MEDICAL MICROBIOLOGY II

LECTURE 1
Collection and Examination of Specimen
for Microbiological
Investigation

Importance of Proper Specimen

Collection
Specimen collection is the backbone of the
investigative process
Isolation and identification of the
microorganism - first step in determining
proper treatment
Microorganism needs to be grown, isolated
and identified
Before all this can happen, a specimen must
be COLLECTED

Importance of Proper Specimen

Collection
Diagnostic results depend on quality of
specimens
Poorly collected / transported specimens will
result in:
1. Failure to isolate causative microorganism
2. Recover contaminants / normal flora

3. Waste of time, resources and man-power

4. Prolongs accurate diagnosis

Importance of Proper Specimen

Collection
5. May lead to harmful therapy
6. Inconvenience or painful for patient to
recollect another specimen

Specimen Collection
A very important step - result of a culture of
bacteria is only as reliable as the method used
to collect and transport the specimen
Knowledge of microorganism growth
requirements are crucial
Specimen should be collected BEFORE starting
on antibiotics

Important questions before collecting

a specimen
Are you suspecting an infection?
If so, what is the nature of infection (e.g.
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 do I need to send the specimen to the
laboratory promptly?
If not, what should I do?

Factors Influencing Specimen Collection

Several factors must be considered:
1. The specimen must be from the actual site of
infection
2. The specimen must be collected with minimal
contamination from adjacent tissues, organs,
or secretions
3. Sufficient quantity must be obtained to
perform the tests requested

Factors Influencing Specimen Collection

4. Appropriate collection devices, specimen
containers and culture media must be used
to ensure optimal recovery of microorganism

Common Specimen Types

These are common specimen seen in
microbiology laboratory:
1. Urine
2. Blood
3. Swab (wound, throat, vaginal, urethral, high
vaginal swab, nasal swab)
4. Sputum
5. Faeces

Less Common Specimen Types

These specimen types are less common:
1. Cerebrospinal fluid
2. Tissue
3. Other body fluid (ascites fluid, synovial fluid,
pleural fluid, peritoneal fluid)
4. Tips (Foleys tip)

Criteria of Containers

Leak-proof
Unbreakable
Sterile (MUST for culture)
High risk specimens should be labeled (e.g.
sputum specimen suspected with tuberculosis,
faecal specimen suspected cholera or typhoid)
Screw-cap or firmly stopped

Factors affecting quality of result

Appropriate samples
Test method
Clinical details
Sample collection
Labeling (incorrect specimen identification)
Transportation / storage

Specimen collection

1. Pre-Analytical

Factors affecting quality of result

2. Analytical
- Equipment

- Accuracy
- Updated knowledge
- Technique
- Quality Control

- Precision
- Calibration
- Reagent
- SOPs
- Specificity

Factors affecting quality of result

3. Post-Analytical
Transcription
Communication

Specimens from Various Body Sites

Specimen for microbiology can be collected
from various body sites:
1.
2.
3.
4.
5.
6.

Blood
Sputum
Throat
Nasopharynx
Cerebrospinal fluid
Body fluids (synovial, pericardial, peritoneal
etc.)
7. Urine
8. Stool
9. Abscess aspirates or exudates

Blood culture
One of the most important investigation
An appropriate procedure in collection,
processing, identifying and timely reporting
can be life saving
Blood collection has to be free of
contamination
2 - 3 cultures should be collected at random
times during 24hrs period

Blood culture
Palpate the skin for a good puncture site
Skin is disinfected with 70% alcohol, followed
by iodine
The disinfectant is allowed to dry
The puncture site should not be palpated after
disinfection
Ideally, 20 - 30mL of blood per culture is
collected from an adult (1 - 5mL from infants
and small children)

Blood culture
Iodine should be cleaned
from the puncture site with
alcohol following the
venipuncture
If collecting a set of tubes
and blood culture, the first
sample should be blood
culture
Clean the top of blood
culture bottle (with 70%
alcohol)

Blood Culture
DO NOT:
Palpate the skin after disinfection
Use iodine to clean the top of bottle

