Plant Tissue Culture Aseptic Techniques You Must Know: Blocking the Invisible Enemies!

What is Plant Tissue Culture?

Plant tissue culture is a collection of techniques used to maintain or grow plant cells, tissues, or organs under sterile conditions on a nutrient culture medium of known composition. It is widely used in plant science, forestry, biotechnology and hobbyists. The method allows for the production of exact copies of plants that produce particularly good flowers, fruits, or have other desirable traits. Essentially, it involves the use of small pieces of plant material, often referred to as explants, which are treated to grow in a nutrient medium under sterile conditions.

Importance of Maintaining Aseptic Conditions

Aseptic conditions are critical in plant tissue culture to prevent contamination from microorganisms such as bacteria and fungi. These microorganisms can rapidly multiply in the nutrient-rich culture media designed for plant growth. By maintaining a sterile environment, we ensure that these unwanted guests do not compromise the growth and development of plant tissues. This is crucial because even a single bacterium or fungal spore can ruin a culture, leading to the loss of weeks or months of work.

Consequences of Contamination

1. Loss of Viability: Contaminants can outcompete plant cells for nutrients and space on the culture medium, leading to weakened or dead plant tissues.
2. Altered Growth: Some bacteria produce plant hormones or hormone-like substances that can radically change the way plant tissues grow, often causing deformities or uncontrolled growth patterns.
3. Biochemical Changes: Contamination can lead to biochemical changes in the culture medium, altering the pH and composition, which can be detrimental to the growth of the target plant tissue.
4. Economic Loss: Contamination not only wastes valuable resources such as media, explants, and labor but also can delay research and commercial projects significantly, leading to financial losses.
5. Spread of Contamination: In a laboratory setting, contaminants can spread from one culture to another, potentially affecting a large number of cultures and exacerbating the loss.

Preventing Contamination

To avoid these detrimental effects, it’s crucial to implement strict aseptic techniques. This includes everything from the sterilization of tools and surfaces to the proper handling of plant materials and culture media. By adhering to these practices, researchers and technicians can maintain the purity and integrity of plant cultures, ensuring successful outcomes in their tissue culture projects.

(We want to take a moment to let you know that this article includes affiliate links to products we love and recommend. If you decide to make a purchase through one of these links, we earn a small commission at no extra cost to you. This helps us keep our little business growing and continuing to bring you great content like this. Thank you for your support and happy gardening!)

Sterilizing Tools

Maintaining sterile tools is essential in plant tissue culture to prevent the introduction of contaminants into the culture environment. Various methods can be employed, each with its specific applications and benefits.

Bead Sterilizer

A bead sterilizer is a device used for the quick sterilization of small tools, such as forceps, scalpels, and scissors. It works by heating glass beads to a high temperature (typically around 250°C (482°F)) which kills microorganisms effectively and quickly.

Advantages of Bead Sterilizers Over Other Methods:

• Speed: Tools can be sterilized in just 5 to 30 seconds, allowing for almost immediate reuse.
• Convenience: No need for preheating or cooling down periods, which are typical with autoclaves.
• Safety: Reduces the use of potentially hazardous chemicals required for some disinfection methods.
• Efficiency: Ideal for small tools and frequent sterilization, minimizing downtime in workflow.
• Cost-Effective: Low operating costs compared to maintaining an autoclave, especially in settings where only small tools need regular sterilization.

However, bead sterilizers are not suitable for materials that cannot withstand high temperatures or are too large to fit into the device. For these, other methods such as autoclaving are more appropriate.

Autoclave

An autoclave uses steam under high pressure to achieve complete sterilization. It is effective for sterilizing bulk tools, media, containers, and other materials that can withstand the required temperatures and pressure.

Operation and Benefits:

• Complete Sterilization: Steam under pressure reaches higher temperatures than boiling water, effectively killing all microorganisms, including spores.
• Versatility: Can sterilize a wide range of tools and materials, including some plastics and all metal tools.
• Reliability: Provides consistent results with proper operation.

