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    Gram PDF

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    The Gram stain procedure demonstrates gram-negative and gram-positive bacteria in tissue samples. It works by staining both types of bacteria with crystal violet initially. For gram-negative bacteria, an acetone wash disrupts the outer cell membrane, allowing the crystal violet to be removed and basic fuchsin stain to take its place, appearing red. Gram-positive bacteria retain the crystal violet stain, appearing blue. The procedure involves multiple staining and washing steps to achieve this differential staining result. An infected appendix is a suitable positive control sample containing both gram-negative and gram-positive bacteria.

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    Gram PDF

    Uploaded by

    Anna
    21 views5 pages
    The Gram stain procedure demonstrates gram-negative and gram-positive bacteria in tissue samples. It works by staining both types of bacteria with crystal violet initially. For gram-negative bacteria, an acetone wash disrupts the outer cell membrane, allowing the crystal violet to be removed and basic fuchsin stain to take its place, appearing red. Gram-positive bacteria retain the crystal violet stain, appearing blue. The procedure involves multiple staining and washing steps to achieve this differential staining result. An infected appendix is a suitable positive control sample containing both gram-negative and gram-positive bacteria.

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    The Gram stain procedure demonstrates gram-negative and gram-positive bacteria in tissue samples. It works by staining both types of bacteria with crystal violet initially. For gram-negative bacteria, an acetone wash disrupts the outer cell membrane, allowing the crystal violet to be removed and basic fuchsin stain to take its place, appearing red. Gram-positive bacteria retain the crystal violet stain, appearing blue. The procedure involves multiple staining and washing steps to achieve this differential staining result. An infected appendix is a suitable positive control sample containing both gram-negative and gram-positive bacteria.

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    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
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    21 views5 pages

    Gram PDF

    Uploaded by

    Anna
    The Gram stain procedure demonstrates gram-negative and gram-positive bacteria in tissue samples. It works by staining both types of bacteria with crystal violet initially. For gram-negative bacteria, an acetone wash disrupts the outer cell membrane, allowing the crystal violet to be removed and basic fuchsin stain to take its place, appearing red. Gram-positive bacteria retain the crystal violet stain, appearing blue. The procedure involves multiple staining and washing steps to achieve this differential staining result. An infected appendix is a suitable positive control sample containing both gram-negative and gram-positive bacteria.

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    of 5

    SURGICAL PATHOLOGY HISTOLOGY Date:

    STAINING MANUAL - MICROORGANISMS Page: 1 of 3


    GRAM BACTERIA - MODIFIED BROWN AND BRENN
    PURPOSE: For demonstrating gram-negative and gram-positive in tissue.

    PRINCIPLE: Both bacteria's, positive and negative, cell wall is composed


    of peptidoglycan,(the gram-positve has a thicker wall) and both will take
    up the crystal violet. The gram-negative has a a layer of
    lipopolysaccharide external to the peptidoglycan wall, which is disrupted
    in the acetone rinse, allowing the crystal violet to be differentiated out.
    This allows the the gram-negative bacteria to take up the basic fuchsin
    stain.

    CONTROL: An infected appendix, or any tissue containing both negative


    and positive gram rods.

    FIXATIVE: 10% formalin

    TECHNIQUE: Cut paraffin sections 4-5 microns.

    EQUIPMENT: A staining rack, coplin jars, dropper bottles, bibulous paper.


    Rinse all glassware in DI water.

    REAGENTS:
    1% Crystal Violet: 0.25% Basic Fuchsin:
    Crystal violet 1.0 gm Basic fuchsin 0.25 gm
    Distilled water 100.0 ml Distilled water 100.0 ml

    Filter into a dropper bottle. Stable Filter into a dropper bottle.


    for 1 year. Solution is stable for 6 months.
    CAUTION: Irritant, possible carcinogen CAUTION: Possible carcinogen.

    Lugol's Iodine: Picric Acid-Acetone:


    See Stock Solutions Picric acid, saturated 100.0 ml
    Acetone 900.0 ml
    CAUTION: Dermal sensitizer, irritant
    to eyes, skin and respiratory system. Discard after use.
    CAUTION: Flammable, corrosive.
    Acetone:
    Purchased through Fisher.
    CAUTION: Flammable.
    MICROORGANISMS
    GRAM STAIN Page: 2 of 3
    SAFETY/PPE: Wear gloves, goggles and lab coat. Avoid contact and
    inhalation of dyes and chemicals.

    Crystal violet/Gentian violet: carcinogen, skin irritant.

    Iodine: Dermal sensitizer, irritant to eyes, skin and respiratory system.


    Corrosive.

    Basic fuchsin: carcinogen, work in a well ventilated area.

    Picric acid: can become exlosive if allowed to dry out. Toxic through skin
    exposure.

