SPECIMEN COLLECTION & PROCESSING IN MICROBIOLOGY

FUNDAMENTALS OF SPECIMEN COLLECTON SPECIMEN REQUISITON

1. If possible, collect the specimen in the acute phase of the infection A complete requisition should include the following:
and before antibiotics are administered. 1. The patient’s name
2. Select the correct anatomic site for collection of the specimen. 2. Hospital identification number
3. Collect the specimen using the proper technique and supplies with 3. Age and date of birth
minimal contamination from normal biota. 4. Sex
4. Collect the appropriate quantity of specimen. 5. Collection date and time
5. Package the specimen in a container or transport medium designed 6. Ordering physician
to maintain the viability of the organisms and avoid hazards that result 7. Exact nature and source of the specimen
from leakage. 8. Diagnosis (may be ICD-10-CM code)
6. Label the specimen accurately with the specific anatomic site and the 9. Current antimicrobial therapy
patient information—patient’s name and identification number.
7. Transport the specimen to the laboratory promptly or make provisions SPECIMEN REJECTION
to store the specimen in an environment that will not degrade the Reject specimen if:
suspected organism(s). 1. The information on the label does not match the requisition form
8. Notify the laboratory in advance if unusual pathogens or agents of 2. Specimen is transported at improper temperature
bioterrorism are suspected. 3. The specimen is transported in an improper medium
4. The quantity of specimen is insufficient for testing (QNS= Quantity Not
SPECIMEN LABELS Sufficient)
1. Patient’s name 5. Specimen is leaking
2. Identifying number (hospital or sample number) 6. Unpreserved specimen is transported more than 2 hours from
3. Birth date collection
4. Date and time of collection 7. The specimen is formalin-fixed which can kill microorganisms present
5. Source 8. Specimen received is for anaerobic culture but from the site known to
6. Initials of the individual that collected the sample have anaerobes as normal flora (ex. Mouth, vagina
9. Specimen is dried up
10. Processing a specimen that would produce a result with questionable
medical value (ex. Foley catheter tip)
 Table 1. Collection, Transport, Storage, and Processing of Specimens Commonly Submitted to a Microbiology Laboratory
Specimen Container Patient Preparation Special Transportation Storage Before Primary Plating Direct Comments
Instructions to Laboratory Processing Media Examination
Abscess (also Lesion, Wound, Pustule, Ulcer)
Superficial Aerobic swab Wipe area with Aspirate if possible, < 2 hrs 24 hrs/RT BA, CA, Mac, CNA Gram Add CNA if smear
moistened with sterile saline or swab along leading optiona suggests mixed gram-
Stu- art’s or 70% alcohol edge of wound. positive and gram-
Amie’s medium negative flora
Deep Anaerobic Wipe area with Aspirate material < 2 hrs 24 hrs/RT BA, CA, Mac, CNA Gram Wash any granules and
transporter sterile saline or from wall o optiona “emulsify” in saline
70% alcohol
Blood or Bone Marrow Aspirate
Blood culture Disinfect Draw blood at time Within 2 hrs/RT <2 hrs/RT Must Blood culture Direct Gram Other considerations:
media set venipuncture site of febrile episode; be incubated at bottles may be stain from brucellosis, tularemia,
(aerobic and with 70% alcohol draw two sets from 37°C on receipt used; positive blood cell wall– deficient
culture bottles bacteria, leptospirosis,
anaerobic right and left arms; in laboratory.
BA, CA BBA- of antibiotics or AFB; blood cultures
bottle) or do not draw more anaerobic of when possible should be collected
than three sets in a antibiotics when before administration
Vacutainer tube 24-hr period; draw possible of antibiotics when
with SPS ≥ 20 mL/set possible
(adults) or 1-20
mL/set (pediatric)
depending on
patient’s weight; or
per manufacturer’s
instructions.
