SPECIMEN PREPARATION
than 2 hours.
Learning Outline:
1. specimen transport, storage &
preservation
2. specimen rejection of prioritization
3. specimen consideration of each
specimen types
General Guidelines
1. The laboratory can make accurate
and useful determinations if
specimen has been collected and
transported properly.
2. Specimens must be labelled
properly.
3. Specimens must be collected during
the acute or early phases ( or within
2 to three days for viral infections),
and before antibiotics are
administered.
4. Specimens for microbiology cultures
should be collected in sterile
containers except for stools.
5. The site should be cleansed properly
before collection.
6. Specimens should be submitted in
two swabs and these specimens are
never recommended for anaerobic
culture.
7. For lesions, wounds and abscess,
the specimen is collected preferably
by needle aspiration rather than by
swabs
8. Swabs are used for Upper
Respiratory Tract (URT), Ear, Eye,
and Genital Tract.
Specimen Transport, Storage and
Preservation
1. Ideally, specimens should be
transported to the laboratory
immediately after the collection
collection Or preferably within 30
minutes of collection and not longer
2. All specimens should be transported
in a leak proof container,
transported within sealable,
leakproof, plastic bags with
separate section for paperwork.
3. Use of special preservatives for
holding media for transportation of
specimens delayed for more than 2
hours is important for the organism’s
survival.
SPECIMEN TRANSPORT
● JEMBEC system- Collection
transport system used for Neisseria
gonorrhoeae specimens; contains
selective agar and a carbon dioxide
(co2)- generating tablet
● Cary blair- stool pathogens
● Amie’s- respiratory samples
● Todd-Hewitt and LIM (Modified
Todd-Hewitt)- S. agalactiae (vaginal
swab)
● Leibovitz-Emory media- viral
transport media
SPECIMEN PRESERVATION
● Boric acid- urine
● Polyvinyl alcohol (PVA) & buffered
formalin- stool for ova and parasite
examination
● Charcoal- added to media like
Stuart’s and Amie’s medium to
absorb fatty acids present in the
specimen that could kill fastidious
(fragile) organisms
● Preservatives should not be added
on fecal specimens
Anticoagulants- These are used to prevent
clotting of specimens including blood, bone
marrow, and spinal fluid.
 ➢ Citrate, EDTA or other
anticoagulants - not used for
microbiology because their efficacy
has not been demonstrated for a
majority of organisms.
1. Heparin
➔ Naturally occurring anticoagulant in
the body
➔ Often used for viral cultures and
isolation of Mycobacterium spp. from
blood.
➔ May inhibit growth of gram (+)
bacteria and yeast.
2. 0.025% Sodium Polyanethol
sulfonate (SPS)
➔ Actions:
○ Inhibits phagocytosis and
complement activation
○ neutralizes aminoglycosides - if the
patient is taking antibiotics such as
aminoglycosides
○ neutralizes bacterial effect of
plasma - plasma contains
antibodies, killer cells, and
macrophages
➔ Neisseria gonorrhoea and some
anaerobic bacteria are particularly
sensitive to higher concentrations
(G. vaginalis, Neisseria, S.
moniliformis, P. anaerobius) - high
concentration of SPS is not effective
for cultures because this will inhibit
bacterial activity of these bacteria
➔ 1% gelatin - counteracts SPS
SPECIMEN STORAGE
Storage:
1. -70 C- Tissues or specimens for long
term storage, including stool for C.
difficile toxin study if testing is
delayed for more than 3 days.
2. -20 C- Serum for serologic studies
may be frozen for up to 1 week.
3. 4 C- urine, stool, viral specimens,
sputa, swabs except genital,
foreign devices like catheter, stool
for C. difficile toxin study up to 3
days.
4. 22- 25 C (RT) - anaerobic culture,
sterile body fluids, genital
specimens, swabs.
5. 37 C- CSF
SPECIMEN PRIORITIZATION AND
REJECTION
Specimen Priorization
● When the specimen is received with
multiple requests but the amount of
specimen is insufficient, the clinician
should inform the laboratory staff
which of the tests should be
prioritized.
● When multiple specimens arrive at
the same time, priority should be
given to those that are most critical
such as CSF, tissue, blood, and
sterile fluids.
