Essential Laboratory Equipment for Cell Culture Dr. Khalid Enan DFG-Sponsored Workshop, Assuit...

Essential Laboratory Equipment for Cell Culture

Dr. Khalid Enan

DFG-Sponsored Workshop, Assuit University, Egypt

10 Sep 2012

Definition of cell culture :

Cell culture refers to the culturing of cells derived from multicellular eukaryotes, especially animal cells

A complex process by which cells are grown under controlled conditions

Used to diagnose infections test new drugs in research.

Basic requirements for cell culture Sterile work area Laminar flow hood Biohazard cabinet

Sterile work area: A separate room should be made available

for clean cell culture work. This room should be free of through traffic

and equipped with an air flow cabinet, which supplies filtered air around the work surface.

Cell culture room

Laminar flow hood Strict sterility is needed. Laminar flow hood offers the best sterile

protection. Suitable only for preparing medium and

other sterile reagents and for culturing non-primate cells.

Laminar-Flow Hood

Biohazard cabinet: Used for potentially hazardous materials

including cell lines, cultures for virus propagation, or toxic drugs).

Biohazard cabinet

All work surfaces, benches and shelves and the base of the airflow cabinets must be kept clean by frequent swabbing with 70% ethanol or an alternative disinfectant.

Incubation facilities: The cell culture laboratory will need to be furnished

with an incubator or hot room to maintain the cells at 30-40 °C. The incubation temperature will depend on the type of cells being cultivated.

It may be necessary to use an incubator which has been designed to allow CO2 to be supplied from a main supply or gas cylinder so that an atmosphere between 2-5% CO2 is maintained in the incubator

Incubators

Fridges and freezer (-20 °C) Both items are very important for storage of liquid

media at 4 °C and for enzymes (e.g. trypsin) and some media components (e.g. glutamine and serum) at -20 °C.

A fridge or cold room is required to store medium and buffers. A freezer will be needed for keeping pre-aliquoted stocks of serum, nutrients and antibiotics. Reagents may be stored at -20 °C.

For prolonged preservation, cells may be preserved in liquid nitrogen or a -70 °C freezer.

Fridges and freezer (-20 °C)

Microscopes A simple inverted microscope is essential

so that cultures can be examined in flasks and dishes.

A very simple light microscope with x100 magnification for routine cell counts in a hemocytometer.

Inverted Microscope

Tissue culture ware (Flasks) A variety of tissue culture plastic ware is

available, the most common being specially treated polystyrene.

Cells can be maintained in flasks (25 cm2 or 75 cm2), tissue culture tube, microtitre plates………

Filter sterilization Non-autoclavable substances, such as

culture media, enzymes, hormones, and bicarbonate buffers, must be sterilized through a 0.22 μm pore size membrane filter. These are obtainable in various designs to allow a wide range of volumes to be filtered (e.g., Millipore, Gelman).

Filter sterilization:

Liquid N2 System It is important to maintain continuity in

cells to prevent genetic drift and to guard against loss of the cell line through contamination and other disasters.

Facilities for counting cells It is possible to monitor cell growth by

eyes (looking for confluence), however, more accurate cell counts are required for most experimental purposes.

The most commonly used device is the Improved Neubauer hemocytometer originally designed for counting blood cells.

Facilities for counting cells

Further required equipment: Centrifuge Centrifuge tubes and universal containers Water bath Vacuum pump Graduated pipettes of various sizes. Disposable Pasteur pipettes. Rubber bulbs or pipette-aid Automatic pipettes (volumes of 1-1000 μl). Plastic tips. Graduated glass or plastic cylinders.

water bath

vacuum pump

graduated pipettes

Pasteur pipettes.

centrifuge tube (Falcon tube)

rubber bulbs

Eppendorf tube

automatic pipettes

Pipet-aid

Centrifuge

Question ?

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