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Thread: Tissue culture information

  1. #1

    Join Date
    Jun 2010
    Posts
    15

    Default Tissue culture information

    Dear All,

    I am from Goa.I want to set up a tissue culture plant. I am very much interested in tissue culture. Can anyone please guide me for the initial setup, like where i will get the materials, information, How to start , whom to contact etc.

    Thanks in advance..

    Sanjay

  2. #2
    Ashwini's Avatar
    Join Date
    Apr 2005
    Posts
    4,466

    Default

    Hi sanjay
    Any laboratory, in which tissue culture techniques are performed, regardless of the
    specific purpose, must contain a number of basic facilities. These usually include the
    following:

    A general washing area

    A media preparation, sterilization, and storage area

    An aseptic transfer area

    Environmentally controlled incubators or culture rooms

    An observation/data collection area.

    Washing Area
    The washing area should contain large sinks, some lead-lined to resist acids and alkalis,
    draining boards, and racks, and have access to demineralized water, distilled water, and
    double-distilled water. Space for drying ovens or racks, automated dishwashers, acid
    baths, pipette washers and driers, and storage cabinets should also be available in the
    washing area.
    Media Preparation Area
    The media preparation area should have ample storage space for the chemicals, culture
    vessels and closures, and glassware required for media preparation and dispensing.
    Bench space for hot plates/stirrers, pH meters, balances, water baths, and media-
    dispensing equipment should be available. Other necessary equipment may include air
    and vacuum sources, distilled and double-distilled water, Bunsen burners with a gas
    source, refrigerators and freezers for storing stock solutions and chemicals, a microwave
    or a convection oven, and an autoclave or domestic pressure cooker for sterilizing media,
    glassware, and instruments.
    In preparing culture media, analytical grade chemicals should be used and good
    weighing habits practiced. To insure accuracy, and exact step-by-step routine should be
    developed for media preparation and a complete checklist required of all media preparers
    even for the simplest media.
    The water used in preparing media must be of the utmost purity and highest quality.
    Tap water is unsuitable because it may contain cations (ammonium, calcium, iron,
    magnesium, sodium, etc.), anions (bicarbonates, chlorides, fluorides, phosphates, etc.),
    microorganisms (algae, fungi, bacterial), gases (oxygen, carbon dioxide, nitrogen), and
    particulate matter (silt, oils, organic matter, etc.) Water used for plant tissue culture
    should meet, at a minimum, the standards for type II reagent grade water, i.e., be free of
    pyrogens, gases, and organic matter and have an electrical conductivity less than 1.0
    μmho/cm.
    The most common and preferred method of purifying water to type II standards is a
    deionization treatment followed by one or two glass distillations. The deionization
    treatment removes most ionic impurities, and the distillation process removes large
    1
    organic molecules, microorganisms, and pyrogens. Three other methods that will
    produce type II purity water are absorption filtration, which uses activated carbon to
    remove organic contaminants and free chlorine; membrane filtration, which removes
    particulate matter and most bacterial contamination; and reverse osmosis, which removes
    approximately 9% of the bacterial, organic, and particulate matter as well as about 90%
    of the ionized impurities.
    Transfer Area
    Under very clean and dry conditions, tissue culture techniques can be successfully
    performed on an open laboratory bench. However, it is advisable that a laminar flow
    hood or sterile transfer room be utilized for making transfers. Within the transfer area
    there should be a source of electricity, gas, compressed air, and vacuum.
    The most desirable arrangement is a small dust-free room equipped with an overhead
    ultraviolet light and a positive-pressure ventilation unit. The ventilation should be
    equipped with a high-efficiency particulate air (HEPA) filter. A 0.3-μm HEPA filter of
    99.97-99.99% efficiency works well. All surfaces in the room should be designed and
    constructed in such a manner that dust and microorganisms do not accumulate and the
    surfaces can be thoroughly cleaned and disinfected. A room of such design is
    particularly useful if large numbers of cultures are being manipulated or large pieces of
    equipment are being utilized.
    Another type of transfer area is a laminar flow hood. Air is forced into the unit through
    a dust filter then passed through a HEPA filter. The air is then either directed downward
    (vertical flow unit) or outward (horizontal flow unit) over the working surface. The
    constant flow of bacteria-free filtered air prevents nonfiltered air and particulate matter
    from settling on the working surface.
    The simplest type of transfer area suitable for tissue culture work is an enclosed plastic
    box commonly called a glove box. This type of culture hood is sterilized by an
    ultraviolet light and wiped down periodically with 95% ethyl alcohol when in use. This
    type of unit is used when relatively few transfers are required.
    Culture Room
    All types of tissue cultures should be incubated under conditions of well-controlled
    temperature, humidity, air circulation, and light quality and duration. These
    environmental factors may influence the growth and differentiation process directly
    during culture or indirectly by affecting their response in subsequent generations.
    Protoplast cultures, low-density cell suspension cultures, and anther cultures are
    particularly sensitive to environmental cultural condition.
    Typically, the culture room for growth of plant tissue cultures should have a temperature
    between 15 and 30 C, with a temperature fluctuation of less than 0.5C; however, a
    wider range in temperature may be required for specific experiments. It is also
    recommended that the room have an alarm system to indicate when the temperature has
    reached preset high or low temperature limits, as well as continuous temperature recorder
    to monitor temperature fluctuations. The temperature should be constant throughout the
    entire culture room (i.e., no hot or cold spots). The culture room should have enough
    fluorescent lighting to reach the 10,000 lux; the lighting should be adjustable in terms of
    quantity and photoperiod duration. Both light and temperature should be programmable
    for a 24-hr period. The culture room should have fairly uniform forced-air ventilation,
    and a humidity range of 20-98% controllable to 3 percent. Many incubators, large
    growth chambers, and walk-in environmental chambers meet these specifications.

