PRINCIPLE OF

SPECIMEN
COLLECTION
AND
HANDLING

dr. Titik Nuryastuti, M.Si, SpMK(K), Ph.D

Department of Microbiology, Fac of Medicine, UGM
 Learning Objectives

Able to understand :
 Procedures, preparation, processing of specimens
collection
 Procedures, preparation, processing and transport
of specimens for microbiology examination
Step of Microbiology Test

Reporting Patient/Client Prep

Sample Collection
Personnel Competency
Test Evaluations
•Data & Laboratory
Management
•Safety
•Customer Service
Sample Receipt and
Accessioning

Record Keeping

Sample Transport
Quality Control
Testing
 PATHOGEN
IDENTIFICATION
Bacteria, Virus, Fungi

Gram Biochemical
Antibiotic
Specimen Culture Gram susceptibility
staining assay
assay
 Specimen collection:
key issues

o Consider differential diagnoses

o Decide on test(s) to be conducted
o Decide on clinical samples to be collected to
conduct these tests
 consultation between microbiologist, clinicians and
epidemiologist
 CLINICAL MICROBIOLOGY
EXAMINATION

Location  representative site:

where infection takes
place

 adequate volume
Amount  minimally contaminated

Timing  before antibiotic

administered 3 days after baru boleh
 Clinical specimen

garbage in garbage out

6
 Timing of specimen collection

 Onset of infection
 Before antibiotic administered
( 3 days free of ab)

(Health Protection Agency (2009). Investigation of throat swabs. National

Standard Method BSOP 9)
 Successful laboratory
investigations

Advance planning
Collection of 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
 Introduction

 Management specimen for microbiology lab:

◦ collection
◦ obtaining
◦ Storage
◦ labelling
◦ transport

 Aim:
◦ Avoid contamination ,
◦ Keep m.o survive
 Labelling
skipping labelling may mix up specimen

Patient identity :
 Name
 Age
 Gender
 Ward
 Type of assay
 Time of spesimen collection
Container label
 Criteria for rejecting
samples
o Mismatch of information on the label and the request
o Inappropriate transport temperature
o Excessive delay in transportation
o Inappropriate of transport medium
1. specimen received in a fixative
2. dry specimen
3. sample with questionable relevance: DD vs type of assay
o Insufficient quantity
o Leakage
 Management speciment
Considering safety of health care
worker and lab technician:
PPE : personal protective
equipment (lab coat,
gloves,mask, etc) according to
the speciment

Close the container lid and

avoid from leakage
Considering patient comfortness and
safety:

Informed consent before taking speciment,

communicate effectively

Appropriate time and site of sampling avoid

resampling
3. Adequat number of live suspected m.o
Appropriate speciment
Appropriate time sampling
Adequat vol
Appropriate storage and transportation
Appropriate assay
Appropriate transport Media
Before AB administration
Anaerob exam : don’t expose to O2
4. Avoid contamination from normal flora and
environment microbe (skin, upper respiratory
tr, urogenital tr, gastrointestinal tr. etc)

Aseptic technique
Sterile and tightly close container

5. Communicate : clinician and clin. microbiologist

.... Fulfill exam. form
 SPECIMEN
 Blood
 Urine
 Sputum: expectorated, induced
 Pharyng swab, nasal swab
 Gastrointestinal : gastric lavage, faeces, rectal
swab
 Wound, abcess, tissue biopsy
 LCS (Liquor Cerebrospinal)
Method :
 Swab, Scraping, aspiration, puncture, lavage/irigation,
endoscopy, biopsy
 Container

Transport medium

Transport for Blood

Urine Container Sputum Extractor

 Specimens Selection and
Collection
 Avoid contamination from indigenous flora
 Select the correct anatomic site from which to obtain the
specimen
 Collect the specimen with appropriates supplies.
 Pay attention in case of anaerobic microbes. Chose the biopsy
of needle aspiration rather than swab. Never refrigerates, but
RT
 Collect adequate volumes
 Place in container to promote the survival
 Label: Name, ID number, source, specific site, date, and time,
give initial of the collector.
Blood specimen collection

