COLLECTION AND TRANSPORT OF SPECIMENS

JITENDRA KUMAR PANDEYMGM medical college ,mumbaiPG,MEDICAL MICROBIOLOGY 3yr

INTORDUCTION: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.

General guidelines for specimen collection:Depending on the type of infection

e.g. bloodAseptic precautionsAnatomic sites and locationsAdequate amount/volumeTissue or other body fluids should be

preferred over swabs, to get quality material

Proper timingClinical laboratory form

An Ideal Request form Name xxxx Age Sex IP/ OP No xyz Time Date Ward xx123 Urgent /

Routine Nature of specimen Investigation needed Doctor/Staff Contact No

1234567

Use of transport mediaProper handling ,labelling and

transportation

Use of proper containerInstruction to the patientBefore the administration of antibioticsAvoid contamination of specimens

SPECIMEN TRANSPORT:Within 2 hours of collection Containers should be leak-proofSeparate section for paperwork Special preservatives or holding

mediaBiohazard label

Triple packaging system

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 :

     Missing or inadequate identification      Insufficient quantity      Specimen collected in an inappropriate

container      Contamination suspected      Inappropriate transport or storage      Unknown time delay      Haemolysed blood sample.

Containers and swab for the collection of specimens:Containers:

For faeces:-• Universal container• Spoon attached to the inside of the screw cap

For urine:-Universal container for small quantitiesFor larger quantities 250 ml wide

mouthed screw-capped bottles are convenientFor sputum:-• Universal container should

not be used• Squat ,wide-mouthed disposable containers should be used

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

For serous fluids:-• Universal container • Addition of 0.3ml of 20% solution

sodium citrate to the container priorto autoclaving (with the cap fitted) is recommended for collection of fluids that may coagulate on standing

• This avoids difficulty in performingcell counts or centrifuging procedure with such fluids

Swabs:-Swabs suitable for taking Specimensof exudates from the throat, nostril , ear , skin, wounds and other accessiblelesions consist of a sterile pledget of 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

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 or jacket and containing either Cary-Blair , Amies transporter pre-reduced (PRAs) medium id used.

Plastic pouch or Bio-bag (transparent) containing a CO2 generating system, palladium catalyst and an anaerobic indicator can also be used.

EYE:Various specimens collected are:A. specimens:1.Conjunctival:- Container: • Aerobic swab moistened with Stuart’s or Amie’s medium 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

Transport :• Within 24hrs/RT• For viral culture place in viral transport media and deliver

promptly to laboratory or refrigeratedfor a short time and then transport on wet ice

• For Chlamydia place in 2-Sp transport medium

2. Corneal scrapings: Container:

Bedside inoculation of BA,CA,SDA,7H10,Thio Patient preparation:

Clinician should instill 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

3. 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 aspirationTransport:Immediately/RT

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

2. Outer ear:Container:• Aerobic swab moistened with Stuart's or Amie’s mediumPatient preparation:• Wipe away crust with sterile salineCollection:• Firmly rotate swab in outer canalTransport:• Within 24hrs/RT

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.

RT is broadly divided into:

A . Upper RT:Container:• Swab moistened with Stuart’s or Amie’s mediumCollection: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

tissue2. Nasal swab:• Use swab moistened with sterile saline.• Insert approx. 2cm into nares• Rotate swab against nasal mucosa

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 timesB. Aspirate :• Is collected with a plastic tube attached to 10

ml syringe or suction catheterC. Washings: • Is obtained with a rubber suction bulb by

instilling and withdrawing 3-7 ml of sterile buffer saline

4.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

5. Throat swab:• 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

B. Lower RT: Container:• Sterile screw-top container Collection: 1.Sputum: Patient preparation:Ask patient to brush teeth and then rinse or gargle withwater 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

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):• 30-50 ml of physiological saliva

is injected through a fiberoptic bronchoscope .

• the saliva is then aspirated 4.Bronchial brush:• Is collected via a protected

catheter bronchial brush as part of a bronchoscopy examination

5.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

BODY FLUIDS:1.Cerebrospinal fluid:Container: • Sterile screw-cap tubePatient preparation:• Disinfect skin before aspirating specimenCollection: • 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 labeled sterile container

• The fluid to be collected at the rate

of 4-5 drops per second.

