wV>i>`>i`
in a biohazard bag.
U"Vi iVii` i >L>] iVi Vi `
be performed in a class II biosafety cabinet or, at a minimum,
behind a plastic shield to protect the technologist and the laboratory environment.

Urine Specimens
Many options exist for test ordering, specimen collection, and
transport of a urine specimen. Collection of an early morning
specimen, or one from a patient who has not urinated for several
hours prior to specimen collection, is considered to be best for
recovery of urinary pathogens, as concentrated specimens decrease
false-negative results due to excess uid intake. Patients should
not be asked to drink in order to produce a specimen, as this leads
to specimen dilution.
It is generally recommended that specimens be collected into a
sterile cup after appropriate cleansing to minimize contamination
with skin ora. For patients who can urinate into a specimen cup,
collection of a mid-stream specimen may decrease contamination
with bacteria that colonize the urethra. Specimens may also be collected from the port of an indwelling catheter, by straight catheterization, via suprapubic aspiration, or by other methods, but never
from bedpans, urinals, or catheter bags. Although not preferred,
collection of urine from a pedibag (pediatric bag) may be the only
means of collecting a specimen from neonates and toddlers.
If not processed within 2 hours after collection, urine specimens
should be refrigerated and processed within 24 hours (3,4). Escherichia coli, a common urinary tract pathogen replicates. about once
every 20 minutes. A generally insignicant bacterial density of
approximately 10,000 CFU/ml can multiply to a signicant density
within hours. In a small study conducted in 1976, the number of
CFU in 32% of 47 positive cultures increased by more than 1 log
unit when held at room temperature and processed 4 to 6 hours
after collection (6). Designated specimen refrigerators at nursing
units and transport coolers with ice packs are essential if specimens
are not transported immediately to the laboratory after collection.
As an alternative to refrigeration, urine may be placed into a preservative tube, such as the BD Vacutainer Culture and Sensitivity
tube, which contains sodium formate, sodium borate, and boric
acid for specimen preservation (Becton Dickinson and Company,
Sparks, MD). This preservative maintains stability at room temperature for up to 48 hours. Although early studies support the
use of a preservative (7,8), some evaluations described a decrease
in CFU/ml when preservative tubes were processed after 24 or
48 hours (9,10).
A recent publication by Eisinger et al. demonstrated a signicant
increase in the false-positive rate for unpreserved, unrefrigerated
urine specimens held between 4 and 24 hours prior to culture
compared to those that were either refrigerated or preserved.
There was no signicant increase in negative cultures or decrease
in CFU for urine cultures from preserved or refrigerated samples,

suggesting that preservation is not toxic to urinary tract pathogens (11). Differences between these results and those from the
early 1980s are likely due to changes in the formulation of the
preservative tubes.
Given the increasing complexities and centralization of health
care systems, the use of preservative tubes for urine culture prior
to transportation to centralized laboratories is gaining acceptance. However, implementation of the use of preservative tubes
can be challenging. First, there is an additional expense associated with materials (the preservative tube and transfer straw cost
about $0.45) and with the personnel time to perform the transfer.
Secondly, once a specimen is placed in preservative it cannot be
shared for the purpose of urinalysis or chemistry tests. Similarly,
the sodium propionate and chlorhexidine in the urinalysis preservative inhibit bacteria and fungi, making the urinalysis tube
unsuitable for culture.
Once the urine sample is in the hands of the laboratory technologist, attention to the details of specimen inoculation is essential.
Bacteria will have settled to the bottom of the preservative tube or
cup; therefore, all specimens must be well mixed prior to inoculation. Many laboratories inoculate 1 l of urine using a calibrated
loop, making accuracy of inoculation an important consideration.
The procedure of dipping the loop into the urine specimen must
be performed with care. The loop should be held vertically and
dipped just below the surface of the specimen. The further the
loop is introduced into the sample, the more uid is in contact
with the shaft. Urine on the shaft will be pulled toward the loop
by gravity. When only 1 l of urine is processed, small amounts of
urine on the shaft can signicantly alter the total volume planted.
Assessment of the impact of sampling depth was conducted at
the Cleveland Clinic with the Walk Away Specimen Processor
(Copan Diagnostics, Brescia, Italy). Increasing the depth of sampling by *2 cm increased colony counts by as much as 2.6 times
with the 1-l loop (12).
Whether 1 l or 10 l is used for inoculation, platinum loops
should be periodically checked for accuracy. All plastic disposable
loops are not equally accurate. Verication of accuracy should be
conducted before placing any manufacturers loop into service.
Specimens should be spread with a non-calibrated loop or other
device to obtain isolated colonies for counting.
Blood Cultures
Optimally, blood cultures should be collected before or during the
febrile period and prior to administration of antibiotics. Collection
from venous catheter lines should be avoided, as they are more
likely to yield contaminants and increase the risk of contamination
of the port. Regardless of timing, the most signicant parameter
contributing to recovery of bloodstream pathogens is volume. Not
infrequently, the number of organisms in the bloodstream may be
<1 per ml (13). For adults, 8 to 10 ml of blood per bottle that contains 40 ml of broth medium is generally recommended, and the
manufacturers recommendations should be closely followed. Blood
culture collection practices require venipunctures at two separate
sites, each consisting of 20 ml, distributed equally into aerobic

