FACULTY OF MEDICINE

BACHELOR OF MEDICINE & BACHELOR OF

SURGERY (MBBS 240)

SESSION 2022/2023
Year 1 Semester 1

GENERAL MODULE
MEDICAL MICROBIOLOGY & PARASITOLOGY

PRACTICAL 2 – Part A
Basic Diagnostic Techniques (Virus)

Date: 10.11.2022
Time: 10 am – 1 pm
PRACTICAL 2: BASIC DIAGNOSTIC TECHNIQUES (VIRUS & FUNGUS)

Specific learning outcomes

At the end of the practical session, students should be able to:

• understand and describe the basic diagnostic technique commonly used in
the diagnosis of viral infection.
• describe the principle of antigen and antibody detections tests, and
molecular technique.
• describe the indication, advantages/disadvantages for the commonly used
technique/test in the diagnosis of viral infection.
• Interpret commonly used technique/test in the diagnosis of viral infection.

This practical session consists of THREE parts: A, B, and C

Part A Introduction/Theory/Video Presentation and Principle of Test
Part B Exhibits and Discussions

PART A: Introduction/Theory/Video Presentation and Principle of Test

Activity: There are a total of 1 video presentation in this section. You may discuss about
the techniques shown in the video with your respective lecturers during the practical
session.

The proper collection, transport and processing of clinical specimens is of utmost

importance in the diagnosis of viral infections.

A. Specimen selection and collection

To ensure the quality of clinical specimens collected, first the selection of specimen
must be appropriate for the suspected infection. Then a proper specimen collection
must be done. Proper collection eliminates or minimizes contamination of the
specimen. The clinical specimen collected should be
1-from a site of pathogen is most likely to be found.
2-collected during acute stage of illness
3-of sufficient quantity.
4- placed or collected into a sterile container or suitable transport medium.

Video V1- Example of Specimen collection (Nasopharyngeal swab)

https://www.youtube.com/watch?v=DVJNWefmHjE
B. Specimen Transport (Transportation of viral specimens)
Viral transport media.
• To stabilise the virus (keep virus alive), inhibit overgrowth of bacteria and fungi
and for maintenance and long-term freeze storage of viruses.
• Commonly used to transport clinical specimens for investigation of viral
infection.

C Specimen processing
Common diagnostic technique used to diagnose viral infections are listed as
below:

i. Viral detection method

• Antigen detection
I. Immunofluorescence
II. Enzyme-linked immunosorbent assay (ELISA)
III. Latex agglutination
• Cell culture
• Molecular
• Electron microscopy

ii. Antibody detection (Serology)

• ELISA
• Indirect immunofluorescent
• Haemagglutination inhibition test (HAI)
 PRINCIPLE OF TESTS

VIRAL DETECTION

The following methods are commonly used for viral detection:

IMMUNOFLUORESCENCE (DIRECT AND INDIRECT)

It is used to detect the microbial antigens and antibodies directed against these
antigens.

a. Direct Immunofluorescence
• In the direct immunofluorescence, antibody labeled with a fluorescent dye
is applied to a tissue section bearing the unknown antigen.
• Unbound antibody is washed away.
• Slide then examined with fluorescence microscope
• Presence of fluorescence indicate presence of antigen (positive result).
Figure 1.

b. Indirect Immunofluorescence
• In the indirect immunofluorescence unlabeled antibody is applied to a
tissue section bearing the unknown antigen.
• Unbound antibody is washed away.
• Fluorescent-labeled anti-immunoglobulin is added to amplify the signal.
• Slide then examined with fluorescence microscope.
• Presence of fluorescence indicate presence of antigen (positive result)
Figure 1.
 indicator

Figure 1: Immunofluorescence microscopy showing positive and negative result

ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA)

• The procedure is performed in a microtiter plate.

• Can be used to detect both antigen and antibody
• Qualitative ELISA- positive or negative result.
• Quantitative ELISA- measure the amount of antibody

Immunoassay for antigen:

• Known antibody is incubated on microtiter plate (Figure 2) and become

absorbed onto the plastic surface.
• Test antigen is added, which binds to the antibody, forming antigen antibody
complex.
• Unbound antigens are washed away.
• Enzyme-labelled specific antibody is then added, which will bind to the
antigen antibody complex.
• Unbound enzyme-labelled specific antibody is washed away.
• Substrate /chromogen is then added.
• The enzyme portion of the specific antibody hydrolyzes the added
substrate/chromogen and produce a colored end-product. (Figure 3)
• A colour change that can be observed with naked eye indicates positive
result (qualitative)

Known antibody added to the microtiter plate and absorbs to

the well surface.

Test antigen is added, binds to the known antibody, forming

antigen antibody complex

Enzyme-labelled specific antibody is added, which will

bind to the antigen antibody complex.
.

Substrate /chromogen is then added.

The enzyme portion of the specific antibody hydrolyzes
the added substrate/chromogen and produce a colored
end-product.
.
 Figure 2: Microtiter plate for ELISA Figure 3: Image showing the microtiter
plate with colour changes seen

LATEX AGGLUTINATION
Latex (Passive) Agglutination

• The latex particles are coated with specific antibody (e.g. virus specific antibody)
• When a specimen containing the virus is mixed with a suspension of the latex
particles coated with the specific antibody, the interaction between antigen and
antibody causes and immediate agglutination of particles, which is visible to the
naked eye as clumping.
CELL CULTURE/VIRAL CULTURE/VIRUS ISOLATION

This is a laboratory technique in which patient samples are inoculated to cell lines
flask or tube (Figure 5), incubated, and then viewed under microscope to look for
cytopathic effect (CPE). CPE is observed when there is destruction of the
monolayer cells of the cell line, and it indicates presence of infecting virus.
Common CPE observed

• Detachment
• Rounding
• Clumping
• Ballooning
• Fusion (syncytium formation)
• Inclusion body formation

Figure 5 : Cell line flask for viral culture

MOLECULAR
Molecular diagnostic methods are based on the detection of nucleic acid
(RNA and DNA) of the microrganism. These methods are highly sensitive and
specific and can be classified into 3 categories:

• Hybridization
• Amplification (eg polymerase chain reaction (PCR))
• Sequencing

Example of molecular method test result is shown in Figure 6.

