ASSIGNMENT 1

ADVANCED ANIMAL BIOTECHNOLOGY

TOPIC- CONTAMINATION & APPLICATION OF
ANIMAL CELL CULTURE

SUBMITTED TO:- Ms.ANUBHA SINGH

DATE :-10/10/2022
 Intoduction-
Microbial contamination of cell cultures is a serious and relentless threat to your
research. Invasive mycoplasma, bacteria, and fungi can kill or drastically alter cells in
culture, leading to disastrous results, lost time, and wasted resources. This brochure
provides an insight into the contaminants that are most likely to invade your cultures,
the good practices to avoid them, and the solutions to eliminate them.

While bacterial and fungal contaminations are eventually detected by the naked eye,
mycoplasma and endotoxins remain invisible.

Detection-

Microbial contamination must be detected as early as possible. Detection methods

depend on the nature of the microbe. They include biological assays, PCR,
fluorescence or chemical staining, optical microscopy, turbidimetry, pH measurements,
or simple visual inspection. Bacteria and fungi can usually be identified by optical
microscopy. Their fast growth rate allows their detection by the naked eye as early as
48 hours (i.e. over the weekend), the contaminated cultures appearing turbid or spotty.
Subsequently, identification of these micro-organisms can be performed with testing
kits. Mycoplasma in cell cultures cannot be detected visually, not even by optical
microscopy.
Prevention-

Knowing the sources of microbial contamination is crucial for minimizing the risk to cell
cultures (see below). Although absolute prevention is impossible, you can take various
measures to prevent infection. Firstly, ensure that you are working in a sterile
environment and using proper aseptic technique. Secondly, quarantine any incoming
cell cultures until these have been confirmed free of contamination.

Elimination –

Typically, once invasive microbes are detected in cell cultures it is recommended to

discard the cells and the media. However, some cell cultures are so precious that they
cannot be lost (i.e. stable clone selection, cell lines derived from explanted tissues,
primary cells) and are not available elsewhere (i.e. not yet frozen).

Types of contamination
Cell culture contaminants are divided into chemical and biological contamination and is
a significant problem when working with cell cultures. Contamination can be introduced
in numerous ways that include; through the atmosphere, work surfaces, operator and
solutions such as reagents.

1. Chemical-
Chemical contamination causes a negative effect on the cell culture due to the presence
of a nonliving object that includes; sera, endotoxins, media, storage vessels, water,
incubators and fluorescent lights .

1.Sera: in order to detect impurities, cell culture-based screening programs are used.
Fetal bovine sera is a common serum used and is not usually toxic, but variation in
growth factor and hormone concentrations in different sera can cause contamination.
To avoid contamination, serum free media is recommended.

2.Endotoxins: affects the growth of cell cultures and are present in sera, culture
additives and water. Endotoxins can be quantified by using Limulus Amebocyte
Lysate assay and by working in an aseptic environment, the presence of endotoxins
is reduced.

3.Media: in order to avoid contamination, high quality reagents should be used. Most of
the chemical contamination arises through cell culture media where the water, sera or
reagents contain impurities.

4.Storage vessels: before using glass or plastic bottles it should be washed and
disinfected thoroughly, as previously used solutions may contain traces of organic
compounds and heavy metals that can cause contamination. In the washing process,
residues from the disinfectant, or dry heat sterilization can leave contaminants on the
instruments.

2.Biological-

There are two categories that biological contamination can be divided into depending on
detection; fungi, bacteria and yeasts are easiest to detect, where viruses, mycoplasmas
and other cell types are harder to detect and cause more damage to the cell culture.

1.Fungi and yeasts: as they are quick colonizers and ubiquitous, they are one
of the most common group of contaminants. To detect the presence of yeasts,
the media will become turbid, and fungi will produce a mycelium that will form clumps.

2.BACTERIAL CONTAMINATIONS

Bacteria can easily be mistaken for cellular debris, especially at the early stages of
contamination. Bacteria are a large and ubiquitous group of unicellular micro-organisms.
They are typically a few micrometers in diameter and can have a variety of shapes,
ranging from spheres to rods and spirals. Bacteria are the most commonly encountered
biological contaminants in cell culture. Despite being detectable using a light
microscope, bacteria can easily be mistaken for cellular debris, especially during the
early stages of contamination.