and anaerobic bottles. If 20 ml of blood cannot be collected, the

aerobic bottle should be inoculated rst, and the remainder placed
into the anaerobic bottle (14). Special bottles for pediatric blood
culture collection may provide better a blood-to-broth ratio than
standard bottles. Weight-based collection guidelines for children
should not exceed 1% of total blood volume (4). Upon receipt of
blood cultures, laboratories should assess the volume. Although not
perfectly accurate, laboratories may weigh an uninoculated bottle
and compare the weight to that of the bottles after blood collection, guring about 1 g for each ml of blood. Cultures received
with low volumes should not be rejected, but notation of the low
volume should accompany culture results to inform practitioners
that results may be suboptimal.
In 2007, a study performed at two university hospitals using stateof-the-art continuously monitoring blood culture systems and
bottles containing either resin or activated charcoal supported
previous ndings regarding volume and the number of cultures
collected. With each blood culture consisting of 20 ml of blood,
73.1% of bacteremias were detected with 1 blood culture, 93.9%
with two cultures, and 96.9% with three cultures (15). Two or three
blood cultures per episode of bacteremia are recommended (14).
Preparation of the skin for venipuncture is the second critical factor
in collection of a quality blood culture. The skin is rst cleansed of
surface oils and dirt with 70% isopropyl or ethyl alcohol. Studies
comparing disinfectants support the use of either tincture of iodine
or chlorhexidine gluconate over iodophores (14). However, iodinecontaining disinfectants must be removed from the skin after
venipuncture, and chlorhexidine gluconate is not FDA approved
for infants <2 months of age. Manufacturers directions for use
of disinfectants must be followed. Most importantly, the drying
time must be strictly observed. If the drying time is insufcient,
disinfection will be inadequate, leading to contaminated cultures.
Health care providers are often unaware that the rubber septum
under the cap of commercial blood culture bottles is not sterile;
therefore, the septum must also be disinfected with 70% alcohol or
iodine and allowed to dry. (Note that alcohol disinfection will not
kill spores.) Laboratories should develop a procedure for periodic
evaluation of blood culture contamination rates, and as a quality
measure, report an overall contamination rate and, perhaps, rates
by nursing unit or clinic. Personal feedback to individual phlebotomists or nurses performing venipuncture is also an effective
means of maintaining high-quality collection. Lower contamination rates are generally observed when dedicated phlebotomy
teams are utilized for collection of blood cultures (16). Historically, contamination rates of <2 to 3% were generally considered
acceptable (14,16); however, some institutions using chlorhexidine
gluconate report rates below 1%.
Specimen collection and processing requirements and interpretation of laboratory results for assessment of catheter-related bloodstream infection (CRBSI) are varied and somewhat controversial
(14). A meta-analysis of methods for diagnosis of CRBSI concluded that comparison of paired quantitative blood cultures (one
collected through the line and one peripheral) is the most accurate method. However, other common methods, such as time to

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positivity of paired blood cultures, the quantitative Maki catheter

tip roll plate method (17), and quantitative culture of a sonicated
catheter tip (18), had acceptable sensitivity and specicity (19) and
are much easier for most laboratories to perform. Either the Maki
method or sonication is supported by the Infectious Diseases Society of America (20). Several recent papers favor the Maki method
as more sensitive than sonication (20-22). Importantly, if catheter
tips are submitted for analysis, they should always be accompanied
by one or more peripheral blood cultures for comparison of results.
Catheter tips should never be cultured if CRBSI is not suspected.
Questions arise regarding the best way to manage blood cultures
when the collection site is separated from the central laboratory,
forcing transport times of more than 30 minutes. Clearly, the
sooner a blood culture is incubated in a continuously monitoring blood culture system the better. If transported, blood culture bottles should remain at room temperature during transport
to prevent the threshold of detection from being missed due to
log-phase growth already under way in the bottle. The Clinical Laboratory Standards Institute publication on blood cultures
recommends that cultures be incubated within a few hours (14).
A recent study of blood culture transport using spiked blood cultures showed that total time to detection (transport plus incubation within the blood culture instrument) was signicantly lower
for blood cultures with a 2-hour transport time than for those with
a 9-hour transport time, although cultures with longer transport
times had shorter time to detection once incubating in the blood
culture instrument (23). Current information supports incubating blood cultures on site if incubation is delayed more than 2 or
4 hours (3,4,14). Studies of the impact of delayed blood culture
incubation on patient care are needed to help health systems plan
appropriate laboratory services.