    Acetone: moderatly toxic on ingestion. May cause dizziness, headaches and


    irritation to respiratory system on inhalation. Defatting and dermatitis on
    skin contact.

    PROCEDURE:
    1. Deparaffinize and hydrate to distilled water.
    2. Place slides on staining rack, drop crystal violet stain onto tissue
    section, stain for 1 minute.
    3. Wash in tap water.
    4. Lugol's iodine, 1 minute.
    5. Wash in tap water.
    6. Blot sections dry, breath on section then quickly pour acetone over
    section until no color runs off.
    7. Wash in tap water.
    8. Place slides on staining rack, drop Basic fuchsin on tissue sections,
    stain 3 minutes.
    9. Wash in tap water, blot gently but not completely dry.
    10. Dip quickly into acetone, 2 dips.
    11. Dip directly into picric acid-acetone mixture until a 'salmon' color.
    12. Dip quickly into two changes of acetone.
    13. Air dry, dip into xylene, and coverslip.

    RESULTS:
    Gram-positive bacteria blue
    Gram-negative bacteria red
    Nuclei red
    Background yellow
    MICROORGANISMS
    GRAM STAIN Page: 3 of 3
    REFERENCES:
    Luna L, Manual of Histologic Staining Methods of the AFIP, 3rd Ed, pp 222,
    McGraw-Hill,NY
    Carsen F, Histotechnology: A Self-Instructional Text, 1990,pp194-
    196,ASCP,Ill
    Crookham,J, Dapson,R, Hazardous Chemicals in the Histopathology
    Laboratory, 2nd ED, 1991, Anatech

    Prepared: By:

    Approved: By:
    Downloaded from WebPath: Internet Pathology Laboratory
    http://www-medlib.med.utah.edu/WebPath/webpath.html
    PROCEDURE CARD
    GRAM BACTERIA - MODIFIED BROWN AND BRENN
    CONTROL: An infected appendix, or any tissue containing both negative and positive gram
    rods.

    PROCEDURE:
    1. Deparaffinize and hydrate to distilled water.
    2. Place slides on staining rack, drop crystal violet stain onto tissue section, stain for 1
    minute.
    3. Wash in tap water.
    4. Lugol's iodine, 1 minute.
    5. Wash in tap water.
    6. Blot sections dry, breath on section then quickly pour acetone over section until no color
    runs off.
    7. Wash in tap water.
    8. Place slides on staining rack, drop Basic fuchsin on tissue sections, stain 3 minutes.
    9. Wash in tap water, blot gently but not completely dry.
    10. Dip quickly into acetone, 2 dips.
    11. Dip directly into picric acid-acetone mixture until a 'salmon' color.
    12. Dip quickly into two changes of acetone.
    13. Air dry, dip into xylene, and coverslip.

    RESULTS:
    Gram-positive bacteria: blue
    Gram-negative bacteria: red
    Nuclei: red
    Background: yellow

    1% Crystal Violet: Lugol's Iodine:


    Crystal violet 1.0 gm See Stock Solutions
    Distilled water 100.0 ml
    CAUTION: Corrosive, irritant.
    Filter into a dropper bottle. Stable for 1 year.
    Acetone:
    CAUTION: Possible carcinogen, avoid
    Purchased through Fisher
    contact and inhalation.
    CAUTION: Flammable.
    Saturated Picric Acid: Picric Acid-Acetone:
    Picric acid 13.0 gm Picric acid, saturated 100.0 ml
    Distilled water 1000.0 ml Acetone 900.0 ml

    Mix well, some precipitate should Discard after use. Stable for 1 year.
    remain at the bottom of the bottle. CAUTION: Flammable, corrosive.
    CAUTION: Toxic, do not allow to dry
    out. DATE:

    DATE: TECH:

    TECH: EXPIRATION:

    EXPIRATION: NONE 1% Crystal Violet/Gentian:


    Crystal violet 1.0 gm
    Picric Acid-Acetone: Distilled water 100.0 ml
    Picric acid, saturated 100.0 ml
    Acetone 900.0 ml Filter into a dropper bottle. Stable
    for 1 year.
    Discard after use. Stable for 1 year.
    CAUTION: Irritant, possible
    CAUTION: Flammable, corrosive. carcinogen.

    DATE: DATE:

    TECH: TECH:

    EXPIRATION: EXPIRATION:

    0.25% Basic Fuchsin: 1% CRYSTAL VIOLET


    Basic fuchsin 0.25 gm
    Distilled water 100.0 ml DATE:
    Filter into a dropper bottle.
    Solution is stable for 6 months. TECH:

    CAUTION: Carcinogen.

    0.25% BASIC FUCHSIN

    DATE:

    TECH:

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