The document discusses guidelines for proper collection, storage, and transport of specimens for microbiological testing. It outlines important competencies including collecting samples appropriately and maintaining confidentiality. Key points covered include using sterile equipment, proper packaging and transport of samples, and factors that could lead to rejection of specimens. Detailed guidance is provided on collecting samples from different body sites like respiratory tract, blood, CSF, and others. Maintaining biosafety during collection and transport is emphasized.

1. Principle of Diagnostic Methods :
Collection, Storage and Transport
of Specimens
UNDER GARADUATE STUDENT’S PRACTICAL ON
BY
GUNJAL PN
ASSIST. PROF.
DEPT OF MICROBIOLOGY
DVVPF’S MEDICAL COLLEGE & HOSPITAL
AHMENDAGAR
7/9/2021 Department of Microbiology 1

2. Competencies
• Following are the competencies for this practical class :
• MI 8.9 – Discuss the appropriate methods of collection of samples
in performance of laboratory tests in detection of microbial agents
causing infectious diseases.
• MI 8.10- Demonstrate appropriate methods of collection of
samples in the performance of laboratory tests in detection of
microbial agents causing infectious diseases.
• MI 8.11 – Demonstrate respect for patient samples sent to
laboratory for performance of laboratory tests in the detection of
microbial agents causing infectious diseases.
• MI 8.12 – Discuss confidentiality pertaining to patient identity in
laboratory results.
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3. Learning Objectives
At the end of the session, the students will be able to
understand:
• Site and type of specimens to be collected for
laboratory diagnosis of microbiological investigations.
• Procedures, preparation, processing and transport of
specimens.
• Procedures, preparation, processing for specimen
storage for laboratory diagnosis.
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4. Introduction
 Specimen collection and transportation are critical
considerations.
 Because any results the laboratory generates is limited by the
quality of the specimen and its condition on arrival in the
laboratory.
 Specimens should be obtained to minimize the possibility of
introducing contaminating microorganisms that are not
involved in the infectious process.
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5. 7/9/2021 Department of Microbiology 5
General guidelines for specimen collection
 Advance planning- Discussion between with microbiologist,
clinician, epidemiologist.
 Collection of Correct, adequate and appropriate specimens
 Sufficient documentation
 Biosafety and decontamination
 Correct packaging
 Rapid transport
 Choice of a laboratory that can accurately perform the tests
 Timely communication of results.

6. An Ideal Request form
A. Name :
B. Age :
C. Sex :
D. IP/ OP No :
E. Time & Date :
F. Ward:
G. Urgent / Routine :
H. Nature of specimen :
I. Investigation needed :
• Doctor/Staff Contact No : 1234567
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7. Use sterile single use equipment.
Disinfect.
Work in a clean, dedicated area.
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Biosafety: protect the patient

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Use personal protective equipment
 Disposable gloves.
 Laboratory coats / gown.
 Mask.
 Protective eyewear / face shields if procedure is
likely to generate aerosols.
If no sharps container: collect sharps immediately to prevent
needle-stick injury.
Have first aid kit readily accessible.
Do not reuse contaminated equipment.
Biosafety: protect yourself

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Biosafety: protect others, the environment
Package samples appropriately for transport.
Decontaminate spills - 10% bleach after wiping the surface clean.
Disinfect working areas for future use - 1% household bleach daily
• Soak contaminated non-disposable equipment/material in 1%
household bleach for 5 minutes.
– wash in soapy water before re-use, sterilize if
necessary
Place waste in leak-proof biohazard bags - ensure safe final
management of waste.
Protect cleaning/decontamination personnel with protective
coat, thick rubber gloves.

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Transport medium
 Allows organisms (pathogens and contaminants) to
survive.
 Non-nutritive - does not allow organisms to
proliferate.
 For bacteria – i.e., Cary Blair.
 For viruses - virus transport media (VTM).

11. Specimen transport
Within 2 hours of collection
Containers should be leak-proof
Separate section for paperwork
Special preservatives or holding media
Biohazard label
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12. Triple packaging system
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• Triple packaging provides three layers of
containment to protect the substances being
shipped.
• These layers are primary, secondary, and outer
containers.
• The following diagram shows the basic concept of
triple packages.