Body Fluids
Fluids present in body compartments for maintenance of normal
tissue homeostasis, including pleural, peritoneal, pericardial, vitreous, amniotic, and synovial uids, are often called normally sterile
body uids. Cerebrospinal uid (CSF) is generally considered a
unique entity, apart from other uids. Fluid samples collected from
drains or aspirated from abscesses or cysts that are not present in
the healthy host are generally handled with less urgency and may
be subjected to less thorough analysis than those from normally
sterile sites. For the purposes of this discussion, normally sterile
body uids are referred to as uids or uid specimens.
All isolates from uid specimens are assessed for clinical signicance. Many patients from whom uids are collected may be
immunosuppressed or receiving antimicrobial therapy, increasing
the need to determine the relevance of all species isolated. Fluids
are considered sterile unless proven otherwise, but if collected
inappropriately, uids can be contaminated by ora from the skin
and mucosa. When collected by aspiration, it is essential that uids are collected with a sterile technique similar to that used when
collecting a blood culture. For patients with complicated anatomy
who may have physical communication between sterile compartments and the mucosa, such as a stula or a perforated bowel, uid
cultures may yield multiple species that may not need to be fully

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identied and tested for antimicrobial susceptibility. Clinical correlation is warranted for many uid samples.
Fluid specimens should be transported to the laboratory immediately at room temperature. Although time-consuming, this may
best be accomplished by use of personnel dedicated to walking
specimens to the laboratory to ensure rapid processing and to prevent loss of an irretrievable specimen in a pneumatic tube system.
Transport time is especially critical when very small volumes are
submitted. Large-volume specimens and those that are purulent
are more likely to maintain organism viability for longer periods.
If anaerobic culture is requested, specimens should be transported
to the laboratory within 15 to 20 minutes or placed in anaerobic
transport medium (4). After disinfecting the rubber septum, uid
is transferred into the vial. Fluid should be placed on top of the
semi-solid agar, not into the agar. Aerobic and/or anaerobic blood
culture bottles may also be inoculated at the point of collection.
The microbial burden in uid specimens and the amount of specimen available for culture can be highly variable. Thus, unlike blood
cultures, no standard culture volume is recommended for uids.
Collection of up to 20 ml of uid may be helpful, depending on
the collection site. The organism burden is often quite low in
continuous ambulatory peritoneal dialysis (CAPD) uid, synovial
uid, and peritoneal uid collected from patients with spontaneous
bacterial peritonitis (24,25). Large-volume specimens should be
centrifuged prior to inoculation. The limit of detection of a culture
is improved if specimens are inoculated into blood culture medium
and incubated for 5 days on a continuously monitoring blood culture instrument. If more than 2 ml can be collected, at least 1 ml of
peritoneal, synovial, or CAPD uid should be placed into a blood
culture bottle(s) (24-26). The literature in support of the use of
blood culture bottles for culture of pleural and other uids is more
limited, but this may also be considered (23,24). Recovery from
blood culture medium with resin or activated charcoal has been
shown to be superior to that from standard bottles (24-26). Blood
culture media provides enrichment, as well as antibiotic-neutralizing capability and inhibition of mediators of the innate immune
system. Specimens should preferentially be inoculated into the
aerobic bottle. For sites in which anaerobic infection is suspected,
such as the abdominal cavity, equivalent amounts of uid may be
placed into the aerobic and anaerobic bottles.
Inoculation of blood culture bottles at the point of collection helps
maintain the specimen during transport and is the preferred practice, as long as 0.5 to 1 ml of specimen is submitted in a separate,
sterile syringe, tube, cup, or anaerobic transport vial for Gram
stain and possible inoculation to solid medium. Fluids that are
not viscous, bloody, or purulent should undergo a cytospin process prior to Gram staining. However, cytospin preparations of
thick or bloody specimens are difcult to read, and thick material
is easily washed away during staining (4).
When volume is insufcient for use of blood culture bottles, thioglycolate, anaerobic brain heart infusion, fastidious broth, brucella
broth, or other broth media may be used. No longer recommended
for all uid specimens, broths are especially helpful when low
numbers of organisms may be present or when specimens such

