1. Essential Laboratory Equipment for Cell Culture Dr. Khalid Enan DFG-Sponsored Workshop, Assuit University, Egypt 10 Sep 2012

2. Definition of cell culture : Cell culture refers to the culturing of cells derived from multicellular eukaryotes, especially animal cellseukaryotesanimal A complex process by which cells are grown under controlled conditionscells Used to diagnose infections test new drugs in research.

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

4. 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.

5. Cell culture room

6. 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.

7. Laminar-Flow Hood

8. Biohazard cabinet: Used for potentially hazardous materials including cell lines, cultures for virus propagation, or toxic drugs).

9. Biohazard cabinet

10. 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.

11. 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

12. Incubators

13. 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.

14. Fridges and freezer (-20 °C)

15. 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.

16. Inverted Microscope

17. 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………

19. 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).

20. Filter sterilization:

21. 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.

23. 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.

24. Facilities for counting cells

25. 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.

26. water bath vacuum pump graduated pipettes

27. Pasteur pipettes. centrifuge tube (Falcon tube)

28. rubber bulbs Eppendorf tube

29. automatic pipettes

30. Pipet-aid

31. Centrifuge

32. Question ?

33. Thank you