Name : Meghana Padia

Roll no .: 143
Semester : III
Course : Microbiology
K.J. Somaiya College of Science and Commerce,
Vidyavihar,
Mumbai -400077.

ANIMAL TISSUE CULTURE LABORATORY & EQUIPMENT

Cell culture has become one of the major tools used in the life sciences. Tissue culture is
the general term for the removal of tissue or organs from an animal and their subsequent placement
into an artificial environment. This environment usually consists of a suitable glass or plastic culture
vessel containing a liquid or semisolid medium that supplies the nutrients essential or survival and
growth. Animal tissue/ cell culture is the very complex process in witch there all cells grown in in
vitro controlled conditions. It was Jolly, who (1903) showed for the first time that the cells can
survive and divide in vitro. Ross Harrison, (1907) was able to show the development of nerve fibres
from frog embryo tissue, cultured in a blood clot. Later, Alexis Carriel (1912) used tissue and
embryo extracts as cultural media to keep the fragments of chick embryo heart alive. Chinese
Hamster Ovary (CHO) cell lines were developed during 1980s.
Recombinant erythropoietin was produced on CHO cell lines by AMGEN (U.S.A.). It is
used to prevent anaemia in patients with kidney failure who require dialysis. After this discovery,
the Food and Drug. A lot of progress has been also made in the area of stem cell technology which
will have their use in the possible replacement of damaged and dead cells. In 1996, Wilmut and coworkers successfully produced a transgenic sheep named Dolly through nuclear transfer technique.
Thereafter, many such animals (like sheep, goat, pigs, fishes, birds etc.) were produced. Growing
tissues of living organism outside the body is made possible in an appropriate culture medium,
containing mixture of nutrient either in solid or liquid form. Commonly used animal cell lines are
IMR-90, Hep - 2, MCF -7 etc for different research are such as cancer research, toxicity testing,
model system, virology , genetic engineering and many more.

Animal tissue culture has special requirements thus, it has to design in that way it lab should
have proper environment and it should also have all the equipment which are needed. Laboratory
setup includes Laminar Air Flow (LAF), Centrifuges, CO 2 Incubators , Inverted microscopes,
freezer and many more.
1. LAMINAR AIR FLOW
It is also known as Microbiological Safety Cabinets. It is one of the important equipment for animal
tissue culture. Cabinet provides clean and sterile environment to the operator to carried out work.
Protection provided with HEPA filters (High efficiency particulate air). These are of two types
vertical and horizontal. Vertical LAF is effective for harmful organisms and horizontal LAF is
designed in such a way that the filtered air flow directly at the operator hence they are not useful
for harmful organisms but are the best for cultures. Whereas in vertical LAF air blows down from
the top of the cabinet. Both types of hoods have continuous displacement of air that passes through
a HEPA (High efficiency particle 0.3 micrometer) filter use for the purpose of removes
particulates from the air. The hoods are equipped with a short-wave UV light that can be turned on
for a few minutes to sterilize the surfaces of the hood, but be aware that only exposed surfaces will
be accessible to the UV light. Do not put your hands or face near the hood when the UV light is on
as the short wave light can cause skin and eye damage. The hoods should be turned on about 10 - 20
minutes before being used. Significance of LAF is it can maintain a working area devoid of
contaminants.
2. CARBON DIOXIDE INCUBATORS
Cell cultures require a strictly controlled environment in which to grow. Specialist
incubators are used routinely to provide the correct growth conditions, such as temperature, degree
of humidity and CO2 levels in a controlled and stable manner. Generally they can be set to run at
temperatures in the range 28C (for insect cell lines) to 37C (for mammalian cell lines) and set to
provide CO2 at the required level (e.g. 5-10%). Copper-coated incubators are also now available.
These are reported to reduce the risk of microbial contamination within the incubator due to the
microbial inhibitory activity of copper. The inclusion of water bath treatment fluid growth in the
incubator water trays will also reduce the risk of bacterial and fungal the water trays. However,

there is no substitute for regular cleaning. Cells are thought to left out of the incubator for as
undersized time as possible and the incubator doors should not be opened for very long.
3. CENTRIFUGES
Centrifuges are used routinely in tissue culture as part of the subculture routine for most
cell lines and for the preparation of cells for cryopreservation. There are different types of
centrifuges based on speed. A low speed centrifuge is needed for most of the cell culture. The
separated beads of cells are disrupted simply by a gentle breaking action. Frequently cells are
centrifuged at 20C because of motor evolves heat which rises the temperature; therefore make use
of low temperature centrifugation is preferred so that the cells should not be exposed to elevated
temperature. By their very nature centrifuges produce aerosols and thus it is necessary to minimize
this risk. Ideally the centrifuge should have a clear lid so that the condition of the load can be
observed without opening the lid. This will reduce the risk of the operator being exposed to
hazardous material if a centrifuge tube has broken during centrifugation. Care should always be
taken not to over-fill the tubes and to balance them carefully. These simple steps will reduce the risk
of aerosols being generated. The centrifuge should be situated where it can be easily accessed for
cleaning and maintenance. Centrifuges should be checked frequently for signs of corrosion.
4. MICROSCOPES
Inverted phase contrast microscopes used for visualizing the cells. In 1850, John Lawrence
Smith, a faculty member at what is now Tulane University, invented the inverted microscope. The
inverted microscope is designed with the light source and the "condenser" lens above the specimen.
The condenser lens concentrates the light. The "objective" and turret of the microscope is on the
bottom. The objective focuses the light to produce a real image. Microscopes must be kept enclosed
and the lights turned down at the same time as not in use. Because the cells are found on bottom of
the tissue culture flask that is by use of an inverted microscope is important to absorb cell culture in
vitro. The culture media remains above the growing cells plates. If such plates are put over the stage
of an ordinary microscope, the growing cells, at bottom cannot be observed. Therefore, the inverted
microscope is used for the intention. It has a ability to maintain a more natural environment for the
specimen, it extends its life. This is the major advantage over compound microscope.
5. VESSELS
Anchorage dependent cells have compulsory of a nontoxic, biologically inert, and optically
visible surface that will allow cells to attach and allow improvement for the duration of growth.

