You’re in the middle of preparing your cell cultures for an experiment but OH NO!
You've noticed that your cell cultures have turned cloudy and smell rotten.
Could it be a sign that something is contaminating your cultures?
If it’s contaminated, are your cells done for or can they still be saved?
In this video, you’ll learn about the types of contamination, detection methods, and solutions
to combat this prevalent issue that occurs in many cell culture labs!
Contamination occurs when cells are exposed to foreign substances
which compromise cell culture quality and can harm the cells.
There are multiple types of biological contaminants.
Today we’ll be focusing on the most common ones: bacterial, fungal, and mycoplasma contamination!
Bacterial organisms are extremely small smaller than the size of eukaryotic cells
This makes them difficult to detect under the microscope until the contamination is full blown
Not only are they small but they're also capable of surviving in many different types of conditions
whether it's hot or cold or wet or dry which makes them challenging to get rid of.
If your cultures are contaminated by the germ gang, you'll notice a cloudy appearance, cell morphology changes,
and the detachment or death of cells.
Sometimes, dark spots may appear which can indicate contamination, but it could also just be debris from the cells.
Here are some indications that the black spots you're looking at are probably cell debris.
If there is no odd media colour change or the particles don't increase in number.
The particles are uneven in shape and size.
There is no directional movement at high magnification.
if the dark spots move with the motion of media, it is most likely cell debris.
Now, here are indicators that the black spots may actually be bacterial contaminants:
If the dark spots move independently from the motion of the media!
All particles jiggle at high magnification due to Brownian motion
but a lot of bacterial contaminants do actually swim!
If you see a particle moving any distance at all in a straight line or circle
you’re likely looking at bacterial contaminants.
If you smell something funky!
When bacterial contamination is full blown, it will start to smell.
At this time it is too late to salvage your cultures
and they should be immediately disposed of properly to avoid spreading the contamination.
So what should you do if you find out you have bacterial contamination?
If you have stock vials of your cells, it is best to dispose of your contaminated cells and start over.
ou’ll also need to dispose of your old media and do some deep cleaning of your incubator and cell culture hood.
If your cells are precious or difficult to procure,
you can try to add stronger antibiotics or BacAway to your culture to help you fight the bacterial army!
If you see small grey, white or greenish colonies floating at the surface of your medium, you’ve got fungal contamination!
Fungi can spread easily through the air in their spore form until they find suitable growth conditions.
If fungal contamination is full blown, your medium will become turbid and cloudy
and your media may start to change color, becoming a lighter pink due to changes in pH.
At higher magnification under a microscope, you’ll also notice long fiber-like structures growing among your cells if it is mold,
or clusters and chains of small round branching ovoid particles if it is yeast.
So, how do you get rid of fungal contamination?
Similar to bacterial contamination, it is best to keep the contaminated culture vessel closed
to avoid spreading spores and dispose of your contaminated cells if you can.
If they’re too precious to throw away,
you can try using antimycotic cocktails or add an antifungal like fungizone which contains amphotericin B.
You’ll also need to throw away your contaminated media
and do a deep clean of your equipment to ensure all fungal spores are removed!
One of the most common unnoticeable types of contamination is mycoplasma!
Mycoplasmas are a type of bacteria that are only 0.2 to 0.3 micrometers in size
which makes them hard to see!
They do not cause noticeable pH or turbidity changes
and they lack a peptidoglycan cell wall which makes antibiotics ineffective to use against them!
Like bacterial cells, once the mycoplasma mafia infect your cell culture it can cause cells
to die as they can take up most of the available nutrients.
But what’s most scary about Mycoplasma is that it can also cause chromosomal aberrations
and change gene expression profiles without actually killing your cells,
therefore affecting your experimental results without your knowledge!
Luckily, there are multiple methods that can be used to detect mycoplasma which includes
histological staining, enzyme activity measurements, ELISA, DNA staining, and PCR.
abm provides a quick and efficient microplasma PCR detection kit that is capable of detecting
over 200 strains of mycoplasma within a few hours!
So what do you do if you end up detecting mycoplasma in your sample?
As always, it’s best to discard contaminated cells as soon as possible
to prevent other cells from being infected.
But if your cells are too important to discard, there are a few ways to save them!
Diluted chemical treatments with tetracycline, macrolides or quinolones
can be used to eliminate mycoplasma while maintaining the quality of your cell culture.
abm also offers a mycoplasma elimination cocktail that can effectively clear cells from
mycoplasma contamination within a few weeks
Another common type of contamination is cell cross contamination.
This occurs when a different cell line gets mixed in with the desired cells that one is working with
due to cells traveling in aerosol droplets or on contaminated equipment and media.
For example, a scientist could be writing up the results for an experiment that they performed on what they thought were human kidney cells,
but unbeknownst to them, their sample could have contained HeLa cells that somehow made their way into the cell culture plate.
Therefore, their results are no longer valid as the experiment was performed on HeLa cells rather than the desired kidney cells
In order to confirm the identity of the cells you are working with,
a short tandem repeat profiling or STR for short can be performed on the cells to verify its origins.
STR utilizes PCR to amplify a microsatellite region on the DNA sample that is unique to a certain species.
The PCR products are then analyzed with software that compares the microsatellite sequence to a database
which is able to confirm the identity of the species that the cell is from with high certainty.
abm offers comprehensive STR profiling to help you ensure you are working with the correct cell line!
With all this unpleasant talk about contamination, there are actually multiple methods
that scientists can utilize to prevent contamination from occurring in the first place.
Practicing aseptic techniques in the lab is one of the most efficient ways of preventing contamination.
Examples of aseptic techniques include wearing proper PPE
so that foreign particles on your skin or clothes will not be able to make it into the cell culture plate.
It is also important to work in a sterile environment like a BioSafety Cabinet
Cabinet which is a laminar flow unit that contains ultra filtered air
that prevents foreign substances from entering the work environment.
Before placing any items in the BioSafety Cabinet,
it is essential to wipe all bottles and surfaces with an ethanol solution to effectively eliminate any microbes,
and, to further prevent bacterial contamination, it is common to add a penicillin or streptomycin solution to cell cultures!
Lastly, in order to prevent cell cross contamination,
it is highly recommended to work with one cell line at a time in the BioSafety cabinet
while practicing good labeling techniques to prevent the mix-up of cells.
Hopefully, you’re now much more aware of some of the common types of contamination
that can occur in a cell culture lab and how to identify, prevent, and deal with them.
Now that you’re up to the task,
you can perform a cell culture experiment without having to worry about foreign organisms contaminating your plate!
To learn more about cell culture quality control and contamination, click on the learning resources links in our video description!
As always don’t forget to subscribe and leave a comment! Thanks for watching!