    These are the details and for detailed information contact the horticulture department


    Regards
    Ashwini

  3. #3

    Join Date
    Jun 2010
    Posts
    15

    Default

    Hi Ashwini,

    Thanks for the valuable information.

    Best Regards,

    Sanjay

  4. #4

    Join Date
    Aug 2008
    Posts
    24

    Default tissue culture

    Sir Ashwini,
    I noted in your info about tissue culture is short and encouraging. Kindly post the cost and if anyone is partner with me.
    Hope this find a resposive message.
    Thank youi,
    Paul Ponniah.
    Please contact..
    Last edited by moderator A; July 2nd, 2010 at 11:18 AM. Reason: Only Business Members are allowed to post contact info. Please click “Upgrade” to become a Business Member.

  5. #5

    Join Date
    Jul 2010
    Posts
    4

    Default

    Quote Originally Posted by Ashwini View Post
    Hi sanjay
    Any laboratory, in which tissue culture techniques are performed, regardless of the
    specific purpose, must contain a number of basic facilities. These usually include the
    following:

    A general washing area

    A media preparation, sterilization, and storage area

    An aseptic transfer area

    Environmentally controlled incubators or culture rooms

    An observation/data collection area.

    Washing Area
    The washing area should contain large sinks, some lead-lined to resist acids and alkalis,
    draining boards, and racks, and have access to demineralized water, distilled water, and
    double-distilled water. Space for drying ovens or racks, automated dishwashers, acid
    baths, pipette washers and driers, and storage cabinets should also be available in the
    washing area.
    Media Preparation Area
    The media preparation area should have ample storage space for the chemicals, culture
    vessels and closures, and glassware required for media preparation and dispensing.
    Bench space for hot plates/stirrers, pH meters, balances, water baths, and media-
    dispensing equipment should be available. Other necessary equipment may include air
    and vacuum sources, distilled and double-distilled water, Bunsen burners with a gas
    source, refrigerators and freezers for storing stock solutions and chemicals, a microwave
    or a convection oven, and an autoclave or domestic pressure cooker for sterilizing media,
    glassware, and instruments.
    In preparing culture media, analytical grade chemicals should be used and good
    weighing habits practiced. To insure accuracy, and exact step-by-step routine should be
    developed for media preparation and a complete checklist required of all media preparers
    even for the simplest media.
    The water used in preparing media must be of the utmost purity and highest quality.
    Tap water is unsuitable because it may contain cations (ammonium, calcium, iron,
    magnesium, sodium, etc.), anions (bicarbonates, chlorides, fluorides, phosphates, etc.),
    microorganisms (algae, fungi, bacterial), gases (oxygen, carbon dioxide, nitrogen), and
    particulate matter (silt, oils, organic matter, etc.) Water used for plant tissue culture
    should meet, at a minimum, the standards for type II reagent grade water, i.e., be free of
    pyrogens, gases, and organic matter and have an electrical conductivity less than 1.0
    μmho/cm.
    The most common and preferred method of purifying water to type II standards is a
    deionization treatment followed by one or two glass distillations. The deionization
    treatment removes most ionic impurities, and the distillation process removes large
    1
    organic molecules, microorganisms, and pyrogens. Three other methods that will
    produce type II purity water are absorption filtration, which uses activated carbon to
    remove organic contaminants and free chlorine; membrane filtration, which removes
    particulate matter and most bacterial contamination; and reverse osmosis, which removes
