samplecollection and transport of sample

SAMPLE COLLECTION AND
TRANSPORT
Dr. Dinesh Kr Jain, MD.,
Assistantprofessor,
Department of Microbiology,
SMS Medical college, Jaipur

General collection criteria
 Completely filled requisition form
 Properly labelled and leakproof container
 Adequate volume of sample
 Specimen must be collected at optimal timing and from actual
infection site
 Avoid contamination with commensal flora
 Instructions for collection of samples should be given in local
language

 Collected before starting antimicrobial chemotherapy
 Proper transportation
 Safety of patient & medical staff

Criteria for rejection of specimens:
• Mismatch identification
• Insufficient quantity
• Specimen collected in an inappropriate container
• Contamination suspected
• Haemolysed/turbid/lipaemic blood sample.

• Inappropriate transportation temperature
• Excessive delay in transportation
• Inappropriate transport medium
• specimen received in a fixative
• dry specimen
• Leakage
• Unacceptable specimen sources

Labeling specimens
 Patient’s name
 Specimen type
 Unique ID number
 Date, time and place of
collection
 Name/ initials of collector

Biosafety: protect medical staff
Use appropriate barrier
precautions: includes
personal protective
equipment
 disposable gloves
 laboratory coats /
gown/mask
 protective eyewear /
face shields

COLLECTION OF VARIOUS SPECIMENS
• BLOOD CULTURE BOTTLE:
• BD BACTEC: includes resin
containing media
• BacT/ALERT: supplemented
with BHI broth containing
activated charcoal particles

ADULT: This must be at least large
enough to hold 50ml of liquid
with 10-20 ml of the patient’s
blood
PEDIATRIC:1-5 ml blood is
taken
Ratio -
patient blood: culture media
1:5

FLUIDS: includes sterile body fluids
(CSF, pleural fluid, peritoneal fluid, asctic fluid,
synovial fluid)
Universal container
1.Without anti-coagulent
2.With anti-coagulent by
Addition of 0.3ml of 20% solution
sodium citrate to the container prior to autoclaving
Volume: 1-5 ml collected under aseptic precautions

For urine:-
Sterile wide mouth container
For sputum:-
Clean, sterile, wide-mouthed
disposable containers
should be used

For faeces:-
Universal container
Spoon attached to the
inside of the screw cap
Volume:1teaspoon(5ml)

Swabs:-
Swabs suitable for taking Specimens
of exudates from the throat, nostril ,
ear , skin, wounds and other accessible
lesions consist of a sterile pledget of
absorbent material, usually cotton-wool
or synthetic fiber, mounted on a thin wire
or stick.

 Cotton swabs: not prefered for culture as
contains fatty acids
 Dacron or rayon polyester: better choice
 Flocked swabs: newer, numerous microscopic
folds

Calcium alginate swabs: for
nasopharygeal
secretions,especially for
chlamydia

Types of swab:
 Baby swab: for small orifices
 Pernasal swab: for nasopharynx
 Post nasal swab: for nasopharyngeal
secretions
 Laryngeal swab: for bronchial
secretion
 High vaginal and cervical swab: for
uterus ,cervix and its lumen

QUALITY ASSURANCE
 Follow standardized operative
procedures for collection of bio-
specimen handling. Samples have to
be made available in all collection
areas wherever required.

Guidelines are issued by national authorities which should be strictly
followed.
• Ideally specimens should be transported to the laboratory as quickly
as possible and processed in the laboratory within 1-2 hours after
collection.
• In case of delay of more than 2 hours the specimen should be
transported in transport media or refrigerated.
TRANSPORT OF SPECIMENS

