Peralatan Dan Metode Sterilisasi

7/27/2019 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



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

Peralatan Dan Metode Sterilisasi

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