Download ECACC Handbook – Fundamental Techniques and Protocols for by Sigma Aldrich PDF

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E. uk 50 Cell Culture Protocols Key Points 1. Trypan Blue is toxic and is a potential carcinogen. Protective clothing, gloves and face/eye protection should be worn. Do not breathe the vapour. 2. The central area of the counting chamber is 1mm2. This area is subdivided into 25 smaller squares (1/25mm2). Each of these is surrounded by triple lines and is then further divided into 16 (1/400mm2). 1mm. 3. 1mm) 4. There are several sources of inaccuracy: • The presence of air bubbles and debris in the chamber.

9. Examine cultures and media daily for evidence of gross bacterial or fungal contamination. This includes medium that has been purchased commercially. uk 36 Cell Culture Protocols 10. Quality control all media and reagents prior to use. 11. Keep cardboard packaging to a minimum in all cell culture areas. 12. Ensure that incubators, cabinets, centrifuges and microscopes are cleaned and serviced at regular intervals. 13. Test cells for the presence of mycoplasma on a regular basis. The Don’ts 1.

Clean the haemocytometer. 5. Moisten the coverslip with water or exhaled breath. Slide the coverslip over the chamber back and forth using slight pressure until Newton’s refraction rings appear (Newton’s refraction rings are seen as rainbow-like rings under the coverslip). Dimensions of a haemocytometer 6. Fill both sides of the chamber with cell suspension (approximately 5-10µl) and view under an inverted phase contrast microscope using x20 magnification. 7. Count the number of viable (seen as bright cells) and non-viable cells (stained blue).

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