peralatan dan metode sterilisasi
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PERALATAN KULTUR
DANMETODE STERILISSI
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Peralatan Kultur
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Laminar Air Flow
Class II
Biological
Safety
Cabinet
Protection of
personnel
environment
product
Class 1 Cabinets
protect the
product only
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Vertical
Laminar AirflowAir Barrier
Exhaust Fan
Exhaust
HEPA Filter
Laminar Flow
Fan
Laminar
HEPA Filter
Class IIBiological
Safety
Cabinet
HEPA filters
Laminar flow
NATA certified
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Carbon dioxide incubator
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Microscope
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Manual cell count (Hemocytometer)
Diagram represent cell count using hemocytometer.
http://www.google.com.sa/imgres?imgurl=http://www.thesciencefair.com/Merchant2/graphics/00000001/BloodCountrSlideB-4005_M.jpg&imgrefurl=http://www.thesciencefair.com/Merchant2/merchant.mvc?Screen=PROD&Product_Code=B4005&Category_Code=BS&usg=__EtNMmLLw00kvEcNwqiYvAeb9OdA=&h=206&w=450&sz=30&hl=ar&start=10&itbs=1&tbnid=PAlwwf6kckrBJM:&tbnh=58&tbnw=127&prev=/images?q=hemocytometer&hl=ar&safe=active&sa=G&gbv=2&tbs=isch:1 -
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Tissue culture Ware
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Tissue culture Ware
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Tissue culture Ware
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centrifuge
autoclave
Micro pipette
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Prevention of Contamination
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Consequences of contamination
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Key concept
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Type of culture contaminant
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Types of contamination in animal cell
culture systems
Biological
Bacteria
Fungi
Cross-contamination by other cell cultures Chemical
Residues left from detergents or disinfectants on
glassware, pipettes, instruments, etc.
Metal ions, other impurities in water
Endotoxin: highly bioreactive part of the cell wall of
some types of bacteria (endotoxin molecules are shed
from bacteria and are left behind even after bacteria die)
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Bacterial contamints
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Fungal and yeast contaminants
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Chemical contaminant
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Viral contaminants
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Insecta and parasites
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Mycoplasma
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Characteristics of microbial
contamination in cell cultures
pH Sudden change in pH is often a strong
indicator of contamination
Turbidity
Media looks cloudy
Microscopic evaluation Can see individual microorganisms, often
because their motion can be seen easily under
the microscope
F h d i f i i i ll
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Further detection of contamination in cell
cultures Mycoplasma
Smallest free-living prokaryotes Not killed easily by many antibiotics
Contamination cant be seen by microscopicevaluation
Mycoplasma testing should be doneroutinely (several tests are available)
Long-term effects of mycoplasma contamination includereduced growth rate, changes in cell shape and
metabolism, and chromosome abnormalities Endotoxin
LAL test: an extract from the blood ofhorseshoe crabs is used to test for endotoxin (horseshoecrab blood contains a protein that binds endotoxin & can be
detected)
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Sterile technique
Procedures by which cultures are manipulated
without infecting the worker or contaminatingthe cultures or the laboratory environment
Important for the cell culture
You want to be sure you are growing only the cellsyou want to grow a single unwanted cell can ruin an
experiment or a multimillion $ production run
Important for the labratory worker Some cultured cells can pose health threats to
workers if they are inhaled, ingested, or absorbed--
sterile technique prevents exposure of the worker to
cultured cells
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Sterile technique: Tissue culture
Working with cells in a laminar flow hood HEPA filter
Disinfect 70% Ethanol is sprayed in hood, onto bottles entering hood
Minimizing contamination through awareness Inoculation loop, pipets, pipet tips, etc. should never touch
contaminating surfaces
Containers holding media and other cell additives shouldbe kept closed until needed, then opened briefly
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Laminar flow
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Sterilization methods Autoclave
Applies heat under high pressure; this increases the boiling point
of water to 121C (normal boiling point of water is 100C) 15-20 min. is sufficient to kill most microbes
Filtration
Large volumes: suction filter
Small volumes: syringe filter
UV radiation
Causes mutations to form in the DNA of microbes, causing
genetic damage and eventual death
Used to sterilize surfaces (such as the surface of laminar flow
hoods)
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Culture Media Sterilization
Media is usually sterilized by filtration
Standard biological filters are 0.22 mm - 0.45 mm;these remove most microbes by trapping them inthe filter
This does not remove all microbes (such asmycoplasm), and will not remove viruses
Unlike bacterial media, animal cell media cannot
be autoclaved, because this would destroy many ofthe growth factors and other molecules needed forcell growth
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Culture Media Sterilization
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Filtration:Air&Fluids
http://www.nalgenunc.com/MF75/filter.htmlhttp://www.airfilterstore.com/iqair/features.htm -
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Changing Medium
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Using a Micropipette
To avoid air bubbles
and extract the correct
amount of solution
utilizing a micropipette,the tip must be
completely submerged
in the solution.
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Forceps and other equipmentshould never be placed incontact with surfaces
Should be kept in a 70%ethanol (alcohol) solution,and flamed over an alcohollamp before contacting
sterile material.
Sterilization Equipment
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Ster
ilizatio
nTim
es
171o C, 60 minutes, dry heat
160o C, 120 minutes, dry heat
149o C, 150 minutes, dry heat
141o C, 180 minutes, dry heat121o C, 12 hours, dry heat
121o C, 15 minutes, moist heat (but
dont start the clock until entire item is
up to tempe.g., large volumes fluid)
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Minimizing contamination
Contamination is a fact of life when dealing
with cell cultures
Very difficult to prevent entirely, but good lab
practices can keep contamination incidents to a
minimum
Proper cleaning and sterilization of glassware,
pipettes, and other lab instruments. Practicing sterile technique when working with
cell cultures
P t ti l f t i ti i
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Potential sources of contamination in
cell culture
Equipment Glassware, instruments, incubators.
Solutions
Media or reagents added into media
Room air
Work surfaces
Operators
Hands, hair, clothing, breath, etc.
Incoming cells
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Sources of Contamination
Bacteria
Fungi
Mould
Yeast Mycoplasma
Other cell types
Free organisms, dust particles or aerosols
Surfaces or equipment
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Aktivities Particles produced/mnts
1. Sitting or standing with no
movement
100,000 0.3um
2. Simple arm movement 500,0003. Everage arm movement with
slight leg movement
1,000,000
4. Average walking 7,500,000
5. Walking fast 10,000,000
6. Boisterous activity 15x106 to 30x106
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Aseptic Technique 1
Controlled environment
Traffic, air flow
Sterile media and reagents
Avoids aerial contamination of solutions
Avoids manual contamination of equipment
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Aseptic Technique 2
Minimise traffic
Clear work area
70% ethanol swab
Minimise work area (field of vision)
Keep work area clean
Do not lean over open vessels
UV irradiation before and after
Only use disposable equipment once
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Aseptic Technique 3
Minimise exposure to air
Flame bottles if on open bench
Avoid repeated opening of bottles
Avoid liquid accumulation around necks and lips ofbottles
Avoid excessive agitation
Only one cell type at a time
Do not open contaminated solutions
No burner in hood
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ContaminationA cell culture contaminant can be defined as some element in the culture
system that is undesirable because of its possible adverse effects on either
the system or its use.1-Chemical Contamination
Media
I ncubator
Serum
water
2-Biological Contamination
Bacteria and yeast
Viruses
Mycoplasmas
Cross-contamination by other cell culture
How Can Cell Culture Contamination Be Controlled?
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Hands Spread Disease