How to Avoid Contamination During Cell Culture?

How to Avoid Contamination During Cell Culture? Complete Guide!

How to Avoid Contamination During Cell Culture? Contamination in cell culture can cause problems. Contaminated cells are a waste and should not be used for any experiment. They are all hard to obtain and grow. The worst thing is that you might end up contaminating other people’s cells and results of experiments.

How to Avoid Contamination During Cell Culture?
How to Avoid Contamination During Cell Culture?

Table of Content

Techniques to Avoid Contamination in Cell Culture

Pre-wetting techniques

One of the most important aspects of cell culture is proper pre-wetting. Pre-wetting techniques can help prevent contamination during the culture process. Moreover, proper labeling of flasks and vials is very important. The right labeling minimizes cross-contamination.

Maintain Sterility

The reagents used for cell culture must be sterile. These reagents should be filtered through 0.2 mm filters. Also, aliquoting can help preserve the sterility of the reagents. This technique allows scientists to discard only a small portion of a stock if it becomes contaminated.

In addition, media should be sterilized thoroughly to minimize the possibility of contamination. Depending on the type of contamination, some bacteria are easy to detect using the unaided eye, while others can only be detected by standard light microscopy.

Microscopic Observations of Cell Culture Medium

Regular microscopic observation of the cultures is important for detecting microbial contamination early, preventing the contamination of neighboring cultures. The sterility of the medium is also preserved when the media is stored at room temperature.

Read more: How to Maintain Sterility in Cell Culture Lab? 

Prior Preparation

Before you start preparing your samples for cell culture, it is important to prepare the cells with proper reagents. The preparation of the cells should be done in an aseptic environment. For example, before preparing the culture medium, the cells should be suspended in a single-cell suspension.

Proper pipetting procedures

Proper pipetting procedures are crucial for the maintenance of sterile environments during cell culture. A simple mistake can result in contamination of a culture. This error has the potential to delay or even prevent a subsequent experiment. To minimize this risk, follow the guidelines listed below:

  • It is important to use a pipette with a sterile tip. This will decrease the chances of contamination. Also, pipette tips should always be positioned on the same side of the hood as the person who will be pipetting.
  • The non-sterile part of the pipette should be placed farther back. It is also important to keep the hood clean by avoiding contact with samples.
  • Proper training and guidance are essential for preventing contamination during the cell culture process. This can be achieved by ensuring that all personnel involved with the process are trained.
  • In addition, a separate laboratory should be designated for cell culturing and maintenance. This can reduce the risk of contamination from bacteria, fungi, and other microorganisms.

Storage of Chemicals

To minimize the risk of cross-contamination, make sure to store the cell culture medium and solutions in separate bottles. The risk of contamination is highest when multiple cell lines are stored in one incubator.

Also, CO2 incubators and autoclave (Validation of an Autoclave? How To Validate the Autoclave?) should be cleaned daily to ensure that all components are sterile. Also, distilled water used to maintain the humidity should be changed regularly. This water is known to contain pathogens.

Read more: Genetic Toxicology-A Brief Guide to Genetic Toxicology

Ensure proper pipetting procedures for each step in cell culture. Avoid rushing through the process because cells have an inherent lag period. This phase can also cause the cell culture to fail, thus wasting precious time and money. If not done properly, mistakes can be made that affect the whole culture.

Types of Contaminations in Cell Culture

Besides the usual bacteria and fungi, another important problem in cell culture is mycoplasma contamination. Mycoplasma can cause considerable damage to cell culture. These microbes are difficult to detect by casual inspection, but can be detected through morphological changes, unusual proliferation rates, and abnormality in cell health. Commercially available mycoplasma detection kits can help you confirm whether or not your cultures are mycoplasma-free.

Read More: How to induce Apoptosis in Cell Culture?

