How to Detect, Avoid and Deal With Contaminated Cell Cultures

Jul 3, 2016 | Cell Culture Techniques

There is nothing more tragic than walking into your tissue culture room only to find a researcher curled up on the floor, clasping a flask, and wailing that their cells have been contaminated. It’s a fate I wouldn’t wish on my worst enemy! Hopefully it has never happened to you and never will but if it has or if you’d like to know what you have to do if it does happen to your lab or if you want to learn how to avoid contamination, read on!

If you’re new to culturing, you might be wondering if contamination is a big deal or not. Simple, usually, it is and here’s why:

Once contaminated, most tissues or cells are only fit for the trash. This is especially bad if your cells were special, hard to obtain, difficult to grow, or worst of all, entrusted to you by another person while they were out.
It leads to wasted time and money.
If you didn’t realize early on, you may not only have a contaminated culture but also contaminated results from your experiments.
You may have contaminated other people’s cultures.
It’s a hit to how much people are going to trust you including your coworkers and boss!

To name a few pretty significant reasons!

In order to deal with contamination and even better to avoid it in the first place, we need to look at the types of contaminants.

Assessing the Damage: Discovering, Assessing and Dealing with Contaminants

How easy the contaminant is to detect is the most important factor in deciding how “bad” your contamination is. Extensive damage can done by tainted cells affecting months or even years of research unbeknownst to you. Here, we’ve graded contamination into three levels. With each of these, good aseptic technique is essential to avoiding an invasion by nefarious solutions or creature.

Minor

About:	Includes most bacteria, yeast and fungi.
Likelihood:	Quite common.
How to detect it:	For bacteria: Look for shape moving around that are distinct from your cells. For yeast and fungi: Look for clumps, mats, budding (yeasts) and colonies on the surface of the media (fungi). If your media contains phenol red: look for changes in the color of your media as this indicates pH changes. If it starts to go orange/yellow, you may have a problem (either contamination or you need to replenish your cell’s media supply more frequently). Look for signs of turbidity or cloudiness of the media. Take 1ml of your culture/potentially contaminated media/new cell/cells fresh from storage and add it to 14 ml of media in a tube. Incubate and examine by eye and under your microscope at 400X magnification.
Dangers:	Easy to mistake for cell debris and thus miss, particularly when at lower levels. To avoid mixing these up remember: Static shapes are usually debris. Debris isn’t usually uniform in size and shape.
How to treat it:	If possible, start again from scratch. If you’d strongly prefer to attempt a salvage operation and save your cells/tissue, this level of infection is usually treatable with an appropriate antibiotic.

Major:

About:	Includes substances and biologics that are harder or nearly impossible to eliminate.
Likelihood:	Less common to rare.
How to detect it:	Chemical contaminants are very difficult to detect. Viruses can be detected using ELISA, PCR, immunostaining and other similar techniques. Insects and arachnids may carry other harder to detect organisms. Parasites are also very difficult to detect.
How to treat it:	There is no sure fire way to remove viruses or chemical contaminants so if possible start from scratch.

More major contamination would include substances and biologics that are harder or nearly impossible to eliminate. These include viruses and parasites.

Potentially Disastrous:

About:	Mycoplasma have no cell walls, are tiny (0.2-0.3 µmeters), can affect virtually every aspect of a cell’s behavior and growth and can exist in very large numbers without being detected. They can cause every kind of disaster from chromosomal breakage to changes in your cell’s morphology. The most common route of transmission is from one contaminated culture to another.Other cell lines: usually HeLa cells!
Likelihood:	Most common forms of contamination as they are highly contagious and so difficult to detect.
How to detect it:	Mycoplasma: PCR and ELISA. Fluorescent DNA stains (requires a fluorescence microscope). Send off to be tested. Cross-contamination: Karyotyping DNA fingerprinting. Isoenzyme analysis.
How to treat it:	You need to autoclave everything. There are cleaning services you can even hire for this purpose of completely eliminating the mycoplasma. Beware! Commercial clean-up kits are not very effective and are often only temporary solutions.

A Note on Antibiotics

Regular use is not necessary and is simply a crutch for those with poor technique! Short term use (1-2 weeks) is fine if you’re not planning on using the cells to create more batches. Long term use may lead to antibiotic resistant strains and may allow low levels of contamination go undetected. Antibiotics can also affect cell’s normal behaviour and thus impact your results.

Bottom line: Do not overuse antibiotics. They do not prevent contamination and are not a substitute for good technique.

Rules of Thumb to Minimize Contamination

Be clean: Practice good technique. Never use tap water or potentially contaminated media (like media or other supplies used by anyone else)
Be suspicious: Never trust that any culture is what it says on the label, even newly bought cells.
Be cautious: Only buy from reputable cell banks. Quarantine incoming samples until their identity can be confirmed and their cleanliness status verified. This is true for cells you both buy and remove from storage.
Be guarded: Never use cells from another lab.
Be proactive: check for contamination regularly.
Be diligent: never leave your cells to someone who isn’t fastidiously organized with impeccable technique.
Be pessimistic: assume that contamination is a matter of time so always be on the lookout for possible weak points in your lab’s set-up. Possible sources include: the water tray in your incubator and your incubator’s air supply.
Be organized: it is best practice to use a new batch of cells every 3 months so freeze several batches of your cells as back ups and maintain careful records of what you have.
Be thorough: when cleaning hoods/biosafety cabinets and incubators, take care to thoroughly remove any detergents.
Be careful: Do not leave your cells under fluorescent light for extended periods as this can provoke toxin production in response to photoactivation of certain ingredients in media.