Specimen Collection

In Infectious Diseases
Dr.T.V.Rao MD
Professor Of Microbiology
Why specimen collection is
Important in Microbiology
Specimen collection in Microbiology to
isolate and identify the causative
agents forms back bone of the
investigative procedures.
In developing world, lack of awareness
and casual attitude among junior staff
hampers the definitive diagnosis.
Specific procedures in collecting
specimens will certainly improve the
quality of services of Microbiology
Departments
Important questions before
collecting a specimen
 Are you suspecting an Infection ?
If so what is the Nature of infection, eg Bacterial,
Viral, Mycological or Parasitological
 Which tests are your priority ?
 When to collect the specimen ?
 How to collect the specimen ?
 Am I choosing the correct container ?
 Why to send the specimens promptly
if not what I should do ?
Fishing for Diagnosis in
Laboratories, Is it worth?
 The physicians and
Microbiologists should
be aware of the clinical
manifestations, before
undertaking the test.
 Microbiological tests are
expensive and technically
demanding
 Causal testing of
Microbiological tests are
counterproductive.
Policies on Specimen Collection.
Every laboratory should formulate guidelines
on procedures for each major category of
specimens and requests.
Every laboratory should assist extra
examinations,outwith the standard
procedures may be required if specifically
requested by the Physician or if the clinical
information provided on the request form
suggests that an unusual infection may be
present.
Why Prop er wri tten
Requ est
 Your request is a legal document.
 Identifies all the outcome of test.
 No interchange of results.
 Short forms are dangerous
 Signature of the Doctor / Nurse is essential in legible
form, can help to contact in case of results which can
save a patient.
 When the patient is serious, write a Tele contact
number which can help in prompt delivery of results
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
Wh en one E xpects the Res ul ts

On sending the sample the Physician will be

anticipating the early reports, the
Microbiologists should promptly dispatch
results in all life saving investigations.
However the Doctors must be
made aware limitation of the
investigations and discuss the pros
and cons of the Lab or ator y reports
Wh en to Repea t
Diagnostic T es ts
On many occasions less than ideal sample is
received in laboratory.
The rejection of clinical samples should be
done with great care and wisdom of only
senior staff who should take the
responsibility.
In the welfare of the patient samples can be
repeatedly collected for better diagnosis, as we
need repeated isolation to confirm uncommon
pathogens.
Wh en to Col lect the
Earl iest Speci me n

 Start collection of specimens for all

cultures before starting an Antibiotic.
 The advice is ideal but may not be
possible, as many prescribe
Antibiotics before considers the
Microbiological diagnostic options.
When to Request Transport
Medium
 When facilities are not available to perform the
desired tests at the place of collection or laboratory
located far away, request the Diagnostic laboratories
to advice on transportation of specimens, and
consider how to preserve and transport in ideal
medium before it is processed
 Popularly used transport medium are
Amie 's transport Medium
Stuart's Transport Medium
V.R Medium
What containers to use

 Containers must be leak proof,

 Unbreakable
 Forcult ur es st er ile
co ntai ner s a M ust
Label Hi gh ri sk
Spe cime ns

 Sputum with
suspected Tuberculosis
 Fecal samples
suspected with
Cholera, Typhoid,
 Anthrax ?
 Serum when suspected
with HIV/ HBV/HCV,
infections
Blood for Culturing
Blood Collection for Culturing
 Most important
investigation
 An appropriate
procedures in
collection and
processing, identifying
and timely reporting
can be Life saving
Col lecti on of Bl ood
 A scientific approaches and
dedicated staff participating
in blood collection will
eliminate the basic failure as
Contamination
 Improper handling of
syringes increases chances
of contamination
 Contamination hampers the
ideal reporting,
 A valuable time is lost
 The goal in blood collection
is avoiding the
contamination
Coll ec ting th e Blo od
fo r Cultu ring
 Teach the staff how to
collect the Blood.
 The nurse are advised
on principles of aseptic
precautions by self as
washing hands and
wearing gloves
 Proper areas of
disinfection with good
antiseptic solutions.
Hyg ienic preca uti ons will
decrease co nta minati on