Guidance for Autoclave Operation:

1. Loading: Arrange items loosely in the autoclave to allow steam to circulate freely.
2. Cycle Selection: Choose the appropriate cycle based on the material type (e.g., dry goods, liquids).
3. Regular Maintenance: Ensure the autoclave is serviced regularly to maintain its efficiency and safety.

Pressure Cooker

For smaller labs or home setups, a pressure cooker can serve as an economical alternative to an autoclave. While not as controlled as an autoclave, it can reach temperatures and pressures sufficient to sterilize many types of tools and media.

Benefits and Operation:

• Cost-Effective: Significantly cheaper than a commercial autoclave.
• Accessible: More likely to be available in non-specialized settings.
• Adequate for Basic Needs: Sufficient for many types of plant tissue culture applications, especially in a non-commercial context.

Using a Pressure Cooker for Sterilization:

1. Water Level: Ensure sufficient water is in the cooker to generate steam but not submerge the items.
2. Heat and Timing: Follow specific guidelines for the duration and temperature necessary to achieve sterilization.
3. Safety: Handle with care, especially when releasing pressure and opening the lid after sterilization.

Air Flow Hoods in Plant Tissue Culture

Importance and Advantages of Laminar Flow Hoods

Laminar flow hoods, or clean benches, are essential in maintaining a sterile environment for plant tissue culture. These hoods provide a continuous wash of HEPA-filtered air across the work area, which is free from particulate contamination. This airflow is designed to sweep away airborne contaminants that could jeopardize the sterility of the culture media and the plant materials being manipulated.

Advantages include:

• Protection Against Contamination: The steady stream of filtered air creates a barrier that prevents airborne microbes from settling in the work area.
• Enhanced Sterility: Provides a controlled environment that significantly reduces the risk of contamination during sensitive processes like subculturing, inoculation, and media preparation.

Using and Maintaining Laminar Flow Hoods

1. Proper Usage:
   • Do Not Block Air Flow: Arrange all tools and materials so that they do not obstruct the airflow. Items should be placed downstream of the work area.
   • Avoid Cross-Contamination: Be mindful of your movements. Avoid rapid or sweeping movements that can disrupt the laminar flow and introduce contaminants from outside the clean area. Actions such as crossing your arms or touching your face can result in contamination, stay vigilant while engaging with sterile work areas.
2. Maintenance:
   • HEPA Filters: Regularly check and replace HEPA filters according to the manufacturer’s guidelines. Avoid touching the filters directly, as oils and debris from hands can damage the filter’s integrity.
   • Cleaning: Wipe down the interior surfaces of the hood with an appropriate disinfectant before and after each use, ensuring that the cleaning agents are compatible with the materials of the hood and do not damage the HEPA filter. Wipe down these surfaces at the beginning of the day, when leaving for periods of time such as lunch and at the end of the day. Do not touch or spray any disinfectants on the filter itself.

DIY Still Air Box: An Alternative to Laminar Flow Hoods

For those without access to a laminar flow hood, a still air box (SAB) is a viable alternative. A still air box provides a work environment that, while not as controlled as a laminar flow hood, still offers a degree of protection against airborne contaminants.

Constructing a Still Air Box:

1. Materials:
   • Large clear Tupperware container or a similar plastic box with lid.
   • Large Latex Gloves
   • A box cutter or drill for making holes.
   • Strong Tape
   • Optional: A small light to improve visibility
2. Steps:
   • Prepare the Box: Cut two arm-sized holes in one side of the Tupperware container. Ensure the edges are smooth to avoid snagging gloves. Tape the edges of the gloves to the outside of the drilled holes making sure you can fit your hands in easily and work without breaking the seal around the gloves. (This video is a similar tutorial as described here: How to Make a Still Air Box for Growing Mushrooms (youtube.com))
   • Sanitization: Clean and disinfect the entire container thoroughly before each use.
   • Setup: Place all necessary tools and materials inside the box before starting your work. Once your arms are in, avoid opening the box until you complete your task.

Limitations and Appropriate Use:

• Limitations:
  • The still air box does not provide active air filtration or circulation, which means it cannot clear airborne contaminants that already exist inside the box.
  • Its effectiveness depends heavily on pre-sanitization and minimal movement during use to avoid stirring up contaminants.
• When to Use:
  • Use a still air box for less critical or short-duration tasks where a laminar flow hood is not available.
  • It is particularly useful for hobbyists or small-scale operations where investment in a laminar flow hood is not feasible.