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Primary Container
Secondary Container
Outer Container
Triple packaging system

14. Category A packaging requirements
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15. Criteria for rejection of specimens
• Several criteria can be considered by a laboratory on
the basis of which the processing of a specimen may
not be done by the laboratory.
• Such a decision must be made in light of the specific
requested investigation.
• Laboratory investigations of a sample are a waste of
time and resources if following criteria are not
fulfilled :
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16. • Missing or inadequate identification Insufficient
quantity.
• Specimen collected in an inappropriate container
Contamination suspected.
• Inappropriate transport or storage.
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Criteria for rejection of specimens

17. Containers and swab for the collection of
specimens
• For Faeces:
• Universal container.
• Spoon attached to the inside of the screw
cap.
• For Urine:
• Universal container for small quantities.
• For larger quantities 250 ml wide mouthed
screw-capped bottles are convenient.
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18. • For sputum:
• Universal container should not be
used.
• Squat ,wide-mouthed disposable
containers should be used.
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Containers and swab for the collection of
specimens

19. • For Blood:
• Without anticoagulant for
serological examination.
• With EDTA for parasitological
examination.
• Blood culture bottle:
• This must be at least large enough
to hold 50ml of liquid medium
,with which it is issued from
laboratory, plus 5-10ml of
patient’s blood
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Containers and swab for the collection of
specimens

20. For serous fluids
• Universal container
• Addition of 0.3ml of 20% solution
sodium citrate to the container
prior to autoclaving (with the cap
fitted) is recommended for
collection of fluids that may
coagulate on standing.
• This avoids difficulty in performing
cell counts or centrifuging
procedure with such fluids.
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21. Swabs
• Swabs suitable for taking Specimens of exudates from the throat,
nostril , ear , skin, wounds and other accessible lesions consist of a
sterile absorbent material, usually cotton-wool or synthetic fiber,
mounted on a thin wire of stick.
• Swabs for special purpose:
• Baby swabs
• Pernasal swabs
• Post-nasal swabs
• Laryngeal swabs
• High vaginal and cervical swabs
• Serum coated cotton wool swab
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22. Containers of anaerobic specimens:
• Syringe and needle for aspiration:
• Tube or vial contains semi-solid holding
medium an atmosphere of 5% CO2 ,a reducing
agent, tube used for putting up the swab.
• Readymade swabs in a plastic tube and
containing either Cary-Blair , Amies
transporter prereduced (PRAs) medium.
• Plastic pouch or Bio-bag (transparent)
containing a CO2 generating system, palladium
catalyst and an anaerobic indicator can also be
used.
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23. EYE
• Various specimens collected are:
• A. specimens:
• 1.Conjunctival:
• Container:
• Aerobic swab moistened with Stuart’s or Amie’s
medium.
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24. • Collection:
• Obtained from superior and inferior tarsal conjunctiva.
• Specimen of both eyes with separate swabs by rolling swab over
each conjunctiva.
• If a viral culture is requested ; a second specimen is collected
• For Chlamydia culture swabs are taken with a dry calcium
alginate swab
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25. • Transport :
• Within 24hrs/RT.
• For viral culture place in viral transport media and
deliver promptly to laboratory or refrigerated for
a short time and then transport on wet ice.
• For Chlamydia place in 2-Sp transport medium.
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EYE

26. 2. Corneal scrapings
• Container: Bedside inoculation of BA, CA, SDA, 7H10, Thio.
• Patient preparation: Clinician should instil local anesthetic
before collection.
• Collection: By using heat sterilized platinum spatula or
calcium alginate tipped swab dipped in sterile trypticase soya
broth.
• Transport: Immediately/RT
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27. Anterior chamber and vitreous cultures
• Collection:
• Aspiration is carried out with a tuberculin syringe
• Fitted with a 25-27 gauge needle for the aqueous.
• 20-21 gauge needle for vitreous aspiration.
• Transport: Immediately/RT
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28. EAR
• 1. Inner ear:
• Container: • Sterile , screw-cap tube or anaerobic
transporter.
• Patient preparation:
• Clean ear canal with mild soap solution before
puncture of the ear drum.
• Collection:
• Aspirate material behind drum with syringe if ear
drum is intact; use swab to collect material from
ruptured eardrum.
• Transport:
• Immediately/RT
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29. 2. Outer ear:
• Container:
• Aerobic swab moistened with Stuart's or Amie’s medium
• Patient preparation:
• Wipe away crust with sterile saline.
• Collection:
• Firmly rotate swab in outer canal.
• Transport:
• Within 24hrs/RT
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30. Respiratory tract(RT):
• Collection of specimen in the case of RTI poses a
number of problems because , there is enormous
commensal flora that colonizes this tract.
• Therefore, the specimen collection is very crucial
and specially in case of viral infections of RT.
• One has to avoid contamination of the
specimens.
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31. • RT is broadly divided into:
• A . Upper RT:
• Container:
• Swab moistened with Stuart’s or Amie’s medium.
• Collection:
• 1.Oral swab:
• Remove the oral secretions or debris from the surface of lesion with swab and
discard.
• Using 2nd swab ,vigorously specimen the lesion avoiding any areas of normal
tissue
• 2. Nasal swab:
• Use swab moistened with sterile saline.
• Insert approx. 2cm into nares.
• Rotate swab against nasal mucosa
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Respiratory tract(RT):

