TOPIC 5

VIROLOGY LABORATORY DIAGNOSES

INTRODUCTION:
Although this course is lecture only, we must be familiar with the laboratory
procedures like specimen collection, how to culture the specimen, and different
laboratory examinations and realizing that its category is Biosafety level 4.

OBJECTIVES:
At the end of the topic the students will be able to:
A. discuss the methods of specimen collection;
B. enumerate some of the transport media and their principles;
C. explain the different types of tissue culturing; and
D. discuss the different testing methods for the presence of viruses.

PRETEST:
____________ 1. The specimen collected at the base of the lesions in chicken pox
____________ 2. The specimen collected to detect the presence of virus at the
convalescent stage of disease
____________ 3. Transport media is needed if the specimen is blood. (True/False).
____________ 4. The change in the morphology of the cells due to virus infection
____________ 5. The cells that came from the African green monkey used in
culturing to multiply the cells to be used in inoculation of virus for preparing vaccine
____________ 6. The fixative is needed before performing microscopy in the cells
(True or False
____________ 7. The technique used to insert foreign nucleic acid (DNA or RNA)
into a cell, typically with the intention of altering the properties of the cell.
____________ 8. The substance present as transport media for specimen collected
as swab.
____________ 9. The process of cell culture by transferring them to a new vessel
with fresh growth medium to provide more room for continued growth
____________ 10. The special media formulated to preserve a specimen and
minimize bacterial overgrowth from the time of collection to the time it is received at
the laboratory to be processed.

Collection of Specimens:
1. Specimens should be taken as early as possible in the disease. Since viral
shedding is usually greatest during early stages of infection. The sensitivity of
viral culture may decrease 3 days after acute onset of symptoms.
2. Take sterile specimens if possible; if it is NOT possible, do not contaminate
the specimen further; collect aseptically.
3. Fluid materials (throat washings, CSF, stools , tissues) should be placed in
sterile containers. Stools should be shipped in ice or preferably frozen in dry
ice. Throat washings should be taken using nutrient broth or sterile saline.
4. Dry materials such as swabs should be put in sterile containers which should
contain a small quantity of fluid, such as nonimmune serum or nutrient broth.
5. Vesicle fluid should be drawn into sterile capillaries and the ends of the tubes
sealed with sterile paraffin, beeswax, or similar material.
Vesicle or blister - a thin-walled sac filled with a fluid, usually clear and
small. Vesicle is an important term used to describe the appearance of many
rashes that typically consist of or begin with tiny-to-small fluid-filled blisters
 23

6. Scabs and crusts should be placed in sterile bottles.

7. Blood for viral culture should be sent citrated or heparinized since virus may
be present in the cellular elements but absent or nearly so in the serum.
Indeed, with the use of immunofluorescent techniques, the putative virus may
be identified with the leukocytes. Such specimen must be examined as soon
as possible after collection and should NOT be frozen.
8. Blood for serologic studies may be sent as serum; it is advantageous to send
a sample during the early part of the illness and one during the convalescent
stage.
9. Tissues taken at autopsy from brain, liver, lung, or other organs, depending
on the suspected disease should be stored at -20oC or lower until sections
have indicated suspicion of a particular viral disease, or until viral cultures are
made. Five to 20 grams of each tissue is sufficient.

Figure Stages of diseases, related to specimen collection

APPROPRIATE SPECIMENS FOR MAXIMUM RECOVERY-

1. Specimens for viral isolation should be collected from affected site :
2. Respiratory mucosa for viral diagnosis of respiratory infections
3. Aspirates or surface swabs for lesions - Aspirated secretions preferred
Swabs – easier; must be made of Dacron or rayon.
- Calcium alginate swabs inhibit the replication of some viruses and can
interfere w/ nucleic acid amplification test; keep swabs moist.
- Tissue samples must be kept moist
4. Stool exam if intestinal mucosa
5. Vesicle or blisters
6. Blood ( buffy coat), CSF If systemic, congenital, or generalized disease –CSF
7. Portal of entry (ORAL, RESPIRATORY TRACT)
8. URINE OR stool- Exit –
Example: Enteroviruses – can cause resp. infection, aseptic meningitis, can
be isolated from urine
 24

purpose of calcium alginate:

