How to Do a Tissue Culture

Tissue refers to a collection of cells with similar characteristics. These cells live in organisms, such as people, plants, and animals. A tissue culture refers to the deliberate growth of tissues and/or cells separate from the donor organism. Tissue cells are taken from the original organism and are cultivated under sterile and controlled conditions in a laboratory. The growth and behavior of the cells are observed, studied, and/or altered in the culture. Scientists study the cells in a liquid or semi-liquid culture medium in an effort to identify their workings, alter their make-up, or channel their growth towards a defined result. The cultures are studied to see how cells develop in different environments. The plan may be to promote growth; for example, growth in plant tissue may lead to larger, more productive plants. Another plan might be to determine what stops growth, perhaps, to curb a disease. While media and conditions may vary from cell type to cell type, the basic process remains the same. Doing a tissue culture is time-consuming, exacting, and meticulous work. The following will show how to do a tissue culture.

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 Steps

Sterilize your work area thoroughly

    1
    Sterilize the laminar hood and the area around it.
        Wipe the work area generously and thoroughly with a cloth dampened with ethanol.
    2
    Use only sterile tools and equipment for cell cultures.
    3
    Open all culture containers, test tubes, etc. inside the laminar flow hood.
        Immediately dispose of tainted materials, such as those opened outside of the laminar hood, according to hazardous materials rules.
    4
    Spray everything with ethanol - except the tissue cultures themselves and the equipment used to move or feed them.
    5
    Clean your gloved hands thoroughly with ethanol after cleaning the equipment and placing it in the hood.
    6
    Follow protocols for disposal of waste.

Choose the culture media

    1
    Select media appropriate to the cell line (for example, plant or animal) and to the intent of the culture (for example, growth, control, or change).
        There are thousands of media to select from, so confirm that the media selected is appropriate to the test.
    2
    Use only media that have been previously sterility tested.

Prepare the culture media

    1
    Prepare media and perform other cell culture work inside the laminar flow hood.
    2
    Prepare the media carefully as instructed by the provider or by the laboratory protocol.

Secure the tissue sample

    1
    Scrape or swab cells from host organism - person, plant, animal, etc.
        This can be more or less invasive surgically, depending on the location of the tissue in the body.
        Tissue samples can be secured by scraping or sectioning cells from plants depending on the purpose.

Prepare the culture

    1
    Perform all functions under a laminar hood.
    2
    Add the medium and the tissue sample to their container.
    3
    Cap or lid the container snugly.
    4
    Record activity as required by lab protocol.
    5
    Store the culture as directed.

Feed the cells

    1
    Replenish media solution every 2-3 days, or the cells may die from lack of nutrition.
    2
    Warm media to 37 degrees Celsius or 95 degrees Fahrenheit before using.
        Use pre-warmed media and do not keep cells out of their incubator for more time than is necessary.
    3
    Use a T-flask to pour off old medium and pour in new.
    4
    Shake T-flask lightly to see if cells are healthy or aggregating, a sign that they are not healthy.
    5
    Proceed with feeding them by adding fresh medium if the cells appear healthy.
    6
    Return the culture to the incubator or refrigerator according to the protocol for the particular cell line.

Follow the growth and morphology (signs of change) of the cells

    1
    Examine the tissue cultures each day.
        Look for growth, notice any change in the color, and/or record any change in the cell count.
    2
    View cells through a microscope.
        The live cells will appear to be quite bright while unhealthy cells will be dull.
        Suspension cells, such as blood cells, will appear smooth and regular.
        Adherent cells, such as those scraped from a plant, attach together in masses.

Tips

    Keep a "tissue log;" record the type of cell, the make-up of the medium, the dates on which the cells were treated, and any observations on the culture.
    Practice maintaining sterility with your least important tissue cultures since small amounts of contamination can ruin your stock of cells.

Warnings

    Treat all tissue cultures as if they are hazardous waste because they may carry viral strains.
    Be careful not to contaminate the tissue culture with bacteria or other cell sources.
    Allow no eating, drinking, or smoking in or near the work area.

 Things You'll Need

    Growth Media are solutions that might contain (1) bulk ions ; (2) trace elements: (3) glucose; (4) amino acids: (5) vitamins; and/or (6) serum to promote growth activities.
    Media containers, petri dishes, microscope slides, T-flasks, and other laboratory devices used to measure, add, or transport fluids
    Laminar vertical flow hood
    Ethanol
    Safety equipment, including gloves, goggles, and mask
    Inverted phase contrast microscope