This is a fantastic DIY Video for a different version of the Still Air Box: Build A Simple Still Air Box Using Household Items! | Plant Tissue Culture at Home – YouTube

Handling Media and Cultures

Keeping Media Containers Closed

Maintaining the sterility of culture media is critical in tissue culture. Even brief exposure to the open air can introduce contaminants that compromise the growth and health of plant tissues.

• Prevent Airborne Contaminants: Always ensure that media containers are only open in a sterile environment, such as under a laminar flow hood, and are closed immediately after use.
• Minimize Exposure: Reduce the duration that media containers are open to as short as possible to limit the opportunity for contaminants to enter.

Handling Plant Cultures Carefully

Proper handling of plant cultures is essential to maintain their sterility and ensure successful growth.

• Gentle Handling: Handle cultures gently to avoid any damage that could expose the internal tissues to contaminants.
• Sterile Implements: Use sterile instruments to manipulate cultures and ensure that instruments are re-sterilized between handling different cultures to prevent cross-contamination.
• Workflow Management: Organize your workspace to move sequentially from one process to another without needing to backtrack, which can increase the risk of contamination.

Storage of Tools and Media

Tools

Proper storage of tools is essential to maintain their sterility after sterilization and before their next use.

• Sealed Containers: Store all sterilized tools in sealed containers that are labeled and dated. Containers should be only opened inside a laminar flow hood to maintain sterility until the moment they are used.
• Organized Workspace: Arrange tools in a manner that they are easy to reach and identify to minimize handling and movement that could lead to accidental contamination.

Media

The way in which culture media are stored can greatly affect their viability and sterility.

• Temperature Control: Store culture media at temperatures that will preserve their integrity. This often means refrigeration for media containing perishable components.
• Protection from Light and Air: Store media in conditions that limit exposure to light and air. Light can cause photodegradation of some components, while air exposure can lead to contamination and changes in pH.
• Use of Sealed Containers: Utilize tightly sealed containers for storing media. If possible, consider dispensing media into smaller, single-use aliquots. This minimizes the exposure of the bulk media every time a portion is needed, thus reducing the risk of contamination.

Importance of Personal Protective Equipment (PPE)

In plant tissue culture, the use of appropriate personal protective equipment (PPE) is crucial for maintaining a sterile environment and protecting both the cultures and the technicians. PPE helps prevent the introduction of contaminants from the outside environment and reduces the spread of microorganisms.

Essential PPE in Tissue Culture Labs:

• Lab Coats: Provide a barrier against contaminants that might be carried on everyday clothing.
• Shoe Covers: Minimize the transfer of contaminants from the floor into the sterile work area.
• Masks: Reduce the risk of contaminants entering the culture from respiratory droplets.
• Gloves: Protect cultures from contaminants that may reside on the skin.

Proper Donning and Doffing of PPE

Donning PPE:

1. Hand Hygiene: Begin by washing hands thoroughly with soap and water or using an alcohol-based hand sanitizer.
2. Shoe Covers: Put on shoe covers before entering the sterile area to avoid bringing in dirt and microorganisms from outside areas.
3. Lab Coat: Don the lab coat, ensuring that it is fully buttoned or zipped up to cover clothing completely.
4. Masks: Place the mask over the nose and mouth, ensuring it fits snugly with no gaps around the edges.
5. Gloves: Gloves should be the last item put on. They should cover the wrist area of the lab coat to create a continuous barrier.

Doffing PPE:

1. Gloves: Remove gloves first to prevent contaminating other parts of the PPE or the environment. Do so by peeling one glove off from the wrist to fingertips and holding it in the gloved hand. Slide fingers under the wrist of the remaining glove and peel it off over the first glove.
2. Masks: Remove the mask by handling only the ties or elastic bands. Avoid touching the front of the mask.
3. Lab Coat: Carefully remove the lab coat without touching the outside surface. Unbutton or unzip, then pull the coat away from the body, touching only the inside.
4. Shoe Covers: Remove shoe covers last to prevent any transfer of contaminants from the floor as you exit the sterile area. Handle only the edges of the shoe covers and avoid touching the bottom.
5. Hand Hygiene: Perform hand hygiene again after all PPE has been removed.