1. to provide homeostasis, calcium alginate is more commonly thought of as
the dressing that can absorb 20 times its weight in exudate, soak up loose
debris from the wound bed, provide an optimal environment for healing, and
provide a painless dressing change
2. When placed on a wound, the sodium and calcium ions interact with serum to
form a hydrophilic gel. Alginates provide a moist wound environment and
can prevent microbial contamination

TRANSPORT MEDIA:
Transport media are special media formulated to preserve a specimen
and minimize bacterial overgrowth from the time of collection to the time it is
received at the laboratory to be processed. Depending on the type of organisms
suspected in the sample, transport media may vary.
a. Antibacterial & Antifungal agents – added in transport systems to inhibit
contamination of bacteria & fungi
b. respiratory swab, tissue samples - are collected w/ viral transport
media (may dry)
c. blood , bone marrow, CSF, amniotic fluid, urine, pericardial fluid, pleural
fluid – collected w/o viral transport media (Fluids)
d. transport container MUST be : unbreakable and able to w/stand freezing
and thawing

Transport media:
1. Saline- added to sterile containers to keep tissues from drying out
2. Trypticase soy broth-buffered isotonic solution w/ some type of protein, such
as albumin, gelatin or serum to protect less stable viruses
3. Leibovitz-Emory,
4. Hank’s BSS
5. Stuarts’s medium, Amies medium – for both bacteria and viruses

Hanks' Balanced Salt Solution :

a. (HBSS) is composed of inorganic salts and supplemented with glucose. The
solution may be used to wash cells and tissue and to maintain cells in a viable
state.
b. provide the cells with water and inorganic ions
c. The solution is buffered with phosphate and maintains a physiological pH and
osmotic pressure

How to prepare Hank's BSS?

1. Prepare 800 mL of distilled water in a suitable container.
2. Add 8 g of NaCl to the solution.
3. Add 400 mg of KCl to the solution.
4. Add 140 mg of CaCl2 to the solution.
5. Add 100 mg of MgSO4-7H2O to the solution.
6. Add 100 mg of MgCl2-6H2O to the solution.
7. Add 60 mg of Na2HPO4-2H2O to the solution.
8. Add 60 mg of KH2PO4 to the solution.
9. Add 1 g of D-Glucose (Dextrose) to the solution.
10. Add 350 mg of NaHCO3 to the solution.
11. Add distilled water until volume is 1 L.
 25

Amies medium:
a. Amies medium - improved transport medium, containing charcoal to
prolong the viability of pathogenic organisms. It is a semisolid media
recommended for use in qualitative procedures for the transport of clinical
swab specimens to the laboratory. (5 Mar 2019)
b. To preserve microbiological specimens especially throat, vaginal, and wound
swab samples
c. Routinely used

LEM:
a. a modified Leibovitz-Emory medium (LEM), viral transport medium in which
agarose was used instead of agar,  
b. LEM - transport media for Herpes Simplex Virus Types
c. Agarose - natural polymer prepared from seaweed (red algae) and consists
of the D-galactose and 3,6-anhydro-L-galactose repeating units
Agarose gel electrophoresis - used to resolve  DNA fragments on the basis
of their molecular weight. Smaller fragments migrate faster than larger ones;
the distance migrated on the gel varies inversely with the logarithm of the
molecular weight

STUART TRANSPORT MEDIUM:

a. a semisolid medium used in the transport and preservation of biological
specimens for the cultivation of diverse organisms such as gonococci,
streptococci, Enterobacteriaceae, etc. It is essentially non-nutritive and
contains sodium thioglycollate to retard oxidation.(24 Oct 2019)
b. nonnutritional medium which maintains the viability of organisms without
significant multiplication. The small agar content provides a semisolid
consistency which prevents oxidation and drying during transport. Methylene
blue is the oxidation-reduction indicator
 26

Culture immediately viral specimens:

1. some viruses like RSV, respiratory syncytial virus – more difficult to recover
even few hours after collection (very sensitive)
syncytia – giant multinucleated cells formed from cell fusion as a result of viral
infection
2. if cannot be processed immediately after collection (1-3 days) – store at 4oC
3. do NOT freeze; unless significant delay of more than 4 days, at -70oC
4. should NOT be stored at -20oC – will facilitate formation of ice crystals that
disrupts host cells and results in significant loss of viral viability

viable – developed, live successfully

Major Methods in diagnostic Virology:

1. Direct detection of the virus in clinical specimen
2. Nucleic acid-based detection
3. Isolation of viruses in cell cultures
4. Serologic assays to detect antibodies to virus

Direct detection of the virus in clinical specimens – NOT as sensitive as culture

method but can offer quick results to allow for rapid therapy; but of valuable
assistance to lab. Scientist; viral detection allows clinicians to decide about therapy &
infection control measures, hospitalization; virology results might be available before
routine bacteriology culture results.
1.a. Microscopy – except for poxviruses, individual virus particles are too small to be
seen by bright-field microscopy. Electron microscopy for greater magnification to
detect virions but expensive, NOT very sensitive, can detect noncultivatable viruses
such as NORWALK viruses in stool filtrates.
 CPE ,Cytopathic effect – virus produce visual changes in infected cells as
detected in cell scrapings from infected site.

Tzanck smear – can detect Cowdry type A from herpes simplex virus , HSV and
varicella-zoster virus VZV lesions
dermatopathology, the Tzanck test, also Tzanck smear, is scraping of an ulcer
base to look for Tzanck cells; sometimes also called the chickenpox skin test
 tzanck smear, which is used to determine whether skin lesions are caused by a
herpes virus, a blister is scraped
the examination of fluid from a bullous lesion for Tzanck cells (altered epithelial
cells, rounded and devoid of intercellular attachments)
Pap smear can reveal human papillomavirus, HPV – associated w/ koilocytosis
Koilocytosis – squamous cells w/ an enlarged nucleus surrounded by a
nonstaining halo
Rabies – detecting negri bodies (eosinophilic cytoplasmic inclusions in neurons)

1.b Immunofluorescence – direct fluorescent antibody(DFA) test – cells from a

patient are fixed to a microscope slide and fluorescence labeled antibodies are
added. If viral antigens are present, the labeled antibody will bind and fluorescence
will be seen microscopically
  DFA (Direct Fluorescent Antibody) assays –
Respiratory specimen - for adenovirus, influenza viruses A & B, measles,
parainfluenza viruses 1 through 4, RSV (resp. syncytial virus),
Cutaneous lesion material - for HSV-1(herpes simplex), HSV-2, & VZV (varicella-
zoster)
From blood - for CMV (cytomegalovirus)
 
 27

1.c Enzyme Immunoassays – many EIA test for viral detection are commercially
available; packaged as microtiter plate assays
- Can detect RSV and influenza A,(respiratory) ,HBV (hepatitis B virus)and HIV-
1 from serum and plasma, HSV from cutaneous lesions and conjunctival swab
  - Other tests, single-test platforms w/ + specimen - detected by calorimetric
changes or optical density changes on membrane or silicon surfaces – for RSV,
influenza viruses A & B from resp. specimen, rotavirus & enteric adenovirus from
rectal swab, & West Nile virus WNV from serum; IF test – negative results are
confirmed by cell culture, gene amplification
 - - EIA tests – often less sensitive than cell cultures or IF test so negative results
are confirmed with cell culture, gene amplification or IF or Nucleic acid-based tests

2. Nucleic acid-based detection

– advantage: faster turn around time TAT, better cell sensitivity than cell cultures and
DFA(direct fluorescent antibody), can be quantitative, and can detect virus
unculturable by cell culture. EX. Norovirus, hepatitis viruses, can detect multiple
viruses simultaneously
a. PCR assay; hybridization assay; branched DNA
3. Isolation of virus in cell cultures
3 methods of isolation in diagnostic virology:
a. Cell culture
b. Animal inoculation
c. Embryonated eggs
 Cell culture (most common) – culture of cells in vitro; cells are NOT
organized into a tissue
Animal inoculation – extremely costly; used in Ref. Lab. or research lab;
identified w/in 24 hr of inoculation
Ex. coxsackie virus require suckling mice for isolation of virus
Embryonated eggs – rarely used; Ex. influenza virus is enhanced but easily
accomplished in cell culture