as synovial uids collected for suspected prosthetic joint infection

(PJI) must be held 14 days for recovery of Propionobacterium acnes.
Each laboratory will need to establish which specimens are placed
into broth, depending on their patient population.
While inclusion of blood culture bottles increases the sensitivity of
detection, the addition of solid agar plates, such as chocolate and
blood agars, may also improve the time to detection and provide
some indication of the number of organisms in the specimen. Use
of solid media along with blood culture bottles increases costs and
generates more plates for technical evaluation. When organisms
are observed on Gram stain, solid media should always be inoculated. A drop of approximately 50 l of uid specimen is generally inoculated, and plates are streaked to obtain isolated colonies.
To increase the yield from fungal cultures, uid specimens of >2 ml
should be centrifuged (3). If <2 ml is available, more uid can also
be planted by placing about 5 large drops on the plate in a daisy
pattern. As any amount of mold or yeast must be evaluated and
sterile-site specimens usually have minimal contamination, obtaining isolated colonies is not as important as improving the yield. If
the volume collected for all testing is <1 ml, it may be necessary to
obtain priorities from the care provider. At a minimum, a chocolate agar plate may be inoculated. Fastidious bacteria and yeast
species will be recovered with this single medium. A comment in
the report to indicate that lamentous fungi may not be recovered
due to low specimen volume is appropriate.
If the volume of material aspirated is so small that it remains in the
bore of the needle, the needle can be ushed at the site of collection
by drawing sterile saline into the syringe and then expressing that
material into a small, sterile tube. Although it is not typically recommended that syringes be sent to the laboratory, when volumes
are very small, receipt of the specimen in the syringe reduces specimen loss. Importantly, the needle must be removed and the syringe
safely capped to prevent needle stick exposures. Again, specimens
at low volumes should be transported to the laboratory immediately to maintain organism viability and prevent leakage or loss.

Abscess, Drainage, Exudate, Wound, Cellulitis, and Tissue

The goal of all specimen collection is to maximize recovery and
minimize contamination with extraneous ora; this is a particularly challenging task for collection of specimens contiguous to
body regions harboring a high density of normal ora, i.e., skin
and mucous membranes. Material from wounds, abscesses, drains,
exudates, and tissues are considered here, together with the application of similar processing guidelines. Samples should be collected only from areas that are clinically infected, deteriorating,
or failing to heal. Specimen collection for non-specic reasons
should be discouraged. The advancing margin or active area of
infection should be sampled, and frank pus should generally be
avoided. Specimens may be collected from surfaces that are contaminated with ora from the skin or mucous membranes or from
the environment. Surface abscesses include specimens such as boils,
furuncles, cysts, and surgical wounds. Examples of surface wounds
include those occurring from trauma, such as bites or lacerations,
abrasions, burns, or vesicles. Prior to collection from a supercial

site, the area should be debrided of necrotic tissue and thoroughly

rinsed with non-bacteriostatic sterile saline. Collection of samples
for assessment of cellulitis is difcult. Injection of a small amount
of sterile saline into the tissue and withdrawing it may be successful, but often a biopsy is necessary to obtain an adequate specimen.
For sampling of wounds or abscesses closed to the surface, the skin
should be cleansed with tincture of iodine or chlorhexidine or an
approved surgical scrub. The sample may then be aspirated with
a needle and syringe. Transfer into a sterile tube or cup for transport to the laboratory is preferred to sending the syringe with the
needle removed. Specimens from drainage tubing may be submitted. However, the drainage uid should be collected fresh from
tubing that has been appropriately disinfected. Drainage uid that
has been sitting in a collection bag is unacceptable.
Material from wounds, abscesses, tissues, drains, etc., should be
transported to the laboratory within 2 hours at room temperature
to prevent loss of fastidious species, to maintain colony counts,
and to prevent overgrowth of contaminating species. If anaerobes
are among the pathogens in the differential diagnosis, anaerobe
transport should be utilized and samples should be rapidly placed
into anaerobe transport medium, at the bedside if possible. The
special considerations for collection and handling of small volumes
of sterile body uid discussed above apply similarly to material
from abscesses and wounds.
Where tissue is concerned, surgeons may struggle with the appropriate size to submit. How much is enough, and how much is too
much? Collection of tissue via punch biopsy typically requires test
prioritization, with immediate delivery to the laboratory to limit
drying of the tissue. Moisture may be maintained in the tissue
by adding a piece of sterile gauze saturated with sterile saline or
adding a few drops of sterile saline to the transport tube or cup.
Consultation with a specialist in Infectious Diseases or Clinical
Microbiology may help with developing the differential and prioritizing which cultures are most critical when there is not enough
to inoculate all the cultures requested. Approximately 1 gram of
tissue, about a centimeter of material, is adequate for inoculation
of all microbiology cultures.
Frequently, specimens are diluted to allow them to be processed.
Stretching a specimen in this manner lowers the pre-test probability of detection of any and all pathogens. In contrast, very large
pieces of tissue or whole body parts, such as amputated toes, are
inappropriate. Providers should be educated that microbiologists
do not have the tools of the gross pathology laboratory or the
knowledge of the surgeon. These samples should be separated in
the operating room or assessed by anatomic pathology. If a large
piece of material is received and the technologist is unsure which
portion to process, pathology residents or pathology assistants
have excellent training in gross anatomy and can aid in the selection process.
Once the sample is received in the laboratory, the technologist may
need to select the most appropriate portion of the sample. Signs of
infection include yellow to tan pus, rust-colored blood, mucus, and
brown discoloration consistent with necrosis. Areas that are very

