Liquid medium for bacterial growth is a common form of medium used in fields such as microbiology and cell biology. Unlike solid culture mediums, it is mainly in a liquid state and can be used to culture microorganisms or animal and plant cells through aeration or shaking. The liquid medium for bacterial growth is prone to contamination by bacteria, fungi, spores, and other microorganisms during the preparation process, which affects the growth and reproduction of the experimental cells. Therefore, effective sterilization methods must be adopted to ensure the normal growth of experimental cells.
Autoclave sterilization is one of the most commonly used sterilization methods in laboratories, suitable for sterilizing heat-resistant objects, utensils, and media. It utilizes high-temperature, high-pressure steam to destroy the cell structures of microorganisms, achieving sterilization purposes.
The operating steps are as follows:
Preparation
Dispense the prepared liquid medium for bacterial growth into appropriate containers such as Erlenmeyer flasks or test tubes. Seal the dispensed medium with cotton plugs, gauze, or sterile breathable sealing film, and place them in a sterilization basket awaiting sterilization. Note that the medium should not be filled too full; generally, the volume should not exceed 2/3 of the container to prevent overflow due to pressure imbalances during sterilization. Ensure there is space between the containers to allow steam to circulate.
Loading the Autoclave
Place the sterilization basket into the autoclave, close the door tightly to prevent accidents caused by high steam temperature and pressure. Open the steam intake valve to start sterilization.
Heating and Rising Temperature
Adjust the exhaust valve of the autoclave to gradually increase pressure and temperature to meet the required sterilization standards. When the steam pressure is 0.105 MPa (about 1.05 atmospheres), the internal temperature can reach 121°C.
Sterilization
Once it reaches the required sterilization temperature and pressure, maintain it for a period, usually at 121-124°C for about 30 minutes, to ensure that bacteria, fungi, spores, and other microorganisms are thoroughly killed. Record the sterilization temperature and time for traceability.
Depressurization and Removal
After sterilization, close the steam intake valve, adjust the exhaust valve, or naturally depressurize to bring the internal pressure to zero. Open the autoclave door and remove the culture medium. Be sure to wear gloves to avoid burns when removing items.
For heat-sensitive liquid medium for bacterial growth, such as certain plant growth regulators or organic substances that decompose at high temperatures, filtration sterilization can be used.
The operating steps are as follows:
Preparation
Choose an appropriate filter sterilizer, typically using a membrane filter with a pore size of 0.45 μm. Sterilize the filter membrane and the container for collecting the filtrate with high temperature to prevent contamination. Check and replace the filter membrane regularly to ensure filtration effectiveness.
Filtration
Under sterile conditions, filter the liquid medium for bacterial growth through the filter sterilizer to remove microorganisms and spores.
Collection
Collect the filtered sterile culture medium into a sterile container and seal it for later use.
In addition to the two commonly used sterilization methods mentioned above, other methods such as ultraviolet sterilization and chemical sterilization can also be used for liquid medium for bacterial growth sterilization. However, these methods usually have limitations. For example, ultraviolet sterilization has weak penetration and is only suitable for air and surface sterilization; chemical sterilization may leave harmful residues, affecting the normal growth of experimental cells. Therefore, ultraviolet and chemical sterilization are less commonly used in practice.
Sterilization Time
The time for autoclave sterilization should not be too long or too short. Prolonged time may damage the nutrients in the liquid medium for bacterial growth, whereas too short may not thoroughly kill microorganisms and spores.
Temperature Control
Strictly control the temperature during sterilization to ensure it reaches the required sterilization temperature and maintains it for enough time.
Aseptic Operation
Ensure aseptic operation throughout the filtration sterilization process to avoid any possible contamination.
Storage Conditions
Use the sterilized liquid medium for bacterial growth as soon as possible. If it needs to be stored, keep it at an appropriate temperature (such as below 10°C) and periodically check for contamination.
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