PERALATAN KULTUR DAN METODE STERILISSI

Peralatan Kultur

Laminar Air Flow

Class II Biological Safety Cabinet
Protection of personnel environment product Class 1 Cabinets protect the product only

Exhaust Fan Exhaust HEPA Filter

Laminar Flow Fan

Laminar HEPA Filter

Class II Biological Safety Cabinet

HEPA filters Laminar flow NATA certified

Air Barrier

Vertical Laminar Airflow

Carbon dioxide incubator

Microscope

Manual cell count (Hemocytometer)

Diagram represent cell count using hemocytometer.

Tissue culture Ware

Tissue culture Ware

Tissue culture Ware

centrifuge Micro pipette

autoclave

Prevention of Contamination

Consequences of contamination

Key concept

Type of culture contaminant

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)

Bacterial contamints

Fungal and yeast contaminants

Chemical contaminant

Viral contaminants

Insecta and parasites

Mycoplasma

Characteristics of microbial contamination in cell cultures

pH

Sudden change in pH is often a strong indicator of contamination

Turbidity

Media looks cloudy Can see individual microorganisms, often because their motion can be seen easily under the microscope

Microscopic evaluation

Further detection of contamination in cell cultures

Mycoplasma

Smallest free-living prokaryotes Not killed easily by many antibiotics Contamination cant be seen by microscopic evaluation Mycoplasma testing should be done routinely (several tests are available) Long-term effects of mycoplasma contamination include reduced growth rate, changes in cell shape and metabolism, and chromosome abnormalities

Endotoxin

LAL test: an extract from the blood of horseshoe crabs is used to test for endotoxin (horseshoe
crab blood contains a protein that binds endotoxin & can be detected)

Sterile technique

Procedures by which cultures are manipulated without infecting the worker or contaminating the cultures or the laboratory environment Important for the cell culture

You want to be sure you are growing only the cells you want to grow a single unwanted cell can ruin an experiment or a multimillion $ production run
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

Important for the labratory worker

Sterile technique: Tissue culture

Working with cells in a laminar flow hood

HEPA filter 70% Ethanol is sprayed in hood, onto bottles entering hood Inoculation loop, pipets, pipet tips, etc. should never touch contaminating surfaces Containers holding media and other cell additives should be kept closed until needed, then opened briefly

Disinfect

Minimizing contamination through awareness

Laminar flow

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)

Sterilization methods

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 in the filter This does not remove all microbes (such as mycoplasm), and will not remove viruses

Unlike bacterial media, animal cell media cannot be autoclaved, because this would destroy many of the growth factors and other molecules needed for cell growth

Culture Media Sterilization

Filtration: Air & Fluids

Changing Medium

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.

Sterilization Equipment

Forceps and other equipment should never be placed in contact with surfaces Should be kept in a 70% ethanol (alcohol) solution, and flamed over an alcohol lamp before contacting sterile material.

Sterilization Times

171o C, 60 minutes, dry heat

160o C, 120 minutes, dry heat

149o C, 150 minutes, dry heat

141o C, 180 minutes, dry heat

121o 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)

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

Potential sources of contamination in cell culture

Equipment

Glassware, instruments, incubators. Media or reagents added into media

Solutions

Room air Work surfaces Operators

Hands, hair, clothing, breath, etc.

Incoming cells

Sources of Contamination

Bacteria Fungi Mould Yeast Mycoplasma Other cell types

Free organisms, dust particles or aerosols Surfaces or equipment

Aktivities
1. Sitting or standing with no movement

Particles produced/mnts
100,000 0.3um

2. Simple arm movement 3. Everage arm movement with slight leg movement 4. Average walking 5. Walking fast 6. Boisterous activity

500,000 1,000,000 7,500,000 10,000,000 15x106 to 30x106

Aseptic Technique 1

Controlled environment

Traffic, air flow

Sterile media and reagents Avoids aerial contamination of solutions Avoids manual contamination of equipment

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

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 of bottles Avoid excessive agitation Only one cell type at a time Do not open contaminated solutions No burner in hood

Contamination
A 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 Incubator Serum water
2-Biological Contamination Bacteria and yeast Viruses Mycoplasmas Cross-contamination by other cell culture

How Can Cell Culture Contamination Be Controlled?

Hands Spread Disease