    approximately 9% of the bacterial, organic, and particulate matter as well as about 90%
    of the ionized impurities.
    Transfer Area
    Under very clean and dry conditions, tissue culture techniques can be successfully
    performed on an open laboratory bench. However, it is advisable that a laminar flow
    hood or sterile transfer room be utilized for making transfers. Within the transfer area
    there should be a source of electricity, gas, compressed air, and vacuum.
    The most desirable arrangement is a small dust-free room equipped with an overhead
    ultraviolet light and a positive-pressure ventilation unit. The ventilation should be
    equipped with a high-efficiency particulate air (HEPA) filter. A 0.3-μm HEPA filter of
    99.97-99.99% efficiency works well. All surfaces in the room should be designed and
    constructed in such a manner that dust and microorganisms do not accumulate and the
    surfaces can be thoroughly cleaned and disinfected. A room of such design is
    particularly useful if large numbers of cultures are being manipulated or large pieces of
    equipment are being utilized.
    Another type of transfer area is a laminar flow hood. Air is forced into the unit through
    a dust filter then passed through a HEPA filter. The air is then either directed downward
    (vertical flow unit) or outward (horizontal flow unit) over the working surface. The
    constant flow of bacteria-free filtered air prevents nonfiltered air and particulate matter
    from settling on the working surface.
    The simplest type of transfer area suitable for tissue culture work is an enclosed plastic
    box commonly called a glove box. This type of culture hood is sterilized by an
    ultraviolet light and wiped down periodically with 95% ethyl alcohol when in use. This
    type of unit is used when relatively few transfers are required.
    Culture Room
    All types of tissue cultures should be incubated under conditions of well-controlled
    temperature, humidity, air circulation, and light quality and duration. These
    environmental factors may influence the growth and differentiation process directly
    during culture or indirectly by affecting their response in subsequent generations.
    Protoplast cultures, low-density cell suspension cultures, and anther cultures are
    particularly sensitive to environmental cultural condition.
    Typically, the culture room for growth of plant tissue cultures should have a temperature
    between 15 and 30 C, with a temperature fluctuation of less than 0.5C; however, a
    wider range in temperature may be required for specific experiments. It is also
    recommended that the room have an alarm system to indicate when the temperature has
    reached preset high or low temperature limits, as well as continuous temperature recorder
    to monitor temperature fluctuations. The temperature should be constant throughout the
    entire culture room (i.e., no hot or cold spots). The culture room should have enough
    fluorescent lighting to reach the 10,000 lux; the lighting should be adjustable in terms of
    quantity and photoperiod duration. Both light and temperature should be programmable
    for a 24-hr period. The culture room should have fairly uniform forced-air ventilation,
    and a humidity range of 20-98% controllable to 3 percent. Many incubators, large
    growth chambers, and walk-in environmental chambers meet these specifications.

    These are the details and for detailed information contact the horticulture department


    Regards
    Ashwini
    Dear Ashwini

    Can i have your contact number.
    Thank you
    Regards
    Vivek
    Last edited by moderator A; July 7th, 2010 at 04:21 PM. Reason: Only Business Members are allowed to post contact info. Please click “Upgrade” to become a Business Member.

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