BBT 2106 Animal Cell Culture Practical Manual

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  Page 1  of 18   BBT2106 ANIMAL CELL CULTURE AND DEVELOPMENT Practical 1: COMMON ASEPTIC TECHNIQUES AND CELL CULTURE MEDIA APPLIED IN THE ANIMAL CELL CULTURE GLASSWARE - DECONTAMINATION, WASHING AND STERILIZATION AIM To describe the procedures adopted in washing and sterilization of glass and polypropylene wares used in common cell culture laboratory. REQUIREMENTS Equipment: Trays for disinfection, Autoclave for decontamination, Autoclave for sterilization, Ultrasonic tanks, Drier and Hot air oven for sterilization. Consumables: Detergent solution, 0.5% Non-Toxic cleaning Solution, Potassium dichromate, 10% HCl, Brush, Aluminium foil, Brown wrapper sheet, Thread. Miscellaneous: Autoclave register, Hot air oven register Designated place of work: Washing & Sterilization room PRINCIPLE Autoclave Moist heat sterilization involves the use of steam in the range of 121-134°C. Steam under pressure is used to generate high temperature needed for sterilization.  Autoclaves use pressurized steam to destroy microorganisms for the decontamination of laboratory waste and the sterilization of laboratory glassware, media, and reagents. Saturated steam is generated when water boils at a high temperature and pressure. When steam condenses on the surface of the material to be autoclaved, latent heat is liberated, that is responsible for the destruction of micro-organisms.  Autoclaves should be tested periodically with biological indicators like cultures of Bacillus stearothermophilus to ensure proper function.  Page 2  of 18   PROCEDURE a) Washing i) GLASSWARE Media Bottles: 1. Bottles are soaked in chromic acid solution for 4 hours. 2. They are washed in running tap water by filling and emptying them 10 times. 3. They are brushed and soaked in warm Non-Toxic cleaning (0.5%) solution for 5-10 minutes. 4. Subsequently, they are rinsed in running tap water and soaked for 1 hour in 10% HCl. 5. They are rinsed with running tap water and immerse in distilled water for ½ day. 6. Again they are rinsed and transferred to next distilled water change. 7. This procedure of immersion in distilled water for ½ day is repeated 2 more times. 8. Finally, they are immersed in double distilled water for ½ day, drained & dried at 80  C.  Bottle Covers and Washer  : Caps and covers are washed thoroughly in running tap water for 10 times and soaked in distilled water. Finally, they are boiled in double distilled water, drained and dried at 45°C for half an hour. Glass Tubes: 1. They are washed in running tap water 10 times and soaked in chromic acid solution for 4 hours. 2. They are removed from chromic acid solution, rinsed and soaked in 0.5% non-Toxic cleaning solution. 3. Rinsed in running tap water 25 times. 4. Soaked in 10% HCl for 1 hour and rinsed in tap water 25 times. 5. The tubes are immersed in distilled water for ½ day and subjected to 2 more changes in distilled water for ½ day each. 6. Finally, they are immersed in double distilled water for ½ day once and drained and dried at 80  C.  Page 3  of 18   ii) FILTRATION UNIT 1. Filters should be dismantled. 2. Wash in running tap water for 10 times. 3. Brush the vessel and filter holder with Non-Toxic cleaning. 4. Rinse in running tap water. 5. Soak in distilled water for 1 hour. 6. Boil in double distilled water; drain the water and air dry. 7. Parts of the filter should be kept together in one place. b) Sterilization Hot air oven 1. The mouth of the bottle is covered with tin foil and paper. Pack the glass tubes in paper and affix date of sterilization. 2. They are sterilized in the Hot air oven at 180  C for 3 hours. 3. The hot case is allowed to cool. 4. The glassware is re-sterilized at 180  C for 3 hours on the consecutive day. 5. The rubber and polypropylene items are not to be sterilized in the hot air oven. Autoclave 1.  Bottle covers and washers   are packed with tin foil and brown wrapper sheet.   2.  Autoclave paper is fixed in a bin and sterilize in autoclave at 121  C, 15 lbs/Sq. in pressure for 30 minutes. Precautions:   All used glass and polypropylene ware should be discarded in 1% hypochlorite solution in the place of work and left in the wash room by concerned technician.   The discard trays along with hypochlorite should be decontaminated in autoclave at 15 lbs for 30 minutes.   Items are removed from the autoclave and allowed to cool.   Prior to washing labels are removed.  Page 4  of 18   Practical 2: MEDIA AND REAGENTS PREPARATION, STERILITY CHECKS AIM To prepare media for the growth and maintenance of cell lines. THEORY The term tissue culture was srcinally applied to explants of tissue embedded in plasma. The term subsequently became associated with the culture of cells in general and is now obsolete in its srcinal sense. Cell culture is the term most correctly used today. It refers to tissue dissociated into a suspension of single cells by both mechanical and enzymatic means. After being washed and counted, the cells are diluted in growth medium and allowed to settle onto the flat bottom surface of a glass or specially treated plastic container. Most types of cells adhere quickly, and under optimum conditions they will undergo mitosis and cell division about once a day until the surface is covered with a confluent cell monolayer. Such cells constitute a primary culture . These cells are usually removed from the surface on which they are growing with a combination of trypsin and a chelating agent such as EDTA, counted, diluted, and replated in new containers with fresh medium. This is called a secondary culture . Cells may be grown in vitro in several ways. Although all cell types have similar base requirement for growth, each tissue type or cell line differs in the type of medium  required for their propagation. REQUIREMENTS S.No Reagents Storage 1. Sodium Bicarbonate Fridge 2. DMSO Fridge 3. EDTA Fridge 4. Fungizone -20 O C 5. FCS -20 O C 6. L  –  Glutamine -20 O C 7. HBSS Fridge 8. Hepes Buffer Fridge

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Jul 29, 2017
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