32. 3. Nasopharyngeal:
• 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.
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33. 7/9/2021 Department of Microbiology 33

34. Laryngeal swab
• Before use the swab is moistened with sterile D/W.
• Patient is made sit and 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 where it should induce reflex
coughing that will expel sputum onto swab.
• Withdraw the swab and replace it in its tube for delivery
to the laboratory.
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35. Throat swab
• 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.
• Transport: Within 24hrs/RT
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36. B. Lower RT
• Container: Sterile screw-top container.
• Collection: 1.Sputum:
• Patient preparation: Ask patient to brush teeth and then rinse or gargle with
water before collection.
• Collected early in the morning before eating.
• Make collection in a disposable wide mouthed screw-capped sterile plastic
container of about 100ml capacity.
• Instruct to wait until he/she feels material coughed into his/her throat.
• Then work it forward into mouth and spit it directly into container.
• Should be collected before starting antimicrobial chemotherapy.
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37. 7/9/2021 Department of Microbiology 37

38. • 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
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2. Transtracheal aspiration(TTA):

39. 3.Bronchioalveolar lavage (BAL):
• 30-50 ml of physiological saliva is
injected through a fiberoptic
bronchoscope .
• The saliva is then aspirated.
• 4.Gastric lavage:
• In the morning before the patient
has taken anything but after a bout
of coughing and swallowing ,
aspirate the fasting stomach
contents with a Ryle’s tube.
• Transport: Within 24hrs/RT
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40. BODY FLUIDS
• 1.Cerebrospinal fluid:
• Container: Sterile screw-cap tube.
• Patient preparation:
• Disinfect skin before aspirating specimen.
• Collection:
• Lumbar puncture to collect the CSF for examination to be collected by
Physician trained in procedure with aseptic precautions to prevent
introduction of Infection.
• The trained physician will collect only 3-5 ml into a labelled sterile
container.
• The fluid to be collected at the rate of 4-5 drops per second.
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41. • The best site for puncture is inter
space between 3 and 4 lumbar
vertebrae.
• The Physician should wear sterile
gloves and conduct the
procedure with sterile
precautions.
• The site of procedure should be
disinfected and sterile air and
water tight (occlusive) dressing
applied to the puncture site after
the procedure
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42. Transportation to Laboratory:
• The collected specimen of CSF to be dispatched
promptly to Laboratory without any delay.
• Delay may cause death of delicate pathogens,
e.g. Meningococci and disintegrate leukocytes.
• Preservation of CSF:
• It is important when there is delay in
transportation of specimens to Laboratory do not
keep in Refrigerator, which tends to kill H.
Influenzae.
• If delay is anticipated leave specimen at Room
Temperature.
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43. 2.Pleural/Peritoneal/Pericardial/ Synovial fluid:
• Container: Sterile screw-cap tube or
anaerobic transporter
• Patient preparation:
• Disinfect skin before aspirating with 2% iodine
tincture.
• Collection: Obtained via percutaneous needle
aspiration or surgery.
• Transport: Immediately/RT.
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44. BLOOD
• Container: Blood culture media set(aerobic and
anaerobic bottle) or vacutainer tube with SPS
(sodium polyanethol sulfonate) prevents blood from
clotting for 2-4 hrs.
• Patient preparation:
• Disinfect venipuncture site with 70% alcohol and
disinfectant such as betadine.
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45. • Collection:
• Select the vein from which blood is to be drawn.
• Disinfect the venipuncture site.
• Allow it to dry.
• With precautions to avoid touching and recontaminating the
venipuncture site.
• Take the specimen of blood and put it immediately through
the hole in the cap of bottle.
• Volume of blood:
• In adult 5-10ml
• In children 1-5ml
• Transport: Within 2hrs/RT
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46. Gastrointestinal tract(GIT)
• 1.Stool:
• Container:
• Clean leak-proof container. Do not clean with disinfectants.
• Collection:
• Collect sample during active phase.
• Pass stool directly into a sterile, wide-mouth, leak proof container with a
tight fitting lid.
• For parasitological investigation mix with 10% formalin or polyvinyl
chloride, 3 parts stool to 1 part preservative
• Transport: Within 24hrs/4 °C.
• If delay is unavoidable and particularly when the weather is warm collect
the specimens in a container holding 6 ml buffered glycerol saline
transport medium.
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47. Transport media for stool specimens
• Cary-Blair - All enteric organisms.
• Stuart - All enteric organisms.
• Amies - All enteric organisms.
