MEDICAL MICROBIOLOGY II

Lesson 8

Laboratory Methods in Diagnostic

Virology

Collection of Specimens for Virology

Specimens should be collected during the
acute stage of illness when viruses are shed in
large numbers
Specimens such as swabs or tissues should not
be allowed to dry before reaching the
laboratory should be placed in about 3 - 4
mL of a viral transport medium (VTM) so that
the viruses do not die due to dehydration

Collection of Specimens for Virology

The VTM has a buffering system to maintain
the pH between 7.2 and 7.4, a low
concentration of protective proteins and
antibiotics to inhibit bacterial and fungal
contaminants
Various types of VTM are commercially
available

Viral Transport Medium

Collection of Specimens for Virology

A simple VTM can be prepared:
Hanks balanced salt solution
Foetal bovine serum
Gentamicin
Amphoterin B

100 mL
2 mL
10 mg
50 g

Collection of Specimens for Virology

Commonly used
specimens include:
1. Throat swab
2. Respiratory
aspiration
3. Nasal washings
4. Mucous membrane
swabs
5. Conjunctival swabs
6. Vesicle fluid

7. Cerebrospinal,
pericardial and
pleural fluid
8. Saliva
9. Urine
10. Stool
11. Tissue
12. Blood
13. Postmortem
specimens

Collection of Specimens for Virology

1. Throat swabs
Swab the tonsillar area and the posterior wall
of the pharynx with a cotton tipped sterile
swab
Place it in 3 - 4 mL of VTM, immediately
delivery to laboratory

Collection of Specimens for Virology

2. Respiratory aspiration
Collect the respiratory secretions into a
plastic disposable suction aspirator using a
fine gauge rubber catheter
In babies, pass it through the nose; in adults
through posterior pharynx

Collection of Specimens for Virology

3. Nasal washings
Instill about 5 mL saline into each nostril and
collect into a screw-capped bottle
A Dacron or rayon swab may also be used; it
should be left in the nostril

4. Mucous membrane swabs

Swab the mouth, lips or genital areas and put
into VTM

Collection of Specimens for Virology

5. Conjunctival swabs
Collect the swabs using dry Dacron or rayon
swabs
Roll a dry cotton swab gently along the lower
conjunctival surface and collect into VTM

Collection of Specimens for

Virology
6. Vesicle fluid
Aspirate several vesicles using a tuberculin
syringe and a 25 gauge needle
If possible, choose fresh, plump vesicles
Send the vesicle fluid in a sterile container to
the laboratory

Collection of Specimens for Virology

7. Cerebrospinal, pericardial and pleural fluid
Place about 1 mL of fluid in a dry, sterile
container

8. Saliva, urine and stool

These specimens should be collected in sterile
containers
Rectal swab may be used if stool cannot be
obtained

Collection of Specimens for Virology

9. Tissue
It should be collected from an appropriate
part of the organ, placed in a sterile container
containing 3 - 4 mL of VTM and kept at 4 C
until it reaches the virology laboratory

10.Blood
Collect 10 mL of blood in a sterile container
containing heparin, EDTA or citrate
For serology, collect blood in a plain sterile
tube

Collection of Specimens for Virology

11.Post-mortem specimens
These should be collected from the suspected
sites of disease and transported to the
laboratory as soon as possible

Transport and Storage of Specimens

for Virology
Specimens for viral diagnosis must be
considered INFECTIOUS and should be
handled with great care
In the laboratory, the specimens should only
be handled in safety cabinets without
releasing aerosols
Every virus isolation specimen should be
treated as URGENT and transported to the
virus laboratory IMMEDIATELY

Transport and Storage of Specimens

for Virology
If must wait for transport, the specimen in a
VTM should be kept at 4 C, but not at 0 C or
in an ice-box
Care must be taken during transport and
storage of specimens that the virus in the
specimen should survive

Transport and Storage of Specimens

for Virology
Factors which can destroy viruses are:
1.
2.
3.
4.
5.
6.

