SPECIMEN MANAGEMENT
In late 1800s, the first clinical microbiology laboratories were
organized to diagnose infectious diseases such as tuberculosis,
typhoid fever, malaria, intestinal parasites, syphilis, gonorrhea, and
diphtheria.
Between 1860 and 1900, microbiologists such as Pasteur, Koch, and
Gram – developed the techniques for staining and the use of solid
media for isolation of microorganism.
In general, a microbiologist will perform one or more of the
following functions:
• Cultivation (growth), identification, and antimicrobial
susceptibility testing of microorganisms
• Direct detection of infecting organisms by microscopy
• Direct detection of specific products of infecting organisms
using chemical, immunologic, or molecular techniques
• Detection of antibodies produced by the patient in response to an
infecting organism (serology)
GENERAL CONCEPTS FOR SPECIMEN COLLECTION AND
HANDLING
Specimens should be obtained to preclude or minimize the possibility
of introducing contaminating microorganisms that are not involved in
the infectious process.
the throats of hospitalized patients on ventilators may frequently be
colonized with Klebsiella pneumoniae (K. pneumoniae is not usually
involved in cases of community-acquired pneumonia, it can cause a
hospital-acquired respiratory infection)
APPROPRIATE COLLECTION TECHNIQUES
 Specimens should be collected during the acute (early) phase of
an illness (or within 2 to 3 days for viral infections) and
before antibiotics are administered
 Swabs generally are poor specimens if tissue or needle aspirates
can be obtained
 Information for the nursing staff and clinicians should include
the following:
o Safety considerations
o Selection of appropriate anatomic site and specimen
o Collection instructions including type of swab or transport
medium
o Transportation instructions including time and temperature
o Labeling instructions including patient demographic
information (minimum of two patient identifiers)
o Special instructions such as patient preparation
o Sterile versus nonsterile collection devices
o Minimal acceptable quality
 Instructions should be written so that specimens collected by the
patient (e.g., urine, sputum, or stool) are handled properly
 when distributing kits for sputum collection - microbiologist
should be able to explain to the patient the difference between
spitting in a cup (saliva) and producing good lower respiratory
secretions from a deep cough (sputum).
SPECIMEN TRANSPORT
 Ideally, specimens should be transported to the laboratory
within 2 hours of collection
 Specimen containers – leak-proof, and the specimens should be
transported within sealable, leak-proof, plastic bags with a
separate section for paperwork; resealable bags or bags with a
permanent seal are common for this purpose. (w/ biohazard
label)
 Many microorganisms are susceptible to environmental
conditions
o oxygen (anaerobic bacteria)
o changes in temperature (Neisseria meningitidis)
o changes in pH (Shigella)
 use of special preservatives or holding media for
transportation of specimens delayed for more than 2 hours is
important to ensure organism viability (survival).
SPECIMEN PRESERVATION
 Boric acid for urine or polyvinyl alcohol (PVA) and buffered
formalin for stool for ova and parasite (O&P) examination 
TO MAINTAIN THE APPROPRIATE COLONY COUNTS
(URINE) OR INTEGRITY OF TROPHOZOITES AND CYST
(O&P)
 Other transport, or holding, media maintain the viability of
microorganisms present in a specimen without supporting the
growth of the organisms (maintains the organisms in a state of
suspended animation so that no organism overgrows another or
dies out)
 Example of common holding media: Stuart’s medium and
Amie’s medium
 Charcoal is added to these media to absorb fatty acids  that
could kill fastidious (fragile) organisms such as Neisseria
gonorrhoeae or Bordetella pertussis.
 Anticoagulants are used to prevent clotting of specimens: blood,
bone marrow, and synovial fluid
 Sodium polyanethol sulfonate (SPS) at a concentration of
0.025% (w/v) is usually used, because Neisseria spp. and some
anaerobic bacteria are particularly sensitive to higher
concentrations.
o it is necessary to have both large (adult-size) and small
(pediatricsize) tubes available for specimens are not
overwhelmed by the concentration of SPS
o Heparin is also a commonly used anticoagulant,
especially for viral cultures, although it may inhibit
growth of gram-positive bacteria and yeast.
