Special considerations for novices in cultivating cells - Database & Sql Blog Articles

Aseptic operation

Sterilization of the sterile room:

1. Regularly clean the sterile room: once a week, first use the tap water to mop the floor, wipe the table, clean the workbench, etc., then wipe with 3 ounces of Sol or chlorhexidine or 0.5% peroxyacetic acid.

2. CO2 incubator (incubator) sterilization: first wipe with 3 ‰ 新洁尔, then wipe with 75% alcohol or 0.5% peracetic acid, then irradiate with UV light

3. Pre-experimental sterilization: open the UV lamp, the three-oxygen sterilizer, and the air purifier system for 20-30 minutes each.

4. Sterilization after experiment: Wipe the stage of the clean bench, the side table, and the inverted microscope with 75% alcohol (3 ‰ 新洁尔灭).

Aseptic preparation of the experimenter:

1. Wash your hands with soap.

2. Wear a gown, a good isolation cap, a mask, and put on the slippers.

3. Wipe your hands with a 75% alcohol cotton ball.

Demonstration of aseptic operation:

1. All bottles of alcohol, PBS, medium, and trypsin brought into the clean bench should be wiped with 75% alcohol on the outer surface of the bottle.

2. Operate near the alcohol lamp flame.

3. The vessel must be sterilized before use.

4. Continue to use the utensils (such as caps, droppers) to be placed high, still overheated when in use.

5. All kinds of operations should be close to the alcohol lamp, the action should be light and accurate, and it should not be touched. If the straw does not touch the waste tank.

6. When taking more than two kinds of liquids, pay attention to replace the straw to prevent cross-contamination.

Cleaning and disinfection of instruments

Glass equipment washout:

First, the decontamination of new glassware:

1. Wash with tap water to remove dust.

2. Drying, soaking hydrochloric acid: drying in an oven, and then immersing in 5% dilute hydrochloric acid for 12 hours to remove dirt, lead, arsenic and the like.

3. Brushing and drying: Immediately after 12 hours, rinse with tap water, then wash with detergent, rinse the tap water and dry in the oven.

4. Soaking acid, washing: Soak 12 with cleaning solution (potassium dichromate 120g: concentrated sulfuric acid 200ml: distilled water 1000ml)

Hours, then remove the vessel from the acid tank and rinse it with tap water for 15 times, then rinse the distilled water 3-5 times and use double steamed water for 3 times.

5. Drying and packaging: After washing, dry first, then wrap with kraft paper (slick paper).

6. Autoclave: Pack the package into the pressure cooker, cover the lid, open the switch and safety valve. When the vapor rises in a straight line, close the safety valve and keep it for 20-30 minutes when the pointer points to 15 lbs.

7. Drying after autoclaving

Second, the decontamination of the old glassware:

1. Brushing and drying: The used glassware can be directly infused into the Sur liquid or the detergent solution. The utensils soaked in the Sur solution (detergent) should be cleaned with water and then dried.

2. Soaking acid, cleaning: After drying, soak in the cleaning solution (acid solution). After 12 hours, remove the container from the acid tank and rinse it with tap water immediately (avoid the protein is dry and adhere to the glass and it is difficult to clean), then use distilled water. Rinse 3 times.

3. Drying and packaging: After washing the dried utensils, take out the packaging with kraft paper (slick paper) to facilitate disinfection and storage, and prevent dust and pollution.

4. High-pressure disinfection: the packaged utensils are placed in the pressure cooker, the lid is closed, the switch and the safety valve are opened, and the steam rises out of the safety valve as the temperature rises. When the vapor emerges in a straight line for 3-5 minutes, the safety valve is closed. The barometer index will rise. When the pointer points to 15 pounds, adjust the electric switch for 20-30 minutes. (Glass culture bottle can be gently covered before disinfection)

5. Drying and standby: Since the vessel will be wetted by steam after autoclaving, it should be placed in the oven for drying. Metal instrument washout:

Metal utensils should not be sour. When washing, wash with detergent, then rinse with tap water, then wipe with 75% alcohol, then tap water, then rinse with distilled water, then dry or air to dry. Put it in an aluminum box and pack it in a pressure cooker for 15 pounds of high pressure (30 minutes), then dry it for later use.

