General Laboratory Practice

(MLS 501)

N. M. Bunza
Department of Medical Microbiology, School of
Medical Laboratory Sciences,
Usmanu Danfodiyo University, Sokoto.

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 Tissue/Cell Culture
 This is the method of choice routinely employed for
cultivation of viruses.

 Tissue and cell culture consists of cells or tissues obtained

from humans, animals, or plants supplied with necessary
nutrients grown in aseptic conditions.

 This technique is made possible by the development of growth

media for animal cells and by the use of antimicrobial agents
that prevent bacterial and fungal contamination.

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 Cells are grown on glass or a treated plastic surface in
a suitable tissue culture medium for Monolayer or in
flasks as suspension cultures.

 For Monolayer, first growth medium, usually balanced salt

solution are taken and the host tissue or cell is inoculated.

 Cultured cells require a constant supply of nutrients to

sustain healthy growth, and so the media must be
replenished regularly.

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 On incubation the cell divide and spread out on the
glass/plastic surface to form a confluent monolayer. After
growth of cells maintenance medium is added.

 Virus is then inoculated to observe the cytopathic effects

(CPE) and other purposes.

 It is vital to prevent any contamination of the culture,

whether chemical or biological, during both media
replacement and subculturing.

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Below are the kinds of cell culture flasks, plates and dishes

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 Advantages of cell culture
 They are used as substitute hosts to study the pattern of viral
infection.
 Use of animals reduced.

 Widely used method to grow virus for some vaccine production

(e.g., chicken pox, polio, rabies, hepatitis B, and measles).
 In vitro models allow for control of the extracellular environment.

 They are used in study of the effects of toxins and contaminants.

 Cancer research, which requires the study of uncontrolled cell

division in cultures
 They are good tools for testing the potency of drugs 6
 Disadvantages of cell culture
 The process requires sophisticated laboratory and trained
technicians with experience in working on a full time
basis.
 It is nearly impossible to recreate the in vivo environment.

 The process is time consuming.

 Contamination

 Dedifferentiation
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 General Method for Cell Culture
 Tissue like Monkey Kidney, Rabbit Kidney is taken and treated
with proteolytic enzymes and by mechanical shaking, tissues
are dissociated into the component cells.
 The proteolytic enzyme digest the binding material that binds
the cells together in a tissue and results into free cells.
 After treatment with proteolytic enzyme , cells are washed,
counted and suspended in the growth medium.
 Growth medium constitutes all those essential elements
required for the growth of cell 8
 Antibiotics are added into the growth medium to prevent
bacterial contaminants.
 Some indicators like phenol red, neutral red etc. are added
into the growth medium.
 Change in indicator colour in growth medium, indicates the
growth of virus in cell culture.
 In such growth medium cells divides and multiply. Then
these cells are dispensed in bottle or petri plates.
 On incubation, cells divides to form a confluent monolayer
sheet of cells within a week. 9
 Cell Culture Media
 It is the environment provided for the growth of the cells in the
laboratory, similar to those conditions that the cells have been exposed
to in vivo.
 One of the most important factors in animal cell culture is the medium
composition.
 Any successful medium is composed of isotonic, low-molecular-
weight compounds known as basal medium and provides inorganic
salts, an energy source, amino acids, and various supplements.
 The 10 basic components that make up most of the animal cell culture
media are as follows:
 inorganic salts nitrogen source
 energy sources vitamins,
 fat and fat soluble component nucleic acid precursors,
 growth factors and hormones, antibiotics,
 pH and buffering systems, O2 and CO2 concentrations.
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 Types of Cell Culture Media
1. Natural media: Natural media consist of naturally occurring
biological fluids sufficient for the growth and proliferation of animal cells
and tissues. These media useful for promoting cell growth are of the
following three types:

❖ Coagulants or clots: The most commonly used clots are plasma clots,
which have been in use for a long time. Plasma is now commercially
available either in liquid or lyophilized state.

 Biological Fluids: various biological fluids used as culture media e.g ;

amniotic fluid, ascitic and pleural fluid, aqueous humour from eye ,
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insect haemolymph , serum, e.t.c
 Tissue Extract:

 Chick embryo extract is most commonly used tissue extract,

 but bovine embryo extract is also used.

 Other tissue extract that have been used are spleen, liver, bone
narrow, leucocytes, etc .
 Tissue extract can often be substituted by a mixture of amino
acids and certain other organic compounds.
 The natural biological fluids are generally used for organ culture

 For cell cultures, artificial media with or without serum are used.

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2. Artificial media

 The media contains partly or fully defined components that are prepared
artificially by adding several nutrients.

 It contains a balanced salt solution designed for immediate survival of

cells.

 Artificial media supplemented with serum or with suitable formulations

of organic compounds supports prolonged survival of the cell culture.