● Acid-fast bacilli, viral, & fungal
specimens can be processed by
batch
Specimen Rejection
Rejection of unacceptable specimens:
● Information on the label does not
match the information on the
requisition or the specimen is not
labeled at all ( patients name or
source of the specimen is different)
● Specimen has not been transported
in the proper medium (Ex.
specimens for anaerobic bacteria
has been transported in aerobic
transports)
 ● The quantity of the specimen is
insufficient for testing
● Specimen is leaking
● 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 microorganisms
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 a
questionable medical value (Ex.
foley catheter tip)
SPECIMEN CRITICAL VALUES
Examples of critical values in Microbiology:
➢ Positive blood culture- streak
immediately in agar; first in blood
culture tube then ipansak sa
machine then after how many days
the machine can detect bacterial
growth if the medium will become
turbid due to CO2 produced. It
should also be gram stained and
cultured in primary media such BA,
CA, and MAC
➢ Positive cerebrospinal fluid, gram
stain or culture
➢ Positive Cryptococcal antigen test or
culture
➢ Positive blood smear for malaria
➢ S. pyogenes from a sterile site
➢ Positive acid-fast smears or positive
Mycobacterium culture
➢ S.agalactiae or Herpes Simplex
virus from a genital site of a
pregnant woman
➢ Detection of significant pathogen (B.
pertussis, Brucelle spp. Legionella
spp.)
SPECIMEN CONSIDERATIONS
1. BLOOD
➔ Highest concentration of microbe
during the height of fever.
➔ Disinfect venipuncture site with 70%
alcohol and then with betadine (3
rounds)
➔ Draw blood at febrile episodes, 2-3
sets in 24 hours, at least 1 hour
apart from the left and right arms
➔ <20 ml per set collected in adults
➔ 5-10 ml per set in children
➔ 0.5 to 1 ml in neonates
➔ Ratio of blood and blood culture
media
● Adults- 1:10
● Children- 5:10
➔ If the patient is on antimicrobial, use
THIOL BROTH to and
antimicrobials or ANTIMICROBIAL
REMOVAL DEVICE (ARD) to
remove them before culture.
2. CSF
➔ Collected through Spinal Tap by the
physician - 3 collection tubes
● 1- Chemistry test (lactate,
glucose) or Serology testing
(antigenic test)
● 2- Microbiology test
● 3- Hematology test
● 4. Microbiology (better due to
less skin contaminant)
➔ Used to isolate bacteria causing
meningitis
● Neonates- Group B Strep
S.agalactiae & Listeria
monocytogenes - cause
 meningitis in elderly and
immunosuppressed patients
● 1 month- 5 yrs old -
Haemophilus influenzae
● 5-29 yrs old- Neisseria
meningitidis
➔ Rapid testing like Gram staining and
cryptococcal antigen testing (CAT) is
recommended
➔ Collect at least 1 ml and not more
than 10 ml by needle aspiration and
placed in a sterile, screw cup
container or anaerobic transporter
➔ Store at 35 to 37 degrees Celsius for
up to 6 hours, except for viruses,
which can be held at 4 degrees
Celsius for up to 3 days
➔ Cytocentrifuge to collect the
sediment for staining or culture
➔ Use acridine orange if there is no
cytocentrifuge
➔ The most common isolates found in
CSF Neisseria meningitidis,
Streptococcus pneumoniae,
Streptococcus agalactiae,
Escherichia coli, Staphylococcus
aureus, Listeria monocytogenes
➔ Normal cells found in CSF are
monocytes and lymphocytes
➔ Normal CSF is clear and liquid. Clots
indicate traumatic tap- wala nadritso
ug kuha, naay dugo pa. Clot without
traumatic tap indicates Tubercular
meningitis (or Tuberculous
meningitis in google)
➔ Viruses can cause meningitis
(Enteroviruses, Poliovirus
Coxsackie)
➔ Cryptococcus neoformans -
organism that can be mistaken as
lymphocyte in CSF, can cause
fungal meningitis
3. THROAT AND NASOPHARYNGEAL
SPECIMEN
➔ Culture must include anaerobic
conditions for Beta hemolytic
Streptococcus ( some are non
hemolytic unless conditions are
anaerobic)
➔ Culture on Todd-Hewitt broth for
fluorescence microscopy of Beta
hemolytic Streptococcus
➔ a swab that is moistened with
Stuart’s or Amie’s medium Is used
for the collection of specimens.