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dry or black should be avoided, as drying and necrosis will both

limit pathogen recovery. If the technologist is unsure of selection
choices, sampling small portions from several suspicious areas and
combining these pieces is reasonable.
Tissue may be placed in a sterile petri dish for selection of appropriate portions and minced into small pieces prior to grinding
(except for fungal cultures). Addition of up to a few milliliters
of sterile saline will facilitate grinding. When fungal culture is
requested, several pieces of minced tissue should be placed onto
the surface of agar medium, in addition to a few drops of ground
specimen. Grinding has been shown to greatly reduce recovery
of Zygomycetes.
Although swabs are easy specimens to collect and transport, they
are usually inferior to tissue or uid for assessment of wounds,
abscesses, exudates, and drainage. The amount of material collected by a swab is frequently inadequate, and swabs are prone to
contamination with skin ora. Swabs with a wooden shaft and dry
swabs should be rejected. Traditional swabs are particularly poor
for recovery of anaerobes and mycobacteria. Microbiologists often
request submission of a swab for Gram stain and an additional
swab for each culture type requested. This approach is helpful,
because it provides more sample volume. However, discrepancies
between results for the Gram stain and culture are more likely to
occur if separate swabs are used, even if they are collected simultaneously. As an alternative, one or more swabs may be eluted into
1 ml of sterile saline. After placing the swab into the tube with
saline, the shaft is broken and the tube is capped to allow vortex
mixing. Saline is a preferred over broth for elution, because it is
less likely to contain non-viable bacteria and adheres better to the
microscope slide.
Another option is use of the E-swab from Copan Diagnostics.
Flocked swabs and the E-swab transport system are a signicant
improvement on traditional swabs. Rather than being wound
around the shaft as for traditional swabs, the bers of ocked swabs
are perpendicular to the shaft, providing a brush-like action for
specimen collection. The E-swab transport tube consists of 1 ml
liquid medium, such as Amies, Stuarts, or viral transport medium.
When the ocked swabs are placed into the liquid medium and
vortexed, specimen is released from between their bers. Compared to traditional rayon or polyester swabs, the E-swab has been
shown to release more organisms from the swab. Similar, and
sometimes better, recovery of bacteria, including several fastidious
species and anaerobes, has been obtained with the E-swabs after
holding them for 24 or 48 hours either at room temperature or
under refrigeration (27,28). Better elution of the sample from the
swab and the ability to work with a larger liquid sample volume
likely contribute to the improved recovery (29-31).
Agar media generally include both non-selective and selective formulations. To avoid carry-over of selective agents, non-selective
media should be inoculated rst if the same swab is used to inoculate multiple plates. Broths are generally not included for wound
and abscess cultures; however, a broth medium may be helpful
for tissue collected from a sterile site from which recovery of
small numbers of organisms or fastidious species can be challeng-

