Chapter 26
Specimen Collection and
Processing
for Microbiology
Wang Hui
General specimen collection
and processing issue
First part of this chapter
Specimen Collection
1.
Successful laboratory diagnosis of a
microbial infection depends on many factors
beginning with a well-collected sample.
2.
Proper specimen selection, collection, and
transport are all essential to ensure that a
specimen is representative of the disease
process and minimally contaminated with
microorganisms present in adjacent tissues.
Site and Timing
1.
Collect the sample from the correct
anatomic site . eg. a superficial sample of a
lesion is not useful in identifying the cause of a deep
wound infection.
2.
The timing of sample collection is
also important. eg, when submitting a
specimen for bacterial culture, samples should be
collected before the administration of antibiotics
Collection Techniques
1.
2.
3.
Sterile technique and equipment.
Sufficient volume
After collection, the specimen must be
placed in an appropriately labeled leakproof container.
Requisition slip
1.
2.
Each specimen must be accompanied by a
requisition slip to evaluate the specimen
appropriately and relay the test results back to
health care provider without delay.
The requisition slip should contain these
information :
patient name, age, gender, identification number,
location, name of health- care provider, time and
date of collection, specimen type, diagnosis, and
test(s) requested.
Transport of Specimens
1.
2.
3.
Rapid, optimally in less than 2 hours.
For delays in transport, most specimens
should be refrigerated.
Exceptions: blood, cerebrospinal fluid
(CSF), and specimens to be examined for
anaerobes, fastidious organisms such as
Neisseria gonorrboeae and Bordetella
pertussis, and Trichomonas vaginalis, all of
which should be maintained at room
temperature.
Specimen rejection criteria(1)
1.
2.
Improper transport temperature
Improper transport container or medium
3.
Prolonged transport time
4.
5.
6.
Unlabeled or mislabeled specimen
Broken or cracked container
Leaking specimen
Specimen rejection criteria(2)
7.
8.
9.
10.
11.
Dried-out specimen
Inappropriate specimen for test requested
Inadequate volume
Specimen in fixative (for culture)
Duplicate sample in 24-hr period (for urine,
sputum, feces culture)
Specimen rejection criteria(3)
1. When specimens are rejected, the health care
provider is notified so that another specimen may
be properly submitted.
2. If information on the requisition is incomplete,
laboratory personnel should ask a responsible
person to provide the information before processing
the specimen further.
3. If a specimen is mislabeled, the sample should be
recollected. Relabeling of a specimen is acceptable
only for difficult to collect specimens, such as
tissue obtained during a surgical procedure or CSF.
Specimen not routinely accepted for
anaerobic culture(1)
1.
2.
3.
4.
Throat, nasopharyngeal, or gingival swabs
Sputa
Bronchial wash, lavage, or brush (except
when collected with a protected double
lumen catheter)
Gastric and bowel contents
Specimen not routinely accepted for
anaerobic culture(2)
5.
6.
7.
8.
Ileostomy and colostomy effluent
Voided or catheterized urine
Female genital tract specimens collected
through the vagina
Surface swabs of ulcers, wound, and
abscesses
Standard Precautions
1.
2.
3.
All specimens should be presumed to
contain transmissible agents and therefore
should be collected and handled using
standard precautions.
Use of gloves, gown, mask, and protective
eyewear when there is a risk of coming in
contact with the specimen
In most clinical laboratories, a special area is
designed for processing clinical samples for
culture.
Clinical Diagnosis by Microbiology
Laboratory method(1)
1.





Direct Examination
Gram stain (general)
acid-fast bacilli (AFB) (mycobacteria )
KOH and/or calcofluor white preparation
(fungi )
wet mount (parasites), etc.
Other techniques for directly examining specimens :
direct fluorescent antibody stains (DFA),
enzyme immunoassays (EIAs),
DNA hybridization or amplification assays, etc.
Clinical Diagnosis by Microbiology
Laboratory method(2)
2.