Figure CPE
 28

Microscopic examination – some viruses are visible in cells as elementary bodies

or as inclusion bodies. May use some special stains. Fixation is important to
demonstrate inclusion bodies.
a. Alkaline or neutral fixatives (neutral formalin or Osmium tetrachloride –
inclusion bodies stain poorly
b. Fixative with acetic acid (Zenker’s, Bouin’s, Carnoy’s) – inclusion bodies
shrink, better seen in stained preparation.
-Carnoy's solution is a fixative composed of 60% ethanol, 30% chloroform
and 10% glacial acetic acid, 1 gram of ferric chloride. Carnoy's solution is also
the name of a different fixation composed of ethanol and glacial acetic acid
(3:1).
-Zenker is usually made with 50g of mercuric chloride, 25g of potassium
dichromate, 10g of sodium sulfate (decahydrate) and distilled water to
complete 1000 ml. Before use, 5 ml glacial acetic acid is added to 100 ml of
the solution. Both the stock solution and the complete Zenker fixative are
stable for many years

Inclusion bodies typically represent sites of viral multiplication in a

bacterium or a eukaryotic cell and usually consist of viral capsid proteins;
have a higher density than many other cell components but are porous.

Figure Perivascular cuffing or inflammation around a blood vessel.

Perivascular inflammatory cell infiltrates in hematoxylin & eosin
stained brain tissue. (100x Magnification)

Figure Negri body in infected neuron

 29

VIRAL DIAGNOSIS:
1.Microscopy:
a. Bright field/Phase-contrast
–ID virus-infected cells (inclusion bodies/unusual morphology)
b. Inverted phase-contrast –detects cytopathic effects (shell vial technique)
CPE - structural changes in a host cell resulting from viral infection. CPE occurs
when the infecting virus causes lysis (dissolution)
examples are rounding of the infected cell, fusion with adjacent cells to form a
syncytia (polykaryocytes), and the appearance of nuclear or cytoplasmic
inclusion bodies.
c. Electron microscopy –viral morphology; use
negative/gold/silver/phosphotungstic stain
2. Nucleic acid-based detection –PCR, SB (RNA), NB (DNA)
3. Direct viral antigen detection –IF, EIA/ELISA, Latex aggn
4. Serological antibody reactions - IF, EIA/ELISA, Latex aggn
5. Virus Isolation/Culture
a. Embryonated eggs – may be used for influenza/pox/herpes (pock
formation)
 30

b. Animal inoculation –expensive; used by reference or research labs

(animal death)

c. Tissue/cell cultures –most commonly used (CPE, HAI/HI, serology,

PCR/hybridization)

 
Cell culture refers to the removal of cells from an animal or plant and their
subsequent growth in a favorable artificial environment. ... At this stage, the cells
have to be subcultured (i.e., passaged) by transferring them to a new vessel with
fresh growth medium to provide more room for continued growth.

TYPES OF CELL CULTURE:

1. Primary cell culture/Tissue cultures –harvested directly from an organ/animal
2. Semi-continuous –up to 50 passages/doublings
3. Continuous –derived from cancer cells; passed indefinitely

Tissue Culture – or organ culture:

Primary Cell cultures – from tissue removed from an animal
a. Tissue finely minced and treated w/ an enzyme such as trypsin to dispense
individual cells further.
b. The cells are then seeded onto a surface to form a monolayer such as in a
flask or test tube.
c. w/ primary cell lines, only minimal cell division occur
d. To maintain the cell viability, they must periodically be removed from the
surface, diluted, and placed into a new container – the process known as
splitting or passaging ,
Ex. using primary monkey kidney cells , PMK
• Primary cells – can only be passaged a few times before new cells must be
obtained
 31

• finite cell culture can divide but limited to 50 generations – diploid, 2 copies
of each chromosomes . a normal genetic make up for eukaryocyte (w/
nucleus)
- HNL, human neonatal lung – ex. of std finite culture for diagnostic virology
 
Continuous cell culture – capable of infinite passage and heteroploid (have an
abnormal and variable number of chromosomes that is NOT multiple of the normal
haploid number. HEp2 (derived from a human laryngeal epithelial carcinoma)
A549 – (derived from human lung carcinoma)
Continuous cell lines are subcultured indefinitely in glass, plastic surfaces, or
suspensions as they are obtained usually from carcinogenic cells.

Cardioviruses replicate in primary or continuous cell lines originating from a variety of

species, including murine (rats or mice), bovine, porcine (pig), human, primate,
guinea pig, and hamster. Baby hamster kidney (BHK-21) and Vero cells are most
commonly used. The virus also replicates in baby mice and chicken embryos and is
pathogenic to many laboratory animals.

