CELL CULTURE CONTAMINATION
 Consequences of contamination
 Types of contaminates
 Microorganisms
 Source of contaminations
 Control method
 How do Biological contaminants
enter cultures
 Contamination investigation
 Sterile VS. Aseptic
 Steps for reducing
contaminations.
BIOSAFETY
 Who may be at risk
 Biosafety Level
 Biosafety Act 2007
 Hazard potential from
mycoplasma
BIOLOGICAL WASTE MANAGEMENT
• Why it is important?
• Whose responsible?
• Types of biological waste
• Waste container requirement
BIOETHICS
• What is bioethics?
• Malaysian Bioethics Council
• Ethical issue in bioethics
Serious problems
When contamination
frequency increases or
entire experiments or
cell cultures are lost.
Minor annoyances
Major catastrophes
Contaminants are
discovered that cell into
doubt the validity of
your past or current
work.
When up to several
plates or flasks are
occasionally lost to
contamination.
CONTAMINATION
PROBLEMS
1) Loss of time, money & effort
2) Adverse effects on the culture
CONSEQUENCES OF
CONTAMINATIONS
3) Loss of valuable products
4) Personal embarrassment
“Contamination can not be totally eliminated, but it can
be managed to reduce both its frequency of occurrence
and the seriousness of it consequences” – John Ryan,
Ph.D (Corning Incorporate).
TYPES OF
CONTAMINANTS
Chemical
Contaminations
Media
Sera/
Serum
Endotoxin
Storage
Vessel
Fluoresce
nt lights
Incubator
Water
Biological
Contamination
Easy to
Detect
Difficult to
Detect
Media
Media
Media
Media
Media
Media
Media
Media
Bacteria




Gram positive & negative.
Media used for cell culture will
maintain bacteria.
Bacteria will compete for
nutrients in media.
Detect by microscopic
observation or effects on the
culture (pH shift, turbidity and
cell destruction).
Fungi



Mold, yeast.
Float in air and are on
surfaces.
Grow prolifically.
Viruses



Enter into cells and destroy them
in their quest to replicate.
May come from natural products
used to grow cells.
Small size, difficult to detect.
Mycoplasma



Can alter their host culture’s cell
function, growth, metabolism.
Bacteria-like microbes, smallest
self- replicating organism know,
lack a cell wall.
Can grow high density in cell
culture, w/o any signs of
contamination- no turbidity, pH
changes or even cytopathic
effects.
Raw
materials
Poorly
cleaned or
maintained
equipment
Operators
(personal
hygiene)
SOUCE OF
CONTAMINATION
Environment
Process
water
Starting materials can be a major source of contamination.
To help prevent this, manufacturers must know what types
and quantities of microbes are likely to be present. This is
one of the reasons your company tests its starting
materials.
Staring materials must always be handles and stored in a
manner to prevent contamination. Some important rules
are:
 Store in clean, dry locations
 Store off the ground and under over
 Always keep containers sealed when not in use
 Be alert to damaged packages
 Never use dirty or wet sampling or measuring equipment
Equipment can also be a source of
contamination.
In order to keep equipment clean, you should:






Store in a clean, dry manner
Drain and store all hoses correctly after use
Remove all product residue
Use approved sanitising procedures
Reinspect equipment before use
Maintain equipment
Process water is a key ingredient of many
medicinal products. Because water is a key
requirement for microbial growth, a lot of effort
is needed to control any water where it is an
ingredient of the product or is used to wash
processing equipment.
The key control over process water are:
 Regular microbiological
monitoring programmers
 Sanitize the system regularly
 Take samples carefully, being
sure not to contaminate the
system or the sample
When products are exposed to the environment, the
environment must be controlled. Some ways of achieving
this include:
 Air handling systems
- Filter the air
- Maintain positive air pressure
- Control temperature, humidity and air flow patterns
 Designated manufacturing zones
- Keep doors and windows shut
- Change outer garments
 Cleaning programmers
- Regularly check air filters
- Clean ducts and air filters regularly
- Keep area clean and dry at all times
People are potentially the target source of
microorganisms and are the hardest factor to
control.
To work in this industry, you must follow the
strictest hygiene practices:





Always wear protective garments/ lab coats.
Never handle products with bare hands.
Wash hands after visiting the toilet.
Report any infections to supervisor.
Don’t eat in the factory.
Contact with non sterile supplier, media or solutions
•Unintentional used of no sterile supplier, medias or solutions (i.e. improper
sterilization or storage, contaminated during use).
•Care must be taken when opening and resealing the packaging of the
consumables
Particulate or aerosol fallour during culture manipulation,
transportation or incubation.
•Aerosols may come from pipetting devices, vacuum pump, refrigeration and
freezes.
Swimming, crawling or growing into culture vessel
•Liquid at the sidewalls of flasks provides a liquid bridge for m/organisms to enter
swim/ grow into the culture.
Accidents and Mistakes
•Ex: a technician retrieves a vial labeled WI-38 from a liquid nitrogen tank thinking
it contained the widely used diploid human cell lines. Once in culture, it was
immediately discovered to be a plant cell line derived from common strain of
tobacco called Wisconsin 38, also designated WI- 38.









Weak spots of the process must broadly examined.
Perform investigation while the process is still active
(visual inspection).
Perform sampling on the contaminated culture.
Quarantine all left over of raw materials/ supplements/
equipment components etc. perform sampling if
necessary.
Review the cleaning and sterilization process.
Review or perform air bioburden test.
Perform personnel monitoring test ( glove print test).
Perform bacterial identification test of contaminated
culture and other samples.
After the investigation completed, assign a most probable
cause diagram (i.e Ishikawa diagram) for cause tracking.
Sterile
• Devoid of all life forms (bacteria, fungi, viruses).
• Difficult to achieve once people start manipulating the
cultures.
Aseptic
• Reduced numbers of life forms.
• Most equipment and reagents provided to you comes
sterile gamma irradiation, autoclaving, high
temperature and high pressure steam, filtration.
Good aseptic
techniques
Strategic use
of cell
repository
Good
housekeeping
by everyone
Strategic use
of antibiotics
Understanding
the nature of
contamination
Contamination
monitoring
program
1.
◦
◦
◦
2.
3.
Apply good aseptic techniques
Use clean lab coats to protect against shedding contaminants from skin
or clothes.
Work with only 1 type of cell line at a time in the hood.
Aliquot sterile solution in small volumes.
Reduce accidents
- Be careful when labeling and choice of acronyms.
- use standardized recordkeeping form.
- Use written protocols/ formulation sheet when preparing
media and solutions.
Keep the lab clean
- Routinely wipe floor and work surface.
- Periodic cleaning and disinfecting of incubator, water bath.
- Autoclaving of any waste that have been in contact with cells.
Routinely monitor for contamination.
- Perform Bioburden and Environment
monitoring.
5. Use frozen cell repository strategically
- To reduce the need to carry large number
of cultures.
- stop biological time for them, preventing
them from acquiring the altered
characteristic that can normally occur in
actively growing cells as a result of
environmental/ age related changes.
6. Use antibiotics sparingly if at all.
4.
Risks for
operators during
cell cultivation.
Risks for the
environment in
general, including
animals, and
plants.
Risks for the
recipient of the
product of the
cells.
WHO MAY
BE AT
RISKS???
Biosafety Level 1 (BSL1)
•BSL1 is the basic level of protection common to most research and clinical lab, and is
appropriate for agents that are known to cause disease in normal, healthy humans.
Biosafety Level 2 (BSL2)
•BSL2 is appropriate for moderate risk agents known to cause human disease of
varying severity by ingestion or through percutaneous or mucous membrane
exposure.
Biosafety Level 3 (BSL3)
•BSL3 is appropriate for indigenous or exotic agents with known potential for aerosol
transmission, and for agents that may cause serious and potentially lethal infections.
Biosafety Level 4 (BSL4)
•BSL4 is appropriate for exotic agents that pose a high individual risk or life
threatening disease by infectious aerosols and for which no treatment is available.
The agents are restricted to high containment lab.
Mycoplasma:
 Extremely small bacteria.
 Cultivating of most mycoplasmas in artificial media requires
several supplements, i.e: cholesterol.
 Difficult in detection and elimination.
 Able to persist in permanent cell- cultures undetected for year.
How do they enter cells???
 Contamination with human commensal mycoplasma from lab
personnel.
 Important to the lab by establishing a primary cell culture of an
infected animal.
 Use of contaminated supplements (serum, cytokines, s/ natant of
infected cells).
 Splitting adherent cells with contaminated trypsine.
Why should we care about them???
 Can change several properties: growth, metabolism,
morphology, and genome structure.
 Compete with host nutrients.
 Inhibit the synthesis of histone and nucleic acid, followed
by chromosomes aberration of the host cells.
 Produce hydrogen peroxide, which is directly toxic to the
cells.
How can we detect mycoplasms???
 Cultured detection is very sensitive, but it takes 2 weeks
and several strain cannot be cultivated.
 DNA staining with intercalating fluorescent substances.
Why it is important???
To ensure the generation and disposal of scheduled
waste is properly managed as well as complies with
Environment Quality (Scheduled Waste) Regulation
2005.
Whose responsible is it and what should they do???
All personnel who generate and handle the waste:
 Arranging the pickup of the waste contractor (e.g
Kualiti Alam).
 Manage and dispose the waste from their work
station to the Temporary Waste Room and record it in
the Waste Log Book Provided.
 Identify the waste category with the labeling.
TYPES OF BIOLOGICAL WASTE:
 Solid non contaminated waste: all non-contaminated materials such as
facemask, bouffant cap, papers, etc are disposed in waste bin that is
lined with black plastic bag.