o Yellow top – maybe trisodium citrate/citric acid/dextrose
(ACD) (not appropriate for use in microbiology) or
SPS
 Specimen Storage
o Several storage methods are used:
 refrigerator temperature [4°C]
 ambient [room] temperature [22°C]
 body temperature [37°C]
 freezer temperature [either –20° or –70°C])
o Urine, stool, viral specimens, sputa, swabs, and foreign
devices such as catheters should be stored at 4°C
o Serum for serologic studies – frozen for up to 1 week at –
20°C
o tissues or specimens for long-term storage should be
frozen at –70°C
SPECIMEN LABELING
• labeled with the patient’s name, identifying number (hospital
number) or birth date, date and time of collection, and source
SPECIMEN REQUISITION
• Requisition is a hard (paper) copy of the physician’s orders and
the patient’s demographic information (e.g., name and hospital
number. Should contain as much information as possible
regarding the patient history and diagnosis
A complete requisition should include the following:
 The patient’s name
 Hospital number
 Age or date of birth
 Sex
 Collection date and time
 Ordering physician
 Exact nature and source of the specimen
 Diagnosis (may be ICD-9-CM code)
 Current antimicrobial therapy
REJECTION OF UNACCEPTABLE SPECIMENS
 The information on the label does not match the information on
the requisition or the specimen is not labeled at all (patient’s
name or source of specimen is different).
 The specimen has been transported at the improper temperature.
 The specimen has not been transported in the proper medium
(e.g., specimens for anaerobic bacteria submitted in aerobic
transports).
 The quantity of specimen is insufficient for testing (the
specimen is considered quantity not sufficient [QNS]).
 The specimen is leaking.
 The specimen transport time exceeds 2 hours post-collection or
the specimen is not preserved.
 The specimen was received in a fixative (formalin), which, in
essence, kills any microorganism present.
 The specimen has been received for anaerobic culture from a
site known to have anaerobes as part of the normal flora (vagina,
mouth).
 The specimen is dried.
 Processing the specimen would produce information of
questionable medical value (e.g., Foley catheter tip)
 Correction of mislabeled specimens must be completed at the
discretion of the laboratories standard operating procedures.
SPECIMEN PROCESSING
 When multiple specimens arrive at the same time, priority
should be given to those that are most critical, such as
cerebrospinal fluid (CSF), tissue, blood, and sterile fluids.
 Urine, throat, sputa, stool, or wound drainage specimens can be
saved for later.
 Acid-fast, viral, and fungal specimens are usually batched for
processing at one time.
 When a specimen is received with multiple requests but the
amount of specimen is insufficient to do all of them, the
microbiologist should call the clinician to prioritize the testing.
 On arrival in the laboratory, the time and date received should
be recorded.
GROSS EXAMINATION OF SPECIMEN
 All processing should begin with a gross examination of the
specimen.
 Areas with blood or mucus should be located and sampled for
culture and direct examination.
 Stool should be examined for evidence of barium (CHALKY
WHITE COLOR)
 Notations should be made on the handwritten or electronic work
card regarding the status of the specimen (e.g., bloody, cloudy,
clotted)
DIRECT MICROSCOPIC EXAMINATION
The direct examination serves several purposes:
1. sputa can be rejected that represent saliva and not lower
respiratory tract secretions by quantitation of white blood cells
or squamous epithelial cells present in the specimen.
2. the microbiologist and clinician can be given an early indication
of what may be wrong with the patient (4+ gram-positive cocci
in clusters in an exudate)
3. the workup of the specimen can be guided by comparing what
grows in culture to what was seen on the original smear
(Example: three different morphotypes (cellular types) are seen
on direct Gram stain but only two grow out in culture – third
organism may be an anaerobic bacterium/ or here are more than
three organisms on the culture plate that were not visible on
Gram stain, indicating possible contamination)
 Direct examinations are usually not performed on throat,
nasopharyngeal, or stool specimens but are indicated from most
other sources due to the presence of abundant normal
microbiota.
 Gram stain, which helps the clinician to visualize rods, cocci,
white blood cells, red blood cells, or squamous epithelial cells
present in the sample.
 The most common direct fungal stains are KOH (potassium
hydroxide), PAS (periodic-acid Schiff), GMS (Grocott’s
methenamine silver stain), and calcofluor white. The most
common direct acid-fast stains are AR (auramine rhodamine),
ZN (Ziehl-Neelsen), and Kinyoun.
SELECTION OF CULTURE MEDIA
 Primary culture media – first are nutritive media, such as
blood or chocolate agars.
 Nutritive media support the growth of a wide range of
microorganisms and are considered nonselective. Can also be
differential, in that microorganisms can be distinguished on the
basis of certain growth characteristics evident on the medium.
 Blood agar is considered both a nutritive and differential
medium because it differentiates organisms based on whether
they are alpha (α)-, beta (β)-, or gamma (γ)- hemolytic.
 Selective media support the growth of one group of organisms,
but not another, by adding antimicrobials, dyes, or alcohol to a
particular medium.
