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Differential Staining Technique

Simple staining depends on the fact that bacterial cells differ chemically from their surroundings and thus can be stained to contrasts with their surroundings. Microorganisms also differ from one another chemically physically, and thus may react differently to a given staining procedure. This is the basic principal upon which differential staining depends. Thus, we may find differential staining as a method of distinguished between types of bacteria.

A. The Gram Stain

The Gram Stain, the most useful staining procedure in bacteriology, is a differential stain. In this procedure bacteria are divided into two groups. The first of these groups is stained purple by the gram stain, while the second group is stained pink. The organisms stained purple was called gram- positive; the organisms stained pink is the gram-negative.

Crystal violet is first applied, followed by the mordant iodine, which fixes the stain. Then the slide is washed with alcohol, and the Gram‐positive bacteria retain the crystal‐violet iodine stain; however, the Gram‐negative bacteria lose the stain. The Gram‐negative bacteria subsequently stain with the safranin dye, the counterstain, used next. These bacteria appear red under the oil‐immersion lens, while Gram‐positive bacteria appear blue or purple, reflecting the crystal violet retained during the washing step.

Another differential stain technique is the acid‐fast technique. This technique differentiates species of Mycobacterium from other bacteria. Heat or a lipid solvent is used to carry the first stain, carbol fuchsin, into the cells.

Then the cells are washed with a dilute acid‐alcohol solution. Mycobacterium species resist the effect of the acid‐ alcohol and retain the carbol fuchsin stain (bright red). Other bacteria lose the stain and take on the subsequent methylene blue stain (blue). Thus, the acid‐fast bacteria appear bright red, while the nonacid‐fast bacteria appear blue when observed under oil‐immersion microscopy.


To learn the gram staining method and to observe the characteristic of gram-positive bacteria and gram-negative bacteria.


  • 1. 24 hours nutrients broth cultures of the following microorganisms for each group of student:

    • (a) E.Coli

    • (b) Staphylococcus aureus

    • (c) Bacillus subtillis

  • 2. Unknown cultures

  • 3. Microscope slides

  • 4. Gram stain reagent sets

  • 5. Immersion oil


    • 1. A separate thin smears of E.Coli, Staphylococcus aureus and Bacillus subtilis was prepared. It was air dried and heat fixed.

    • 2. The smears were covered with crystal violet for one minute.

    • 3. In a slow running tap water, the smears were rinsed slowly to remove the crystal violet for five seconds.

    • 4. Next, the smears were rinsed with the iodine reagent and then the same reagent was applied for one minute.

    • 5. The iodine reagent was rinsed as in step three.


    The alcohol was rinsed immediately as in step three.

    • 8. The smears were covered with safranin reagent for 30 seconds.

    • 9. It was rinsed again and blotted dry with a piece of blotting paper.

      • 10. Each smear was observed under the oil-immersion

    objective and the results were recorded.


    Bacillus Unknown Culture subtilis A Staphylococcus aureus
    Bacillus Unknown Culture subtilis A Staphylococcus aureus
    Bacillus Unknown Culture subtilis A Staphylococcus aureus


    Unknown Culture





    E.Coli 40x
    E.Coli 40x

    E.Coli 40x

    E.Coli 100x Unknown B 40x
    E.Coli 100x
    E.Coli 100x
    E.Coli 100x Unknown B 40x
    Unknown B 40x
    Unknown B
    Unknown B 100x DISCUSSION What determines Gram-positive and Gram-negative bacteria is due to difference in cell

    Unknown B 100x


    What determines Gram-positive and Gram-negative bacteria is due to difference in cell wall composition. Because the cell walls of Gram-negative cells have a higher content of lipids and a thinner layer of peptidoglycan, the alcohol used in the decolorizing step made the Gram-negative cells incapable of retaining the methylene blue-iodine complex. On the other hand, Gram-positive cells have a thicker peptidoglycan that traps the methylene blue-iodine complex, making it less vulnerable to decolourization. The Gram stain technique was used on unknown number 20. The unknown appeared to be Gram negative and cocci shaped. Since this is an unknown, the literature value is unknown. Possibilities of difference in

    literature and experimental value could be due to excess primary and mordant iodine dyes being flushed out entirely by the 95% Ethanol, or that the mordant iodine dye was not left on long enough.


    Gram staining is used to determine the gram positive bacteria will stain to a purple colour while the gram negative bacteria stain to be in pinkish colour.


    1. Are there any chemical differences between the cell wall of gram positive and gram- negative bacteria, which might explain differences in the rate of decolourization?

    The cell wall in bacteria contains peptidoglycan, a

    polymer of N-acetyl glucosamine, N-acetyl muramic acid and amino acid. Gram positive cell walls contain a thick layer of peptidoglycan layer that encircles the cells. While the gram negative cell walls contain a thin layer of peptidoglycan between the cytoplasmicmembrane and the outer membrane. Gram-negative bacteria stained with crystal violet are decolorized by 95% alcohol within 2 min, whereas Gram-positive bacteria require at least 3 min treatment. Aqueous solutions of safranin, neutral red, and carbol fuchsine replace crystal violet from stained Gram- positive bacteria more quickly than alcohol alone, and alcoholic solutions of these counterstains are in most cases still more effective. Treatment of crystal violet- stained organisms with alcoholic safranin for 15 sec will distinguish Gram-positive bacteria (violet) from Gram- negative bacteria (pink). Alcohol containing very low concentrations of iodine generally decolorizes crystal violet-stained Gram-positive bacteria more quickly than

    alcohol alone. Increasing concentrations of iodine in alcohol reduce the rate of decolorization of stained bacteria, but stained Gram-negative bacteria are still readily decolorized. The addition of iodine to alcohol increases the rate of extraction of crystal violet by alcohol from Gram-negative organisms, but delays extraction of dye from Gram-positive organisms, and this applies when counterstain is also present. A two- solution modification of Gram staining is described in which crystal violet-stained bacteria are treated with an

    alcoholic solution of safranin, carbol fuchsin, and iodine.


    (a) Does the age of the cultures effect the gram stain? Yes

    • (b) Why? Because old culture of gram positive cells may not

    retain stain as well as younger cultures and could give false negative results.


    • (a) Based on your experience in the laboratory do you feel

    that the gram stain is a simple procedure?

    I feel that the gram stain is not a simple procedure

    • (b) Why?

    For gram stain, we use more than one stain such as crystal violet, iodine, alcohol and safranin to our smears. Unlike the simple staining, we stained either crystal violet, methylene blue or carbol fuchsine to our smears. Furthermore, the cell wall of the bacteria that react in gram stain has thicker or thinner cell wall to know which bacteria is a gram positive or gram negative.


    What is the influence of pH on the gram stain reaction? pH affects the uptake of crystal violet by the bacteria. Crystal violet at low pH and safranin do not stain the walls of bacteria. When organisms of a gram positive are stained with crystal violet at a high pH and then are washed at a low pH, the dye disappears from the walls. Organisms with stained walls resist both decolorizer and counterstain after treatment with iodine and thus appear gram positive.