• Aseptic technique  apply

antiseptic before sample taken
(alcohol 30 seconds, povidone
iodine 2 min)
• During fever (suggested)
• Closed system  minimize
contamination of skin flora
• 8-10 ml adult
• 1-2 ml pediatric
• Room temperature
 Blood speciment

 2 sites (left and right arm, at the same

time)
 Special case: Endocarditis
 3 sites during 24 h

 Aim : To confirm pathogen or skin

contaminant
 Cerebrospinal fluid (CSF)

Collection
o Lumbar puncture
o Sterile tubes
Aseptic conditions
Trained person
 CSF
2 hours transport

Handling and transportation

Bacteria
 Not suitable using transport medium
 immeddiately to inoculate
 transport at ambient temperature

Viruses
 transport at 4-8oC (if up to 48hrs or -70oC for
longer duration)
 Ulcus swab speciment
•Irigation as much as possible, discard necrotic tissue,
minimize contaminant flora by using skin antiseptic
• The specimen should be collected from the advancing
margin of the lesion, the bottom of the wound, or the
abscess wall : NOT taking pus for culturing (pus will
be “steril”)

•Keep in 4o C
 Pharing swab

• Represent speciment of upper resp tract

• Put in transport medium
 Nasopharyngeal swab

 Tilt head backwards

 Insert flexible fine-shafted
polyester swab into nostril
and back to nasopharynx
 Leave in place a few
seconds
 Withdraw slowly; rotating
motion
 Mostly for viral
examination WHO/CDS/EPR/ARO/2006.1
 Nasal swab
 Prepare sterile cotton bud and
dip it into sterile NaCl (0.9%
saline)
 Insert it to a 1cm of nasal cavity
 Swab mucose of nasal septum
 Keep the specimen into
transport medium
 To investigate carier state of
resistant microbe: MRSA
(methicilline resistant Staph
aureus)
 SPUTUM SPESIMENT
(Lower Resp tract)
Quality : sputum
 sputum or saliva..?
 Gastric washing: swallowed sputum, useful for
children
Good quality
 Presence of pmn
 Less epitheliel cell :
 Morning sputum is preferable
 Urine collection

 Semi quantitative assay: number of bacteria/ml urine

 Sampel collection:
◦ Mid stream urine ( Clean catch urine)
◦ Supra pubic puncture – most ideal, mainly for
anaerob culture, but invasive method
◦ Urine catheter. NOT from catheter bag
 Transport :
◦ maximal 2 h after obtained
◦ 2-24 h : keep in refridge 4C
◦ More than 24h: reject
Urine collection
 Stool samples
Collection:
Freshly passed stool samples
 avoid specimens from a bed pan
Use sterile or clean container
 do not clean with disinfectant
During an outbreak - collect from 10-20 patients
 Rectal swabs
Advantage
 convenient
 adapted to small children, debilitated patients
and other situations where voided stool sample
not feasible
Drawbacks
 no macroscopic assessment possible
 less material available
 not recommended for viruses
 Speciment for fungi
examination
 Clean skin/nail using
steril saline
 Scrap skin lesion, not
only by skin swab
 Cut nail lesion as far as
possible
 Collect in sterile petri
dish
 Transport in room temp
 Transport of specimen

 Ideally speciment examined in the lab

SOON
 If delayed: keep in refridge (4C). Except
Blood and other blody fluid
 Transport media can be used for some of
them (maks 2x24 h, in room temperature)
 Transport medium

 Allows organisms (pathogens and contaminants) to

survive
 Non-nutritive - does not allow organisms to
proliferate
 For bacteria – i.e., Carry Blair, Stuart, Amies, BGS,
Alcalie pepton
 For viruses - virus transport media (VTM)
 Transport Media
Stuart’s
 Originally formulated for transport of Neisseria
gonorrhoeae.
 Used charcoal-impregnated swabs which caused
difficulty in Gram stain interpretation.
 Non-nutritive” medium.
Good for most specimens.
 Some gram-negative rods can utilize
glycerophosphate in the medium, thus overgrowing
the culture
 Amies medium

 Modified Stuart’s medium.