DO:
Only palpate before disinfection
Only use alcohol / chlorhexidine

Respiratory Samples
Common respiratory infections are localised in
oropharynx, nasopharynx, and nasal cavities
Causes: sore throat, nasal discharge, fever,
sinusitis, whooping cough, influenza, measles,
infectious mononucleosis
Most throat infections are due to virus, but
some will have secondary infection by bacteria
Among the common respiratory samples are
sputum, ETT aspirates, throat swab, and
bronchial lavage

Sputum
Mixture of bronchial exudates and inflammatory
exudates coughed up into the mouth and
expectorated
Most common specimen sent for lower
respiratory infection investigation
Difficulties in collecting suitable specimen - busy
or uninstructed staff may collect saliva only
Sometimes, repeat specimens may be required
to isolate the causative pathogen

Sputum
Patient instruction
Sputum (expectorated)
Rinse mouth and gargle with water before
collection

Quality of sample
Good quality sample: good isolated bacterial
growth
Unacceptable sample: salivary, clear fluid

Sputum
Collection should be made in a disposable,
wide-mouthed screw capped plastic container
of 50 - 100 mL capacity
Collect sputum before antibiotics are given
Ideal to collect specimen when patient wakes
up, and with the first cough

Throat Swab
Sample collection
The tongue should be depressed and need to
ensure to not touch cheek, tongue, and teeth
A plain cotton swab can be used to collect as
much exudates as possible from tonsils,
posterior pharyngeal walls and other areas that
are inflamed
The swab has to be returned to its tube with
care not to soil the rim

Throat Swab
If it cannot be transported immediately, it
should be refrigerated (2 - 8 C)

One swab / investigation

Nose swab
Nasal swabs are taken to
diagnose healthy carriers
e.g. for H1N1 screening
A deep nasal swab generally
yields the same information as
throat swab

CSF
Collected aseptically by a physician
Collect about 3 - 5mL in a labeled sterile screw
cap container
Must be transported immediately to lab as it
needs to be processed immediately
Delay may kill the pathogen or disintegrate
leukocytes
Specimen cannot be exposed to heat or
refrigeration

CSF
If delay is unavoidable, do not
refrigerate (will kill H. influenza)
If delay is anticipated, keep at room
temperature.
3 sets: for Microbiology, Biochemistry
and Haematology
First sample (or most turbid) is for
Microbiology
Greater volume increase the chance
of organism recover

Body Fluids
Collected by a physician with a
needle and syringe or aspirated into a
sterile container
The use of swabs may inhibit growth
of anaerobes or increase the
likelihood of contamination with
indigenous bacterial flora from
adjacent tissues
Greater volumes increase the
chances of organism recovery

Urine
Clean catch, mid-stream urine (MSU) most common collection method
Do not stop flow of urine
Proper cleansing of the urethral area is
important, especially in women to ensure urine
is not contaminated with commensal bacteria
that colonize in the urethral orifice and
perineum area

Urine
Collect specimen in a disposable widemouthed plastic container with screw cap
Transport immediately, otherwise refrigerate
at 2 - 8 C

Stool
The specimen may be collected from faeces
passed in a clean bed pan or other clean
receptacle
NEVER collect stool specimen from the toilet
Ensure faeces not mixed with urine,
disinfectant or heavily soiled toilet paper
Specimen should be collected into a
disposable, sterile, wide-mouthed, screw cap
plastic container

Stool
Transport immediately to lab
If the specimen cannot be plated within 1
hour of collection, it should be mixed with a
transport medium (e.g. buffered glycerol
saline, Cary-Blair transport medium)
The change in pH and temperature over time
is detrimental to Shigella spp.

High Vaginal Swab / Vaginal Swab

Specimen commonly collected for diagnosis of
vaginitis, vaginosis, or uterine sepsis
Also used for examination for gonorrhea
The swab is inserted into the cervix and
rotated several times before withdrawing it
Transport immediately or use Amies transport
medium
Never refrigerate

Swab from Wound, Skin and Deep Sepsis

Swab from wound, skin or deep sepsis are
usually sent to the lab
Swabs are actually inefficient sampling device
and tends to desiccate the specimen and traps
the bacteria which are then not released onto
the plate
The ideal sample is pus or exudates
Swabs should be collected in a small screw
capped bottle, firmly stopped tube or syringe,
or sealed capillary tube

Pus
Surgically obtained pus / fluid by a physician
Pus better than swab
Aspirates from closed abscesses (syringe
aspirates better than swab)
Must be immediately transported to lab

Tissue
Surgically obtained
Tissue in sterile container with small amount
of sterile saline or,
Tissue in sterile container without any
additional fluid
Immediate transport to the lab

Anaerobic Culture
Acceptable specimens:
1.
2.
3.
4.