Body Fluids
Amniotic, Sterile, screw- Disinfect skin with Needle aspiration < 15 min < 24 hrs/RT May use an Gram (vaginal May need to
abdominal, cap tube or iodine preparation Plate as soon aerobic and fluid is concentrate by
ascites anaerobic before aspirating as received. anaerobic blood recommended) centrifugation or
Incubate blood
(peritoneal), transporter or specimen. culture bottle set filtration – stain and
culture bottles
bile, joint direct at 37°C on for body fluids culture sediment
(synovial), inoculation into
pericardial, blood culture receipt in BA, CA, thio, CNA,
pleural bottles laboratory. Mac (peritoneal)
< 24 hrs/4°C:
Pericardial fluid
BBA, BBE, LKV
and fluids for
fungal cultures anaerobic

Bone Sterile, screw- Disinfect skin Take sample from Immediately/RT Plate as soon BA, CA, Mac, thio Gram May need to
cap container before surgical affected area for as received. homogenize
procedure biopsy
Cerebrospinal Fluid (CSF)
Sterile, screw- Disinfect skin with Consider rapid < 15 min < 24 hrs BA, CA (Routine) Gram—best Add thio for CSF
cap tube iodine or testing (e.g., Gram BA, CA, thio sensitivity by collected from shunt
chlorhexidine stain; cryptococcal Routine (shunt) cytocentrifugati Recommended to also
before aspirating antigen) incubate at on (may also collect blood culture
specimen 37°C, except want to do AO if
for viruses, cytocentrifuge
which can be not available)
held at 4°C for
up to 3 days.
Ear
Inner Sterile, screw- Clean ear canal with Aspirate material < 2 hrs 24 hrs/RT BA, CA, Mac (add Gram Add anaerobic culture
cap tube or mild soap solution. behind drum with thio if prior plates for
anaerobic syringe if ear drum antimicrobial tympanocentesis
transporter intact; use flexible therapy) specimens
shaft swab to
collect material BBA (anaerobic)
from ruptured ear
drum
Outer Aerobic swab Wipe away crust Firmly rotate swab < 2 hrs/ RT 24 hrs/4°C BA, CA, Mac Gram
moistened with with sterile saline in outer canal.
Stuart’s or
Amie’s medium
Eye
Conjunctiva Aerobic swab Sample both eyes; < 2 hrs/ RT 24 hrs/RT BA, CA, Mac Gram, AO, Other considerations:
moistened with use separate swabs histologic stains Chlamydia trachomatis,
Stuart’s or premoistened with (e.g., Giemsa) viruses, and fungi
Amie’s medium sterile saline
Aqueous/ Sterile, screw- Prepare eye for < 15 min/ RT < 24 hrs/RT Set BA, Mac, 7H10, Gram/AO Other considerations:
vitreous fluid cap tube needle aspiration up immediately Ana fungal media; some
on receipt. anaesthetics may be
inhibitory to some
organisms
Corneal Bedside Clinician should < 15 min/RT < 24 hrs/RT BHI 10% Sheep Gram/AO Other considerations:
scrapings inoculation of instill local blood, CA, SDA Acanthamoeba spp.,
BHI 10% anesthetic before Must be with antibiotics The use of 10- herpes simplex virus
collection incubated at mm frosted ring and other viruses,
28°C (SDA) or slides assists Chlamydia trachomatis,
37°C with location of and fungi
(everything specimen
else) on receipt because of the
in laboratory. size of the
specimen
Foreign Bodies
IUD Sterile, screw- Disinfect skin < 15 min/RT Plate as soon Thio
cap container before removal as received
IV catheters, Sterile, screw- Disinfect skin with Do not culture < 15 min/RT Plate as soon BA, Thio,
pins cap container alcohol before Foley catheters; IV as received, if prosthetic valves
removal catheters are possible; store
cultured <2 hrs/4°C
quantitatively by
rolling the segment
back and forth
across agar with
sterile forceps four
times; ≥ 15
colonies are
 associated with
clinical significance.
GI Tract
Gastric Sterile, screw Collect in early AM Most gastric < 15 min/RT < 24 hrs/4°C BA, CA, Mac, HE, Gram/AO Other considerations:
aspirate cap tube before patient eats aspirates are on CNA, EB AFB
or gets out of bed. infants or for AFB Must be
neutralized with
sodium
bicarbonate
within 1 hr of
collection
Gastric biopsy Sterile, screw Rapid urease test < 15 min/RT 24 hrs/4°C Skirrow, BA, BBA H&E stain Other considerations:
cap tube or culture for optional: urea breath test;
(normal saline Helicobacter pylori. immunostaining antigen test (H. pylori)
<2 hrs
transport
medium
recommended)
Rectal swab Swab placed in Insert swab ~1-1.5 < 2 hrs/RT < 24 hrs/RT BA, Mac, XLD HE, Methylene blue Other considerations:
enteric cm past anal Campy, EB for fecal Vibrio, Yersinia
transport sphincter; feces leukocytes enterocolitica,
medium should be visible on Escherichia coli
swab O157:H7, N.