Continuous immortalized cell lines are comprised of a single cell type that can be
serially propagated in culture either for a limited number of cell divisions
(approximately thirty) or otherwise indefinitely. Cell lines of a finite life are usually
diploid and maintain some degree of differentiation.

Vero cells are a lineage of cells used in cell cultures. The 'Vero' lineage (cell line)
was isolated from kidney epithelial cells extracted from an African green monkey key
(Chlorocebus sp.; formerly called Cercopithecus aethiops sabaeus, this group of
monkeys has been split into several different species).The lineage was developed
on 27 March 1962, by Yasumura and Kawakita at the Chiba University in Chiba,
Japan; used to grow certain viruses (e.g., poliovirus and influenza)
The cells are amplified through subcultures in TC-flasks using animal origin-free
trypsin-like enzyme. Vero cells are recognized by the World Health Organization (WHO)
and Chinese Pharmacopoeia in producing vaccines (Sinopharm, use in Dubai, KSA),(
2021)

TC Flask-Treated Cell Culture Flasks

Sinopharm (Vero Cell)- Inactivated, COVID-19 vaccine

HeLa cells as the first immortal human cell line in 1952 (Gey et al., 1952),
continuous cell lines have become widely used as indispensable and inexpensive
tools for basic biological research, chemical metabolism and toxicity tests, and
production of biological compounds such as vaccines,

Continuous cell lines are maintained either by the serial subculture or by storing in
deep freeze at -70°C.

Continuous Cell Line

1. treatment with chemical mutagens,
2. infection with tumorigenic viruses, or.
3. transfection with oncogenes.

Transfection, technique used to insert foreign nucleic acid (DNA or RNA) into a cell,
typically with the intention of altering the properties of the cell.
 32

Figure The African green monkey, Cercopithecus aethiops sabaeus,

source of vero cells

Cell culture media generally comprise an appropriate source of energy and

compounds which regulate the cell cycle. A typical culture medium is composed of a
complement of amino acids, vitamins, inorganic salts, glucose, and serum as a
source of growth factors, hormones, and attachment factors; placement into an
artificial environment conducive to their survival and/or proliferation; and incubator
that maintains correct pH and osmolality;  growth medium or culture medium is a
liquid or gel
DMEM: Endothelium, fetal alveolar epithelial
IMDM: Bone marrow, hematopoietic progenitor ...
Media: Tissue or cell line
MEM: Chick embryo fibroblast, CHO cells, emb...
DMEM – Dulbecco’s Modified Eagle Medium widely used basal medium for
supporting the growth of many different mammalian cells. Cells successfully cultured
in DMEM include primary fibroblasts, neurons, glial cells, HUVECs, and smooth
muscle cells, as well as cell lines such as HeLa, 293, Cos-7, and PC-12, Antibody
and cell culture
IMDM -Gibco Iscove's Modified Dulbecco's Media are a highly enriched synthetic
media well suited for rapidly proliferating, high-density cell cultures, including Jurkat,
COS-7, and macrophage cells.
MEM - Minimum Essential Media ... Gibco Minimum Essential Media (MEM) is
patterned after Eagle's Media and is well suited for the growth of a broad spectrum;
most commonly used of all cell culture media. MEM can be used with a variety of
suspension and adherent mammalian cells, including HeLa, BHK-21, 293, HEP-2,
HT-1080, MCF-7, fibroblasts, and primary rat astrocytes.
Types of Cell Culture Media
Animal cells can be cultured either using a completely natural medium or an
artificial/synthetic medium along with some natural products.

Table 1. Types of natural and artificial media in cell culture.

MEDIA TYPE EXAMPLES USES

Natural media Biological Fluids plasma, serum, lymph, human
 33

placental cord serum, amniotic fluid

Extract of liver, spleen, tumors,
leucocytes and bone marrow, extract
Tissue Extracts
of the bovine embryo and chick
embryo
Clots coagulants or plasma clots
Form the basis of
Balanced salt solutions PBS, DPBS, HBSS, EBSS
complex media
Primary and
Basal media MEM DMEM
Artificial media diploid culture
Supports a wide
Complex media RPMI-1640, IMDM range of
mammalian cells
https://www.labome.com/method/Cell-Culture-Media-Review.html

Artificial media

Artificial or synthetic media are prepared by adding nutrients (both organic and
inorganic), vitamins, salts, O2 and CO2 gas phases, serum proteins, carbohydrates,
cofactors [1]. Different artificial media have been devised to serve one or more of the
following purposes: 1) immediate survival (a balanced salt solution, with specific pH and
osmotic pressure); 2) prolonged survival (a balanced salt solution supplemented with
various formulation of organic compounds and/or serum); 3) indefinite growth; 4)
specialized functions.