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 occlusive dressing applied to the puncture site after the procedure.

Transportation to Laboratory:

The collected specimenof CSF to be dispatched promptly to Laboratory , 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. Influenza If delay is anticipated leave at Room

Temperature.

2.Pleural/Peritoneal/Pericardial/ Synovial fluid:

Container:• Sterile screw-cap tube or anaerobic

transporter Patient preparation:• Disinfect skin before aspirating with 2%

iodine tinctureCollection:• Obtained via percutaneous needle aspiration

or surgeryTransport:• Immediately/RT

BLOOD:Container:• Blood culture media set(aerobic and

anaerobic bottle)or vacutainer tube with SPS

Patient preparation:• Disinfect venipuncture site with 70% alcohol and disinfectant such as betadine

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 bottleVolume of blood:• In adult 5-10ml• In children 1-5mlTransport:• Within 2hrs/RT

Gastrointestinal tract(GIT):1.Stool: Container:• Clean leak-proof container Collection:•Pass stool directly into a sterile, wide-mouth, leak proof container with a tight fitting lid.•Pass stool into a clean, dry bedpan, and transfer into a sterile leak proof container with a tight fitting lid.

Stool for ova and parasites should be placed in preservative immediately after collection.

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

Cary-Blair All enteric organismsStuart All enteric organismsAmies All enteric organismsBuffered glycerol saline

All enteric organisms except VibriosCampylobacter

Alkaline peptone water

Vibrios

V-R fluid Vibrios

Transport media for stool specimens

2.Rectal swab:Container:• Swab placed in enteric transport

mediumCollection: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

3.Duodenal aspirates: Container:• Sterile, screw-cap tube Patient preparation:• Collect in early AM 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

• After a predetermined period , during which the capsule reaches the duodenum and dissolves, the string now covered with duodenal contents is retracted .

Transport:• immediately/RT

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 containerTransport:• Within 24hrs/4°C

2.Catheter specimen of urine (CSU):

Container:• Sterile, screw-cap containerPatient preparation:• Clean urethral area (soap and water) and rinse

(water)

Collection: • Insert catheter into bladder • Allow first 15ml to pass• Then collect remainderTransport:• Within 24hrs/4°C

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

HAIR,NAILS, OR SKIN SCRAPINGS

(FOR FUNGUS CULTURE)Container:• Clean, screw-top tubePatient preparation:• Nails or skin: wipe with 70% alcoholCollection:• Hair: Collect hair with intact shaft• Nails: Send clippings of affected area • Skin: Scrape skin at leading edge of lesionTransport:• Within 24hrs/RT

ABSCESS:(also lesions, wounds, pustule, ulcer)A . Superficial: 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

B . Deep: 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

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

2 . High vaginal swab:Container:• Swab moistened with Stuart’s or Amie’s

medium• Or JEMBEC transport systemPatient preparation:• Remove exudatesCollection:• Swab secretions and mucous membrane of

vaginaTransport:• Within 24hrs/RT

3 . Urethral swab:Container:• Swab moistened with Stuart’s or Amie’s mediumPatient 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 urinatedTransport:• Within 24hrs/RT

B . Males:1.Prostrate:Container:• Swab moistened with Stuart’s or Amie’s medium• Or sterile screw-cap tubePatient preparation:• Clean glans with soap and waterCollection:• Collect secretion on swab or • In tubeTransport:• Within 24hrs/RT for swabs• Immediately/RT if in tubes

2 . Urethra:Container:• Swab moistened with Stuart’s or Amie’s

medium• Or JEMBEC transport systemCollection:• Insert flexible swab 2-4cm into urethra and

rotate for 2 sec.• Or collect discharge on JEMBEC transport

systemTransport:• Within 24hrs/RT for swab• Within 2hrs for JEMBEC transport system

REFRENCES: Specimen Collection In Clinical Microbiology Dr. V. L.Malhotra, Dr. Neelam Khandpur Bailey And Scott's Diagnostic Microbiology

(12th Edition) Koneman’s Colour Atlas Of Diagnostic

Microbiology (6th Edition) Textbook Of Microbiology- Ananthanarayan

And Paniker’s(8th Edition) Mackie And McCartney Practical

Microbiology(14th Edition) Internet

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