 The staff should be

advised how to disinfect
the skin over vein, to use
a fresh sterile syringe for
the venepuncture with
fresh sterile needle before
inoculating culture bottle
 The staff should disinfect
their hands before doing
the procedure.
 P ro per hand ling of Syring e
is esse ntial to ob ta in a
bloo d sp ecim en
 The staff should hold
the needle by its butt,
not shaft. Either with
sterile forceps or with
fingers covered with a
dry sterile rubber
glove, and protect self
with potentially
infective pathogens
Do not collect from existing or
indwelling catheters
The staff are warned that
contamination is very
likely if the specimen is
collected from an
indwelling peripheral
venous catheter instead
of from a fresh
venpuncture.
Always collect the Blood
specimens in Hygienic areas
 All procedures in
relation to processing of
the samples should be
done in a sterile
environment, or
bacteria free areas.
 Despite insistence on
aseptic precautions,
most laboratories
report finding
contamination in 1-5%
of the blood cultures.
Cerebrospinal fluid
examination
 Specimen collection for
CSF Examination
 Lumbar puncture
to collect the CSF
for examination to
be collected by
Physician trained
in procedure with
aseptic precautions
to prevent
introduction of
Infection.
 Procedure to collect CSF

 The trained physician will

collect only 3-5 ml into a
labeled sterile container
 Removal of large volume
of CSF lead to headache,
 The fluid to be collected at
the rate of 4-5 drops per
second.
 If sudden removal of fluid
is allowed may draw down
cerebellum into the
Foramen magnum and
compress the Medulla of
the Brain
CSF needs a New and Sterile
container

 Fresh sterile screw

capped container to be
used.
 Reused containers, not
to be used,
contamination from the
previous specimens
misrepresent the present
specimen.
Lumbar puncture for CSF collection

 The best site for puncture is

inter space between 3 and 4
lumbar vertebrae
( Corresponds to highest point of iliac crest )