Table 2. advantages and disadvantages of using serum in the media, cell culture

ADVANTAGES OF SERUM IN MEDIA DISADVANTAGES OF SERUM IN MEDIA

Serum contains various growth factors and
Lack of uniformity in the composition of
hormones which stimulates cell growth and
serum
functions.
Testing needs to be done to maintain the
Helps in the attachment of cells
quality of each batch before using
May contain some of the growth inhibiting
Acts as a spreading factor
factors
Acts as a buffering agent which helps in
Increase the risk of contamination
maintaining the pH of the culture media
Presence of serum in media may interfere
Functions as a binding protein with the purification and isolation of cell
culture products
Minimizes mechanical damages or
damages caused by viscosity

. Centrifugation-Enhanced Shell Vial Culture:

a. simple method, more rapidly identifies viruses than a traditional cell culture
b. Cells are grown on a round coverslip in a shell vial. A shell vial is a small
round, flat-bottom tube, generally w/ a screw cap.
 34

c. The shell vial is inoculated w/ the clinical sample and then centrifuged to
promote viral absorption.
d. The shell vial is incubated for 24 – 48 hours , after w/c the coverslip is
removed and the IF technique performed (modification – to use flat-bottom
microtiter plates).

https://www.google.com/url?sa=i&url=

Figure Shell vial culture

CELL CULTURE PREPARATION:

1. 35°C –temperature used for dissociation of cells
2. After distribution of cells, tubes are incubated at 36-37°C until confluence
(join)of cells
3. 35°C –incubation for isolation of viruses (33°C for rhinoviruses and
myxoviruses)
4. Round bottom tubes –read under conventional microscopes
5. Shell vial –contains a monolayer of cells; read under inverted microscopes
6. Roller drum –used for incubating tubes with gentle agitation

Principles of Serologic Diagnosis:

1. Neutralization – to employ dilutions of serum and aliquots (portion) of virus, to
determine that titer of serum sufficient to prevent the growth of the virus.
2. Cytopathic neutralization – to discover the maximum dilution of serum to
prevent cytopathic change in tissue culture.
3. Hemagglutination and hemadsorption inhibition – dilution of serum is
necessary to prevent the phenomena.
4. Complement fixation – same as mentioned
5. Metabolic inhibition – uses living cells in a medium with a pH indicator.
Normally, as cells metabolize, the pH falls because of excretion of waste
products. But cells infected by virus will generally cause a rise in pH. Immune
serum in dilution is added to the system in order to discover the maximal
dilution necessary to neutralize the change.
6. Gel diffusion techniques may be used
 35

Serologic assays: has limited info:

1. to detect antiviral antibodies – IgM (Immunoglobulin M)
2. First- measure host response rather than directly detecting the virus
3. Second – antibody-producing capabilities of human hosts vary widely;
immunocompromised individuals may NOT produce enough antibodies to be
detected
4. Third – the antibody level does NOT necessarily correlate w/ the acuteness or
activity level of the infection
5. Result: acute/convalescent
Convalescence – recovering health and strength gradually after
sickness or weakness
6. Presence of IgG - indicates an acute infection
7. Interpretation is also difficult because of passive transfer of antibodies, such
as transplacental or transfusion transmission