 MacConkey agar, for example, contains the dye crystal violet,
inhibits gram-positive organisms. Also, differentiates between
lactose-fermenting and nonfermenting gram-negative rods by
the color of the colonial growth.
 Columbia agar with colistin and nalidixic acid (CNA) is a
selective medium for gram-positive organisms because the
antimicrobials colistin and nalidixic acid inhibit gram-negative
organisms.
 In some cases (sterile body fluids, tissues, or deep abscesses in a
patient on antimicrobial therapy), backup broth (also called
supplemental or enrichment broth) medium is inoculated, along
with primary solid (agar) media, so small numbers of organisms
present may be detected.
SPECIMEN PREPARATION
Such procedures include:
 Homogenization (grinding) of tissue
 Concentration by centrifugation
 Filtration of large volumes of sterile fluids, such as ascites
(peritoneal) or pleural (lung) fluids.
 Decontamination of specimens, such as those for legionellae or
mycobacteria.
 Swab specimens are often vortexed (mixed) in 0.5 to 1 mL of
saline or broth for 10 to 20 seconds to dislodge material from the
fibers.
INOCULATION ON SOLID MEDIA
 Urine cultures and tissues from burn victims are plated
quantitatively; everything else is usually plated
semiquantitatively.
 Plates inoculated for quantitation are usually streaked with a
1:100 or 1:1000 loop.
 Plates inoculated for semiquantitation are usually streaked out in
four quadrants.
 Semiquantitation is referred to as streaking for isolation, because
the microorganisms present in the specimen are successively
diluted out as each quadrant is streaked until finally each
morphotype is present as a single colony.
 Graded: (4+ many, heavy growth) f growth is out to the fourth
quadrant, (3+ moderate), (2+ few or light), (1+ rare).
INCUBATION CONDITIONS
 28° to 30°C for fungi and 35° to 37°C for most bacteria, viruses,
and acid-fast bacillus
 A number of different environmental conditions exist:
o Aerobes grow in ambient air, which contains 21% oxygen
(O2) and a small amount (0.03%) of carbon dioxide
(CO2).
o Anaerobes usually cannot grow in the presence of O2, and
the atmosphere in anaerobe jars, bags, or chambers is
composed of 5% to 10% hydrogen (H2), 5% to 10% CO2,
80% to 90% nitrogen (N2), and 0% O2.
o Capnophiles, such as Haemophilus influenzae and
Neisseria gonorrhoeae, require increased concentrations of
CO2 (5% to 10%) and approximately 15% O2 (achieved
by a candle jar (3% CO2) or a CO2 incubator, jar, or bag.)
o Microaerophiles (Campylobacter jejuni, Helicobacter
pylori) grow under reduced O2 (5% to 10%) and increased
CO2 (8% to 10%).
SPECIMEN WORKUP
One of the most important functions that a microbiologist performs is
to decide what is clinically relevant regarding specimen workup.
EXTENT OF IDENTIFICATION REQUIRED
 As health care continues to change, one of the most problematic
issues for microbiologists is the extent of culture workup.
 Complete identification of a blood culture isolate, such as
Clostridium septicum as opposed to a genus identification of
Clostridium spp., will alert the clinician to the possibility of
malignancy or other disease of the colon.
 A presumptive identification of Escherichia coli if a gram-
negative, spot indole-positive rod is recovered with appropriate
colony morphology on MacConkey agar (flat, lactose-
fermenting colony that is precipitating bile salts) is probably
permissible from an uncomplicated urinary tract infection.
 In the final analysis, culture results should always be compared
with the suspected diagnosis.
CRITICAL (PANIC) VALUES
 Positive blood cultures
 Positive spinal fluid Gram stain or culture
 Streptococcus pyogenes (group A Streptococcus) in a surgical
wound
 Gram stain suggestive of gas gangrene (large boxcarshaped
gram-positive rods)
 Blood smear positive for malaria
 Positive cryptococcal antigen test
 Positive acid-fast stain
 Detection of a select agent (e.g., Brucella) or other significant
pathogen (e.g., Legionella, vancomycin resistant S. aureus, or
other antibiotic-resistant organisms as outlined by the facility
and infection control policies).
EXPEDITING RESULTS REPORTING:
COMPUTERIZATION
 The hardware and software together make up the complete LIS.
 Between the HIS and LIS, most functions involved in ordering
and reporting laboratory tests can be handled electronically
 Order entry, patient identification, and specimen identification
can be handled using the same type of bar coding that is
commonly used in supermarkets.
 The LIS also takes care of results reporting and supervisory
verification of results, stores quality control data, allows easy
test inquiries, and assists in test management reporting by
storing, for example, the number of positive, negative, and
unsatisfactory specimens.