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Animal Cell Culture Equipment
Animal Cell Culture Equipment
CULTURE
EQUIPMENT
LINDA K.
INTRODUCTION
A horizontal air flow hood is cheaper and provides the best sterile protection for
cultures, but is only suitable for media preparation and for culturing nonprimate cells.
In animal cell culture, for handling hazardous materials such as primate/human cell
lines, virus producing cultures, radioisotopes and carcinogenic/toxic drugs, a
biohazard cabinet is used.
In contrast to a LAF hood, a biohazard cabinet also filters the exhaust air (HEPA) to
protect both the lab worker and the surrounding environment from pathogens.
HORIZONTAL VS. VERTICAL LAF HOOD
PIPETTE CYLINDER
A pipette cylinder is also called pipette hod and is a cylindrical storage jar for pipettes
and is made up of polypropylene. It may be filled with disinfectant.
They are free-standing and are distributed around the lab, one per workstation.
There should be sufficient numbers of pipette cylinders in reserve, so that cylinders
are allowed to stand for 2 hours in disinfectant before washing.
PIPETTE CYLINDER
ASPIRATION PUMP
An aspiration pump uses suction from a peristaltic pump to remove spent medium or
other reagents from culture flasks. The effluent is collected directly through a suction
line (pump tubing) into a vented container filled with a disinfectant (hypochlorite).
There is minimal risk of discharging aerosol into the atmosphere if the vent carries a
cotton plug or micropore filter.
The effluent should be left to stand for at least 2h before the reservoir is emptied.
Instead of a peristaltic pump, a vacuum pump may also be used.
ASPIRATION PUMP
SERVICE CARTS
Service carts are moveable carts to place items that are to be used at the laminar flow hoods.
They can conveniently fit into the lower space between adjacent hoods and are easily
removed for maintenance of the hoods.
They are also used to carry materials to and from the hoods and larger carts are useful for
clearing soiled glassware and used items from the aseptic area to the washup.
INVERTED MICROSCOPE
Inverted microscopes have the light source and condenser on the top, above the
stage pointing down, while the turret with the objective lenses are below the stage
pointing up.
Inverted microscopes are useful for observing living cells at the bottom of a tissue
culture flask.
The stage should be large enough or removable to accommodate large roller bottles
between it and the condenser.
A microscope with a phototube and camera is useful for digital recording and viewing
of cultures.
INVERTED MICROSCOPE
CENTRIFUGE
Pipetting aids
Pipettors
Graduated Bottle Dispenser (large volume dispensing)
Cornwall syringe (for repetitive dispensing)
PIPETTING AID
A pipetting aid is a motorized pipetting device and has enhanced speed, accuracy and
reproducibility compared to simple pipetting methods (with a rubber bulb for
example).
It may be obtained with a separate or built-in pump and can be operated with
rechargeable batteries or is mains operated.
Pipetting aids usually have a filter at the pipette insert to minimize the transfer of
contaminants.
PIPETTING AID
PIPETTORS
Pipettor, Pipette or Pipetman™ are used to dispense fluid and enable sterile and
accurate liquid handling.
Pipettes that dispense between 1 and 1000µl are termed micropipettes, while
macropipettes dispense a greater volume.
Routine subculture, which should be rapid and secure from contamination, but need
not be very accurate is best performed with a conventional glass or disposable plastic
pipette.
The tip of the pipette always needs to be sterile.
Tips can be brought loose and can be packaged and sterilized in the lab.
GRADUATED BOTTLE DISPENSER
Traditionally used for repetitive dispensing, the cornwall syringe takes the liquid via
one tube and expells it via another tube using a simple two-way valve.
CELL COUNTER
The cell counter is used when more than 2 or 3 cell lines are carried and are tools for
counting live and dead cells in a culture.
Essential for precise growth kinetics.
Both manual and automated cell counters are available:
Manual cell counting is generally accomplished using a hemocytometer, a glass slide
etched with a counting grid.
Automated cell counters operate either via electrical impedence ("Electrical Sensing
Zone" method), direct imaging (either on a disposable slide or in a flow chamber), or
flow cytometry.
CELL COUNTER
Video
CCD CAMERA & MONITOR
High resolution charge-coupled device (CCD) video cameras can be used to record and
digitize images for subsequent analysis & publication.
The addition of a video recorder will allow real-time or time-lapse recordings.
To discuss cultures with colleagues, a 300 or 400mm monitor is adequate.
CCD CAMERA & MONITOR
DISSECTING MICROSCOPE
A dissecting microscope is used for the dissection of small pieces of tissue (e.g.:
embryonic organs or tissue from smaller invertebrates).
A dissecting microscope is also useful for counting monolayer colonies and for
counting and picking small colonies in agar.