 The tertiary container usually made of wood or cardboard.
Shock absorbent material is placed between this and the
secondary container.
• The outside of the tertiary container (box) is labelled, Urgent,
Fragile and if infectious should be marked with BIOHAZARD LABEL.
 The secondary container should be durable, waterproof
and made of metal or plastic with a tightly fitting screw
cap.
Method of Transportation
• For hand carried transportation over a short distance, the
specimen should be placed upright in appropriate racks.
• For long distance transportation, packaging is done in three
containers.
 The primary container (test tube, vial) must be fitted with a leak
proof screw-cap, sealed with adhesive tape

samplecollection and transport of sample

Container: Blood culture media set(aerobic and anaerobic bottle)
or vacutainer tube with SPS(sodium polyanethatol sulfonate)
Patient preparation: Choose the vein from which the blood
is to be drawn by touching the skin before it has been disinfected.
Volume of blood:In adult 5-10ml and In children 1-5ml
Transport: Within 2hrs
Storage: if delay, then stored at room temperature for 24 hrs
BLOOD:
Collection:
• Using 70% alcohol, cleanse the skin over
the venipuncture site in a circle
approximately 5 cm in diameter, rubbing
vigorously.
• Allow to air-dry.
• Starting in the center of the circle, apply
2% tincture of iodine (or povidone-iodine)
Insert the needle into the vein and
withdraw the blood.

Abscess (also surgical site Lesion, skin
lesion, Wound, Pustule, Ulcer)
• Superficial-
– Container: Aerobic swab
moistened with normal sterile
saline.
– Collection:
• Wipe area with sterile saline or
70% alcohol.
• Swab along leading edge of
wound.
• Two swabs one for direct
microscopy and one for culture
are taken
– Transport: Within 2 hrs
– Storage: if any delay stored for 24
hrs at room temperature and
transported in Amies transport
media.

Body fluids- Amniotic/ abdominal/ascites(peritoneal)/
bile/synovial/ pericardial/pleural
• Container : Sterile, screw-cap
• Patient preparation: Disinfect skin before aspirating specimen.
• Collection : Aseptically perform percutaneous aspiration with syringe
and needle to obtain pleural, pericardial, peritoneal, or synovial fluid
• Transport : within 15 minutes.

Cerebrospinal fluid
• Container- Sterile, screw-cap
tube
• Patient preparation- Disinfect
skin before aspirating specimen
with antiseptic solution and
alcohol.
• Specimen collection-
– Barrier precaution is
mandatory.
– Best site: L3-L4.
– Trained physician aseptically
collects 3-10 ml fluid at the
rate of 4-5 drops per second.
– in patients with shunt-
aspiration of CSF from the
Ommaya reservoir or by
collection from the ventricular
drain or shunt.

– Three sepaparte tubes
submitted:
• Tube 1- cell counts and
differential stains
• Tube 2- gram’s stain and culture
• Tube 3- protein and glucose
• Transportation and storage:
– Dispatch the specimen to lab
as soon as possible (<15
mins),
– Any delay may cause death of
pathogens (meningococci) and
disintegration of leucocytes.
– Do not refrigerate (kills
H.inflenzae).
– If any delay , best kept at 37˚C.
– Never refrigerated.

EAR
• Outer Ear:
– Container: swab
moistened with normal
sterile saline.
– Patient preparation: Wipe
away crust with sterile
saline.
– Collection: Firmly rotate
swab in outer canal.
– Transport: as soon as
possible.
– Storage : 24hrs.

• Middle Ear :
– Container: Sterile , screw-cap
tube
– Patient preparation:
• be collected by an otolaryngologist,
using sterile equipment.
• external ear should be cleansed
with a mild germicide such as 1 :
1000 aqueous solution of
benzalkonium chloride to reduce
the numbers of contaminating skin
flora before obtaining the
specimen.
– Collection: Aspirate material
behind drum with syringe, if ear
drum is intact; use swab to collect
material from ruptured eardrum.
– Transport: Immediately.

EYE
1.Conjunctival:-
– Container: Sterile swab
moistened with normal saline
– Collection:
• Using a Kimura spatula,
gently scrape superior and
inferior tarsal conjunctiva
• For Chlamydia culture
swabs are taken with a dry
calcium alginate swab.
• Avoid contamination from
skin and eyelid margins.
– Transport : within 2 hrs
• For Chlamydia place in
Sucrose Phosphate (SP)
transport medium

2. Corneal scrapings:
– Collection:
• By using heat sterilized
platinum spatula.
• Bedside inoculation on
BA,CA,SDA,7H10,Thio or
calcium alginate-tipped
swab dipped in sterile
trypticase soya broth
Clinician should instill local
anesthetic before collection
– Transport: Immediately
– Storage : Must be Incubated
on receipt in laboratory