Rubber and plastic:

Rubber and products are usually treated by washing them with detergent, then washing them with tap water and distilled water separately, then drying them in the oven, and then performing the following treatment procedures according to different qualities:

1. Needle filter cap can not solubilize acid solution, soak for 6-12 hours with NaOH, or boil for 20 minutes, install two membranes before packaging, pay attention to the glossy side (concave up) when installing the filter, then Loosen the screw slightly, put it into an aluminum box and sterilize it in a pressure cooker for 15 lbs for 30 minutes, then dry it for later use. Note that the screw should be tightened immediately when it is taken out of the clean bench.

2. After the rubber stopper is dried, boil it with 2% sodium hydroxide solution for 30 minutes (the used rubber plug is treated with boiling water for 30 minutes), washed with tap water and dried. Then, the hydrochloric acid solution was further inoculated for 30 minutes, and then washed with tap water, distilled water, and three distilled water, and dried. Finally, it is placed in an aluminum box for autoclaving and dried for use.

3. The rubber cap, the centrifuge cap can only be soaked in 2% sodium hydroxide solution for 6-12 hours after drying (remember the time should not be too long), wash and dry the tap water. Then, the hydrochloric acid solution was further inoculated for 30 minutes, and then washed with tap water, distilled water, and three distilled water, and dried. Finally, it is placed in an aluminum box for autoclaving and dried for use.

4. The plastic head can be soaked in 75% alcohol for 5 minutes, then it can be used after UV irradiation.

5. Plastic culture bottle, culture plate, cryotube:

6. Other disinfection methods: Some items can neither be dried and disinfected, nor can they be steam-sterilized. They can be immersed and disinfected with 70% alcohol. The plastic petri dish is opened and placed on a clean bench surface and directly exposed to ultraviolet light for disinfection. Ethylene oxide can also be used to disinfect plastic products. After disinfection, it takes 2-3 weeks to wash off the residual ethylene oxide. It is best to disinfect plastic products with 20,000-100,000 rad r-rays. In order to prevent the cleaning equipment from being disinfected and disinfected

Confused, can be marked with 密 ink after paper packaging. The method is to use a water-filled pen or a brush to smear ink, and make a mark on the wrapping paper. Usually, the ink has no traces. Once the temperature is high, the writing will appear, so that it can be determined whether they are disinfected. Preparation of dense ink: chlorinated diamond (CoC12·6H2o) 2g, 30% hydrochloric acid 10m1, distilled water 88m1.

Precautions:

1. Strictly implement the operation procedure of the pressure cooker: When autoclaving, first check whether there is distilled water in the pot to prevent it from drying out under high pressure. The water should not be too much because it will make the air flow smoothly blocked, which will reduce the high pressure disinfection effect. Check that the safety valve is open to prevent explosions during high pressure.

2. When installing the filter, pay attention to the smooth side: pay attention to the smooth side of the filter is the front, to face up, otherwise it will not be filtered.

3. Pay attention to the protection of the human body and the complete soaking of the utensils: A. Wear acid-resistant gloves when soaking the acid to prevent acid splashes from harming the human body. B. Prevent the acid from splashing on the ground when the vessel is taken from the acid tank, which will corrode the ground. C. The vessel should be completely immersed in the acid solution, and no air bubbles should be left to prevent the acidity from being completely.

Subculture cell culture notes:

1. Strict aseptic operation

2. Moderate digestion: The time of digestion is affected by many factors such as the type of digestive juice, preparation time, and amount added to the culture bottle. During the digestion process, attention should be paid to the change of cell morphology. Once the cytoplasm is retracted, the connection becomes loose. Or if there is a sign of floating up, it is necessary to stop digestion immediately.

Attachment: EDTA (0.02% disodium edetate) digestive solution formula:

EDTA 0.20 g, NaCl 8.00 g, KCl 0.20 g, KH 2 PO 40.02 g, glucose 2.00 g, 0.5% phenol red 4 ml, and distilled to a volume of 1000 ml. 10 lbs of 20 min autoclave, adjust the pH to 7.4 when in use. Note that EDTA cannot be neutralized by serum. After use, the flask should be thoroughly cleaned, otherwise the cells will easily detach from the wall when cultured.

Cell recovery

The principle of cell resuscitation - rapid melting: cells in frozen liquid at -196 ° C must be rapidly thawed to 37 ° C, so that the ice crystals frozen outside the cell will melt rapidly, avoiding the ice crystals slowly melt into the cells to form recrystallization, on the cells Cause damage.