gonorrhoeae, Shigella,
Campylobacter, herpes
simplex virus and
carriage of group B
streptococci
Stool (feces) Clean, leak- Routine culture Within 24 hrs/ 24 hrs/4°C BA, Mac, XLD, HE, Methylene blue See considerations in
routine culture proof container; should include RT in holding Campy, EB; for fecal previous rectal swabs
transfer feces Salmonella, media < 48 hrs/RT or optional: Mac-S; leukocytes;
to enteric Shigella, and 4°C chromogenic agar optional: Shiga Do not perform routine
transport Campylobacter; toxin testing stool cultures for
 medium (Cary- specify Vibrio, Unpreserved < patients whose length
Blair) if Aeromonas, 1 hr/RT of stay in the hospital
transport will Plesiomonas, exceeds 3 days and
exceed 1 hr Yersinia, whose admitting
Escherichia coli diagnosis was not
O157:H7, if needed. diarrhea; these patients
should be tested for
Follow-up may Clostridium difficile
include Shiga toxin
assay as
recommended by
CDC.
O&P O&P Collect three Wait 5-10 days Fresh Indefinitely/RT Liquid specimen
transporters specimens every minimum (up to 2 nonpreserved should be
(e.g., 10% other day at a weeks) if patient liquid examined for
formalin and minimum for has received specimens the presence of
PVA) outpatients; antiparasitic should be motile
hospitalized compounds, examined within organisms
patients (inpatients) barium, iron, 30 minutes of
should have a daily Kaopectate, passage;
specimen collected metronidazole, Milk semiformed
for 3 days; of Magnesia, Pepto- within 1 hour of
specimens from Bismol, or passage.
inpatients tetracycline Specimen in
hospitalized more fixatives,
than 3 days should 24 hrs/RT
be discouraged
Female Genital Tract
Bartholin cyst Anaerobic Disinfect skin with Aspirate fluid; < 2 hrs/RT 24 hrs/RT BA, CA, Mac, TM, Gram
transporter iodine preparation consider chlamydia Ana
before collection. and GC culture.
Cervix Swab Remove mucus Collect 1 hour after < 2 hrs/RT 24 hrs/RT BA, CA, Mac, TM Gram
moistened with before collection of patient’s last
specimen urination. Remove
 Stuart’s or exudate from
Amie’s medium urethral opening.
Cul-de-sac Anaerobic Submit aspirate < 2 hrs/RT 24 hrs/RT BA, CA, Mac, TM, Gram
transporter Ana
Endometrium Anaerobic Surgical biopsy or < 2 hrs/RT 24 hrs/RT BA, CA, Mac, TM, Gram
transporter transcervical Ana
aspirate via
sheathed catheter
Urethra Swab Collect 1 hour after Collect discharge < 2 hrs/RT 24 hrs/RT BA, CA, Mac, TM Gram Other considerations:
moistened with patient’s last by massaging chlamydia,
Stuart’s or urination. Remove urethra against mycoplasma
Amie’s medium exudate from pubic symphysis or
urethral opening. insert flexible swab
2-4 cm into urethra
and rotate swab for
2 seconds; collect
at least 1 hr after
patient has urinated
Vagina Swab Remove exudate. Swab secretions < 2 hrs/RT 24 hrs/RT BA, TM Gram Examine Gram stain for
moistened with and mucous bacterial vaginosis,
Stuart’s or membrane of Culture is not especially white blood
Amie’s medium vagina. recommended for cells, clue cells, gram-
or JEMBEC the diagnosis of positive rods indicative
transport If a smear is also bacterial vaginosis; of Lactobacillus, and
system required, use a inoculate selective curved, gram-negative
second swab. medium for group rods indicative of
B streptococcus Mobiluncus spp.
(LIM broth) if
indicated for
pregnant women
Male Genital Tract
Prostate Swab Clean urethral Collect secretions < 2 hrs/RT for 24 hrs/RT for BA, CA, Mac, TM, Gram
moistened with meatus with soap on swab or in tube swab; swab; plate CNA
 Stuart’s or and water and immediately if in secretions
Amie’s medium massage the tube/RT immediately if
or sterile, prostrate through in tube
screw-cap tube the rectum.