Isolate, Culture and Identification
A combination of media types is used to
isolate bacteria and fungi (include enriched,
nonselective, selective, or differential
media);
Viruses can only be cultured within
mammalian cells, three main categories:
primary, low-passage finite and continuous
cell lines
Culture of parasites is generally not
performed.
Clinical Dignosis by Microbiology
Laboratory method(3)
3.



Lengths of culture time
Most routine bacterial cultures are incubated
for 2 to 3 days.
Mycobacterial and fungal cultures are incubated for as long as 6 weeks.
Viral cell cultures are incubated for varying
lengths of time depending on the specimen
source and the growth rate of the viruses that
are typically recovered from that site
Clinical Dignosis by Microbiology
Laboratory method(4)
4.



The condition of incubation
35℃ for bacteria and viruses
30℃ for fungi
Various atmospheric conditions may be
utilized including ambient, CO2 enriched,
microaerophilic and anaerobic.
Specific recommendations for
each specimen type
The second part of this chapter
Blood - specimen collection(1)
1.
2.
3.
In general, blood for culture should not be
obtained using an intravascular device.
When performing a venipuncture, the skin
must be adequately disinfected to minimize
contamination with normal skin flora.
Blood should be collected and incubated into
the blood culture bottles using the same
needle.
Blood - specimen collection(2)
4.
5.
6.
Blood specimens should be collected before
administering antimicrobial agents.
Optimally, the specimen should be collected
just before a fever spike; however, practically, the specimen should be collected
immediately after the spike.
For adults, 20 to 30mL of blood should be
collected per venipuncture. Less blood is
required for children .
Blood - specimen collection(3)
7.
8.
9.
For adult patient, two sets of cultures should
be collected per febrile episode to help
distinguish probable pathogens from possible contaminants
No more than four sets should be submitted
in a 24-hour period.
Inoculated blood culture vials should be held
at room temperature until they reach the
laboratory.
Blood culture(1)
1.
2.
3.
Cultures for rapidly growing bacteria and
yeast are usually incubated for 5 to 7 days.
Cultures for mycobacteria and slowly growing fungi are held for as long as 42 days.
Many types of blood culture systems are available, including both manual and automated. Each system utilizes a noninvasive
method (i.e., colorimetric, fluorescent, or
manometric methods for detecting CO2 or
other gases) to monitor growth.
Blood culture(2)
4.
5.
6.
As soon as growth is detected from the
blood specimen, a stain is performed (Gram,
acid-fast, or Giemsa stain) to determine the
type of microorganism present.
Positive stain results are considered a critical
value and called directly to the patient’s
health care provider .
Then the specimen should be subcultured to
solid media.
Culture of catheter tips
1.
2.


