Different types of contaminants in plant tissue culture

Did you know that after all the sterilization procedures, there is still a chance of finding contaminations in plant tissue cultures?

That's possible due to contaminants like endophytic bacteria. Bacteria that is already present in plant system and shows up at certain growth stages.

But first: what is contamination in plant tissue culture?

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 quite difficult to get rid of contamination completely as plants are highly susceptible to get pathogen infection. Especially in the case of plant tissue culture.

Why tissue cultures show more susceptibility to contaminations?

The culture media contains 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 biggest bottlenecks in the world of plant tissue culture.

The composition of different ingredients in a tissue culture media is an important step that plays a significant role in the success of your plant tissue culture protocol. To understand contaminations in tissue culture it would be beneficial if you know about different ingredients used in tissue culture media. You can read more in-depth about different components of tissue culture media in our blog on "8 components of tissue culture media".

Let us now discuss different kinds of contaminations that are possible in plant tissue culture:

Types of contamination

The contamination in tissue cultures can appear due to airborne microbes, contaminated lab equipment, or contaminated water. However we, humans, are the most dangerous source for bringing contaminants inside a lab. Hence, if you maintain and follow precise protocols to keep your lab clean, you can have a contaminant-free laboratory.

Though sources of contamination can be tricky to find, once you identify them, it should be your priority to eliminate them. Contaminants may express themselves immediately or can remain latent for long periods of time. This often makes it difficult to identify the source of contamination.

Bacterial and fungal contaminants are relatively easy to identify. But mycoplasma infections show subtle visual symptoms and are difficult to identify. Viruses are quite more challenging as they show even more subtle symptoms.

Contaminants in plant tissue culture are divided into two broad groups:

• Chemical contaminants
• Biological contaminants

Chemical contamination

We speak of chemical contamination when non-living substances present in the laboratory or culture protocol impact tissue culture growth negatively. But where can you find such contaminants?

• The majority of chemical contaminations are present in tissue culture growth medium.
• If there is non-purified water or overly pure water. It is proven that highly pure water is very reactive and will lead to the leaching of toxic chemicals from the equipment used during the tissue culture process. These contaminants can then end up in media or deposited on storage vessels and pipettes during washing and rinsing.
• If there are endotoxins from bacteria.
• If there are free radicals developed in media recipes when they get exposed to fluorescent lights. These free radicals are toxic to cultures. When you leave media on lab benches for longer periods or store media in walk-in cold rooms with lights on, this will cause gradual deterioration of your media quality.
• If there are heavy metals, maybe from water or different chemical ingredients.
• Deposits on glassware, pipettes, instruments, etc., left by disinfectants or detergents.

Chemical contamination can also happen due to mistakes in media preparation. Mistakes in reading bottle labels or wrong weighing of ingredients can lead to poor culture growth. Even essential nutrients can become toxic at high concentrations.

Media present in glass or plastic bottles that have previously contained solutions of heavy metals or organic compounds can be another source of contamination. These contaminants can leach into the media when we don't clean these bottles properly.

The important thing to keep in mind is that chemical contaminants are additive in nature. This simply means that small contaminant amounts from different sources when combined together in any medium can lead to severe toxicity of cultures. However, it is possible that these small contaminant amounts are not toxic individually.

Biological contaminants

Different biological contaminants causing contamination in plant tissue culture processes are:

• Bacteria
• Yeast
• Molds
• Viruses
• Mycoplasmas
• Cross-contamination from other cell lines

We can classify biological contaminants into two groups based on the difficulty of detecting them in cultures:

• Those that are usually easy to detect: bacteria, molds, and yeast; and
• Those that are more difficult to detect, and as a result potentially more serious culture problems: viruses, mycoplasmas and other microorganisms.

Bacteria and fungi

Bacteria, molds, and yeasts are present virtually everywhere and are able to quickly colonize and flourish in the rich and relatively favorable environment available for tissue cultures. These microbes are the most commonly encountered cell culture contaminants, because of their size and fast growth rates.

We can easily detect these microbes in cultures within few days when we don't supply antibiotics to the culture medium. The best way to keep your plants free of microbial contamination is to perform daily observations. This practice can help you to protect your cultures in the long run.

However, if you regularly supply antibiotics to the media, then these organisms can develop resistance and convert into slow-growing or low infection-causing organisms. Eventually, they become very difficult to detect just by visual observations. As a result, many laboratories use microscopes for the identification of these microbial cells.

Mycoplasmas

If you are wondering which contaminant should cause you the most concern, then here it is: mycoplasma. They are severe contaminants and are famous as one of the notorious contaminants found in tissue culture.

They are difficult to detect and have a profound negative effect on cultures. While they are classified as bacterias, however, they lack a cell wall and are quite smaller than other bacterias. So they easily enter filter membranes that we sometimes use during sterilization.

Viruses

Viruses are quite difficult to identify. Unlike other common contaminates, their symptoms take time to show up and are hard to visually identify. There are no cloudy appearances or pH changes in the media when a viral infection is present.

Although the viruses can cause severe damage to the host, they are usually self-limiting. Therefore, the major risk viruses pose is for us, the people working in the laboratory. Also, we can end up becoming carriers of viruses to different species of plants, if we are not careful enough. This is why it is always suggested to strictly follow all the precautionary measures in a laboratory.

So these are the different contaminants that can cause serious damage to your plant tissue cultures. How you can detect them and what you can do to prevent them, are for another discussion. For more articles on this topic, keep checking this space!

By Nancy Bhatia | 30-June-2021

References

• https://safety.fsu.edu/safety_manual/supporting_docs/Understanding%20and%20Managing%20Cell%20Culture%20Contamination.pdf
• https://study.com/academy/lesson/cell-culture-contamination-types-identification.html
• https://www.technologynetworks.com/cell-science/how-to-guides/how-to-prevent-cell-culture-contamination-htg-299231