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    FST-305 Cereal Milling & Legumes

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    Rishav Gautam
    The document describes how to perform a gram staining procedure to differentiate between gram positive and gram negative bacteria. It details the materials and reagents needed, the principle behind gram staining, the staining reaction steps involving crystal violet, iodine, alcohol and safranin dyes, and the procedure to complete the gram stain.

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    FST-305 Cereal Milling & Legumes

    Uploaded by

    Rishav Gautam
    3 views2 pages
    The document describes how to perform a gram staining procedure to differentiate between gram positive and gram negative bacteria. It details the materials and reagents needed, the principle behind gram staining, the staining reaction steps involving crystal violet, iodine, alcohol and safranin dyes, and the procedure to complete the gram stain.

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    FST-305 CEREAL MILLING & LEGUMES

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    The document describes how to perform a gram staining procedure to differentiate between gram positive and gram negative bacteria. It details the materials and reagents needed, the principle behind gram staining, the staining reaction steps involving crystal violet, iodine, alcohol and safranin dyes, and the procedure to complete the gram stain.

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    3 views2 pages

    FST-305 Cereal Milling & Legumes

    Uploaded by

    Rishav Gautam
    The document describes how to perform a gram staining procedure to differentiate between gram positive and gram negative bacteria. It details the materials and reagents needed, the principle behind gram staining, the staining reaction steps involving crystal violet, iodine, alcohol and safranin dyes, and the procedure to complete the gram stain.

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    of 2

    Food microbiology practicals, Dept.food science &tech.

    AIM : To perform gram staining of given sample.

    MATERIAL REQUIRED : Glass slides, bunsan burner , cotton , sample , microscope.

    REAGENTS REQUIRED: Crystal violet dye, iodine, alcohol (95% ethyl alcohol), saffranin
    dye

    PRINCIPLE: Gram staining is most widely staining technique used in m/o examination. It
    was discovered by Danish scientist and physician Hans Christain Joachin Gram in 1884. This
    technique differentiates bacteria in 2 groups i.e. Gram positive and Gram negative bacteria.
    The procedure is based on the ability of m/o to retain colour of the stain during Gram
    reaction. Gram negative bacteria are decolourised by alcohol losing the colour of primary
    stain , purple. Gram positive bacteria are not decolourised by alcohol and will remain as
    purple. After decolourisation stop , a counter stain is used to impart pink colour to the gram
    negative m/o.

    Gram positive bacteria have a thick mesh like cell wall which is made up of peptidoglycan
    (50-90%) of cell wall, which stain purple. Gram negative bacteria have a thinner layer of
    peptidoglycan (10% of cell wall) and lose the crystal violet iodine complex during
    decolourisation with alcohol rinse but retain the counter stain safarin thus appearing reddish
    or purple.

    STAIN REACTION :

    1. Application of crystal violet to heat fixed smear :


    CV dissociates in aqueous solution into CV+ and Cl- ions. These two penetrate the cell
    wall and cell membrane of both gram positive and gram negative . CV+ interact with
    negative component of bacterial cell and stain it purple.

    2. Addition of gram iodine :


    Iodine acts as a mordant and a trapping agent. A mordant is a substance that increase
    the affinity of cell wall for a stain by binding to primary stain , thus forming a
    insoluble complex that get trapped in cell. During the reaction CV-I compex is formed
    and all the cells turn purple.

    3. Decolourization with ethyl alcohol :


    Alcohol dissolve the lipid outer membrane of gram negative bacteria, thus leaving the
    petidoglycan layer exposed and increase the porosity of cell wall. The CV-I complex
    is then washed away from the peptidoglycan layer leaving gram negative bacteria
    colourless.In gram positive bacteria , alcohol has dehydrating effect on cell wall
    causing cell wall to shrink , then CV-I complex get tightly bound into multi layered
    leaving the cell with purple colour.
    1

    4. Counter stain with safranin dye :


    The decolourised gram negative cell can be visible with a suitable counter stain which
    is usually positively charged safarnin, which stained it pink.

    D.A.V college, jalandhar


    Food microbiology practicals, Dept.food science &tech.

    PROCEDURE :1. Prepared very thin smear of sample on glass slide and heat fixed it.

    2. Flooded the smeared slide with crystal violet dye. Avoid over flooding and kept it for 1
    minute.

    3. Washed the slide under running tap water.

    4. Applied iodine solution gently all over the slide and kept for 1 minute.

    5. Washed it under tap water.

    6. Applied 95% ethyl alcohol all over the slide drop wise and kept for 10 second.

    7. Immediately rinsed with water.

    8. Finally, flooded the sample with saffranin dye to counter stain and kept for 45 seconds.

    9. Washed the slide with running water.

    10. Observed it under microscope.

    D.A.V college, jalandhar

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