Title: Identification and Elimination of Contaminations in Cell Culture and Polymerase Chain Reaction Labor
1Identification and Elimination of Contaminations
in Cell Culture and Polymerase Chain Reaction
Laboratories
- Yih-Horng Shiao, Ph.D.
- Laboratory of Comparative Carcinogenesis
- National Cancer Institute at Frederick
- Maryland, USA
June 25, 2005
2The consequences of contamination
- Hazardous to humans
- Inaccurate experimental results
- Loss of cells and samples
- Waste of time, money, and other resources.
3Cell Culture Contamination
4Chemical contaminants and sources
- Exogenous Metals (glassware), reagent residues
(glassware), endotoxin (culture media, sera, and
water), other water impurities, CO2 impurities,
disinfectant residues, etc. - Endogenous Free radicals (photo-activation of
tryptophan, riboflavin, or HEPES buffer by
fluorescent light)
5Microorganism contamination and detection
Fungus
6Microorganism contamination and detection
Cells
Mycoplasma
Mycoplasma
Scanning electron microscopy
Hoechst 33258 stain
PCR-based detections for mycoplasmas and
viruses
HIV
Transmission electron microscopy
7Frequent mycoplasma contamination (Studies in
1990s)
- United States 11-15 of cell cultures
- Netherlands 25
- Former Czechoslovakia 37 (100 of the cultures
from labs without routine testing but only 2
from labs having mycoplasma screening regularly) - Argentina 65
- Japan 80
8Cell line cross-contamination(Surveys in 1970s
and 1980s)
- A study in 1967 showed that 20 commonly used
human cell lines were contaminated with HeLa
cell. - A report in 1976 demonstrated that 14 of 246
cell lines were wrong species and 25 were
replaced completely by HeLa cell. - In a 1981 survey, over 60 cell lines were
actually HeLa cell, 16 were contaminated by
non-HeLa cells, and 12 were interspecies
contamination.
9Sources of biological contamination
- Humans
- Newly arrived cell line
- Glassware
- The neck and outside of culture flasks and dishes
- Sera, culture media, and other reagents
- Airborne particles and aerosols
- Laminar-flow hood and safety cabinet
- Water bath and incubator
- Work surface
- Tubing and container for waste collection
10Cell culture management (1) Aseptic technique
and procedure
- Exercise procedures with the highest ethical and
moral standards. - Wear protective equipments (lab coat, gloves,
etc.). - Swab work surface, biosafety cabinet, and reagent
bottles with disinfectant before and after use. - Disinfect spill, splash, and any suspected areas
immediately. - Use sterile disposable tubes,
- pipettes, and culture vessels.
- Avoid generation of airborne particulates and
aerosols.
11Cell culture management (2) Aseptic environment
- Disinfect water bath, incubator, tubing and
container for waste collection routinely. - Replace HEPA filter on schedule.
- Keep laminar-flow hood on all the time.
- Minimize the number of entrance and frequency of
entering and exiting the cell culture room.
12Cell culture management (3) Monitoring and
surveillance
- Quarantine and test all incoming cell lines for
contamination, except those from reliable
sources. - Perform tests of microorganism contamination and
cell-specific markers for all active cell lines
and freeze aliquots of clean passages
periodically. - Monitor the performance of biosafety cabinets.
- Conduct annual safety training and refreshment
courses (classroom or on-line) to all personnel. - Record and document all monitoring and
surveillance items.
13Cell culture management (4)Curing for
contaminated cells
- Discard and heat-destroy all contaminated cells
because contamination alters cell behaviors and
functions. - If cells are irreplaceable, antibiotics can be
used to eradicate some bacteria and mycoplasmas.
However, the experimental results need to be
interpreted cautiously.
14Types of disinfectants
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16PCR contamination
17Air flow during PCR
Heated lid
Air flow
Heating/cooling block
PCR mixture
18Sources of PCR contamination
- Humans
- Carryover products, especially from PCR using the
same primer set over and over. - Vector DNA containing insert of a target gene and
other positive controls - Dusts and aerosols
- PCR reagents, pipetters, and tubes
- Work surface
- Instruments
19Detection of PCR contamination
- Negative controls
- ? Include no template controls throughout the
entire RNA and DNA analyses, beginning
from nucleic acid extraction. - ? Set up more than two negative controls each
time to detect random contamination. - Sequence polymorphism
- ? Unique gene sequence can be used to detect
contamination. - Repetition
- ? If the sample cannot be repeatedly
amplified, it may indicate contamination.
20Good practices in PCR laboratory
- Be vigilant to avoid carrying vectors, genomic
DNA, and PCR products onto human body. - Use different sets of reagents, equipments, and
supplies for pre-PCR and post-PCR experiments.
Never bring items in the post-PCR areas into
pre-PCR room. - Wipe work surface with 10 Chlorox or other
DNA-destructing agents before and after use. - Aliquot reagents.
- Change gloves often and prevent static build-up
on the gloves. Keep the working areas free of
dusts. - Limit the PCR cycle number.
21PCR laboratory set-up
- Physically separate Pre-PCR from post-PCR room,
and each room has independent heater, ventilation
and air conditioner. - Need a biological cabinet with UV lamps in the
pre-PCR room to provide clean area for steps,
such as DNA extraction and PCR preparation. - It is optional to install a dead-air biological
cabinet in post-PCR room for steps, such as
opening of PCR tube, gel electrophoresis, and
staining, to contain PCR products and to destroy
the products with cabinet UV.
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24PCR cabinets
25Special measures to cure PCR contamination
- Discard contaminated samples and reagents.
- Pre-PCR
- ? Enzymatic digestion (endonuclease, DNase I,
and exonuclease) - ? UV irradiation
- Post-PCR
- ? Isopsoralen followed by UV
- ? Incorporation of dUTP followed by Uracil
DNA glycosylase and heat treatment
26Conclusion
- Be alert and conscious to all potential
contaminants. - Practice safety procedures with the highest
ethical and moral standards. - Follow the schedule for monitoring and
surveillance. - Take refreshment courses or training periodically.