Dehydration
Heat
Freezing at temperatures near 0 C
Sudden pH changes
Oxidising agents
Ultraviolet light

Transport and Storage of Specimens

for Virology
Aspirates, fluids and tissues should be sent to
the laboratory in a sterile, leak-proof
container
Swabs should never be left to dry and should
be placed in VTM immediately
In the laboratory, the specimens should be
refrigerated (4 C) until they are inoculated
into cell cultures

Transport and Storage of Specimens

for Virology
A specimen may be kept at 4 C for up to 96
hours
If the delay is longer, it should be stored at 70 C
Specimens expected to contain viruses such as
enteroviruses, adenoviruses or poxviruses
should be stored at -20 C

Cultivation of Viruses

Viruses CANNOT grow on inanimate media

They are obligate intracellular parasites
They need LIVING cells for replication
There are 3 methods:
1. Animal inoculation
2. Inoculation of embryonated eggs
3. Inoculation of organs, tissue fragments or cell
monolayers

Animal Inoculation
In the past, the only known method of
cultivation of viruses were by inoculation of
human volunteers
Then, in the past few decades, animal
inoculation has been employed for virus
isolation
Laboratory animals include monkeys, rabbits,
guinea pigs, rats, hamsters and mice

Animal Inoculation
The choice of animals and route of inoculation
(intracerebral, intraperitoneal, subcutaneous,
intradermal or intraocular) depends on the
type of virus to be isolated
Animal inoculation can also be used to
observe pathogenesis, immune response,
epidemiology and oncogenesis.
Growth of a virus in the inoculated animal
may be indicated by visible lesions, disease or
death

Animal Inoculation
Sometimes, serial passage into animals may
be required to obtain visible evidence of viral
growth
This method requires special experience
especially in the handling of animals and
inoculation into the various routes

Inoculation of Embryonated Eggs

An embryonated egg provides an aseptic
environment that can contain various types of
viruses
A large number of viruses can be grown in the
different areas of the embryonated egg
The inoculation of eggs can be performed with
relatively simple equipment

Inoculation of Embryonated Eggs

Inoculation of Embryonated Eggs

For inoculation, an 8 - 11 days old hens egg,
preferably with a white shell, is used
Duck eggs can be used in some cases
After inoculation, virus replication takes 2 - 7
days
The contents of the egg are harvested and
inspected for evidence of virus growth

Inoculation of Embryonated Eggs

Most viruses either:
produce morphological changes at the site of
inoculation
kill the embryo
produce haemagglutinins

Egg inoculation is also useful for cultivation of

chlamydiae and rickettsiae

Inoculation of Embryonated Eggs

Involves 4 steps:
1.
2.
3.
4.

Candling
Drilling the egg shell
Inoculation
Harvesting the fluids

Inoculation of Embryonated Eggs

1. Candling
Is the inspection of an egg over a lamp
A special candling lamp or egg may be held
over a strong incandescent lamp
Egg are candled after 3 - 5 days of laying to
check if the egg is fertile or not
Infertile eggs which shows a dead embryo are
discarded
Eggs are candled again on the 10th or 11th

Inoculation of Embryonated Eggs

The air space is marked with a pencil
Then, a suitable site for inoculation is selected
depending on the type of virus

Candling

Inoculation of Embryonated Eggs

2. Drilling the egg shell
The egg shell is disinfected by wiping with
dilute alcoholic antiseptic solution
A small hole is cut at the selected site using a
sterile motor driven flexible shaft or dental
handpiece
Care MUST be taken not to damage the EGG
MEMBRANE

Inoculation of Embryonated Eggs

3. Inoculation
The inoculum is injected through the hole on
the shell onto the desired site, using a fine
needle
The inoculum has to be injected slowly to
avoid spillage of the inoculum
The opening of the inoculation is sealed with a
sealing mixture prepared by 2 parts of molten
paraffin (55 C) and 1 part of petroleum jelly
The egg is incubated at 36 C

Inoculation of Embryonated Eggs

Routes of inoculation: The egg can be inoculated
by 4 routes
a) Intra-amniotic inoculation

A hole is drilled just above the amniotic cavity,

located by candling
The needle is then inserted slowly until the
amniotic sac moves

Inoculation of Embryonated Eggs

Inoculation of Embryonated Eggs

The needle is thrust through the amniotic

membrane and the fluid is injected slowly
A small inoculum (0.1 mL) is used
The opening is sealed immediately
This route is useful for influenza,
parainfluenza and mumps viruses