3.Viruses: very small and hard to detect as one cannot visually see the changes they
make to the cell culture. Their small size also makes it challenging to remove the virus
from the media. Even though viruses only affect their host cells, they present a high risk
of infecting laboratory workers.

4.Mycoplasma: the most common contaminant and are highly infectious. They lack a
cell wall, are the smallest self-replicating organism and has fastidious growth
requirements. These characteristics allows the organism to grow at high densities
without showing any visible signs of contamination. They are not benign in the culture
and are able to alter cellular function, metabolism, growth and morphology, and are
resistant to most antibiotics.

Mycoplasma compete with host cells for nutrients and biochemical precursors. As a
consequence, they alter many cell functions, such as cell metabolism and cell growth,
ultimately leading to cell death. A microarray analysis on contaminated cultured human
cells has revealed the severe effects that mycoplasma can have on the expression of
hundreds of genes, including some that encode receptors, ion channels, growth factors,
and oncogenes.

5.Cross contamination by other cell types: increasing problem in today’s laboratories as

cell lines may not come from reputable cell banks or aseptic techniques are not
followed. The most common cross contamination is with the HeLa cell line as once
introduced, they aggressively displace the original cells.

3.Morphology-
1. Fungi/yeasts: eukaryotic multicellular organisms that consist of mycelia that branch to
form hyphae and lack a flagellum. Fungi can grow up to 2-10 μm in diameter. The cell
wall contains chitin, the cell membrane consists of ergosterol and they divide sexually,
asexually, or both. Fungi exists in two forms; hyphae or yeasts. Dimorphic fungi
consist of both yeast and mycelia.

Yeasts produce asexually and are round/oval in shape and closely resemble bacterial
colonies.

2.Bacteria: are ubiquitous, unicellular prokaryotic organism that divides by binary fission
and contains no true nucleus or organelles. The cell wall consists of peptidoglycan
and the cell membrane consists of cholesterol. Bacteria can grow up to 0.2-1.5 μm in
diameter and 3-5 μm in length. Due to the structural difference in the cell wall, bacteria
can either be gram positive or gram negative.

Bacteria are classified into three different shapes; coccus, bacillus and spiral.

3.Viruses: acellular, obligate intracellular parasite that consists of a protein capsid, a

lipid envelope and a nucleic acid genome.Viruses need abhost in order to multiply, and
cannot undergo binary fission or mitosis. Contains no cell wall and reaches a size of 20-
300 nm.

4.Mycoplasma: bacteria that lack a cell wall and range from filamentous to spherical
cells.Reproduces by binary fission and can start replicating at a size of 300nm. Contains
a plasma membrane, circular double stranded DNA and ribosomes, and are able to
survive without oxygen. The most common form of mycoplasmas is coccus, but can
also be found in the form of filaments, spiral and diploform.

4.Detection-
It is important to monitor the cell cultures to avoid the risk of contamination as mutations
can occur, chromosomes can change in number or rearrange and cell characteristics
may be altered.

Minor: includes bacteria, yeast and fungi-

1.Bacteria: identify unique shapes that are different from the original cells.
2. Yeast and fungi: identify colonies located on the surface for the presence of fungi and
budding for yeasts.
3. Media may be cloudy or turbid
4.Indication by change in colour due to change in pH when using phenol red. If the
media turns yellow/orange there is a chance that the media is contaminated.

Major: substances that are difficult to eliminate, such as viruses.

5.Use PCR, ELISA and/or immunostaining techniques

Potentially disastrous; includes mycoplasmas and cross contamination
6. There are two methods to detect mycoplasmas; direct and indirect. Direct culture is a
sensitive and effective method when detecting for mycoplasmas, however, it is time
consuming and is not 100% effective.