Ensuring Compliance

Regular training and reminders about the correct procedures for donning and doffing PPE are essential. It is also beneficial to have clear, visual guidelines posted in dressing and undressing areas to reinforce best practices and ensure all staff members are compliant with these critical procedures.

Maintaining Cleanliness in the Lab

Regular cleaning and disinfection of the lab, particularly the flow hoods and other critical surfaces, are essential components of maintaining aseptic conditions in plant tissue culture.

Routine Cleaning of Flow Hoods and Lab Surfaces

1. Flow Hoods:
   • Frequency: Wipe down the interior surfaces of the flow hood at the start and end of each day, and also before leaving for any extended period, such as lunch breaks. This practice prevents the build-up of contaminants that can occur even when the hood is not actively being used.
   • Procedure: Start by cleaning the hardest to reach areas and move towards the exit of the hood to avoid re-contaminating cleaned surfaces.
   • Materials: Use a soft cloth or non-shedding wipes to avoid leaving residues that could harbor microbes or interfere with the laminar flow.
2. Lab Surfaces:
   • Wipe down all benchtops, handles, drawers, and commonly touched surfaces with an appropriate disinfectant. Pay special attention to high-touch areas to reduce the risk of cross-contamination.

Choosing Appropriate Disinfectants

Different surfaces and materials in the lab may require specific types of disinfectants to ensure effective cleaning without damaging the surfaces:

• Alcohol Solutions (70% ethanol or isopropanol) are effective for quick sanitization of stainless steel, glass, and other non-porous surfaces. Alcohol evaporates quickly, leaving no residue.
• Chlorine Bleach Solutions are suitable for more thorough disinfection of sinks and drains where biological material may accumulate. However, bleach can be corrosive and should be used cautiously on metal surfaces.
• Quaternary Ammonium Compounds are good for plastic surfaces and equipment, as they are less corrosive than bleach and have good cleaning properties.
• Hydrogen Peroxide is useful for surfaces sensitive to bleach and can be used as an alternative for disinfecting incubators and laminar flow hoods.

Regular Cleaning Schedules

Establish and maintain regular cleaning schedules that include:

• Daily, weekly, and monthly tasks.
• Deep cleaning sessions for the entire lab, which should be scheduled less frequently but are essential to maintain overall cleanliness and function.

Disinfecting Storage Areas

Storage areas for tools and media also require regular disinfection to prevent them from becoming sources of contamination:

• Tools: Ensure that storage cabinets for sterilized tools are wiped down regularly. If tools are stored in drawers or bins, these should also be cleaned and disinfected routinely to maintain sterility.
• Media: Media storage refrigerators and cabinets should be cleaned monthly to prevent any spills from becoming sources of microbial growth. Check for expired media and dispose of them properly to avoid accidental use.

Ensuring Compliance

• Documentation: Keep logs of cleaning activities to ensure that all required cleaning procedures are followed and completed on schedule.
• Training: Regularly train lab personnel in proper cleaning techniques and the use of disinfectants, emphasizing the reasons behind each practice to foster compliance.

Additional Tips for Maintaining Aseptic Conditions

No-touch Technique

The no-touch technique is a critical practice in maintaining sterility in the lab. This technique involves manipulating sterile items and materials in such a way that direct hand contact is minimized or eliminated.

• Implementation: Use tools like forceps, which have been sterilized, to handle sterile items. When possible, set up the workspace so that items are transferred directly from the sterilizer to the workspace without being touched.
• Benefits: This method greatly reduces the risk of contaminating sterile components, such as culture media and plant tissues, thereby enhancing the overall success of the culture processes.

Workflow Optimization

Organizing the lab workspace to ensure a logical flow from dirty areas to sterile areas is essential for minimizing the risk of contamination:

• Design: Arrange the lab so that all activities involving non-sterile materials are done well away from the areas where tissue culture and media preparation occur.
• Physical Barriers: Use physical barriers if possible, such as different bench spaces or clear demarcations on benchtops, to designate clean and dirty areas.
• Directional Workflow: Always work in a direction that moves from clean to less clean areas, ensuring that movement does not need to backtrack into sterile zones after visiting non-sterile areas.