Inoculation of Embryonated Eggs

b) Intra-allantoic inoculation

A site above the allantoic cavity is selected by

candling
The same techniques are followed as for the
intra-amniotic cavity
This is the simplest method of inoculation
with a relatively large yield and is suitable for
the preparation of vaccines
Influenza and paramyxoviruses grow well in
the allantoic cavity

Inoculation of Embryonated Eggs

c) Yolk-sac inoculation

The position of the embryo is determined by

candling
A hole is drilled in the shell at the centre of
the air space at the blunt end
A long needle is inserted and the inoculum is
deposited just below the centre of the egg
To ensure correct position, pull back the
plunger until the yolk sac is pulled up with it

Inoculation of Embryonated Eggs

The opening of the inoculation is sealed
Yolk sac inoculation is also useful for cultivation
of fastidious groups of bacteria such as
Chlamydia and Rickettsia species which do not
grow on inanimate media

Inoculation of Embryonated Eggs

d) Chorio-allantoic membrane (CAM) inoculation

A small triangle is marked at a site where there

are no major blood vessels
This can be located by candling
The shell along the triangle is cut to expose the
CAM
A new air is prepared at the site of inoculation
by reducing the original air sac
A small hole is cut at the blunt end above the
air sac

Inoculation of Embryonated Eggs

The egg is placed horizontally

A gentle suction is applied with a rubber teat
A new air space develops at the top of the
horizontal egg at the site of the triangular cut
A pipette is filled with about 1 mL inoculum
and inoculated through the gap in the CAM and
the shell
The egg is rocked gently to disperse the
inoculum evenly over the membrane
Both the openings are sealed by replacing the
cut triangles with the sealing mixture

Inoculation of Embryonated Eggs

4. Harvesting the fluids
In order to harvest the virus infected fluids
and other structures, the shell must be
opened with great care to avoid unwanted
dissemination of the virus
The use of safety cabinets is recommended
Never use a drill to open infected eggs
because it will create aerosols

Inoculation of Embryonated Eggs

After the desired incubation period, the air sac
portion at the blunt end, which is already
marked during candling is opened
Forceps or a pair of scissors are used for
cutting
The contents of the egg is collected in a sterile
petri dish
The fluid from the inoculated area is aspirated
with a syringe

Inoculation of Embryonated Eggs

After removing the contents of the egg, pull
out the CAM gently with tweezers
Wash the CAM in saline 2 - 3 times
Inspect it for the presence of lesions or pocks
The harvested fluids is tested by direct method
or other methods for the detection and
identification of the virus

Inoculation of Embryonated Eggs

Inoculation of organs, tissue fragments

or cell monolayers
The 1st application of tissue culture in virology
was by Steinhardt et. al. in 1913
They used it for the maintenance of vaccinia
virus in the fragments of rabbit cornea
The major obstacle in the development of
tissue culture was contamination by bacteria
It was overcome when antibiotics became
available

Inoculation of organs, tissue fragments

or cell monolayers
After that, major progress was achieved by
Enders and others in 1949 by growing polio
virus in the tissues of non-neural origin
Since then, a large number of human viruses
have been grown and maintained in tissue
cultures

Inoculation of organs, tissue fragments

or cell monolayers
There are 3 types of tissue cultures:
1. Organ culture
2. Explant culture
3. Cell culture

Inoculation of organs, tissue fragments

or cell monolayers
1. Organ culture
Essentially cultured tissue pieces in which the
architecture and physiology of the tissue is
retained
Such small bits of organs can be maintained in
vitro for a few days
Organ cultures are necessary for the growth
of some fastidious viruses, e.g. ferret trachea
can be used for the isolation of some
rhinoviruses and coronaviruses

Inoculation of organs, tissue fragments

or cell monolayers
2. Explant culture
This technique is particularly important for
the isolation of viruses in the latent stage
The fragments of the tissue are placed in a
test tube or a petri dish in a drop of plasma or
fibrin clot
These explants are then covered with
growth medium
Adenoid tissue explant cultures were used for
the isolation of adenoviruses

Inoculation of organs, tissue fragments

or cell monolayers
3. Cell culture
This is the most widely used technique for
growing viruses
By the action of proteolytic enzymes such as
trypsin, a tissue is dissociated into its
component cells
After washing, cells are suspended in growth
medium containing essential amino acids,
vitamins, salts, glucose and a buffer