3. Anterior chamber and
vitreous cultures:
– Container: Sterile, screw
cap tube
– Collection:
• Aspiration is carried out
with a tuberculin syringe
fitted with a 25-27 gauge
needle for the aqueous
and 20-21 gauge needle
for vitreous aspiration
– Transport: Immediately

Respiratory tract
• Upper respiratory tract-
1.Oral swab-
– Container: sterile swab
– Collection:
• Remove the oral secretions or
debris from the surface of
lesion with swab and discard
it.
• Using 2nd swab ,take the
sample from the lesion
avoiding any areas of normal
tissue.
– Storage: if any delay stored for
24 hrs at room temperature.

2. Throat swab:
– Container: albumin coated or
charcoal coated or plain cotton wool
swabs should be used.
– Collection
• Depress the tongue with a tongue
depressor .
• Introduce the swab between the
tonsillar pillars and behind the
uvula without touching the lateral
walls of the buccal cavity.
• Swab back and forth across the
posterior pharynx.
• Any exudates or membrane
should be taken for specimen.
– Storage: if any delay stored for
24hrs at room temperature.

3. Laryngeal swab:
– Container: Swab moistened with
sterile distilled water.
– Collection:
• Patient is made to sit .
• Holding the tongue fully
protruded with help of a piece
of gauge.
• Pass the swab back through
the mouth wire mid-line and
downwards over the epiglottis
into larynx to induce reflex
coughing that will expel
sputum onto swab.
• Withdraw the swab and
replace it in its tube for
delivery to the laboratory.
– Transport: within 2 hrs

4. Nasal swab:
– Container:
• swab moistened with
sterile saline.
– Collection:
• Insert approx. 2cm of
into nares.
• Rotate swab against
nasal mucosa.

5. Nasopharyngeal:
– Container: Swab moistened with normal sterile
saline.
– Collection:
A. Swabs:
• To collect nasopharyngeal cells, all mucus is
removed
• Small flexible nasopharyngeal swab is
inserted along the nasal septum to the
posterior pharynx
• Rotate slowly for 5 sec. against the mucosa
several times
B. Aspirate :
• Is collected with a plastic tube attached to 10
ml syringe or suction catheter
C. Washings:
• Is obtained with a rubber suction bulb by
instilling and withdrawing 3-7 ml of sterile
buffer saline.
• Prenasal swab in charcoal transport media
for whooping cough.
– Storage: if any delay stored for 24hrs at room
temperature.

Lower respiratory tract
• Following
samples are
taken:
– Spontaneously
produced or
induced sputum,
– Endotracheal
aspirate,
– Transtracheal/
Translaryngeal
aspiration,
– Brochoalveolar
lavage,
– Gastric lavage,

Container: Sterile wide mouthed screw-top container
Collection:
1.Sputum:
• Ask patient to rinse or gargle with water before collection.
• Early morning sputum samples should be obtained as they
contain pooled over night secretions.
• Instruct patient to inhale deeply 2-3 times, cough up deeply
from the chest and spit in the specimen container by bringing it
close to the mouth of about 100 ml capacity.m
• May be watery because of saline nebulization.
• To prevent contamination ,patient should be instructed to press
the rim of the container under the lower lip to catch the entire
expectoration.
• Tightly screw on the cap of the container.
• Aerosol-induced sputum specimens have been collected from
children as young as 5 years of age or ambulatory patients.

– Storage: if any delay stored for 24hrs at room temperature.
2. Endotracheal aspirate:
– By introducing a catheter through the larynx into the trachea.
– If endotracheal tube is in place or there is tracheostomy,
aspirating tracheal secretions is easy.
– Collected in Lukens trap.

2.Transtracheal aspiration(TTA):
– Obtained by inserting a small plastic catheter into the trachea via
a needle previously inserted through the skin and cricothyroid
membrane
– This technique is rarely used any more.
3.Bronchioalveolar lavage (BAL):
Container: Sterile leak proof screw-top container
Collection:
– 30-50 ml of physiological saline is injected through a fiberoptic
bronchoscope into a lung segment .
– Sailne is then aspirated .
4.Bronchial brush:
Container: Sterile wide mouthed screw-top container
Collection:
– Uses a telescoping double catheter plugged with polyethylene
glycol at the distal end to protect a small bronchial brush as part
of flexible bronchoscopy .
– Transported immediately, if not should be refrigerated.