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ing. Examples for which broth media might be especially helpful

include tissues from the heart and brain.
Although this reviews focus is not on culture workup, specimen
collection and transport procedures have downstream effects on
subsequent testing. A Gram stain should be performed on all
specimens unless volume is truly prohibitive. For thin specimens,
a drop may be placed on the slide. A smooth and even layer of
material may be achieved for thicker specimens by using the pull
smear technique. Material is placed on the slide, and then a second slide is placed on top to create a sandwich. The two slides are
then gently pulled apart to generate a thin, even layer. Swabs may
be rubbed gently on the surface of the slide, rolling the swab to
release material from the surface, as needed.
Gram stain assessment should include the numbers of squamous
epithelial cells, other cells, neutrophils, and organism morphologies as indicators of specimen quality, disease state, and contamination with supercial cells and bacteria. These parameters should
drive the approach to culture workup. Two Gram stain scoring
systems have been described. They are the Q score and the Q234
score (32-34). The scores compare the number of organism morphologies and cellularity on Gram stain to the growth in culture
to determine the extent of workup of each organism. Whether
these scoring systems or another approach tailored to the needs
of the clinical practice are chosen may depend on the experience
of the technical staff and the needs of practitioners.

Stool
Fecal specimens should be collected into a clean, dry container and
should not be contaminated with urine or barium. Shigella species
are especially labile in stool. Therefore, samples that cannot be
processed within 1 hour should be placed into Cary Blair transport
medium. The stability and transport temperature requirements
recommended by the manufacturer should be followed. Swabs
are inferior samples and should not be accepted unless absolutely
necessary..
Bacterial pathogens are not shed intermittently as parasites may
be; therefore, collection of a single specimen is usually adequate
for detection of most enteric bacterial pathogens. Patients hospitalized for more than 3 days are unlikely to experience onset of
diarrhea due to bacterial pathogens unless they have consumed
food brought in from an outside source. Requests for stool culture on patients hospitalized more than 3 days should be discussed
with the microbiology director as there may be some exceptions
for immunocompromised patients (5).
In general, detection of enteric bacterial pathogens by culture
tends to be a labor-intensive task with relatively low yield. As
molecular detection methods become more widely available, the
cost-benet ratio for assessment of stool samples for bacteria may
improve.

Lower Respiratory Tract

The most important factor contributing to a meaningful respiratory culture is collection of a quality specimen. Expectorated
sputum is the most convenient specimen for the diagnosis of

pneumonia, but careful collection must occur. The mouth should

be rinsed with water to minimize contaminating ora and epithelial cells and not with bactericidal mouthwashes. While in a
sitting position, the patient should cough deeply from the lungs,
expectorating sputum into a sterile cup or suction trap. If expectorated sputum cannot be produced, respiratory therapists may be
requested for procurement of a better sample. Induced sputum,
endotracheal tube aspiration, and specimens from bronchoscopy
are all appropriate specimens for assessment of lower respiratory
tract infection. Sputum and endotracheal specimens should be
assessed for quality by microscopic examination of a Gram stain.
Specimens with numerous squamous epithelial cells and few neutrophils are of poor quality and should not be cultured. Guidelines
for specimen rejection may be found in MCM and CMPH (3,4).
To prevent overgrowth of contaminating ora and loss of labile
organisms, such as Streptococcus pneumoniae, specimens from the
lower respiratory tract should be transported to the laboratory
within 2 hours and refrigerated if the transport time is longer.
Specimens should be processed to media that include selection
for gram-negative organisms and permit growth of Haemophilus
species.

Hardware and Devices

When a patient has symptomatic infection, procurement of uid or
tissue or even a swab from the infected site for culture or molecular
amplication should be possible. Cell counts, anatomic or cytopathology stains of tissue and cells, and markers of inammation,
such as C-reactive protein or the erythrocyte sedimentation rate,
may contribute to a diagnosis.
Culture is losing its place as the gold standard because in some circumstances it lacks sensitivity. The improved sensitivity of molecular amplication methods and the explosion in use of molecular
tests are instructive on this point; however, even with the advent of
molecular diagnostics, infection at some sites remains confounding.
This is especially true for diagnosis of infection when prostheses
and hardware are in place. These materials may acquire a biolm
that promotes a persistent, often low-level infection. One such
example is the prosthetic joint, for which processing with sonication is becoming the gold standard.
In 2007, Trampuz and colleagues described a method for sonication of hip and knee prostheses for diagnosis of PJI (35). After
placing the hardware or device into a sealable, sterile container
and covering it with sufcient broth or Ringers solution, vortexing
and/or sonication can be performed. An aliquot of sonicated uid
can then be inoculated to agar medium and distributed with a plate
spreader. Alternatively, the hardware may be placed in broth and
incubated overnight prior to subculture. Subsequently, additional
publications have demonstrated the increased sensitivity of culture
of the sonicated uid over standard tissue culture (36) while others
have shown reduced specicity (37). The American Academy of
Orthopaedic Surgeons recommends collection of multiple tissue
and/or uid specimens for diagnosis of PJI of the hip and knee
(38). Cultures should be incubated for 14 days for recovery of P.
acnes, particularly from shoulder joints (39,40). Given the need