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 specimen
of CSF to be dispatched
promptly to Laboratory ,
delay may cause death
of delicate pathogens, eg
Meningococci and
disintegrate leukocytes
 Pr eser va ti on o f 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.
Upper Respiratory
Infections
What are Upper Respiratory
Infections
 The commonest respiratory infections are
localised in Oropharynx, Nasopharynx, and
nasal cavity,
 Causes
Sore thraot,nasal discharge and often fever.
Infect larynx,otitis media,sinusitis,conjunctivitis
or keratitis.
 May present with serious diseases whooping
cough, influenza , measles and infectious
mononucleosis.
Aeti ological ag en ts in Up per
Res pi rato ry Infec tio ns
 In most cases the primary infections are caused by
virus, difficult to isolate.
 But many infections are caused by concomitant
carriage or secondary infection with one of the
potential pathogens present in the Nasopharynx
1 Pneumococcus .Haemophilus influenza
Staphylococcus aureus, and Streptococcus pyogenes.
 Drug resistant coli form bacilli or yeasts may dominate
the throat flora in patients receiving antibiotics.
Specimen collection
in Throat Infections
 A plain cotton wool
swab should be used
to collect as much
exudates as possible
from tonsils, posterior
pharyngeal wall and
other area that is
inflamed or bears
exudates
Cooperation of the patient and
ideal techniques contributes
better results
 If cooperated by
patient, the swab
should be rubbed with
rotation over one
tonsillar area of the
soft palate and uvula,
the other tonsillar area
and finally the
posterior pharynx
Collecting the Swab
 An adequate view of
throat should be
ensured by good
lighting conditions
and the use of a
disposable wooden
spatula or a tongue
depressor to pull
outwards and so
depress the tongue.
Tra nsp or tation o f Throa t
Swa bs
The swab should be
replaced in its tube with
care not to soil the rim
If it cannot be
transported immediately
to laboratory it should
be placed in a
refrigerator at 4ºc until
delivery or preferably
submitted in a tube of
transport medium
Nasal specimens
 A deep nasal swab
generally yields the same
information as throat
swab.
 Nasal swabs are taken to
detect healthy carriers
than diagnose deep
infection
 Deep nasal are taken to
diagnose S.pyogenes and
Diphtheria bacillus.
Specimens in sinusitis
 Puscollected or
aspirated from
sinus, or a saline
wash out should be
examined in a
Gram film and by
culture on aerobic
and anaerobic blood
agar plates.
Collection of Ear Swabs
Acute otitis Media
 Acute otitis Media –
as long as eardrum
remains intact, none
of the infected
exudates can be
collected on an ear
swab , though culture
of the throat swab
may give a
provisional indication
Chronic suppurative otitis media
 Swabs of the
discharge in the
external meatus
should be cultured
to guide the choice
of antibiotics for
systemic and
topical therapy.
Oti ti s e xte rna
 A swab should be taken
from the meatus and
cultured aerobically on
blood agar and
MacConkey agar plates
for the bacteria.
 All specimens should
also cultured on
Sabouraud’s agar plate
with Nystatin 50 units for
Candida and Aspergillus.
Eye Swabs
 Obtaining a adequate
specimen is difficult.
 It is best to make
smears and seed
culture plates beside
the patient
immediately after
collecting the material
from the eye.
Collection of Eye swabs
 It is ideal to pick up the
material with a loop or on
the smoothly rounded tip
of a thin glass rod or on
the thin serum coated
swab
 Clinical material from
Conjunctiva, i.e. from
everted eyelid,
 The margin of the eyelid
should be avoided.
 Specimens for
Lower Respiratory
Infections.
Lo wer Res pirato ry
Inf ec tion s
 Sputum is the material from the lower respiratory
infections most commonly submitted for
bacteriological examination. The sputum is a mixture
of bronchial secretions and inflammatory exudates
coughed up into the mouth and expectorated
 There are several difficulties both in collecting a
suitable sample and interpreting the results of the
culture
 Busy and uninstructed staff may send collection of
saliva to the laboratory.
 On several occasions repeat sample may be
Instr uction fo r
co llec ti ng sp utu m
 Make the collection in
a disposable and wide
mouthed screw
capped plastic
container of 50 – 100
ml capacity.
 Collect sputum before
antibiotics are given.
 Ideal to have when
patient wakes up and
Pre cau tions in handl ing
th e sp ecimens
 Avoid spilling the
material over the rim.
 Tightly screw on the
cap of the container.
 Wipe off any spilled
material on its outside
with tissue paper
 Deliver the specimen
quickly to laboratory
Spu tum Ex amin ation for
Tuberc ul osis
 Specimen should be
collected with
biosaftey precautions.
 Several specimens
should be collected
before coming to
negative conclusions.
Specimens for Urinary
Tract Infections
Col lecti ng Ur ine fo r exam inati on
 Collect the Mid
stream specimens of
Urine
 Do not collect
spontaneously passed
urine without
instructions, which
can lead to
contamination with
commensals bacteria
Colonized on urethral
orifice and perineum
Specimen Collection
 The urine collected in a
wide mouthed container
from patients
 A mid stream specimen is
the most ideal for
processing