• Buffered All enteric organisms except Vibrios &
glycerol Campylobacter.
saline -
• Alkaline Vibrios
peptone
water -
• V-R fluid - Vibrios
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48. 2.Rectal swab:
• Container:
• Swab placed in enteric transport medium.
• Collection:
• Pass the tip of a sterile swab approximately 1 inch beyond the anal
sphincter.
• Carefully rotate the swab to sample the anal crypts and withdraw the
swab.
• Place the swab in transport medium.
• Transport: • Within 24hrs/4°C.
• Not recommended for Viruses.
• No microscopic examination possible.
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49. Urinary tract infection(UTI)
• 1.Urine:
• Container: Sterile, screw-cap container.
• Patient preparation:
• 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
• Males:
• Clean glans with soap and water, then rinse with water, retract
foreskin; after several ml have passed, collect midstream.
• Collection:
• After several ml have passed, collect midstream in a urine
container.
• Transport: Within 24hrs/4°C
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50. 2.Catheter specimen of urine (CSU)
• Container: Sterile, screw-cap container.
• Patient preparation:
• Clean urethral area (soap and water) and rinse (water).
• Collection:
• Insert catheter into bladder.
• Allow first 15ml to pass.
• Then collect remainder.
• Transport: Within 24hrs/4°C
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51. 3.Suprapubic bladder aspiration
• It is used primarily for neonates and small children
but may be safely used in adults.
• A full bladder is required for this.
• Overlying skin id disinfected.
• Bladder is punctured above the symphysis pubis with
a 22-gauge needle on a syringe.
• About 10ml of urine is aspirated.
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52. Abscess
• Abscess(also lesions, wounds, pustule,
ulcer)
• A . Superficial Abscess:
• Container:
• Anaerobic swab moistened with Stuart’s
or Amie’s medium
• Patient preparation:
• Wipe area with sterile saline or 70%
alcohol.
• Collection: Swab along the leading edge
of wound.
• Transport: Within 24hrs/RT
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53. B . Deep Wound abscess
• Container:
• Anaerobic transporter.
• Patient preparation:
• Wipe area with sterile saline or
70% alcohol.
• Collection:
• Aspirate material from wall or
excise tissue.
• Transport: Within 24hrs/RT
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54. Hair, nails, or skin scrapings (for fungus culture)
• Container: Clean, screw-top tube.
• Patient preparation:
• Nails or skin: wipe with 70% alcohol.
• Collection:
• Hair: Collect hair with intact shaft.
• Nails: Send clippings of affected area.
• Skin: Scrape skin at leading edge of
lesion.
• Transport: Within 24hrs/RT
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55. Genital tract
• A . Females:
1.Cervical swab:
• Container:
• Swab moistened with Stuart’s or
Amie’s medium.
• Patient preparation:
• Remove mucus before collection of
specimen.
• Collection:
• Swab deeply into endocervical canal.
• Transport: Within 24hrs/RT
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56. 2 . High vaginal swab
• Container:
• Swab moistened with Stuart’s or Amie’s medium .
• Patient preparation:
• Remove exudates.
• Collection:
• Swab secretions and mucous membrane of
vagina.
• Transport: Within 24hrs/RT
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57. 3 . Urethral swab
• Container:
• Swab moistened with Stuart’s or Amie’s
medium.
• 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.
• Transport: Within 24hrs/RT
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58. B . Males
• 1.Prostrate:
• Container:
• Swab moistened with Stuart’s or Amie’s medium.
• OR sterile screw-cap tube.
• Patient preparation:
• Clean glans with soap and water.
• Collection:
• Collect secretion on swab or
• In tube
• Transport: Within 24hrs/RT.
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59. 2 . Urethra:
• Container:
• Swab moistened with Stuart’s or Amie’s
medium.
• Collection:
• Insert flexible swab 2-4cm into urethra and
rotate for 2 sec.
• Transport: Within 24hrs/RT for swab
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60. Infection control precautions
Precautions Use Requirements
Contact
precautions
Patients known or suspected to have
serious illnesses easily transmitted
by direct patient contact or by
contact with items in the patient's
environment
•Gloves
•Gown
Droplet
precautions
Barrier to stop infections spread by
large (>5 microns), moist droplets
produced by people when they
cough, sneeze or speak
•Contact precautions
•Well-fitting mask
•Eye protection
Airborne
precautions
Patients known or suspected to have
serious illnesses transmitted by
airborne droplet nuclei
•Contact precautions
•Droplet precautions
• N95 mask
•Isolation room
(In hospital)

61. Expected Questions
• Describe the specimen collection method for
urine specimen collection.
• Describe the specimen collection method for
CSF specimen collection.
• Describe the specimen collection method for
blood specimen collection.
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62. Thank You!
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