Pus from closed abscesses

Blood
CSF
Peritoneal fluid

Anaerobic Culture
Unacceptable
specimen:
1. Gastrointestinal
specimens
Faeces
Rectal swab
Gastric / small
intestine
contents

2. Urogenital
specimens
Urine
Urethral swab
Vaginal swab

3. Respiratory
specimens
Throat swab
Coughed sputum

Anaerobic Culture
Collection technique
Exclusion of air
Blood (anaerobic) culture before all other
samples
Rapid transport to the laboratory

Specimen for Viral Studies

Specimen are best collected during the time of
first presentation of symptoms
Specimen should be collected with a Dacron
or rayon swab
Cotton swabs are toxic to viruses
Specimen must not dry out
Usually will be transported in a modified
Stuart transport medium to the laboratory

Specimen for Viral Studies

Other specimens should be collected into viral
transport media such as Hanks balanced salt
solution, 0.2M sucrose-phosphate, or 2% fetal
calf serum with Eagles minimum essential
medium
Some viral transport medium contain:
Antibiotics: inhibit growth of normal bacterial
or fungal flora
Gelatin media: stabilize protein

Specimen for Viral Studies

Urine and stool should be collected in clean
containers with no preservatives
Blood should be collected into heparin or
EDTA tubes
All specimen for viral studies/recovery should
be transported at refrigerated temperatures
(0 - 4 C), regardless of source

Labeling of Specimen Container

Label specimen container and form:
1.
2.
3.
4.
5.
6.

Patient name
Identification number
Medical record number
Source / site
Date / time of collection
Collector

Rejection Criteria
Leaking container
Unlabeled container, wrong labeled,
mismatched samples
Specimen received in fixative
Specimen unsuitable for request
Specimen received in non-sterile or
contaminated containers
Prolonged transport

Rejection Criteria
24 hrs urine or 24 hrs sputum collections
Saliva instead of sputum
Unrefrigerated urine specimens 2 hrs or more
post-collection
Dried specimen
Anaerobic culturing of MSU, upper respiratory
tract specimens, superficial skin or stool
specimens

Transportation
All specimens must be transported to the lab
promptly
Anaerobic specimens must be transported in
an anaerobic transport system
If delay in transportation, most specimens can
be kept at 2 - 8 C
Exception are specimens that likely contain
temperature-sensitive organisms (e.g.
Neisseria), blood culture bottles, and
cerebrospinal fluid

Transportation
Generally, swabs are the least desirable
collection and transport method
However, organisms can be successfully
cultured if the swab is handled and
transported properly (e.g. not allowed to dry
out)
Swabs are inappropriate for culturing
anaerobes

Transportation
If swabs are used, it is essential that an
anaerobic culturette be used
Use of aerobic culturette for the culture of
anaerobes is criteria for specimen rejection

Transport Medium
A transport medium should be:
non-nutritive
semi-solid
reductive
able to hamper self-destructive enzymatic
reactions within the cells
must inhibit toxic oxidation reactions

Generally, 2 commonly used: Stuarts transport

medium and Amies transport medium

Transport Medium

Transport Medium
1. Stuart transport medium - semisolid medium,
used to transport and preserve specimens for
the cultivation gonococci, streptococci,
Enterobacteriaceae, etc.
2. Amies transport medium - modification of
Stuart's transport medium by replacing
glycerophosphate with an inorganic
phosphate buffer and adding charcoal to the
medium. This modified medium gave a higher
percentage of positive results than Stuart
transport medium

Transport Medium
For the collection of the
specimens, use sterile
cotton-tipped swabs or
wooden sticks
Push the swab down one
third of the medium depth
When the cap is screwed
down, the swab is forced
to the bottom of the
medium

Transport Medium
The cap should be firmly screwed
The medium has to be kept cool during
transportation but do not freeze
The cultures on transport swabs must not be
kept at room temperature for more than 24
hours
For optimum results, the time lapse between
sample collection and inoculation onto culture
medium should be reduced to the minimum

THE END