 Replaced glycerophosphate with a balanced
salt solution.
 Retained charcoal but incorporated it into the
medium rather than the swab.
 Better transport system for most specimens.
 Cary & Blair
 Similar to Stuart’s but modified for fecal specimens.
 pH increased from 7.4 to 8.4.
 Removed charcoal from formula.
 Good for stool specimens.
 Recommended for fecal specimens suspicious for
Campylobacter sp (food borne disease) and for
other enteric pathogens.

Alkaline Peptone Water

• Specific for Vibrio cholera due to very high pH (pH9)
 Specimen Refrigeration

CAN be refrigerated before inoculation into the media:

 Urine, Respiratory Exudates
 Stools/Feces, Wounds swab

CANNOT be refrigerated before inoculation :

 Spinal Fluids (LCS) and Other Body Fluids
 Genital/Cervical for Gonococcus Isolation
 Blood
Jenis Spesimen laboratorium (WAJIB)

• Upper Resp : nasopharyngeal AND oropharyngeal swabs

• Lower respiratory (sputum, if possible)) for those patients with
productive coughs.
• Induction of sputum is not indicated
• Blood sampel (2x, acut phase/first week, and convalescence
phase/week 3-4
Lower respiratory tract

Bronchoalveolar lavage, tracheal aspirate

• Collect 2-3 mL into a sterile, leak-proof, screw-cap sputum collection
cup or sterile dry container.
• Refrigerate specimen at 2-8°C and ship overnight on ice pack.
Sputum
• Have the patient rinse the mouth with water and then expectorate
deep cough sputum directly into a sterile, leak-proof, screw-cap
sputum collection cup or sterile dry container. Refrigerate specimen
at 2-8°C and ship overnight on ice pack.
Upper respiratory tract
Nasopharyngeal swab AND oropharyngeal
swab (NP/OP swab) MANDATORY
• Use only synthetic fiber swabs with
plastic shafts.
• Do not use calcium alginate swabs or
swabs with wooden shafts, as they may
contain substances that inactivate some
viruses and inhibit PCR testing.
• Place swabs immediately into sterile
tubes containing 2-3 ml of viral
transport media.
• NP and OP specimens should be kept in
the same vials.
• Refrigerate specimen at 2-8°C and ship
overnight on ice pack.
Nasopharyngeal swab:
• Insert a swab into the nostril parallel to the
palate.
• Leave the swab in place for a few seconds to
absorb secretions.
• Swab both nasopharyngeal areas with the
same swab.
Oropharyngeal swab (e.g., throat swab):
• Swab the posterior pharynx, avoiding the
tongue.
Nasopharyngeal wash/aspirate or nasal aspirate
• Collect 2-3 mL into a sterile, leak-proof,
screw-cap sputum collection cup or sterile
dry container. Refrigerate specimen at 2-8°C
and ship overnight on ice pack.
Nasopharyngeal wash/aspirate or nasal aspirate
• Collect 2-3 mL into a sterile,
leak-proof, screw-cap sputum
collection cup or sterile dry
container.
• Refrigerate specimen at 2-
8°C & send to lab on ice
pack.
Serum
Minimum volume required:
• Children and adults: Collect 1 tube (3-5 mL) of
whole blood in a serum separator tube.
• Infant: A minimum of 1 mL of whole blood is
needed
• Serum separator tubes should be stored
upright for at least 30 minutes, and then
centrifuged at 1000–1300 relative centrifugal
force (RCF) for 10 minutes before removing the
serum and placing it in a separate sterile tube
for shipping (such as a cryovial).
• Refrigerate the serum specimen at 2-8°C and
ship overnight on ice-pack.
Collect appropriate samples