for culture of multiple tissue or uid specimens and for incubating each culture for a prolonged time, performing a single, albeit
labor-intensive, hardware sonication culture may be both a more
sensitive and a more cost-effective culture technique.
The literature supporting sonication of joint prostheses and culture of vascular catheter tips is fairly well developed, and laboratory methods continue to be rened. In contrast, there are few
published data to support any particular culture practice for most
other types of hardware and interpretation of signicance is likewise, not established. The established procedures for culture of
prosthetic joints and vascular catheter tips suggest that recovery of
organisms from a biolm on hardware may be worthwhile. The key
is to nd the balance between recovering rare biolm organisms
and avoiding reporting low numbers of culture contaminants.
Several studies of culture methods for cardiac rhythm management
devices, such as pacemakers and implantable cardioverter-debrillators, have been conducted. Sonication of devices has improved
sensitivity of detection in patients with infection. However, devices
from uninfected patients have sometimes been colonized without
infection, decreasing the positive predictive value of the method
(41,42).
Culture of other types of hardware with a sonication method
have also been reported. As infected ureteral stents may not be
detected with standard urine cultures, the Maki roll-plate method
was assessed for culture of stents and was reported to be superior to sonication (43). Positive sonication cultures correlated
with the degree of capsular contracture for patients with breast
implants (44). Sonication culture of catheter tips from ventricular
drains improved recovery of organisms in comparison to culture
of CSF. However, sonication of catheter tips from ventriculoperitoneal shunts was not more sensitive than CSF culture (45).
Finally, a procedure similar to that used for prosthetic joints has
been applied to hardware removed from spinal implants (46). As
more patients experience infection due to placement of implants
or hardware, laboratories will likely continue to receive requests
for culture. Development of standardized culture or molecular
methods is needed.

Molecular Tests
The molecular test menu is increasing rapidly, and laboratories
may provide molecular assessment for specic pathogens in almost
every specimen. Samples submitted for molecular analysis should
be handled with strict adherence to sterile technique to prevent
contamination with even the smallest amount of foreign nucleic
acids. If samples have been accessed with non-sterile items, the
specimen should not be used for molecular testing.

Quality Measures
Throughout this review, critical aspects of specimen collection,
transport, and processing that are critical to quality outcomes have
been described. Although direct observation is time-consuming
and difcult to manage, periodic observation of processing can
be very useful and is required annually for laboratory personnel
by accrediting bodies.

Clinical Microbiology Newsletter 36:14,2014 | 2014 Elsevier

111

Another way to evaluate the quality of specimens and processing

procedures is to incorporate quality measures. Table 2 presents a
list of quality measures that might be useful. Some of these have
been touched upon throughout the review. For each quality measure, dening the data to be collected is the rst step. Data are
monitored and used to report back to laboratory staff and those
involved in direct patient care with the goal of process improvement.
As an example, for contaminated blood cultures, it has been suggested that overall rates should be <2 to 3% (14,16). The rates
should be monitored at least annually, and changes from baseline should be addressed. Reporting to infection control, nursing

units, and phlebotomy is an essential part of maintaining awareness and continuous quality. Laboratories can work with phlebotomy and nursing to target areas or individuals for retraining
when needed. Recognizing and rewarding those with improved
rates may contribute to employee satisfaction, as well as provide
better patient care.
Several of the measures in Table 2 require counting occurrences,
such as the number of urine or stool specimens received or not
received in transport or the number of blood cultures of low volume. For laboratories with limited laboratory information system
(LIS) support, periodic hand tallies might be one way to collect
these data. Another approach is to incorporate a comment code

Table 2. Examples of quality measures for pre-analytics

Measure

Process

Reporting

Blood cultures with low

volume

Incorporate a code into the LIS that informs the

provider of low volume. Count the times the
code is used per number cultures, stratify by
nursing unit or phlebotomist, and express as
a percentage of the total. Establish expected
rates, depending on patient population.

Provide feedback information to phlebotomist or

nursing staff by individual, nursing unit, or clinic
when rates are exceeded. Retrain on specimen collection as needed. Encourage collection of 8 to 10
ml/bottle.

Contaminated blood cultures

'HQHDFRQWDPLQDWHGEORRGFXOWXUH'HWHU
mine the number of contaminated cultures as
a percentage of all cultures and by phlebotomist, nurse, or patient care technician. Establish
baseline rates and quality goals. Possible goal:
<2-3% contaminated.