Performed to determine the source of a
bacteremia.
Semiquantitative catheter tip culture method
:
The segment is rolled across a blood agar
plate four times
Cultures yielding organisms present in more
than 15 CFU are considered to be
significant, potentially indicating a catheterrelated in-fection.
Cerebrospinal fluid (CSF)(1)
1.
2.
Cerebrospinal fluid (CSF) is submitted for
microbiological analysis when meningitis or
encephalitis is suspected.
For meningitis, the likely infection agent
differs depending on the duration of symptoms. The most likely bacterial agent of
acute meningitis will also vary with the age
of the individual and whether the disease is
comunity or nosocomially acquired.
Cerebrospinal fluid (CSF)(2)
3.
4.
Most infectious cases of encephalitis are a
result of viral infection, both arthropod and
nonarthropod borne.
Parasitic infections of the central nervous
system also occur, with varying clinical
presentations.
Probable infectious cause of
meningitis
Duration of symptoms
< 24 hr
1-7 days
≥4 wk
Probable Pathogen
Pyogenic bacteria
Enteroviruses , Pyogenic bacteria
Mycobacterium tuberculosis
Treponema pallidum
Brucella spp , Candida spp
Leptospira interrogans
Borrelia burgdorferi
Cryptococcus neoformam
Coccidioides immitis
Histoplasma capsulatum
CSF - specimen collection
1.
2.
Obtained by lumbar spinal puncture
Generally at least 0.5mL of CSF (smear,
culture, antigen tests )
For mycobacterial culture, at least 3mL
(greater volumes increase recovery)
CSF – transportation
1.
2.
3.
Transported to the laboratory promptly
and processed as soon as possible.
If a delay in processing is unavoidable,
the specimen should be held at room
temperature.
If greater than 1.0mL of CSF is
received for a given test the fluid is
centrifuged to allow the test to be
performed on the concentrate sediment
CSF-laboratory diagnosis
1.
2.
3.
4.
5.
6.
7.
8.
Gram stain
antigen tests
India ink test (Cryptococcus neoformans )
dark-field microscopy of a concentrated
specimen (Leptospires)
acid-fast bacilli (AFB) (mycobacteria )
bacterial culture
yeast and fungi culture
viral culture
Gastrointestinal Tract(1)
1.
2.
Feces, and in some cases rectal swabs, are
submitted to the laboratory primarily to
determine the etiologic agent infections
diarrhea or food poisoning.
Feces should be collected in a clean
container with a tight lid and should not be
contaminated with urine, barium, or toilet
paper. Optimally be examined within 2
hours of collection.
Gastrointestinal Tract(2)
3.
4.
Rectal swabs should be placed in a tube
transport system containing modified Stuart’s medium.
Unpreserved stool specimens should be
maintained at refrigerator temperature during storage and transport.
Gastrointestinal Tract(3)
5.
It is becoming standard practice to reject
stool specimens for bacterial culture and
parasite examination from patients who have
been hospitalized longer than 3 days . For
such patients, examination for the toxins
produced by Clostridium difficile is recommended.
Gastrointestinal Tract -laboratory
diagnosis
1.
2.
3.
4.
Bacterial culture
selective and differential medium (Mac
Conkey agar, Hektoen enteric or xyloselysine-desoxycholate agar,etc)
Fungal culture of stool is not recommended.
Viral culture
To detect parasites, stool is examined
microscopically for the presence of
protozoa, helminth eggs, and larvae.
Genital Tract(1)
1.
2.
Genital tract specimens are sent to the
laboratory for determining the cause of various clinical syndromes, including vulvovaginitis, bacterial vaginosis, etc.
Many specimens will be contaminated with
the normal microbiota of the genital tract or
skin; therefore, the microbiologist must
differentiate the normal flora from potential
pathogens.
Genital Tract(2)
3.
Organisms such as N. gonorrhoeae, C.
trachomatis, and Haemophilus ducreyi are
always pathogenic, whereas organisms such
as the Enterobacteriaceae, S. aureus, and
group B streptococci are pathogenic only in
some clinical situations.
Genital Tract -laboratory
diagnosis(1)
1.
2.
3.
4.
direct Gram stain (only a few situations )
eg., gram-negative diplococci within polymorphonuclear leukocytes
wet mount preparation of vaginal secretions
clue cells :epithelial cells covered with small
coccobacillary bacteria
vaginal pH
normal≤4.5
whiff test positive :generation of a pungent,
fishy odor on addition of 10% KOH to the
specimen
Genital Tract -laboratory
diagnosis(2)
5.


bacterial culture: depend on the source and
the organisms likely to cause disease at that
site
Tissue and aspirates should be plated to
media capable of recovering fastidious
organisms.
Specimens from the cervix, vagina, and
urethra should at a minimum be evaluated
for N. gonorrhoeae and C. trachomatis by
culture or a direct detection method.
Genital Tract -laboratory
diagnosis(3)
6.
7.
Fungal culture of female genital tract
specimens is not productive.
Viral culture remains the gold standard for
detection of HSV.
Lower Respiratory Tract
1.
2.
3.
primarily to determine the etiologic agent of
pneumonia
Specimen types:
sputum (expectorated or induced), tracheal
aspirates, transtracheal aspirates, bronchial
washes,
bronchial
brushings,
and
bronchoalveolar lavage fluids.
delivered promptly to the laboratory. if
delays are unavoidable , refrigerated .
Lower Respiratory Tract
-laboratory diagnosis(1)
1.



