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    Lab 5 - Microbiology: (Gram Staining)

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    This document describes a lab experiment on gram staining to differentiate between gram-positive and gram-negative bacteria based on their cell wall structure. Staphylococcus aureus was used as the gram-positive test bacteria. It was stained dark purple indicating a gram-positive result due to its thick peptidoglycan layer firmly retaining the crystal violet dye. Gram-negative bacteria have a thin peptidoglycan layer, allowing the dye to be washed away by alcohol, leaving them stained red or pink. The gram stain technique is an important preliminary step for bacterial classification and identification under a microscope.

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    Lab 5 - Microbiology: (Gram Staining)

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    207 views6 pages
    This document describes a lab experiment on gram staining to differentiate between gram-positive and gram-negative bacteria based on their cell wall structure. Staphylococcus aureus was used as the gram-positive test bacteria. It was stained dark purple indicating a gram-positive result due to its thick peptidoglycan layer firmly retaining the crystal violet dye. Gram-negative bacteria have a thin peptidoglycan layer, allowing the dye to be washed away by alcohol, leaving them stained red or pink. The gram stain technique is an important preliminary step for bacterial classification and identification under a microscope.

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    This document describes a lab experiment on gram staining to differentiate between gram-positive and gram-negative bacteria based on their cell wall structure. Staphylococcus aureus was used as the gram-positive test bacteria. It was stained dark purple indicating a gram-positive result due to its thick peptidoglycan layer firmly retaining the crystal violet dye. Gram-negative bacteria have a thin peptidoglycan layer, allowing the dye to be washed away by alcohol, leaving them stained red or pink. The gram stain technique is an important preliminary step for bacterial classification and identification under a microscope.

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    © All Rights Reserved
    Download as DOCX, PDF, TXT or read online from Scribd
    Download as docx, pdf, or txt
    207 views6 pages

    Lab 5 - Microbiology: (Gram Staining)

    Uploaded by

    api-383698554
    This document describes a lab experiment on gram staining to differentiate between gram-positive and gram-negative bacteria based on their cell wall structure. Staphylococcus aureus was used as the gram-positive test bacteria. It was stained dark purple indicating a gram-positive result due to its thick peptidoglycan layer firmly retaining the crystal violet dye. Gram-negative bacteria have a thin peptidoglycan layer, allowing the dye to be washed away by alcohol, leaving them stained red or pink. The gram stain technique is an important preliminary step for bacterial classification and identification under a microscope.

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    The key takeaways are that Gram staining is used to differentiate between bacteria based on differences in their cell wall structure and composition. Gram-positive bacteria have a thick peptidoglycan layer that retains crystal violet dye, while Gram-negative bacteria have a thin peptidoglycan layer and stain pink or red.

    The purpose of Gram staining is to differentiate between bacteria on the basis of their cell wall structure. It is a key procedure used to identify unknown bacteria under a light microscope.

    Gram-positive bacteria have a thick peptidoglycan layer within a single cell membrane, while Gram-negative bacteria have a thin peptidoglycan layer between an inner and outer cell membrane. Gram-positive bacteria stain purple, while Gram-negative bacteria stain pink or red.

    LAB 5 - MICROBIOLOGY

    ( GRAM STAINING )

    INTRODUCTION

    The gram stain is a differential stain commonly used to differentiates bacteria on the basis of

    their cell wall structure. The gram stain is key procedure in the identification of bacteria based

    on staining characteristics, and enable the bacteria to be examined using light microscope.

    Most bacteria can be divided into 2 groups based on the cell wall structure.

    1. Gram positive cell walls have single membrane enclosed by thick, cross link

    peptidoglycan. When the dye and mordant complex forms, it adheres firmly to thick

    peptidoglycan layer. Alcohol cannot wash out the dye-mordant complex because it

    firmly attached in the thick peptidoglycan layer. It has stain dark purple due to

    retaining the primary dye ( Crystal Violet ) in the cell wall. Staphylococcus aureus is

    the example for gram positive bacteria.

    2. Gram negative cell walls have a thin layer of peptidoglycan in the periplasmic space

    within its inner and outer lipid membrane. When dye and mordant complex forms, it

    does not adhere to the thin layer of peptidoglycan. The dye-mordant complex is easily

    removed the peptidoglycan layer with alcohol. It will stain red or pink due to retaining

    the counter staining dye ( Safranin ). Escherichia coli is the example for gram negative

    bacteria.
    OBJECTIVE

     To differentiate between the 2 major categories of bacteria which is gram positive and

    gram negative.

     To understand how gram stain affects the gram (+) and gram (-) based on the

    structural differences of their cell wall.

    METHODOLOGY

    1. Firstly, we added 1 drop of sterile water to the slide.

    2. Next, we chose staphylococcus aures and smear it to the slide.

    3. Then, allow the smear to dry using heat or air dry.

    4. We covered the smear with primarily stain ( Crystal Violet ) for 1 min. After 1 minute,

    we washed the slide with water.

    5. We added the mordant ( iodine ) also for 1 min, after that wash it with water.

    6. Using 95% ethanol, we decolorized the smear until the purple color has stopped

    leaching off the slide. Wash it immediately with water.

    7. We covered the smear with counter staining dye ( Safranin ) for 30 seconds.

    8. We washed both the top and bottom of the slide with water.

    9. Lastly, using the light microscope to view the smear up to 100×.


    RESULT

    Gram positive bacteria - staphylococcus aures

    Color : Dark Purple

    Categories : Gram-positive

    Morphology : Cocci
    DISCUSSION

    In this lab, we want to differentiate between the two major categories of bacteria

    which is grap positive and gram negative. We chose S. aures as our experimental species.

    S.aures is a facultative anaerobic that can grow without need of oxygen and coccal (round)

    bacterium. Although S. aures is not always pathogenic, it is a common cause of skin and

    respiratory infections also food poisoning. After we examined the bacteria under light

    microscope, we identifying the bacteria to be gram- postive bacteria as it stains purple in

    colour.

    Gram-positive differ from gram-negative through their cell walls structure and

    composition. Gram-positive bacteria have a thick cell walls made up of peptidoglycon layer

    beyond the plasma membrane which is cross linked by short chains of peptides. It is then

    resulting in the shape and rigidity of the cell wall. The thick layer of peptidoglycan allows to

    retain the dye-mordant complex ( crystal violet and iodine ). The alcohol cannot wash out the

    dye-mordant complex because it firmly secured in the thick layer of peptidoglycon. As a

    result the cells are stain as purple.


    CONCLUSION

    Staining is a technique used to enhance the clarify of the microscopic image. The most

    commonly used staining procedure in microbiological laboratory. Gram stain is a very

    important preliminary step in the classification and characterization of bacteria. Gram stain

    procedure enables bacteria to retain the color of stains based on the differences in the

    composition of their cell walls. From this experiment, we can differentiate between the two

    major categories ( gram-positive and gram-negative ).

    REFFERENCES

    1. Laboratory Manual, Dr Shakinaz Desa

    2. http://vlab.amrita.edu/?sub=3&brch=73&sim=208&cnt=1

    3. http://www.microscopemaster.com/gram-stain.html

    4. https://microbeonline.com/gram-staining-principle-procedure-results/

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