Solid contaminated/ biohazard: All solid contaminated/ biohazard waste
will be collected in a waste bag and is line with biohazard waste
container (Red Biohazard Bag). It must be label accordingly.
Liquid contaminated/ biohazard waste: After decontamination, liquid
biohazard waste is collected is a liquid waste container and label the
liquid waste container accordingly before being send to disposal.
Biohazardous Sharps: Place all sharps in a puncture- proof container
either a commercially available sharps container or a study cardboard.
Seal the container or box and attach a strip of autoclave tape.
Solid and Liquid Waste Container Requirement
 The waste containers and bags used must be compatible
with the type of waste contained.
 Biohazard liquid waste containers and waste bags cannot
be reuse and should be completely disposed.
 Do not fill the containers and bags completely. Each
container must have at least 1 inch of headspace above
the waste to avoid spillage.
 Tie or seal the waste bags to prevent leakage or expulsion
of content during storage, handling and transport.
 Bio hazardous liquid waste container shall be rigid, heat
resistance (suitable for autoclaving), leak proof and has
tight- fitting cover. Do not use metal container for nay
liquid waste.
 Place sharps in a dedicated puncture resistance container
before dispose it in the waste bag.
What is bioethic???
The study of controversial ethics brought about by advances in
biology and medicine. Bioethicists are concerned with the ethical
questions that arise in the relationships among life sciences,
biotechnology, medicine, politics, law and philosophy.
Malaysian Bioethics council
 Launched on 22 May 2012 by the Minister of Science,
Technology and Innovation YP Datuk Seri Panglima Dr. Maximus
Johnity Ongkili.
 Made up of representatives from noted academic experts, and
representatives from government and non- government offices.
 Aims to provide advice, as well as resolve and manage bioethical
issues in the country.
 Focusing on the impact of science and technology on the
environment, society, public health, culture, laws and religion.
1)



The use of fetal bovine serum: ethical or scientific
problem???
Fetal bovine serum (FBS) is a common component of
animal cell culture media.
FBS is harvested from bovine fetuses taken from pregnant
cows during slaughter. FBS is commonly harvested by
means of a cardiac puncture without any form of
anaesthesia. Fetuses are likely exposed to pain and/ or
discomfort and therefore current practice of fetal blood
harvest is inhumane.
Apart from moral concerns, several scientific and
technical problems exist regarding the application of FBS
in cell culture. Efforts should be made to reduce and
preferably replace FBS by synthesis alternatives. On moral
and scientific grounds the most promising alternative to
FBS is the use of defined media.
2) Stem Cells Controversy
 Not all stem cell research involves the creation, use or
destruction of human embryos. For example, adult stem
cells, amniotic stem cells and induced pluripotent stem
cells do not involve human embryos at all.
3) Human Biopsy Materials
 These aspects extend from the initial contact with the
donor or their next- of- kin, through isolation and supply
procedures, to use in the research facility.
 It should be sup plied through officially recognized human
tissue banks, and collected according to acceptable ethical
standards and in compliance with national laws.