Figure 6 : Test result for SARS-CoV-2 molecular detection test.

ELECTRON MICROSCOPY (EM)

The electron microscope uses electrons instead of light to visualize small

objects, and in virology study, they are used to study the morphology of viruses.
It is not a routine diagnostic method.

Advantages of EM
Helpful for virus detection that do not grow readily in cell culture.
Helpful to recognize characteristic of new virus morphology
Disadvantages of EM
Labor intensive, insensitive, operator dependent, require high viral titre.

ANTIBODY DETECTION (SEROLOGY)

• The detection of IgM , IgG,

• Detection of rising titres of antibody between acute and convalescent stages
of infection
• Can be done by the following methods.

INDIRECT FLUORESCENT TEST FOR ANTIBODY DETECTION

• Indirect fluorescent tests can be used to measure antibodies in serum.

• When measuring antibody levels, known antigen is employed as a tissue
smear, section, or cell culture on a slide or coverslip.
• This is incubated in serum suspected of containing antibodies
(antiserum)/patient serum.
• The serum is then washed off, leaving only specific antibodies bound to the
antigen.
• These bound antibodies may then be visualized by incubating the smear in
FITC (fluorescent dye)-labelled antiglobulin.
• When the unbound labelled antibody is removed by washing and the slide
examined, the presence of fluorescence indicates that antibody was
present.
ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA)

Immunoassay for antibody

• Known antigen in saline is incubated on microtiter plate and become

absorbed onto the plastic surface.
• Test antibody is added, which binds to the specific antigen, forming antigen
antibody complex.
• Unbound antibodies are washed away.
• Enzyme-labelled anti-human antibody is then added, which will bind to the
antigen antibody complex.
• Unbound enzyme-labeled anti-human antibody is washed away.
• Substrate /chromogen is then added.
• The enzyme portion of anti-human antibody hydrolyzes the added
substrate/chromogen and produce a colored end-product.
• A colour change that can be observed with naked eye indicates positive
result (qualitative) Figure 3.
• The amount of test antibody can be measured by assessing the amount of
colored end-product by optical density scanning of the plate by a reader
(quantitative).
 Known antigen added to the microtiter plate and absorbs to
the well surface.

Test antibody is added, binds to the specific antigen, forming

antigen antibody complex

Enzyme-labelled anti-human antibody is added, which will

bind to the antigen antibody complex.

Substrate /chromogen is then added.

The enzyme portion of anti-human antibody hydrolyzes the
added substrate/chromogen and produce colored end-
product.
complex.

HAEMAGGLUTIONATION INHIBITION

• The procedure is performed in a microtiter plate.

• Some viruses (e.g. influenza) have haemagglutinin molecules.
• When the virus particles are mixed with red blood cells, they cause
agglutination (Figure 7).
• In the presence of specific antibody, haemagglutination is inhibited (Figure 8).
• This test can therefore be used to detect the presence of antibodies to specific
virus virus in a patient’s serum.

o POSITIVE RESULT: A button at the bottom of the well (Figure 9)

o NEGATIVE RESULT: A carpet over the bottom of the well (Figure 9)
 Figure 7

Figure 8

Figure 9
PART B: EXHIBITS AND DISCUSSION
Study the following exhibits and answer the following questions with your respective
lecturers during the practical session.
There are 9 exhibits.

VIROLOGY

For virology, you are provided with Exhibit V1- V9.

Exhibit V1 Viral Transport Medium.

Exhibit V2 Image showing test that was done on a nasopharyngeal swab from
a patient with respiratory symptom
Exhibit V3 Image showing the latex agglutination test for rotavirus
Exhibit V4 Electron micrograph of adenovirus
Exhibit V5 Electron micrograph of rotavirus
Exhibit V6 Microphotograph showing uninoculated cell culture
Exhibit V7 Microphotograph showing cell culture inoculated with HSV virus
Exhibit V8 Microphotograph showing cell culture inoculated with HSV virus
Exhibit V9 Image showing test that was done on a serum sample from a
patient with fever and rash.

EXHIBIT V1: Viral Transport Medium

a. What is the purpose of the viral transport medium?

EXHIBIT V2: Image showing test that was done on a nasopharyngeal swab from a
patient with respiratory symptom

a. State the name of test.

b. Interpret the test result.

EXHIBIT V3: Image showing latex agglutination test for rotavirus

This test was done on a stool sample of a patient presented with diarrhoea.
a. Interpret the test result.
EXHIBIT V4: Electron micrograph of adenovirus

EXHIBIT V5: Electron micrograph of rotavirus

Briefly describe the morphology of the viruses shown in Exhibit V4 and V5.

EXHIBIT V4

EXHIBIT V5
EXHIBIT V6: Uninoculated cell culture

EXHIBIT V7: Microphotograph showing cell culture inoculated with RSV virus

EXHIBIT V8: Microphotograph showing cell culture inoculated with HSV virus
Describe your findings in Exhibit V6, Exhibit V7 and Exhibit V8.

Exhibit V6

Exhibit V7

Exhibit V8
EXHIBIT V9: Image showing test that was done on a serum sample from a patient
with fever and rash.

a. State the name of the test.

b. Interpret the finding of the above test