2.
Gram-stained smear
low-power magnification to determine the
number of squamous epithelial cells and/or
neutrophils present
oil immersion to determine the relative
amounts of organisms present
Intracellular organisms should be specifically noted.
culture ,selective and nonselective media, In
addition, a medium capable of recovering
fastidious organisms
Lower Respiratory Tract
-laboratory diagnosis(2)
3.
4.
bronchial brush specimens (0.01 to
0.001mL)
a smear for Gram staining is prepared by
cytocentrifugation, and 0.01mL of the specimen is plated to appropriate media using a
pipette or calibrated loop.
bronchoalveolar lavage (10 to 100mL )
a smear is prepared by cytocentrifugation
and Gram stained (presence or absence of
intracellular organisms), a 0.001-mL aliquot
of the specimen is inoculated onto agar
media
Upper Respiratory Tract
1.
2.
Nasopharyngeal aspirates, washings, and
swab specimens are primarily used for the
diagnosis of viral respiratory infections but
may also be submitted to diagnose pertussis,
diphtheria, chlamydia infections, and
candidiasis, as well as identify carriers of N.
meningitidis or S. aureus.
Throat swab specimens are generally
collected to diagnose group A streptococcal
pharyngitis or to detect shedding of viruses
such as enteroviruses, HSV, or CMV.
Tissues
1.
2.
procured at great expense and considerable
risk to the patient; therefore, for optimal
evaluation enough material should be
collected to allow both histopathologic and
microbiologic examination.
After collection, tissues should be placed in
a sterile container and transported rapidly to
the laboratory to prevent drying.
Tissues -laboratory diagnosis
1.
homogenized by mincing with a sterile
scalpel, grinding with a mortar and pestle or
tissue grinder
2.
Gram stain or other stains
examined for presence of microorganisms,
leukocytes, and squamous epithelial cells
3.
routine culture, liquid medium and enriched
agar medium
bone marrow aspirates, in collection tubes
for the lysis centrifugation blood culture
system or in a sterile container
4.
Urine
1.
2.
Acceptable methods of urine collection include midstream clean catch, catheterization,
and suprapubic aspiration. Foley catheter
tips should not be accepted for culture.
promptly to the laboratory and processed
within 2 hours of collection . If delays are
unavoidable, refrigerated.
Urine -laboratory diagnosis
1.


2.
Screening urine specimens
Gram stain
dipstick tests that combine nitrate reductase
and leukocyte esterase
Quantitative bacterial culture
0.001-mLplastic or wire calibrated loop
blood and Mac-Conkey agars
Skin and Subcutaneous Lesions(1)
1.
2.
3.
Ideally, the infected material is aspirated
with a needle and syringe. For transport, the
material is expelled into sterile container that
is tightly capped and promptly delivered to
the laboratory.
If an aspirate cannot be obtained, swab
specimens of exudate collected from the
deep portion of the lesion are acceptable.
For bacterial and fungal cultures, swabs may
be placed in tube transport system containing modified Stuart’s medium.
Skin and Subcutaneous Lesions(2)
4.
5.
6.
To recover anaerobes, an additional swab
specimen must be collected and placed in an
anaerobic transport system.
For viral culture, the specimen (aspirate or
swab) should be placed in viral transport
medium and kept on ice.
If a delay in processing is unavoidable,
specimens may be stored in the refrigerator,
except those for recovery of anaerobes
(room temperature )
Skin and Subcutaneous Lesions
-laboratory diagnosis
1.
2.
3.
Gram-stain
appropriate media for culture
If detection of mycobacteria is requested,
specimens should be decontaminated and
concentrated. The sediment is used to
prepare a smear for staining for AFB and to
inoculate mycobacterial media.