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Tissue Culture Techniques Lab

Tissue Culture Techniques Lab. Islamic University _Gaza Faculty of science Department of Biotechnology By: Mahmoud W. El-Hindi 2013-2014. Course Syllabus. Course objectives:. Describe the equipments used in animal tissue culture .

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Tissue Culture Techniques Lab

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  1. Tissue Culture Techniques Lab Islamic University _Gaza Faculty of science Department of Biotechnology By: Mahmoud W. El-Hindi 2013-2014 By: Mahmoud El-Hindi

  2. Course Syllabus Course objectives:. • Describe the equipments used in animal tissue culture . • Understand the safety procedures need for tissue culture . • Understand techniques used in tissue culture . By: Mahmoud El-Hindi

  3. Course division:. By: Mahmoud El-Hindi

  4. Lab 1: Aseptic Technique • Aseptic technique is a combination of procedures designed to reduce the probability of infection. • Contamination by microorganisms remains a major problem in tissue culture. • Bacteria, mycoplasma, yeast, and fungal spores may be introduced via the operator, the atmosphere, work surfaces, solutions, and many other sources. By: Mahmoud El-Hindi

  5. Cont. • Aseptic technique aims to exclude contamination by establishing a strict policy of practice and ensuring that everyone using the facility adheres to it. • Contamination can be minor and confined to one or two cultures, can spread among several cultures and compromise a whole experiment, or can be widespread and wipe out your. By: Mahmoud El-Hindi

  6. Cont. • Mycoplasmal infection, invisible under regular microscopy, presents one of the major threats. • Undetected, it can spread to other cultures around the laboratory. • It is therefore essential to back up visual checks with a mycoplasma test, particularly if cell growth appears abnormal . By: Mahmoud El-Hindi

  7. Maintaining Sterility : • Correct aseptic technique should provide a barrier between microorganisms in the environment outside the culture and the pure, uncontaminated culture within its flask or dish. • All materials that come into direct contact with the culture must be sterile and its non sterile surroundings. By: Mahmoud El-Hindi

  8. Cell culture contaminants two types: • 1-Chemicals: difficult to detect caused by endotoxins, metal ions or traces of disinfectants that are invisible. • 2- Contamination by microorganisms remains a major problem in tissue culture. • Bacteria, mycoplasma, yeast, and fungal spores may be introduced via the operator, the atmosphere, work surfaces, solutions, and many other sources. By: Mahmoud El-Hindi

  9. Safety • For best results in tissue culture, we want to work to keep microbial (bacteria, yeast and molds) contamination to a minimum. Guidelines to follow: • Work in a culture hood set-aside for tissue culture purposes. • Most have filtered air that blows across the surface to keep microbes from settling in the hood. • Turn off the UV/antimicrobial light and turn on the hood 30 minutes prior to entering the hood. By: Mahmoud El-Hindi

  10. Cont. • Wash hands with soap and water before beginning the procedure and rewash if you touch anything that is not sterile or within the hood. • Do not breathe directly into your cultures, bottles of media, etc. This also means to keep talking to a minimum. • No singing or chewing gum. By: Mahmoud El-Hindi

  11. Cont. • Use only sterilized pipets, plates, flasks and bottles in the hood for procedures. • Change pipettes for each manipulation. • If the tip of the pipette touches something outside of the flask or bottle, replace with a new one. By: Mahmoud El-Hindi

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  14. Culture Medium Sterilization By: Mahmoud El-Hindi

  15. Culture flask Culture Plate By: Mahmoud El-Hindi

  16. Inverted microscopy Large stage so plates and flasks can be used. Magnification; 5X, 10X, 20X, 40X By: Mahmoud El-Hindi

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  20. ASEPTIC TECHNIQUE IN VERTICAL LAMINAR FLOW • Clean and swab down work area, and bring bottles, pipettes, etc. • Carry out preparative procedures first (preparation of media and other reagents), followed by culture work. • Finally, tidy up and wipe over surface with 70% alcohol. By: Mahmoud El-Hindi

  21. Cont. Sterile: • _ Eagle’s 1×MEM with Hanks’ salts and HCO3, without antibiotics . . . . . . 100 mL • _ Pipettes, graduated, and plugged. • If glass, an assortment of sizes, 1 mL, 5 mL, 10 mL, 25 mL, in a square pipette can, or, if plastic, individually wrapped and sorted by size on a rack _ Culture flasks . . 25 cm2 . . . . . . . . . 10 By: Mahmoud El-Hindi

  22. Cont. Non sterile: • _ Pipetting aid or bulb . • _ 70% alcohol in spray bottle. • _ Lint-free swabs or wipes. • _ Absorbent paper tissues. • _ Pipette cylinder containing water and disinfectant. • _ Scissors. • _ Marker pen . By: Mahmoud El-Hindi

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  25. Good Luck By: Mahmoud El-Hindi

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