1. Thorough Sterilization of Culture Media and Equipment

Autoclave Sterilization: The culture media and all tools should undergo autoclave sterilization (typically at 121°C for 15-20 minutes) to ensure the elimination of potential microorganisms. For materials that are heat-sensitive, alternative sterilization methods, such as filtration, can be used.

Ultraviolet Sterilization: Before using the tools and in the working environment, expose them to UV light for 20-30 minutes to eliminate airborne microorganisms.

2. Thorough Disinfection of Explants

Pre-treatment of Explants: Explants are often the main source of contamination. Therefore, surface disinfection should be carried out before introducing them into the culture. Common disinfectants include 70% alcohol and sodium hypochlorite (NaClO). A typical process involves soaking the explants in 70% alcohol for 30 seconds to 1 minute, followed by disinfection in 0.1%-0.5% sodium hypochlorite solution for 10-20 minutes, and then rinsing several times with sterile water.

Control of Disinfection Time: Ensure appropriate disinfection time. Over-disinfection may damage the explants, while under-disinfection may fail to fully eliminate contaminants.

3. Aseptic Technique

Aseptic Protection for Operators: Lab personnel must wear lab coats, masks, and gloves to prevent the introduction of bacteria from breath or hands. During operations, keep hands and tools clean, avoiding contact with non-sterile surfaces.

Use of Alcohol Lamp or Sterilizer: On the workbench, tools such as tweezers and scissors can be quickly sterilized by passing them through an alcohol lamp flame or using an infrared sterilizer.

Sterilization of Reused Tools: After each use, tools should be immediately disinfected to prevent contamination from using unsterilized tools.

4. Cleaning of the Environment and Laminar Flow Hood

Regular Cleaning of the Laboratory: The laboratory and laminar flow hood must be kept clean. The workbench should be wiped with 75% alcohol before and after each use, and regular UV sterilization should be conducted.

Air Filtration: Use high-efficiency particulate air (HEPA) filters to ensure that microorganisms in the air do not enter the working area, particularly during closed-environment culture processes.

5. Use of the Laminar Flow Hood

Maintenance of the Laminar Flow Hood: Ensure that the airflow and filtration system of the hood are functioning properly, and replace the HEPA filters regularly to prevent contamination.

Sterilization of Workbench Surfaces: Before beginning operations, spray and disinfect the workbench surface, containers, and tools with 75% alcohol.

6. Reducing Explant Browning

Explant browning can lead to contamination of the culture medium or explant material. This is mainly caused by phenolic compounds secreted by damaged tissues. Solutions include:

Antioxidant Treatment: Using antioxidants, such as ascorbic acid or citric acid, can reduce browning.

Minimizing Physical Damage: Avoid excessive mechanical damage to explants to prevent tissue trauma and browning.

7. Regular Monitoring and Prompt Contamination Management

Regular Inspection of Cultures: Regularly check the culture medium and explants during the culture process. If any signs of contamination, such as mucus or discolored fungal spots, are observed, remove and handle them promptly to prevent cross-contamination.

Isolating Contaminants: Contaminated materials should be immediately removed from the culture room to prevent affecting other cultures.

By following these methods, the risk of bacterial and fungal contamination in plant tissue culture can be significantly reduced, thereby improving the success rate of cultivation.