• Lower respiratory tract samples are preferred because the lower

respiratory tract is the primary site of infection.
• Respiratory samples - Combined nasopharyngeal and oropharyngeal
swab in ambulatory patients and sputum (if produced) and/or
tracheal aspirate or bronchoalveolar lavage in patients with more
severe respiratory disease.
• Serum for serological testing - acute and convalescent samples may
be submitted in addition to respiratory samples
• Use universal/viral transport medium for swabs; sterile container for
sputum and aspirates; clotted blood container for serum
• A single negative test result, especially if from upper respiratory tract
specimen, does not exclude infection. Repeat sampling and testing of
lower respiratory tract samples is recommended for case with severe
disease or in whom 2019-nCoV is strongly suspected.
 Persiapan Pengambilan Spesimen
2019-nCoV
1. Dokumen : Formulir Form 2019-nCoV
2. Peralatan Pelindung diri (APD)
3. Peralatan Pengiriman Spesimen Saluran
Pernafasan
a. Virus Transport Media (VTM)
Dapat digunakan dengan beberapa merk komersil yang sudah
siap pakai atau dengan mencampur beberapa bahan (Hanks
BBS; Antifungal dan Antibiotik dengan komposisi tertentu)
untuk disatukan dalam 1 wadah steril.
b. Swab Dacron atau Flocked Swab
c. Tongue Spatel
d. Kontainer Steril untuk Sputum
e. Parafilm
f. Plastik Klip
g. Marker atau Label
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 VTM (Viral Transport Medium)
• Steril
• Larutan Isotonik
• Mengandung Protein
• Mengandung Antibiotik
• Buffer pH
• Indikator (Phenol Red)*
Contoh VTM :
Hanks Balanced Salt Solution (HBSS), Virocult, MEM, UTM, dll
(Tersedia komersial)
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 Perhitungan jumlah spesimen
1 orang pasien dalam pengawasan diambil
spesimen :
1 spesimen Orofaring
Minimal 2 X
1 spesimen Sputum
pengambilan
1 spesimen serum

Jumlah total sampel minimal

untuk 1 pasien = 5 - 6
spesimen pengambilan
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 • Pastikan Anda mengenakan sepasang sarung
tangan dan respirator N95 yang sesuai
• Buka swab steril di bagian poros plastik
• Minta pasien untuk memiringkan kepalanya ke
belakang. Perkirakan jarak dari hidung pasien
ke telinga untuk menguur kedalaman dari
insersi gagang swab
• Masukkan swab dengan lembut ke lubang
hidung dan arahkan ke belakang (tidak ke atas)
ke arah nasofaring sampai sedikit terasa ada
tahanan
• Putar swab 2-3 kali dan tahan di tempatnya
selama 2-3 detik
• Tarik perlahan dan segera masukkan ke dalam
tabung UTM
• Patahkan tangkai swab di bagian poros plastik
supaya tabung UTM bisa ditutup

Pengambilan swab nasofaring

 • Tetap menggunakan sarung tangan yang sama, dan
memegang VTM dengan usap nasofaring, ambil
Pengumpulan swab orofaring swab kedua yang dikerutkan dan buka di poros
plastik
• Minta pasien untuk memiringkan kepala ke
belakang dan membuka mulut lebar-lebar
• Pegang lidah ke bawah dengan penekan lidah
• Minta pasien mengatakan "aahh" untuk
meninggikan uvula
• Usap setiap tonsil terlebih dahulu, kemudian faring
posterior dalam Gerakan "angka 8"
• Hindari mengusap langit-langit lunak dan jangan
menyentuh lidah dengan ujung swab
• Tempatkan swab ke dalam tabung UTM yang sama
dengan swab nasofaring
• Tutup tabung dengan rapat dan tempatkan di
plastik Ziploc
• Lepaskan sarung tangan dan masker N95
• Cuci tangan dengan sabun dan air
 PAN AMERICAN HEALTH ORGANIZATION, Regional Office of WHO