 The artificial media may be grouped into the following four classes:
 serum-containing media,
 serum-free media,
 chemically defined media, and
 protein-free media.
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 Contaminants That Can Affect Cell
Culture Results
1. Bacteria

2. Endotoxins

3. Organic compounds

4. Ions

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 Types of tissue culture
 Organ cultures:
a. Small bits of organs are used for the cultivation of virus.
b. are mainly done for highly specialized parasites of certain
organs e.g. tracheal ring culture is done for isolation of
coronavirus.
 Explant culture:
a. Fragments of minced tissue can be grown as ‘explants’
embedded in plasma clots
b. is rarely done.
 Cell culture:
a. is mostly used for the identification and cultivation of viruses.
b. Different types of cell cultures are used for different viruses
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1. Primary Cell Culture:
 A primary cell culture is defined as a culture of cells obtained
from the original tissue that have been cultivated in vitro for
the first time, and that have not been subcultured
 These cells are typically slow growing, heterogeneous and
carry all the features of the tissue of their origin.
 Primary cell culture are widely acknowledged as the best cell
culture systems available since they support the widest range
of viruses
 Derived from normal animal or human cells
 Since they are directly obtained from original tissue they have
the same karyotype as the original tissue.
 They are capable of only limited growth in culture and cannot
be maintained in serial culture.
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 Primary cell cultures are used during cultivation of viruses for
vaccine production.
 However, they are very expensive and it is often difficult to obtain
a reliable supply.
 An increase in cell numbers in a primary culture results in
exhaustion of the substrate and nutrients, which can influence
cellular activity and lead to the accumulation of high levels of
toxic metabolites in the culture.
 Primary cells can be classified into two types:
 Anchorage-dependent or adherent cells.
 Anchorage-independent or suspension cells.
 Examples: Monkey kidney cell culture, Human amnion cell
culture, Chick embryo fibroblast, Heterogeneous population of
cell.
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 Advantages and Disadvantages of PCC
 These cultures represent the best experimental models for in
vivo studies.
 They share the same karyotype as the parent cells.
 However, they are difficult to obtain and have limited
lifespans.
 Potential contamination by viruses and bacteria is also a major
disadvantage.

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2. Diploid cell lines (Semi-continuous cell lines):
 These are normal cells derived from animal or human cells.
 cells of single type (fibroblast cells) that can be
subcultivated for limited number of times
 They can be sub-cultured up to 50-100 passages by serial
transfer and the cell strain is lost.
 They retain their original diploid chromosomal number.
 They are used for the isolation of some fastidious viruses
and the production of viral vaccines.

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 Examples: Human embryonic lung strain (HL-8), Rhesus
embryo cell strain (W1-38), skin fibroblasts etc.

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3. Continuous cell lines (Heteroploid cultures):
 These are the cells of single type derived from cancer
cells.
 These cells divide rapidly with a generation time of 12–
14 hours
 They can be serially cultured indefinitely
 They can be maintained either by serial subculture or
by storing in deep freeze at -70°c.

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 Due to derivation from cancer cells, they are not useful for
vaccine production

 Its importance lies in the identification and cultivation of the

virus.

 Examples: HeLa (Human Carcinoma of cervix cell line), HEP-

2 (Human Epithelioma of larynx cell line), Vero (Vervet
monkey) kidney cell lines, BHK-21 (Baby Hamster Kidney cell
line), LLC-MK2, MDCK.

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 Detection of virus growth in cell cultures
Viral growth in cell culture can be detected by the following
methods:
1. Cytopathic Effects (CPE):
 morphological changes in cultured cells, seen under microscope
 The infecting virus causes lysis of the host cell or the cell dies
without lysis due to an inability to reproduce
 Viruses causing CPE are called cytopathogenic virus
 CPE helps in the presumptive identification of viruses

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 Many CPEs can be seen in unfixed, unstained cells under the
low power of an optical microscope, with the condenser down
and the iris diaphragm partly closed.

 However, with some CPEs, the cells must be fixed and stained
then viewed under light microscopy.

 CPE include cell lysis or necrosis, inclusion bodies formation,

giant cell formation, and cytoplasmic vacuolization.

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 Types of Cytopathic Effects

a) Total destruction

b) Subtotal destruction
c) Focal degeneration

d) Swelling and clumping

e) Foamy degeneration

f) Syncytium

g) Inclusion bodies
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2. Metabolic Inhibition:
 The normal cells produce acid during normal metabolism.
 However, virus infected cells fail to produce acid due to
inhibition of normal cellular metabolism.
 This can be detected by use of phenol red indicator in cell
culture.
 If the virus grows in cell culture, acid production will be
stopped and phenol red will not show colour change.

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3. Haemadsorption:
 Haemagglutination is the phenomenon of clumping of
RBCs. Viruses such as mumps, measles, influenza and
parainfluenza can able to agglutinate the RBCs.
 Their presence can be indicated by addition of guinea pig
erythrocytes to the culture.
 If the viruses are cultivated in the cell culture, the
erythrocytes will adsorb onto the cell surface also called
‘haemadsorption.

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4. Immunofluorescense:

 Immunofluorescence (IF) technique is widely used for the

rapid diagnosis of viral infections by the detection of virus
antigen in clinical specimens, as well as the detection of
virus-specific antibody.

 The technique makes use of a fluorescent- labelled antibody

to stain specimens containing specific virus antigens, so that
the stained cells fluoresce under UV illumination.

 Fluorescent dye such as fluorescein isothioacynate and

rhodamine are generally used to tag with antibodies.

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 Its very useful in testing for rabies virus in clinical specimen
within few hours with 100% accuracy.

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5. Interference:

 growth of a non-cytopathogenic virus can be tested by

inoculating a known cytopathogenic virus

 growth of first virus will inhibit the infection by second

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