Report hospital-wide contamination rate to Infection Prevention. Provide feedback information to

phlebotomist or nursing staff by individual, nursing
unit, or clinic on a regular basis. Retrain
on skin disinfection, as needed.

Transport time for blood

cultures: time from collection
to incubation

Requires a continuously monitoring blood

culture system with an interface to retain collection time and incubation time stamps.
Determine time to incubation for all specimens.
0RVWKHOSIXOLILQIRUPDWLRQFDQEHVWUDWLHG
by location. Establish baseline times and goals.
Possible goal: <4 h transport time for 90% of
specimens.

Report back to courier or hospital transport teams on

a regular basis. Involve nursing if it is determined
that specimens are held at nursing stations. Retrain
as needed.

Urine culture contamination

rate

'HQHDFRQWDPLQDWHGXULQHFXOWXUH,QFRUSRU
ate a code into the LIS for specimens that are
contaminated. Determine the percentage
of contaminated cultures by nursing unit.
Establish baseline and goals. Possible goal:
<20% contaminated or improvement from
baseline.

Report to nursing units or clinics on a regular basis.

Retrain on specimen collection and transport as
needed.

Urine cultures in preservative

tubes

Incorporate a code into the LIS for specimens

with >2 h transport time not received in preservative or, more simply, the number not in preservative. Count the specimens as a percentage
of the total. Stratify data by clinic or nursing
unit. Establish baseline and goal. Possible goal:
>95% of specimens received in preservative.

Report to nursing unit or clinic when baseline or

goal is not met. Retrain on collection practices as
needed. Focus retraining efforts on collection sites
with longer transport times.

Rejected samples

Incorporate a single code into the LIS that is

added for all rejected samples. Prepare a daily
report listing rejected specimens that includes
specimen collection information and the reason the specimen could not be processed. Track
units or providers with consistent issues.

$SURYLGHUPXVWEHQRWLHGRIDQ\VSHFLPHQWKDWLV
XQWIRUSURFHVVLQJ.HHSLQJWKHOLVWDOORZVFRQVLVtent problems to be addressed with providers.

Rejected wound or sputum

samples

Incorporate a code into the LIS that is added

when wound or sputum samples have many
epithelial cells and are rejected for culture.
Count the rejected cultures by clinic or nursing
unit, and express as a percentage. Establish
baseline and goals based on patient population.

Report to nursing units or clinics when the percentage of rejected samples increases over baseline.
Retrain on specimen collection practices as needed.

112

Clinical Microbiology Newsletter 36:14,2014 | 2014 Elsevier

into the culture report to indicate when a sample is not received in

transport or when blood culture is low volume, etc. The number
of times the code is used can be determined electronically, and the
data can be stratied by collection site or phlebotomist. Compilation of data such as these can be used as an overall quality indicator, which can be reported back to those collecting or processing
specimens to promote improvements and assess progress.
Another useful metric is transport time. Electronic time stamps at
the times of specimen collection, receipt, processing, and reporting
can be recorded and used to calculate metrics, such as transport
time, time to processing, or time to reporting. These data can be
incorporated as part of the routine measures used to regularly
assess laboratory quality. A metric indicating the expected transport time can be established. For example, 90% of blood cultures
received within 4 hours could be a specimen transport goal. Alternatively, median transport times can be monitored, as this statistic
is more representative of the typical transport time when large
outliers exist that substantially alter the mean (average) transport
time. Such information can then be used to improve the quality
of specimens received from particular clinics or nursing units, as
feedback to the providers and transportation personnel can help
to frame their important roles in the process.
Specimen receipt (time stamp) data may also be used to indicate
the number of specimens received throughout each hour of the day.
With this information in hand, stafng can be adjusted to match
peak specimen delivery times, ensuring that specimens do not
encounter processing bottlenecks once delivered to the laboratory.

5.

6.
7.

8.
9.
10.

11.

12.

13.

14.

Summary
It is the responsibility of the laboratory staff to be well informed
of best practices for specimen collection, transport, and processing and to ensure the ancillary health care staff is following the
practices. An up-to-date test directory and a specimen collection
guide that includes relevant information for providers submitting
specimens are essential. Continuous education of those involved in
specimen collection is fundamental. Collection of data and provision of feedback to the technical staff and health care teams using
the laboratory can improve the quality of care provided and help
prevent breaches in best practice.

15.

16.

17.

18.

Acknowledgement
I gratefully acknowledge Gerri S. Hall for review of the manuscript.

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