Nasopharyngeal Swab
a. H. influenza- enriched chocolate
agar with Staphylococcus streak
across the inoculum
b. N. meningitidis- enriched chocolate
c. B. pertussis- Charcoal Cephalexin
Medium
d. MRSA- MSA
4. SPUTUM
➔ Prior to collection, rinse mouth or
gargle with water, instruct to cough
deeply into container
➔ First morning specimen is preferred
for AFB microscopy
➔ Sterile, screw-cap container
➔ Aerosole-induced specimens are
collected by allowing patients to
breathe aerosolized droplets of a
solution containing 15% sodium
chloride and 10% glycerin for
approximately 10 minutes or until a
strong cough reflex is initiated. (For
patients nga dili kacough ug hukaw)
➔ Used to diagnose lower respiratory
tract infections (ex. pneumonia)
➔ A direct Gram stain is performed to
determine the quality of the
specimen. acceptable specimens
 are cultured on SBA, MAC, and
chocolate agars.
➔ A general rule for an acceptable
specimen might be <10 squamous
epithelial cells and >25
Polymorphonuclear neutrophils
PMNs/ low power field
➔ Common significant sputum isolates:
streptococcus pneumoniae,
Klebsiella pneumoniae,
Staphylococcus aureus,
Pseudomonas aeruginosa,
Haemophilus influenzae, Legionella
pneumophila, Mycoplasma
pneumoniae
5. Urine
➔ Sterile, screw-cap container
➔ Plate quantitatively using calibrated
loops (1:1000 or 1:100)
➔ Females: clean area with soap and
water, then rinse with water; hold
labia apart and begin voiding in
commode; after several ml have
passed collect midstream
➔ Male: clean glans with soap and
water, then rinse with water,
retract foreskin; after several ml
have passed, collect midstream
➔ Midstream clean catch- specimen of
choice for urine culture
➔ Catheterized urine- allow first 15ml
to pass
➔ Suprapubic urine- for anaerobic
culture
➔ Escherichia coli- most common
agent for uti in both ambulatory and
hospitalized patients
➔ S. saprophyticus- second most
common; most common cause of
UTI in young, sexually active
females
6. STOOL
➔ If bacterial infection is suspected,
three specimens should be
collected- once a day for three days
if parasites are suspected, three
specimens collected within 10 days
should be sufficient for microscopic
detection of ova and parasites
➔ Clean, leakproof container; transfer
enteric transport medium )Cary blair)
if transport will exceed 1 hour
➔ Protein culture should include
Salmonella, Shigella,
Campylobacter, specify Vibrio,
Aeromonas, Plesiomonas,
Yersinia, Escherichia coli O157:H7 if
needed
➔ Follow-up may include Shiga toxin
assay as recommended by CDC
➔ Culture the stool as soon as possible
or refrigerate specimen
➔ Put rectal swab in enrichment broth
or transport medium( glycerol-saline)
7. GENITAL
➔ For diagnosis of venereal diseases
or STDs
➔ infections caused by T. pallidum, N.
gonorrhoeae, C. trchomatis, G.
vaginalis, T. vaginalis, C. albicans,
and Herpes simplex virus
➔ Specimens: cervical (female,
urethral (male), rectal
➔ The vagina contains normal flora
that changes with age: Lactobacillus
spp. ( during childbearing years),
Staphylococci and Corynebacteria
(earlier and late in life
➔ Molecular techniques are commonly
used for detecting both Neisseria
gonorrhoea and C. trachomatis
8. GASTROINTESTINAL TRACT
➔ Gastric biopsy- rapid urease test
and culture Helicobacter pylori
 -causative agent of gastric ulcer and concentration of 10 cells per 1 ml of
gastric cancer specimen is required.
➔ Gastric aspirate- collected early in
the morning before rising and having
first meal
➔ Sample must be neutralized within 1
hour
➔ Best specimen for infants
➔ Examine for AFB

9. LESIONS/WOUNDS/ABSCESS
➔ Superficial- swab along the outer
edge using swab moistened with
transport media
➔ Deep- aspirate with needle and
syringe and place in an anaerobic
transport system
➔ 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.
➔ Acid-fast, viral, and fungal
specimens are usually batched for
processing at one time.

COLLECTION CONSIDERATIONS

Specimen Processing:
➢ Homogenization( grinding) of tissue
➢ Concentration by centrifugation or
filtration of large volumes of sterile
fluids such as ascites (peritoneal) or
pleural (lung) fluids
➢ Decontamination of specimens,
such as those for Legionella 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.
➢ To visualize bacterial cells by light
microscopy, a minimum