 Female patients passes
urine with a labia
separated and mid stream
sample is collected
How Urine Specimens collected in
young and infants
 Non invasive methods are
safe and ideal
 Follow the Broom hall
method,
By tapping just above the
pubis with two fingers
placed on supra pubic
region after 1 hour of feed,
tapping on at the rate of 1
tap/second for a period of 1
minute, if not successful
tapping is repeated once
again.
The child spontaneously pass
the Urine and to be collected
in a sterile container
Transport of Urine for
Culturing
 All collected specimens
of urine to be transported
to laboratory with out
delay
 Delay of 1 – 2 hour deter
the quality of diagnostic
evaluations.
 If the delay is anticipated
the specimens are at
preserved at 40c
 In field conditions Boric
acid can be added at a
concentration of 1.8 %
Genital Tract
Infections
Genital Inf ec tions in
women
 Genital infections
present with,
urethritis, vaginosis,
genital ulceration,
cervicitis, uterine
sepsis, salphingits,
oophoritis, and
pelvic inflammatory
disease.
Collection of specimens
 The specimen commonly
collected for the
diagnosis of vaginiti’s,
vaginosis or uterine
sepsis is high vaginal
swab
 The swab is inserted into
upper part of the vagina
and rotated there before
withdrawing it.
Sp eci men c ol lecti on in
Go norrhoea
 An endocervical swab
must be collected for
examination for
gonococci.
 A vaginal speculum must
be used to provide a clear
sight of the cervix and
swab is rubbed in and
around the introitus of
the cervix and withdrawn
without contamination
from vaginal wall.
Spec imen s f rom oth er
genital ar eas
 Other swabs should be
collected from any
exudate discharged from
the meatus of the urethra
or a Bartholin's gland.
 Rectal or pharyngeal
swabs should be
considered depends on
sexual habits of the
patient
Transportation of specimens
 All the swabs to be promptly
transported to laboratory, in cases of
delay or in cases of delicate microbes
to be transported in Amie's transport
medium.
 If possible two swabs to be collected
and submitted for each site.
Sp eci men c ol lecti on in Men
 The infection in men
are mostly caused by
the same organism as
in women.
 Urethritis is
commonest
presentation may be
caused by Gonococci
or Non-gonococcal.
 May present with
Genital Ulcers.
Go no coccu s i nf ectio n in Me n
 The specimen is
collected by milking
the urethra and
urethral discharge is
smeared on slides and
inoculated on warmed
plates of heated blood
agar or selective
medium for isolation
of Gonococci
Other Genital infections
 When prostatitis is
suspected and there is
no spontaneous
discharge from
urethra, massage of
the prostate per
rectum may express
some exudate for
examination, and
culture.
Collection of specimens in
Chancres
 The examination of
chancre requires the
careful collection of
exudates and its
preparation for dark
ground microscopy.
 Many patients need
clotted blood for
specific serological
investigation.
Wound, Skin, and Deep
Sepsis
Colle ct ion o f s pec im ens
 Pus or exudates is often
submitted on a swab for
laboratory investigation.
 The swabs are inefficient
sampling device and
tends to desiccate the
specimen and trap the
bacteria which are then
not released on to culture
plate
Ideal s am ples from
wounds
 The ideal sample is pus or
exudates should be
submitted in a small screw-
capped bottle in firmly
stoppered tube or syringe
or a sealed capillary tube.
 Fragments of excised
tissue removed at wound
toilet or curettings from
infected sinuses and other
tissues should be sent in a
sterile container.
Gastrointestinal Infections
Collection of specimens
 Whenever possible, a
specimen of faeces
should be collected.
 A rectal swab is
unsatisfactory, unless
it is heavily charged
and visibly stained
with faeces collected
from rectum, not anus
Collection of Faeces
 The specimen may be collected from
faeces passed into a clean bed pan, not
mixed with urine, or disinfectant or
from the surface of heavily soiled
toilet paper.
 The specimen is collected into 25 ml
screw capped wide mouthed
disposable container.
Transportati on of
speci men
 Collect 1-2 ml of faeces, and apply the cap
tightly.
 Take care not to soil the rim or outside of the
bottle.
 Transmit the container quickly to laboratory.

 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
Specim en c ollecti on for
Vi rol ogi cal di seases
Direct virus diagnosis depends on the detection of
virus particles, viral antigen or viral nucleic acid
in specimen taken from the site of infection.
Specimens should be delivered promptly to the
laboratory so that no deterioration in the quality
of the cells occur.
However the specimen collection techniques alter
depending on the Aetiological agent and site of
involvement.
Tra ini ng t he
Parame dical Sta ff
Nursing staff and Medical Assistants
are the strengths of specimen collection
in the Developing world

All the progressive Medical Institutes should train

their staff in proper and scientific methods in
specimen collection and a fraction of Morbidity and
Mortality can be reduced.
Dr.T.V.Rao MD
Created for awareness on specimen
collection in developing world among
the Medical and Paramedical staff
Dr.T.V.Rao MD
email
doctortvrao@gmail.com