These consist of petri dishes, multi well plates, micro titer plates, roller bottles, and screw cap flasks
etc.

6. WORK SURFACES AND FLOORING :

In order to maintain a clean working environment the laboratory surfaces including bench-tops,
walls and flooring should be smooth and easy to clean. They should also be waterproof and resistant
to a variety of chemicals (such as acids, alkalis, solvents and disinfectants). In areas used for the
storage of materials in liquid nitrogen, the floors should be resistant to cracking if any liquid
nitrogen is spilt. Windows should be sealed. Work surfaces should be positioned at a comfortable
working height.
7. HOMOGENIZER :
Homogenizer is a cartridge fitted with a specialized membrane that utilizes centrifugal
force to reduce the viscosity of a lysate. It provides convenient, consistent homogenization of cells
or tissue prior to nucleic acid isolation and is particularly effective for homogenizing plant tissue.
8. STORAGE :
A cell culture laboratory should have storage areas for liquids such as media and reagents,
for chemicals such as drugs and antibiotics, for consumables such as disposable pipettes, culture
vessels, and gloves, for glassware such as media bottles and glass pipettes, for specialized
equipment, and for tissues and cells. Glassware, plastics, and specialized equipment can be stored at
ambient temperature on shelves and in drawers however, it is important to store all media, reagents,
and chemicals according to the instructions on the label. Some media, reagents, and chemicals are
sensitive to light while their normal laboratory use under lighted conditions is tolerated, they should
be stored in the dark or wrapped in aluminum foil when not in use.
Refrigerators - For small cell culture laboratories, a domestic refrigerator (preferably one without a
autodefrost freezer) is an adequate and inexpensive piece of equipment for storing reagents and
media at 28C. For larger laboratories, a cold room restricted to cell culture is more appropriate.
Make sure that the refrigerator or the cold room is cleaned regularly to avoid contamination.
Freezers -Most cell culture reagents can be stored at 5C to 20C; therefore an ultra deep freezer
(i.e., a 80C freezer) is optional for storing most reagents. A domestic freezer is a cheaper
alternative to a laboratory freezer. While most reagents can withstand temperature oscillations in an

autodefrost (i.e., self-thawing) freezer, some reagents such as antibiotics and enzymes should be
stored in a freezer that does not autodefrost.
9. CRYGENIC STORAGE
Cell lines in continuous culture are likely to suffer from genetic instability as their passage
number increases; therefore, it is essential to prepare working stocks of the cells and preserve them
in cryogenic storage Do not store cells in 20C or 80C freezers, because their viability quickly
decreases when they are stored at these temperatures. There are two main types of liquid-nitrogen
storage systems, vapor phase and liquid phase, which come as wide-necked or narrow-necked
storage containers. Vapor phase systems minimize the risk of explosion with cryo storage tubes, and
are required for storing bio hazardous materials, while the liquid phase systems usually have longer
static holding times, and are therefore more economical. Narrow-necked containers have a slower
nitrogen evaporation rate and are more economical, but wide-necked containers allow easier access
and have a larger storage capacity.
10. CELL COUNTER
A cell counter is essential for quantitative growth kinetics, and a great advantage when more
than two or three cell lines are cultured in the laboratory. The Countess Automated Cell Counter is
a bench-top instrument designed to measure cell count and viability (live, dead, and total cells)
accurately and precisely in less than a minute per sample, using the standard Trypan Blue uptake
technique. Using the same amount of sample that you currently use with the hemacytometer, the
Countess Automated Cell Counter takes less than a minute per sample for a typical cell count and
is compatible with a wide variety of eukaryotic cells.
11. MEDIA PREPARATIONA AND STERILIZATION AREA :
Media preparation area should include space for keeping glasswares, media components ,
chemicals and reagents. It should have space for pH meter, stirrer, water baths, weighing machine,
hot plates. Other necessary equipment include distillation unit, vacuum source , Bunsen burner with
gas source , autoclave for sterilizing media, glasswares etc.The water used for preparing deionized
water because tap water may contain cations, anions, gases, particulate matter, micro organisms
(bacteria, virus, yeast and mold) which is unsuitable for media preparation.
12. CULTURE ROOM :
All type of cell culture required different conditions such as temperature, humidity, air
circulation, light quality and duration. These all factors influence the growth and differentiation of

cultured cell. Animal tissue culture laboratory equipment also includes confocal microscope, flow
cytometer, waste containers, media sera and reagents and cell lines

REFERENCES :
1. Nema, R. Khare, S. (2012). An animal cell culture: Advance technology for modern
research. Scientific research. Vol 3, 219-226.
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http://www.sigmaaldrich.com/technical-documents/protocols/biology/design-andequipment.html (Accessed: 30 September 2016).
3. Shriram,V.Yshika Yashu. (2014) Available at: http://www.slideshare.net/vshriram1/animaltissue-culture (Accessed: 30 September 2016).
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September 2016).
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