Nasopharyngeal and Oropharyngeal Swabs https://www.paho.org/hq/index.php?option=com_

content&view=article&id=7918:2012-videos-sample-collection&Itemid=40295&lang=en

Specimen Packaging : https://www.paho.org/hq/index.php?option=com_content&view=

article&id=7919:2012-videos-packaging&Itemid=40295&lang=en

Personal Protective Equipment: https://www.paho.org/hq/index.php?option=com_content&view=

article&id=7917:2012-videos-proper-use-ppe&Itemid=40295&lang=en
 Waktu Pengambilan Spesimen
• Waktu pengambilan serum
• Pertama kali saat pasien dinyatakan suspek
• Darah ke 2, diambil 10-14 hari kmdn/ menjelang
pasien dipulangkan (<10 hr).
• Waktu pengambilan spesimen saluran nafas:
• Diambil pada hari 1, 2, 3, setelah pasien dinyatakan
suspek dan dirawat di RS
• Spesimen diambil lagi:.
• Pada hari ke 5, 7 dst hingga pemeriksaan PCR
negatif
• Bila kondisi pasien memburuk
Pengiriman spesimen

• Dalam kota: Dilakukan oleh petugas kesehatan setempat dengan

melengkapi formulir yang dipersyaratkan, dalam 1x24jam
• Luar kota: Kurir door to door
• Port to port: harus melibatkan petugas KKP
• Pemeriksaan dilakukan di Laboratorium Pusat Penyakit Infeksi,
Balitbangkes, Jakarta Pusat
 PENGEPAKAN DAN PENGIRIMAN
SPESIMEN
• Spesimen pasien harus dilakukan tatalaksana
sebagai UN3373, "Substansi Biologis, Kategori B",
ketika akan diangkut/ditransportasikan dengan
tujuan diagnostik atau investigasi.
• Semua spesimen harus dikemas untuk mencegah
kerusakan dan tumpahan.Adapun system yang
digunakan adalah dengan menggunakan tiga lapis
(Three Layer Pacakging) sesuai dengan pedoman
dari WHO dan International Air Transport
Association (IATA).

62
PENGEPAKAN/PENGIRIMAN
1. Masukkan cryo tube berisi serum ke dalam plastik
klip per Spesimen Jika menggunakan Pot maka
setiap pot dapat diisi beberapa cryo tube
2. Demikian juga untuk Usap Tenggorok harus dikirim
dalam plastik klip secara terpisah (per
pasien/spesimen)

63
 PENGEPAKAN/PENGIRIMAN
4. Masukkan seluruh spesimen dimasukkan ke dalam
cool box yang berisi Ice pack yang terlebih dahulu
dibekukan. Suhu pengiriman dijaga 2-8°C
5. Ice packs sebaiknya ditempatkan pada sisi kiri-
kanan (ditambahkan juga bagian atas-bawah jika
memungkinkan).
6. Harus dapat dipastikan bahwa spesimen tetap
terjaga kondisi suhunya tetap dingin saat diterima di
laboratorium

64
4 degree celcius

65
PENGEPAKAN/PENGIRIMAN
7. Jangan lupa masukkan juga formulir kuisioner
yang telah diisi dan diberi label kedalam cool
box dengan terlebih dahulu dimasukkan dalam
wadah plastik

66
 PENGEPAKAN/PENGIRIMAN
8. Ke dalam cool box juga bisa dimasukkan kertas pengganjal (bisa
berupa kertas koran yang diremas remas). Kemudian ditutup.

9. Tutup Cool box dengan selotip dan beri label pada sisi kanan dan
atau kiri cool box, yang ditujukan ke Laboratorium rujukan.

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 GOOD QUALITY SPECIMENS
IS NEEDED
FOR RELIABLE MICROBIOLOGIC
DIAGNOSES