Cytopathic effect (CPE) on Cell Cultures

1. For HSV – grows rapidly on many different cell lines w/in 24 hr; produces
focal CPE in w/c adjacent cells become infected , and plaques , or clusters of
infected cells
2. HSV - grow in rabbit kidney
3. CMV produces HSV-like CPE but grows more slowly and ONLY on diploid
fibroblast
4. VZV grows on several types of cells, including diploid fibroblast, A549, and
Vero cells
5. Enteroviruses produce small, round, infected cells that spread diffusely on
PMK, diploid fibroblast, human embryonal rhabdomyosarcoma (RD) and
A549 and PMK. Rounding may be diffused or focal, appearing like cluster of
grapes
6. Resp. viruses may NOT produce characteristic CPE
7. RSV can produce classic syncytial formation HEp2 or even MKC cells
8. Influenza virus usually inoculated onto PMK, LLC-MK2 (rhesus monkey
kidney), or MDCK (canine kidney) cells do NOT produce CPE.
9. Hemagglutination or hemadsorption test is DONE to detect these viruses.
a. Cells infected w/ influenza virus express a viral hemagglutinin (H) protein on
their surface that binds RBC
b. In hemadsorption test – a suspension of RBC is added to the infected cell
monolayer - - If Influenza virus is present, RBC will “stick” or adsorb to the infected
cells
c. In Hemagglutination assay – supernatant from from the infected monolayer
containing influenza virus is mixed w/ suspension of RBC - - will produce RBC
agglutination because influenza virus has H protein on their surface

Fluorescent antibody stains that detect viral antigen like those used
directly in clinical specimens, can also be used to screen cell cultures before
the final negative result is reported. (Besides IF, EIA and nucleic acid
amplification assays can be applied to detect and identify viruses in cell
cultures.)

Indications for Serologic Assays:

1. Dx of infections w/ nonculturable agents such as hepatitis viruses
2. Determination of immune status in regard to rubella, measles, VZV, HAV,
HBV
3. Monitoring of patients who have immunosuppression or have had transplants
4. Epidemiologic or prevalence studies
 36

POSTTEST:
____________ 1. The specimen collected at the base of the lesions in chicken pox
____________ 2. The specimen collected to detect the presence of virus at the
convalescent stage of disease
____________ 3. Transport media is needed if the specimen is blood. (True/False).
____________ 4. The change in the morphology of the cells due to virus infection
____________ 5. The cells that came from the African green monkey used in
culturing to multiply the cells to be used in inoculation of virus for preparing vaccine
____________ 6. The fixative is needed before performing microscopy in the cells
(True or False)
____________ 7. The technique used to insert foreign nucleic acid (DNA or RNA)
into a cell, typically with the intention of altering the properties of the cell.
____________ 8. The substance present as transport media for specimen collected
as swab.
____________ 9. The process of cell culture by transferring them to a new vessel
with fresh growth medium to provide more room for continued growth
____________ 10. The special media formulated to preserve a specimen and
minimize bacterial overgrowth from the time of collection to the time it is received at
the laboratory to be processed.

ANSWER KEY:
1.Tzanck smear
2. Serum
3. False
4. Cytopathic effect
5. Vero cells
6. True
7. Transfection
8. Calcium alginate
9. Passaging or splitting
10. Transport media

REFERENCES:

https://www.leicabiosystems.com › specimen reparation 101

https://www.hmpgloballearningnetwork.com › site › content The Wonder of Calcium

Alginate | 93039

https://www.mantacc.com › transport-medium-for-bacter . . .Popular Transport

Medium For Bacterial And Viral Sample at home ...

https://www.sciencellonline.com › hank-s-balanced-salt-s Hank's Balanced Salt

Solution | ScienCell Research ...

https://www.aatbio.com/resources/buffer-preparations-and-recipes/hbss-hanks-
balanced-salt-solution

https://microbenotes.com › Culture Media Amies Transport Medium | Culture Media

|            Microbe Notes
 37

https://www.ncbi.nlm.nih.gov › articles › PMC376331 Transport Media for Herpes

Simplex Virus Types ... - NCBI - NIH

https://www.sciencedirect.com › topics › chemistry › agarose

Stuart Transport Medium – Condalab https://www.condalab.com › int

https://www.britannica.com › ... › Conditions & Diseases Cytopathic effect |

microbiology | Britannica

https://www.sciencedirect.com/topics/engineering/continuous-cell-line

https://www.who.int/publications/m/item/sinopharm-vero-cell---inactivated-covid-19-
vaccine 23 May 2021

https://microbeonline.com/continuous-cell-line/

https://www.thermofisher.com › gibco-cell-culture-basics Introduction to Cell Culture |

Thermo Fisher Scientific - US

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Culture Media - Thermo Fisher ...

https://microbeonline.com/continuous-cell-line/22 May 2021.

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