Urethra Swab Insert flexible swab < 2 hrs/RT for 24 hrs/RT for BA, CA, TM Gram Other considerations:
moistened with 2-4 cm into urethra swab; within 2 swab; put chlamydia,
Stuart’s or and rotate for 2 hrs for JEMBEC JEMBEC at mycoplasma
Amie’s medium seconds or collect system 37°C
or JEMBEC discharge on immediately on
transport JEMBEC transport receipt in
system system. laboratory
Hair, Nails, or Skin Scrapings (for fungal culture)
Clean, screw- Nails or skin: wipe Hair: collect hairs Within 72 Indefinitely/RT SDA, IMAcg, CW
top tube with 70% alcohol with intact shaft hrs/RT SDAcg
Nails: send
clippings of
affected area

Skin: scrape skin at

leading edge of
lesion
Lower Respiratory Tract
BAL, BB, BW Sterile, screw- Anaerobic culture < 2 hrs/RT 24 hrs/4°C BA, CA, Mac, CNA Gram and other Other considerations:
top container appropriate only if special stains as quantitative culture for
sheathed requested (e.g., BAL, AFB, Legionella,
(protected) catheter Legionella DFA, Nocardia, Mycoplasma,
used acid-fast stain Pneumocystis,
Cytomegalovirus
Sputum, Sterile, screw- Have patient brush Have patient collect < 2 hrs/RT 24 hrs/4°C BA, CA, Mac, PC, Gram and other Other considerations:
tracheal top container teeth and then rinse from deep cough; OFPBL-cystic special stains as AFB, Nocardia
aspirate or gargle with water specimen should be fibrosis requested (e.g.,
(suction) before collection. examined for Legionella DFA,
suitability for acid-fast stain
 culture by Gram
stain; induced sputa
on pediatric or
uncooperative
patients may be
watery because of
saline nebulization.
Upper Respiratory Tract
Nasopharynx Swab Insert flexible swab < 15 min, RT 24 hrs/RT BA, CA, Other considerations:
Nose moistened with through nose into without chromogenic agar add special media for
Stuart’s or posterior transport media; Corynebacterium
Amie’s medium nasopharynx and < 2 hrs/RT using diphtheriae, pertussis,
rotate for 5 transport media Chlamydia, and
seconds; specimen Mycoplasma
of choice for
Bordetella
pertussis.
Pharynx Swab Swab posterior < 2 hrs/RT 24 hrs/RT BA or SSA Other considerations:
(throat) moistened with pharynx and add special media for
Stuart’s or tonsils; routine C. diphtheriae,
Amie’s medium culture for group A Neisseria gonorrhoeae,
streptococcus (S. and epiglottis
pyogenes) only (Haemophilus
influenzae)
Tissue
Anaerobic Disinfect skin. Do not allow < 15 min/RT 24 hrs/RT BA, CA, Mac, CNA, Gram May need to
transporter or specimen to dry Thio; homogenize
sterile, screw- out; moisten with Anaerobic: BBA,
sterile, distilled
cap tube LKV, BBE
water if not bloody.
Urine
Clean-voided Sterile, screw- Females: Preserved 24 hrs/4°C BA, Mac Optional: Check for Plate quantitatively at
midstream cap container Clean area with within 24 hrs/ chromogenic agar pyuria, Gram 1:1000; consider
(CVS) soap and water, RT plating quantitatively at
 Containers that then rinse with stain not 1:100 if patient is a
include a water; hold labia Unpreserved <2 recommended female of childbearing
variety of apart and begin hrs/RT age with white blood
chemical voiding in cells and possible
urinalysis commode; after acute urethral
preservatives several mL have syndrome
may also be passed, collect
used. midstream.

Males:
clean glans with
soap and water,
then rinse with
water; retract
foreskin; begin
voiding in
commode; after
several mL have
passed, collect
midstream
Straight Sterile, screw- Clean urethral area Insert catheter into < 2 hrs/RT 24 hrs/4°C BA, Mac Gram or check Plate quantitatively at
catheter (in cap container (soap and water) bladder; allow first for pyuria 1:100 and 1:1000
and out) and rinse (water) 15 mL to pass; then Preserved <24
collect remainder. hrs/RT Culture of Foley
catheters is not
recommended.
Suprapubic Sterile, screw- Disinfect skin Needle aspiration Immediately/RT Plate as soon BA, Mac, Ana, Thio Gram or check Plate quantitatively at
aspirate cap container above the as received for pyuria 1:100 and 1:1000
or anaerobic symphysis pubis
transporter through the
abdominal wall into
the full bladder.