5. Gastric lavage:
• Limited to
– senile,
– Non-ambulatory patients,
– children younger than 3 years of age (specimen of choice);
– patients who fail to produce sputum by aerosol induction.
• Levine collection tube is inserted through a nostril.
• When the tube has been fully inserted, a syringe is attached to the
end of the tube and filtered distilled water is injected.
• Syringe is then used to withdraw 5 to 10 mL of gastric Secretions.
• Take Morning sample before the patient has taken anything.
• Should be processed within 4 hours.

6. Sinus Aspirates-
– An otolaryngologist will obtain material from maxillary,
frontal, or other sinuses using a syringe aspiration
technique.
– Container : sterile screw-capped container.
– Transport within 2 hours.
– Refrigerated upto 48 hours.
NOTE: unacceptable for smear or culture because this material will
be contaminated with aerobic and anaerobic normal respiratory flora;
sinus washings or aspirates surgically collected are the specimens of
choice.

Bone
• Container : Sterile, screw-cap container.
• Patient preparation : Disinfect skin before
surgical procedure.
• Collection : Take sample from affected area
for biopsy.
• Transport : immediately.

1. Stool
• Container - Clean, leakproof container.
• Collection- Freshly passed stool samples, avoid
specimens from a bed pan & mixed with urine.
• Transport- . Unpreserved sample should be
transported in <1 hr . If it has to be transported
to other laboratory then Cary Blair medium is
used.

2. Rectal Swab
• Advantage
Although its not an ideal method, rectal swab is adopted
for collecting in small children, debilitated patients
and other situations where voided stool sample is not
feasible.
• Drawbacks
-no macroscopic assessment possible
- less material available
- not recommended for viruses
NOTE: Rectal swabs may be more effective than feces for
recovery of certain strains of Shigella & Clostridium difficile
because these organisms are susceptible to cooling and drying.

• Collection- Insert swab , 1-1.5 cm past anal
sphincter; feces should be visible on swab.
• Transport -Within 2 hrs. If any delay in transport, it
has to be transported in Cary Blair medium

3.Gastric Aspirate
• Container: Sterile, screw-cap tube
• Patient preparation: Collect in early morning before
patient eats or gets out of bed.
• Collection:
 Ask the patient to swallow a weighted gelatin capsule
containing a tightly wound length of string, which is left
protruding from the mouth and taped to the cheek- string
test(Entero test).
 After a predetermined period , during which the capsule
reaches the duodenum and dissolves, the string now
covered with duodenal contents is retracted .
• Transport: should be done Immediately

4.Gastric Biopsy
Performed to detect H.pylori to detect its urease
activity.
• Container- Sterile, screw-cap tube (normal saline ,2
hrs transport medium recommended)
• Transport- within 1 hr
• Storage- if any delay, kept for 24 hrs at 4°C
NOTE: Gastric specimens for culture are rarely
obtained and limited to only those few situations for
which diagnosis may not be possible by other
means.

URINE COLLECTION TECHNIQUE
INVASIVE PROCEDURE NON- INVASIVE PROCEDURE
Suprapubic bladder aspiration Mid stream urine
Urinary Catheters Tapping

1.Midstream Urine
• The purpose of midstream is to collect urine that has been
in bladder.
• Withhold urine for 4-6 hours before giving sample or give
early morning sample.
• Container: Universal Sterile, screw-cap container
• Patient preparation:
Females:
Clean area with soap and water, then rinse with water,
hold labia apart and begin voiding ; after first part of urine has
passed, collect midstream.
Males:
Clean glans with soap and water, then rinse with water,
retract foreskin; after first part of urine has passed, collect
midstream

Collection:
after passing urine, collect midstream in a
labelled urine container
Transport: within 2 hours.

