【Experimental Principle】 The main difference between cell culture technology and other general laboratory work is the requirement to maintain aseptic operation，Avoid the influence of microorganisms and other harmful factors。A general standard cell culture chamber should include a preparation chamber、Preparation room and culture room。The three chambers are both interconnected and relatively independent，Each completes different operations in the culture process。 【Objective】 (1) Understand the setting and equipment of the culture room。 (2) Learn the concept of aseptic and the essentials of aseptic operation。 【Operation Steps】 1. Laboratory Setup (1) Preparation Room (2) Preparation Room (3) Culture Room The culture room should be completely closed，Maintain a constant temperature，The following points should be paid attention to in the design： (1) The location of the culture room is best located on the shaded side，Places where direct sunlight cannot be reached，Prevent the indoor temperature from rising。 (2) The height of the ceiling should not exceed 2 meters，In order to ensure the effective sterilization effect of the UV lamp， (3) The door is generally sliding door，to prevent air movement。 (4) Ceiling、The floor and surrounding walls should be smooth and free of dead corners，To tile or paint，It's designed like this，First, it is easy to clean and disinfect，In addition, it is not easy to accumulate dust in the corners of the wall。 2Commonly used laboratory equipment: (1) Preparation room equipment, (1) Double distilled water retort：Prepare double distilled water。 (2) Acid tank：Hold lotion，Soak glassware。 (3) Oven：Washed glassware is oven dried。 (4) Pressure cooker：glassware、Dissecting utensils、Partial liquid、Sterilization of plastic utensils, etc。The effective sterilization pressure and time are different for different items。 (5) Locker 1：Place unsterilized items。 (6) Locker 2：Place sanitized items。 (7) Packing table：For packaging before sterilization。 Preparation room precautions： (1) Prevent the still from being roasted dry。 (2) Water seeping out from the placed pool。 (3) Do not splash acid on clothing or the ground。 (4) Do not block the pressure cooker exhaust valve and safety valve。 (5) Sterilized items should be stored separately from unsterilized items。 (2) Equipment of the liquid preparation chamber (1) Balances (torque balances and electronic balances)：Weigh (2) pH meter： (3) Magnetic stirrer： (3) Equipment of cell culture room (1) Ultra-clean workbench： There are many types of clean benches，There is a one-sided single、Single-sided double、Double-sided double or double-sided quadruple, etc，It works by utilizing a blower，Enables the air to pass through highly efficient air filtration，Slowly pass through the countertop，This results in a sterile and homogeneous air in the working environment。 Depending on the laminar flow method，Ultra-clean workbenches are mainly divided into two types: horizontal laminar flow type and vertical laminar flow type，The rationale is much the same，The indoor air is initially filtered through a coarse filter，It is pressed into the static pressure box by a centrifugal fan，Then pass through the high-efficiency air filter，The resulting clean air flow passes through the sterility at a certain uniform cross-sectional velocity，So as to form a dust-free and sterile high-cleanliness working environment。However, the direction of the airflow is different between the two types of stations。 There are several issues that should be paid attention to when using a clean bench： A. The purification workbench is best installed in a dust-free room，It is best to be in a well-isolated sterile room，In order to avoid excessive dust, the filter will be easily blocked，Reduces the purification effect，Shorten the service life。 B. Newly installed or long-term unused workstations，The workbench and surroundings must be cleaned with a vacuum cleaner or a tool that does not produce fibers before work，Then use drug sterilization or ultraviolet sterilization for sterilization。 C. Every time the workbench is used，The countertop should be scrubbed with 75% alcohol first，And treat the microorganisms accumulated in the purification work area with 30~50min ultraviolet sterilization lamp in advance。After turning off the UV lamp, the blower should be started and let it run for two minutes before the incubation operation。 D. Unnecessary items should not be stored in the purification work area，to keep the clean airflow pattern undisturbed。 E. Generally，The HEPA filter is replaced every three years。Replacing the high-efficiency filter should be operated by a professional，to keep the seal well。The coarse filter should be removed and cleaned regularly，The time should be determined according to the cleanliness of the environment，It is usually done every 3~6 months。 F. Every time you use the purification table, you should remove the items on the work surface in time，And scrub the countertop with sprinkler to keep it clean at all times。 (2) 4 °C freezer and low temperature freezer： (3) CO2 incubator： A. A concentration of CO2 on cell growth，In particular, primary culture and single-cell culture have a facilitating effect (5%)。 B. A certain concentration of CO2 can maintain a constant pH of the culture medium。Suitable for open cultures，After the incubator is sealed，It can be cultivated in a general incubator，However, when using Petri dishes or multi-well plates，Air with high humidity and high CO2 content is required。 C. It increases humidity，The box is equipped with a water bath，Humidity in the incubator can be guaranteed。 D. The incubator is equipped with a UV lamp。 E. The ratio of CO2 to air can be adjusted by means of a gas flow meter。 F. Preservatives (dioxacetic acid) can be added to the water。It can prevent high humidity in the CO2 incubator，Prone to mold。 (4) Ozone generator： (4) Centrifuge： (5) CO2 cylinders： (6) Liquid nitrogen tank： (7) Lockers：Used to store sundries (8) UV lamps： (9) Air purification system (10) Inverted microscope 3. Aseptic operation (1) Sterilization of the clean room： (1) The ultraviolet lamp should be turned on when no one is there； (2) Wear a sterile gown when entering the sterile room、Wear a hat and mask； (3) Clean and disinfect once a week； Read More …
