Plant Tissue Culture Contamination: Causes, Identification & Prevention

Contamination is one of the most common challenges in plant tissue culture. Even when a culture looks perfectly clean immediately after inoculation, bacteria or fungi may appear several days or even weeks later.

Understanding where contamination comes from is one of the most important skills a tissue culture grower can develop.

Contamination usually comes from one of three places: the plant material itself, the working environment, or the equipment and materials used during culture.

External contamination

The surface of a plant naturally carries microorganisms. Leaves, stems, roots and corms may contain fungal spores, bacteria and other microbes that are harmless to the plant outdoors but become a serious problem inside a nutrient-rich culture vessel. This is why explants are surface sterilized before they are introduced into culture. Plant material that has been growing in soil is generally more difficult to sterilize than material taken from cleaner above-ground growth.

Internal contamination

Some microorganisms live inside plant tissues rather than on the surface. These are sometimes called endophytic microorganisms. Because they are protected inside the plant, surface sterilization may not remove them. A culture can therefore remain clear for several days before bacteria suddenly begin growing from the explant. This is one reason why establishing a clean mother plant before tissue culture can significantly improve success rates.

Contamination from the working environment

Air contains fungal spores, bacteria and dust particles. Whenever a culture vessel is opened, there is an opportunity for microorganisms to enter. The risk can be reduced by working inside a laminar-flow cabinet or a properly prepared still-air workspace and by keeping culture vessels open for as little time as possible.

Contamination from tools

Scalpels, forceps and other tools must be sterile when they touch an explant. A tool that touches a non-sterile surface can transfer microorganisms directly into a culture vessel. Good sterile technique therefore involves thinking constantly about which surfaces are sterile and which are not.

What does contamination look like?

Different contaminants can look very different.

Bacterial contamination

Bacteria often appear as:

cloudy areas in the medium
wet or slimy growth around the explant
translucent films
yellow, cream or white colonies
rapid deterioration of plant tissue

Some bacterial contamination is extremely subtle and may initially appear only as a faint haze around the base of the explant.

Fungal contamination

Fungi are usually easier to recognize.

Common signs include:

white fluffy growth
grey or black mould
fine thread-like structures
rapidly expanding colonies
coloured spores

A single fungal colony can spread across an entire vessel surprisingly quickly.

Yeasts

Yeast contamination may appear as small cream-coloured colonies or cloudy growth in the medium. It can sometimes be mistaken for bacterial contamination.

Why contamination sometimes appears late

A culture that looks clean after three days is not necessarily sterile. Microorganisms may be present in very small numbers and need time to multiply before becoming visible.

Internal bacteria can also emerge gradually from the plant tissue. For this reason, new cultures should normally be observed for several weeks before they are treated as reliably clean.

How to reduce contamination

Successful contamination control relies on several layers of protection:

Use healthy mother plants whenever possible.
Thoroughly clean plant material before sterilization.
Use an appropriate surface-sterilization procedure for the type of explant.
Sterilize culture media and vessels properly.
Work in a clean environment.
Sterilize tools between explants.
Minimize the time culture vessels remain open.
Avoid touching sterile surfaces.
Keep contaminated cultures away from clean cultures.

No sterilization protocol works perfectly for every species. A soft young shoot and a soil-grown corm may require very different treatment.

The goal is to find a balance between killing microorganisms and keeping the plant tissue alive.

Should contaminated cultures be saved?

In most hobby tissue culture situations, heavily contaminated cultures should be removed from the growing area. Opening a contaminated vessel near clean cultures can release fungal spores or bacteria into the workspace.

If an explant is particularly valuable, it may sometimes be possible to remove clean internal tissue and attempt re-establishment, but this should be done carefully and separately from clean cultures.

The most important rule

Contamination is not simply a failure. Every contaminated culture gives information.

Record:

the plant species
explant type
sterilization method
sterilization duration
date of inoculation
type of contamination
how long it took to appear

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