Understanding and Managing Cell Culture Contamination
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Understanding and Managing Cell Culture Contamination (archive)
A very detailed PDF technique bulletin written by John Ryan, Ph.D., Corning Incorporated. It addresses some common contamination problems in cell culture and solutions to control and eliminate the cell culture contamination.
Content:
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . .1
What Are the Major Cell Culture
Contaminants? . . . . . . . . . . . . . . . . . . . . . . . . . . 2
What Are the Sources of Biological
Contaminants? . . . . . . . . . . . . . . . . . . . . . . . . . 8
How Can Cell Culture Contamination Be Controlled? . . . . . . . . . . . . . . . . . . . . . . . . 11
A Final Warning . . . . . . . . . . . . . . . . . . . . . . . 20
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . 21
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Cell Culture Protocols and
Technical Articles . . . . . . . . . . . . . . . . . . . . . . .22
Last update 25-Jun-2005, Rating Very Good of 5 votes.
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Turn on the UV lamp for few minutes to sterilize the surface.
The laminar flow hood should be turned on about 10-20 min before being used.
Wipe down all surfaces with ethanol before and after each use.
Decontamination of Cultures with Antibiotics
When an irreplaceable culture becomes contaminated, researchers may
attempt to eliminate or control the contamination. First, determine
if the contamination is bacteria, fungus, mycoplasma, or yeast.
Isolate the contaminated culture from other cell lines. Clean
incubators
and laminar flow hoods with a laboratory disinfectant, and check HEPA
filters.
Antibiotics and antimycotics at high concentrations can be toxic to
some cell lines. Therefore, perform a dose response test to determine
the level at which an antibiotic or antimycotic becomes toxic. This
is particularly important when using an antimycotic such as Fungizone
or an antibiotic such as tylosin. The following is a suggested
procedure
for determining toxicity levels and decontaminating cultures.
1.Dissociate, count, and dilute the cells in antibiotic-free media.
Dilute the cells to the concentration used for regular cell passage.
2.Dispense the cell suspension into a multiwell culture plate or
several small flasks. Add the antibiotic of choice to each well in a
range of concentrations. For example, we suggest the following
concentrations for FungizoneŽ. 0.25, 0.50, 1.0, 2.0, 4.0, and 8.0
ug/ml.
3.Observe the cells daily for signs of toxicity such as sloughing,
appearance of vacuoles, decrease in confluency, and rounding.
4.When the toxic antibiotic level has been determined, culture the
cells for two to three passages using the antibiotic at a
concentration one to two-fold lower than the toxic concentration.
5.Culture the cells for one passage in antibiotic-free media.
6.Repeat step 4.
7.Culture the cells in antibiotic-free media for four to six passages
to determine if the contamination has been eliminated.
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To the point & informative.
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very useful information
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thorough and referenced
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very good
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Related resource
Fungi contamination in cell culture work
Index of Typical Cell Culture Contaminants
Mycoplasma Detection and Elimination
Detection of Mycoplasma by Culture Protocol
Mycoplasma Detection Using DNA Staining
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