4 methods of sterilization used in plant tissue culture

If you have been reading about plant tissue culture, then by now you know how important sterile conditions are for a successful in vitro culture practice. However, did you know about the different methods laboratories around the world use to sterilize laboratory articles, explants, and equipment?

Contamination in plant tissue culture consists of impurities, chemicals, or pathogens. These make the cultured tissues unfit for further development of healthy plantlets. It is also difficult to get rid of contamination completely as plants are highly susceptible to get pathogen infections.

There are different contaminations that can occur in your tissue culture experiment. Micro-organisms could be present on beakers, pipettes, or on your culture vessels. A single fungal spore or bacterial cell that comes into contact with the culture media or explants rapidly contaminates plant tissue culture materials and instruments as well. If you want to know more about different micro-organisms that cause contaminations, then do consider reading our article on "Different types of contaminants in plant tissue culture".

The tissue culture media also contain sugar and other elements that are more susceptible to attract microbes like bacteria, fungi, etc. Thus, plant tissue cultures are naturally prone to contaminations and hence, are hard to avoid. Over the years, scientists have come up with different solutions to reduce existing contaminants. But contaminations are still one of the largest bottlenecks in the world of plant tissue culture, leading to loss of yield and productivity among cultured plants.

Let us now discuss different methods of sterilization that are popularly used for eradicating or avoiding contaminations:

Sterilization by heat

Sterilization of media and glassware

Many laboratories use autoclaves for sterilizing media which works on the principle of moist heat. Moist heat sterilization is a procedure in which heated, high-pressure steam is used to sterilize an object (at 121-degree °C, 1.03 bar pressure for 15-20 minutes). You can sterilize labware, glass articles, pipettes, and culture media with the help of an autoclave. You can get rid of nearly all microbes with this method. However, autoclaves are labor-intensive and time-consuming when the overall process is considered. An autoclave takes around 2-3 hours of time for heating up, sterilizing, and cooling down.

Nowadays, most industries prefer highly efficient media preparator machines which have an automated workflow to prepare sterilized media. These machines use different heating techniques with great precision and take quite less time as compared to an autoclave. Research has shown that a very fast sterilized medium will not only increase the resistance against diseases of your plantlets, but will also reduce the growth cycle time of some and increase the multiplication rate of others.

Sterilization of metallic tools

Metallic instruments may rust and become blunt with the use of moist sterilization. Therefore, objects like scalpel holders/blades, forceps, etc. are sterilized using dry heat methods. This sterilization is achieved by using instruments like glass bead sterilizers and bunsen burners, the former being more efficient and safer than the latter.

*Cycle time from 60° C, 15 minutes of sterilization at 121° C, and back to 60° C.

Sterilization by filtration

Certain media ingredients are thermo-labile such as plant extracts, hormones, etc. and they tend to decompose due to the high temperature involved in the sterilization process via heating. Thermo-labile substances are easily deactivated or are destroyed in the presence of heat. Hence, using a syringe/needle and filter membrane, these thermo-labile ingredients can be added to the media batch in a media preparator, simply by injecting it through the septum connection on the lid.

Using this method, you can force a liquid solution through a membrane to sterilize. The size of the pore in the filter decides what size of microorganisms will be filtered out. However, the pore size of the filter membrane should not be larger than 0.2 microns. During this sterilization, all the particles and micro-organisms that are bigger than the pore diameter of the filter get removed.

Air sterilization

In simple terms, there are millions of particles floating in the ambient air. They may comprise microbes such as viruses, bacteria, fungi, and many more. Eventually, these floating particles will land on objects such as culture vessels, explants, etc., and cause contaminations. For this very reason, it is necessary for you to ensure that clean and sterile air is flowing over your tools, containers, and specimens. In order to achieve this goal, you need proper air treatment equipment such as laminar flow cabinets. They possess HEPA filters (high-efficiency particulate air filter) and hence, the air passes through this filter and thereby traps fungi, bacteria, and other dust particles. 

Explant sterilization

Explant sterilization is a crucial step in the tissue culture process. The explants that you obtain from the stock plant need to be free from any kind of micro-organisms. This method involves several steps of treatment for efficient results. Initially, you need to clean your explants after extracting them from the stock plants. You can decide on which type of cleaning protocol to use, based on how contaminated your explant is. You also need to consider the sensitivity of the chosen explant to the cleaning agents/disinfectants. A too strong cleaning agent will kill your explant, while a too weak agent will not do the job.

Let us now talk about some common sterilizing agents.

Common explant sterilization agents

Mercuric chloride: It is rarely used in labs because of its high toxicity to plants as well as humans. So, if you are using it, follow extensive care.

Sodium hypochlorite: You can also call it bleach. It is one of the most commonly used agents for surface sterilization of explants. Bleach is diluted up to 10-20% for sterilization in tissue culture processes. This gives you a solution with the final concentration of 0.5-1.0% of bleach. The time duration required to sterilize the explant varies for each type of explant. But, usually 30-40 minutes are enough to serve the purpose of sterilization.

Calcium hypochlorite: It is commercially available in the form of powder and before using it for sterilization, it must be dissolved in water. You also need to filter this solution. It is normally used in a concentration of 3.25%.

Hydrogen Peroxide: It is also a commonly used chemical for surface sterilization. The most favorable concentration of this agent is 3%. However, you should take all precautions while using this agent for sterilization.

Ethanol: It is also known as isopropyl alcohol. The ethanol concentration between the range of 70-95% is extensively used for sterilizing. However, ethanol is also toxic to plants. Hence, you should keep explants in ethanol for only one second as a longer duration in ethanol damages the explants.

And lastly, you also need to sterilize the surface areas in your laboratory. This means that all the surfaces such as tabletops, floor, cabinets, cupboards, etc. should be sterilized with ethanol, bleach, or a cleaning solution on a regular basis.

We hope you got a glimpse of some important methods to follow in order to maintain a clean and safe environment for culturing plants in a laboratory. For more informational posts on different aspects of plant tissue culture, keep checking this space!

Also if you like this article, do share it with others as well. Let us build a wonderful community of plant tissue culture enthusiasts.

By Nancy Bhatia | 9-September-2021

References

1. Stanisavljević, Aleksandar & Bošnjak, Dejan & Ivna, Štolfa & Vukovic, Rosemary & Kujundžić, Toni & Drenjančević, Mato. (2017). Sterilization of different explant types in micropropagation of CAB-6p and Gisela 6 cherry rootstock. Poljoprivreda. 23. 31-37. 10.18047/poljo.23.2.5.
2. https://www.sigmaaldrich.com/technical-documents/protocols/biology/explant-sterilization.html
3. Robert H. Smith (2013). Plant Tissue Culture- Techniques and Experiments. Elsevier Inc.
4. Kyte, Kleyn, et al (2013) Plants from test tubes: An introduction to micropropagation. Timber press, Inc.
5. Bhojwani, S.S., & Dantu, P.K. (2013). Plant Tissue Culture: An Introductory Text. Springer India
6. Meristem culture and subsequent micropropagation of Chilean strawberry (Fragaria chiloensis (L.) Duch.). Retrieved from https://biolres.biomedcentral.com/articles/10.1186/s40659-017-0125-8