Specimen storage:
• must be immediately refrigerated or preserved chemically.
• Bacterial counts in refrigerated (4°C) urine remain constant
for as long as 24 hours.
• Chemical preservation: Urine transport tubes(BD Urine
Culture Kit[Becton Dickinson Vacutainer Kits,Franklin
Lakes,Nj]) containing boric acid, sodium borate, and sodium
formate have been shown to preserve bacteria without
refrigeration for as long as 24 hours.
• Another method(Starplex Scientific,Inc, Cleveland,Tn) uses a
10 mL sterile conical vial containing a boric acid tablet that
maintains organism viability for up to 72 hours.
• Both boric acid products preserve bacterial viability in urine
in the absence of antibiotics.

2.TAPPING
• A non invasive method of stimulating urine flow
in a baby
• Tapping just above the pubis with two fingers at 1
hour after a feed: 1 tap/second is given for 1 min,
an interval of 1 min is allowed, then tapping is
resumed in this cycle.
• The method was described by Broomhall et al
(1985) and has been favourably appraised by
Taylor et al (1986)

Other voided urine specimen
• For urethritis- Initial portion of voided urine is of
interest. A swab may be inserted into distal
urethra to collect the specimen.
• Primary application is in diagnosing urethritis
caused by N.gonorrhoeae and Chlamydia
trachomatis.
• For diagnosis of prostatitis, prostatic secretions
are specimen of choice.

2.Straight Catheter(in and
out)
• Container: Sterile, screw-cap container
• Transport: Within 2 hours
• Patient preparation- Clean urethral area
(soap and water) and rinse (water)
• Collection- Insert catheter into bladder;
allow first 15 mL to pass; then collect
remainder.

Indwelling Catheters(e.g. foley catheter)
• Restricted to patients who are unable to produce adequate midstream
sample.
• Urine should be collected using aseptic technique.
• The catheter tubing should be clamped off above the port to allow the
collection of freshly voided urine.
• The catheter port or wall of the tubing should then be cleaned
vigorously with 70% ethanol, and urine aspirated via a needle and
syringe.
• Sample should not be obtained from catheter bags.
• Foley catheter tips are unsuitable for culture because they are invariably
contaminated with urethral or colonizing organisms.

3.Suprapubic Bladder Aspiration
It is used primarily for neonates and small children but may be safely
used in adults who are
 Comatose
 with urine retention
 urethral trauma/stricture
 periurethral infection or phimosis.
A full bladder is required for this.
• Patient preparation-Overlying skin is disinfected
• Collection- Bladder is punctured above the symphysis pubis with
a 22-gauge needle on a syringe and about 10ml of urine is
aspirated
• Transport-should be done immediately

FEMALES
1. Cervical swab:
• Container: Swab moistened with normal sterile saline
• Patient preparation: Remove mucus before collection
of specimen
• Collection: Swab deeply into endocervical canal
• Transport: Within 1-2 hours. If any delay then latest
upto 24 hours.

2 . High vaginal swab:
• Container: Swab moistened with normal saline
• Patient preparation: Remove exudates
• Collection: Swab secretions and mucous
membrane of vagina

3 . Urethral swab:
• Container: Swab moistened with normal sterile saline
• Patient preparation: Remove exudates from urethral opening
• Collection:
 Collect discharge by massaging urethra against pubic symphysis or
 insert flexible swab 2-4cm into urethra and rotate swab for 2 sec.
 Collect at least 1 hr after patient has urinated.

Males
1.Prostrate:
• Container: Sterile screw-cap tube or Swab
moistened with normal sterile saline
• Patient preparation: Clean glans with soap and
water
• Transport: Within 1-2 hours if in swab or
Immediately if in tubes

2 . Urethra:
• Container: Swab moistened with normal sterile
saline
• Collection: Insert flexible swab 2-4cm into urethra
and rotate for 2 sec. Or collect discharge on
JEMBEC transport system
• Transport: Within 24hrs for swab or within 2hrs
for JEMBEC transport system.

Intra Uterine Device
• Container- Sterile, screw-cap container
• Patient preparation- Disinfect skin before
removal.
• Transport- within 15 min

IV catheters, tip of shunts, pins)
• Container- Sterile, screw-cap container
• Patient preparation- Disinfect skin with alcohol before
removal
• Transport- within 15 min and should be processed in <2
hours.
NOTE: Do not culture Foley catheters; IV catheters are
cultured quantitatively by rolling the segment back and forth
across agar with sterile forceps four times; >15 colonies are
associated with clinical significance.

samplecollection and transport of sample

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