Bacteria, fungi, and yeast can infect cells and media. The bacteria often produce endotoxins, which cause a fever in humans and inhibit cell culture growth. When bacteria die, endotoxins are released into the environment. These toxins can be present in almost any environment where bacteria can grow. Viruses can also infect cells and cause contamination.

Preventing techniques to Avoid Contamination in Cell Culture

Avoiding overuse of antibiotics

While antibiotics can be useful for short-term therapeutic purposes, routine, long-term use in cell culture creates more problems than they solve. Unlike other drugs, antibiotics can cause toxicity in cells and can impair their morphology and function. This can compromise the results of experiments. To avoid these problems, it is important to follow guidelines and avoid using antibiotics during cell culture.

Here are some guidelines:

Antibiotics are commonly used in cell culture to control contamination and maintain aseptic conditions. They also allow researchers to select cells that contain certain genetic modifications or to prevent loss of cell lines. Which antibiotics to use depends on the cell type and the contaminants to be avoided. Selectable markers are also helpful for guiding antibiotic selection.

Antibiotics can interfere with the metabolism of eukaryotic cells, making the risk of contamination even greater. When used for extended periods of time, antibiotics can also create antibiotic-resistant bacterial strains. These are a major concern for cellular research. To avoid this, it is important to practice good aseptic techniques and to sterilize instruments and media thoroughly.

When to Use Antibiotics?

In general, antibiotics should be used only when necessary and only as a last resort. Antibiotics should be removed as quickly as possible after use. In cases where they cannot be avoided, the best course of action is to use a lower concentration. This can reduce the risk of antibiotic-induced genetic changes.

Antibiotics are necessary for some treatments, but overuse and misuse of antibiotics are leading to the development of antibiotic-resistant bacteria. This can lead to higher medical costs, prolonged hospital stays, and increased mortality. Therefore, the world needs to change its attitude towards antibiotic use. Without a change in behavior, antibiotic resistance will continue to increase and become an even greater threat to public health.

Sterilization of lab equipment

Among the most important preventive measures to reduce cell culture contamination is the proper sterilization of lab equipment. Sterilization of equipment can be achieved by regularly cleaning it with a disinfectant that eliminates bacterial spores. Equipment that is not sterilized may harbor microbial spores or even bacterial DNA. In addition to sterilizing equipment, it is important to wash hands frequently. This includes before working with cultures and before entering the lab.

Sterilization of Media and Equipment

Sterilization of lab equipment and media is essential for ensuring that cell culture media remain free from contamination.

A typical workflow involves setting up an aseptic work area in a biological safety cabinet, using gloves, and disinfecting the equipment with bleach or 70% ethanol.

Sterilization is also important for avoiding contamination by viruses, which are difficult to identify visually. The process of disinfecting lab equipment and instruments should be thorough. Bleach is a common disinfectant that is easily available and cheap. It inactivates vegetative bacteria, fungi, and lipid and non-lipid viruses. However, this disinfectant is very toxic without the proper ventilation system, fume hood, or personal protective equipment.

Sterilizing lab equipment is essential to ensure that the media used for cell culture is free from bacteria and fungi. Pipettes and serological pipettes are essential for microbiology experiments and should be sterile. They should also be equipped with a cotton wool plug to prevent overfilling and contamination. Glass serological pipettes are recommended for experiments involving organic solvents.


For optimal results, sterilize equipment and workbenches regularly. The process of sterilizing the biosafety cabinet should be done in an enclosed area that is cleaned and disinfected monthly. This helps keep the cell culture samples free from contamination. For labs with multiple lines, it is important to avoid working on more than one line at the same time, as this can cause cross-contamination.

What We have Learnt?

Cell culture is a crucial process in science and a successful experiment depends on keeping the cells free from contamination. Infections in the medium can come from a variety of sources, including airborne particles, contaminated work surfaces, or unsterile supplies. To avoid contamination, a cell culture laboratory must adhere to strict best practices and use advanced technologies. In addition to using gloves, sterile equipment should be kept free of clutter.