Inoculation of organs, tissue fragments

or cell monolayers
Serum such as foetal calf or newborn bovine
serum is added as supplement
Antibiotics are also added to prevent bacterial
contamination
A change in pH is indicated by phenol red
incorporated in the medium
The cells suspension in growth medium is
dispensed in flasks or tubes
The cells adhere to the surface of the flasks

Inoculation of organs, tissue fragments

or cell monolayers
When incubated under appropriate conditions,
they divide and redivide to form a confluent
sheet of cells in a single layer - monolayer
It is achieved by a mechanism known as
contact inhibition occurs when cells come in
contact with surrounding cells which inhibits
further multiplication
Formation of monolayer to cover the surface
of the culture vessel usually takes 3 - 7 days
depending on the type of cells used

Types of Cell Culture

3 types of cell culture are used in virology
depending on their origin, chromosomal
characters and the number of generations
they can be maintained
1. Primary cell cultures
2. Diploid cell cultures (semi-continuous cell
culture or cell strains)
3. Continuous cell lines

Types of Cell Culture

1. Primary cell culture
Prepared from an organ which is minced into
small pieces, treated with an enzyme and then
used for the preparation of a monolayer
Capable of only limited growth in culture
Only a few serial passages can be made
Advantage: Large number of cultures can be
prepared if sufficient animal organs are
available

Types of Cell Culture

Disadvantage: Primary cell cultures can
contain latent viruses in the donor animal
The commonly used primary cell cultures are
monkey kidney, human embryonic kidney,
human amnion and chick embryo cell cultures
Useful for isolation of viruses such as
enteroviruses, myxo- and paramyxoviruses
Used for large scale production of vaccines

Types of Cell Culture

2. Diploid cell cultures
These cultures mostly consist of a single type
of cells which may undergo 30 - 50 passages
before senescence or death of the culture
The human embryonic lung or kidney
fibroblasts make excellent diploid cell strains,
e.g. MRC5, W138 are fibroblast strains
Diploid cultures are maintained by creating a
pool of frozen cells stored in liquid nitrogen

Types of Cell Culture

Serial passages of primary cell culture are made
and large portions of the initial 8 - 10 passages
are stored in liquid nitrogen
When the diploid cell strains start dying, the
subcultures can be restarted from the frozen
portions cells from the same source can be
used for many years giving constant results
Used for selective isolation of some viruses and
preparation of vaccines herpes simplex,
cytomegalovirus, varicella-zoster, and rhinovirus

Types of Cell Culture

3. Continuous cell lines
These cells may be serially sub-cultured
indefinitely
These cells have a malignant character, and
the number of chromosomes is different from
that of the original host
They have very fast growth rate and contact
inhibition is absent

Types of Cell Culture

Commonly used cell lines are HeLa (human
cervical cancer), HEp2 (human epithelial), BHK21
(baby hamster kidney) RK13 (rabbit kidney) and
Vero (African green monkey kidney)
HeLa, HEp2 and Vero cells: Cultivation of
poliovirus, coxsackie virus, adenovirus and herpes
simplex virus
RK13 and BHK21: Isolation and propagation of
rubella virus

Types of Cell Culture

Cannot be used for preparation of vaccines
because vaccines grown in cancer cells are not
considered to be safe

Diagnostic Methods
There are several methods by which viral
disease can be diagnosed in the clinical lab
There are 3 categories:
I. Direct methods - electron microscopy,
immune electron microscopy,
immunological methods and nucleic acid
hybridisation
II. Isolation and identification of the
causative agent
III. Serological diagnosis

Serological Diagnosis
The procedures commonly used include:
1. Indirect immunofluorescence (IF) test
2. Enzyme linked immunosorbent assay (ELISA)
or enzyme immunoassay (EIA)
3. Neutralisation (NT) test
4. Complement fixation (CF) test

They are employed for the identification of

viral isolates by using a known antiserum
against the suspected virus

Serological Diagnosis
Serodiagnosis of viral infections involves
detection of antibodies to viral antigens in
patients serum in sufficiently high titres
The antibody can be titrated by using a range
of dilutions
IF and ELISA are the most commonly used
because they are sensitive, easy to perform
and less time consuming than other tests such
as CF and NT

THE END