EQUIPMENTS FOR INCUBATION
Incubator
Humid CO2 Incubator
Temperature Recorder
Roller Racks
Magnetic Stirrer
INCUBATOR
The incubator should be 50 to 200l or 1.5 to 6ft³ per person and should have forced
air circulation, temperature control to within +/- 0.2°C and a safety thermostat that
cuts off if the incubator overheats.
It should be resistant to corrosion (stainless steel) and easily cleaned.
A double chamber or two incubators stacked is preferable to one large incubator,
because it can accommodate more cultures with better temperature control.
Many incubators have a heated water jacket to distribute heat evenly around the
cabinet.
Incubator shelving is usually perforated to facilitate the air circulation.
INCUBATOR
HUMID CO2 INCUBATOR
A magnetic stirrer is a lab device that employs a rotating magnetic field to cause a stir
bar immersed in a liquid to spin very quickly, hence stirring it.
For the stirrer to be used in enzymatic tissue disaggregation or suspension culture,
the motor should not heat the culture and the speed must be controlled to 50 rpm.
It should be capable of stirring up to 10l of fluid and should maintain several cultures
simultaneously.
EQUIPMENTS FOR PREPARATION &
STERILIZATION
STERILIZATION PREPARATION
Soaking Baths or Sinks Balances
Pipette Washer pH meter
Pipette Drier Hot Plate Magnetic Stirrer
Water Purifier Automatic Dispensers
Sterilizing & Drying Ovens Conductivity Meter
Steam Sterilizer (Autoclave) Osmometer
Glassware Washing Machine
SOAKING BATH/SINK
Soaking baths/sinks should be deep enough so that all glasswares (except pipettes
and the largest bottles) can be totally immersed in detergent during soaking, but not
so deep that the weight of the glass is sufficient to break smaller items at the bottom.
400mm wide, 600mm long and 300mm deep
PIPETTE WASHER
Purified water is required for rinsing glassware, dissolving powdered media and
diluting concentrates.
Purified water should not be stored but should be recycled through the apparatus
continually to minimize infection with algae or other microbes.
STERILIZING & DRYING OVENS
It is preferable to sterilize pipettes and other glassware by dry heat to avoid the
chemical contamination by steam condensate and the corrosion of pipette cans.
It requires a high-temperature (160-180°C) fan powered oven to ensure even heating.
STEAM STERILIZER (AUTOCLAVE)
When the amount of glasswares gets too great, the purchase of an automatic washing
machine is considered.
BALANCES
The pH meter is used to measure the pH during media preparation, stock solution
preparation etc.
HOT PLATE MAGNETIC STIRRER
Magnetic stirrer with a hot plate is needed to accelerate the dissolution of some
reagents.
AUTOMATIC DISPENSER
A simple check of ionic concentration can be made with a conductivity meter against
a known standard such as normal saline (0.15M).
OSMOMETER
Storage in liquid nitrogen freezer is the best method for preservation of cultured cells.
The frozen cells are transferred rapidly to the cryofreezer when they are at or below -
70°C.
Cryofreezers differ in design depending on the size of the access neck, storage system
employed and location of liquid nitrogen.
For a small lab, a 35l freezer with a narrow neck & storage in canes and canisters or in
drawers in a rack system should hold about 500-1000 ampoules.
An appropriate storage vessel is also needed to hold a backup supply of liquid
nitrogen.
CRYOFREEZER
CONTROLLED RATE FREEZER
Some cells may need different cooling rates or complex, programmed cooling curves.
A programmable freezer (e.g.: Cryomed Planer) enables cooling rates to be varied by
controlling the rate of injecting liquid nitrogen into the freezing chamber, under the
control of a preset program.
LABORATORY BACKUP
Most labs have one or more computers, which may be networked (connected to each
other).
They are used for entering records for cell line maintenance, primary culture and
experiments and make later retrieval and analysis easier.
UPRIGHT MICROSCOPE
An upright microscope is needed for chromosome analysis, mycoplasma detection
and autoradiography.
CONFOCAL MICROSCOPE
PCR CYCLER
Techniques in cell line validation such as mycoplasma detection & DNA profiling rely
on amplification and detection of specific DNA sequences.
They utilize the polymerase chain reaction and need a thermal cycler.
SPECIALIZED EQUIPMENT
Syringes & Needles: Syringes are required for filtration in conjunction with syringe
filter adapters and needles may also be needed for extraction of reagents from sealed
vials.
Sterilization Filters: Most labs use disposable filters. Commonly 25mm syringe
adapters and 47mm bottle top adapters or filter flasks are used.
Paper Towels & Swabs: Provided in a central location and beside each workstation or
hood for easy access.
Disinfectants: All discarded biological material should go into a disinfectant to prevent
growth of potential contaminating organisms in waste containers.
Chlorine-based disinfectants are used in liquid concentrate form (Clorox, Chloros) or
as tablets (Precept).