Traffic Control in Lab

The movement of people within and around the tissue culture lab can disrupt air patterns and introduce contaminants:

• Limit Access: Restrict access to the tissue culture room to essential personnel only. This control helps in maintaining the cleanliness and integrity of the aseptic areas.
• Schedule Coordination: Manage schedules to minimize the number of people in the lab at any one time, thereby reducing air disturbance and potential contamination.

Regular Audits

Regular audits of aseptic techniques help ensure that all personnel are adhering to established protocols and identify any areas where practices may need improvement:

• Frequency: Conduct audits on a regular basis, such as quarterly or semi-annually.
• Checklist: Develop a comprehensive checklist that covers all aspects of aseptic technique, from personal hygiene to the correct use of equipment and handling of materials.
• Feedback and Action: Provide immediate feedback and corrective action plans for any non-compliance found during audits.

Training and Refreshers

Ongoing education and training are key to maintaining high standards of aseptic technique in the lab:

• Regular Sessions: Hold training sessions at regular intervals (e.g., annually) to refresh staff on proper aseptic techniques and introduce new procedures or updates.
• New Employee Training: Ensure that all new lab personnel undergo thorough training in aseptic techniques as part of their onboarding process.
• Simulation and Role-playing: Include practical sessions where staff can practice techniques in a controlled setting, which helps in reinforcing correct practices.

Maintaining strict aseptic practices is not just a routine part of plant tissue culture; it’s the backbone of its success. The meticulous nature of these practices directly influences the health and viability of plant cultures by shielding them from the myriad of microorganisms that thrive in less controlled environments. By adhering to rigorous sterilization processes for tools and surfaces, employing proper protective equipment, and carefully handling media and cultures, lab personnel can significantly reduce the risk of contamination. This in turn ensures that the cultures develop as expected, free from the setbacks of microbial invasions.

The dynamic nature of microbial threats, coupled with the evolving standards and technologies in tissue culture, means that what works today might need adaptation tomorrow. Continuous evaluation and improvement of aseptic techniques are crucial. Every technician and scientist involved in tissue culture should see themselves as a guardian of these sterile environments, always vigilant and always ready to adapt their methods for the sake of their cultures’ purity and productivity.

Encouragement for Ongoing Improvement:

1. Stay Informed: Keep up to date with the latest developments in aseptic techniques and technologies. Advances in materials, equipment, and methodologies can offer new ways to enhance sterility.
2. Regular Feedback and Learning: Encourage an environment where feedback on aseptic practices is regularly sought and given. Learning from mistakes and near misses can prevent future occurrences and foster a culture of continuous improvement.
3. Participate in Training and Workshops: Engage with workshops and training sessions not just as a participant, but also as a contributor. Sharing experiences and insights can help improve practices not only within your own lab but across the wider scientific community.

By embedding these practices into the daily routine and striving for continual improvement, tissue culture labs can maintain the high standards necessary for successful plant tissue culture. Ultimately, the goal is to ensure that every culture thrives in its intended outcome, contributing to advancements in research, agriculture, and biotechnology.

We would love to hear from you! Leave us a comment below about your tissue culture experiences and what you would like to see us cover next!

Up Next: Exploring the Incredible World of Callus Tissue Culture: The Future of Farming!

For tissue culture supplies and a detailed description of each product, visit our Tissue Culture Supplies Page.

Visit our Retail Shop for some amazing rare and variegated plants to add to your collection!

Coming soon!

In collaboration with Plant Cell Technology and Xplant Laboratory, we are happy to announce that we will be hosting our first Tissue Culture Masterclass this summer on August 10th-11th! Visit out Main Page and submit the form for more details. You won’t want to miss this exciting opportunity!

Check out this introductory video for more on the Master Class!

(Disclosure: This post may contain affiliate links, meaning I get a commission if you decide to make a purchase through my links, at no cost to you. These links will be seen throughout the post and listed at the bottom for easy reference.)