Guide lines for use of Cell Culture Core facility · 3.Why is the vertical laminar air flow hood...
Transcript of Guide lines for use of Cell Culture Core facility · 3.Why is the vertical laminar air flow hood...
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Guidelines for use of Cell Culture
Core
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Content•General rules
•Pointers for aseptic technique
•Work area and equipment
•Safety considerations
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General rules for facility use
• Reagents and culture flasks must be labeled with your name, PI’s initials and date.
• Always sign equipment logs when using core equipment.
• Do not share you access code.
• Deposit waste in proper containers.
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Aseptic TechniqueWhat is it?
Aseptic technique is everything you do to reduce the probability of contamination.
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Important Pointers for Aseptic
Techniques
• Clean Work Area: a clean hood and well organized work area.
• Personal Hygiene: good hand washing habits , use of gloves and other personal protective equipment,(PPE) such as caps lab coats and face mask.
• Sterile handling: Swabbing with 70% ethanol and capping
• Handling bottles and flasks; culture flasks should be laid down horizontally and like bottles, held at an angle during manipulations. Never reach over any open container.
• Pipetting: Use disposable plastic pipettes come in a convenient size range (e.g.. 1 ml, 5 ml ,10 ml and 25ml)
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Work area and equipment
Biosafety Laminar air flow hoods
Types:
1. Horizontal air flow
2. Vertical air flow
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Horizontal
Horizontal air flow –used for
media preparation and other
sterile reagents and for culture
of non primate cell lines.
Provides no protection to user.
Air flow is towards user
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Vertical
Vertical air flow -used mainly for
potentially hazardous materials
( any primate, including human,
cell lines, virus producing cultures,
radioisotopes, carcinogenic and
toxic drugs.) Usually equipped with
UV light.
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A Class II Vertical Type A2 laminar flow
hood is the type used in cell culture
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Incubators
• For most mammalian cells
-Temperature at 37˚C
-Humidity at 95%
-Carbon Dioxide at 5%
Water is heated in a jacket surrounding
the inner area to distribute heat
evenly to the interior of the chamber
A water pan is placed with treated water
to maintain humidity.
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Centrifuge
Cell culture procedures require the
use of a refrigerated centrifuge
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Inverted microscope
• Specially designed for viewing living cells
• Light source is above and phase objectives
of 4X, 10X, 20X and 40X are located below.
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Equipment for
Cryopreservation
The cell core facility has two ultra low temperature freezers
(-140°c) and one (-80°c) low temperature freezer .
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The importance of freezing cells
• Cross contamination of other cell lines
• Senescence and the resultant extinction of cell line
• Genetic drift due to genetic instability
• Contamination of microorganisms
• Need for distribution to other users
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Basic safety considerations
• Minimum standard applied to any cell
culture lab is category 2 containment.
• Safety applies both ways;
-protect your cells and protect yourself
• General safety rules
• Aseptic vs. antiseptic technique
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Safety hazards of
the cell culture lab
• Broken glass
• Electrical
• Radiation
• Frost bite
• Chemical toxicity
• Infection
• Biohazards
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Basic safety procedures for cell
culture
• Be aware of the potential for biohazard
• Use of proper personal protective equipment (PPE).
• Wash hands before handling cultures and before leaving the lab
• Decontaminate work surface before and after use
• Properly dispose of biological and chemical waste
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QuizShort answers please
1.What is the main advantage of good aseptic technique in the cell culture?
2.Give three examples of basic equipment of a cell culture lab.
3.Why is the vertical laminar air flow hood better for cell culture than a horizontal air flow hood?
4.Name three things you can practice to reduce the probability of contamination.