5.Prevention (aseptic technique)

Contaminants can come from all sorts of places; including clothes, shoes, mouth and
nose,and hands. Therefore, lab coats and gloves should be worn at all times and in
addition,proper personal equipment should be worn. When entering and exiting the lab,
hands should be washed. Work in a biosafety cabinet when handling sterile objects.
Keep the work area clean and wipe with ethanol before and after use. Before using
media and equipment, look for visible signs of contamination. Always work in an aseptic
manner. To prevent contamination, only one cell culture should be worked with at a
time.

6.Treatment/elimination
When bacteria, fungi or yeasts have caused contamination, the most suitable method is
to discard the culture, reagents and media that were used and decontamination should
only be carried if the cell cultures are vital to the experiment or cannot be replaced, and
this needs to be done under professional supervision and in quarantine. If the
contamination cannot be eradicated, then the culture and materials used with it must be
autoclaved.

7.Conclusion
There are many factors that affect the integrity of cell culture work and therefore the
results of the experiment. Being such a complex and sensitive method of work,
contamination is an issue for most laboratory workers interacting with cell cultures and
is essential that preventative steps are implemented. Through a greater understanding
of the nature of contamination and basic preventative strategies a better controlled
environment can be created to reduce the effects of contamination although it may
never truly be eliminated.
 APPLICATION OF ANIMAL TISSUE CULTURE

Application # 1. Model Systems:

(1) Basic cell biology and biochemistry,

(2) The interactions between disease-causing agents and cells,

(3) The effects of drugs on cells,

(4) The process and triggers for aging, and

Application # 2. Toxicity Testing:

Cultured cells are widely used alone or in conjunction with animal tests to study the
effects of new drugs, cosmetics and chemicals on survival and growth in a wide variety
of cell types. Especially important are liver- and kidney-derived cell cultures.

Application # 3. Cancer Research:

Since both normal cells and cancer cells can be grown in culture, the basic differences
between them can be closely studied. In addition, it is possible, by the use of chemicals,
viruses and radiation, to convert normal cultured cells to cancer causing cells. Thus, the
mechanisms that cause the change can be studied. Cultured cancer cells also serve as
a test system to determine suitable drugs and methods for selectively destroying types
of cancer.

Application # 4. Virology:
One of the earliest and major uses of cell culture is the replication of viruses in cell
cultures (in place of animals) for use in vaccine production. Cell cultures are also widely
used in the clinical detection and isolation of viruses, as well as basic research into how
they grow and infect organisms.

Application # 5. Cell-Based Manufacturing:

While cultured cells can be used to produce many important products, three areas are
generating the most interest. The first is the large-scale production of viruses for use in
vaccine production. These include vaccines for polio, rabies, chicken pox, hepatitis B
and measles.
The second is the large-scale production of cells that have been genetically engineered
to produce proteins that have medicinal or commercial value. These include monoclonal
antibodies, insulin, hormones, etc. The third is the use of cells as replacement tissues
and organs. Artificial skin for use in treating burns and ulcers is the first commercially
available product. However, testing is underway on artificial organs such as pancreas,
liver and kidney.

Application # 6. Genetic Counselling:

Amniocentesis, a diagnostic technique that enables doctors to remove and culture fetal
cells from pregnant women, has given doctors an important tool for the early diagnosis
of fetal disorders. These cells can then be examined for abnormalities in their
chromosomes and genes using karyotyping, chromosome painting and other molecular
techniques.

Application # 7. Genetic Engineering:

The ability to transfect or reprogram cultured cells with new genetic material (DNA and
genes) has provided a major tool to molecular biologists wishing to study the cellular
effects of the expression of these genes (new proteins). These techniques can also be
used to produce these new proteins in large quantity in cultured cells for further study.
Insect cells are widely used as miniature cells factories to express substantial quantities
of proteins that they manufacture after being infected with genetically engineered
baculoviruses.

Application # 8. Gene Therapy:

The ability to genetically engineer cells has also led to their use for gene therapy. Cells
can be removed from a patient lacking a functional gene and the missing or damaged
gene can then be replaced. The cells can be grown for a while in culture and then
replaced into the patient. An alternative approach is to place the missing gene into a
viral vector and then “infect” the patient with the virus in the hope that the missing gene
will then be expressed in the patient’s cells.