Bacterial Morphology and Staining Techniques

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Running head: “Bacterial Morphology and Staining Techniques” 1

“Bacterial Morphology and Staining Techniques” 6

“Bacterial Morphology and Staining Techniques”

Student’s Name

Western Governors University

“Bacterial Morphology and Staining Techniques”

A1. Introduction


Gram-positive cell’s exterior consists of a dense cell wall made up of net like polymer called peptidoglycan. It is located next to to an interior plasma membrane. The 60 to 90% of cell wall of a Gram-positive bacterium is peptidoglycan. The peptidoglycan layer is strengthened by another molecule called teichoic acids that is situated perpendicular to the peptidoglycan network and provide structural backing. The size of the cell wall of Gram-positive cells ranges from 20 to 80 nm in thickness and is cross-linked in two dimensions which makes it a sturdy and firmer framework (Hands-on-Labs, 2015).

The thick cell walls of Gram-positive bacteria retain primary stain of crystal violet. The retention of primary stain is directly related to wall thickness and not to peptidoglycan. This is evident because many of yeast cells that have thick walls but lack peptidoglycan, retain these stains as well (Hands-on-Labs, 2015).

Physiological damage or aging can lead to leak out the stain-dye complex from Gram-positive cells. Therefore, Gram staining must be performed on cultures within 24 hours. As Gram-positive cells age (24-48 hours old), the crystal violet-iodine dye complex leaks and is washed away during the step of decolorization. These types of cells are called as Gram-variable cells and they can also give the impression of Gram-negative or as both kinds of cells during staining process (Hands-on-Labs, 2015).

Gram Negative

Gram-negative bacteria’s cell wall is thinner but more complex than the cell wall of Gram-positive bacteria. The size of the cell wall of Gram-negative cells ranges from 2 to 8 nm in thickness. It consists of various polysaccharides, proteins, and lipids and only 10 to 20% of the cell wall is peptidoglycan. They have a single coating of peptidoglycan and an apparent space between cell wall and cell membrane which is known as periplasmic space. The fine cell wall is cross-linked in one area and is flexible but is more vulnerable to destruction (Hands-on-Labs, 2015).

The cell wall of gram-negative bacteria fails to hold the crystal-violet and iodine dye complex during the procedure of decolorization. This is because of their thin cell walls and due to comparatively large extents of lipoproteins and lipopolysaccharides in their walls (Hands-on-Labs, 2015).

A2. Materials and methods

First, apply a thin smear of bacteria on a slide and then leave it for air dry. After that, pass the slide through flame for fixing the specimen in place (Hands-on-Labs, 2015).

Step 1: Crystal Violet

After the specimen is fixed in place, put few drops of the primary stain crystal-violet dye in the specimen for 1 minute to provide them a color of purple. And then rinse it with water (Hands-on-Labs, 2015).

Step 2: Iodine

Next, add Gram’s Iodine as a mordant to the purple colored specimen for one minute to conserve the color of crystal-violet. And, rinse the slide with water. Here, all the cells remain purple due to retention of crystal violet stain (Hands-on-Labs, 2015).

Step 3: Decolorizer

The decolorizing step distinguishes between the Gram-negative and Gram-positive cells. The specimen is rinsed quickly with 95% ethanol or an ethanol-acetone solution. The alcohol will soften the cell membrane, exposing the thin cell wall, and permitting the crystal violet dye to sweep away in gram-negative cells. The cell walls of Gram-positive bacteria are dense and extensively cross-linked, which allow the dye complex to stay bound and secured in the step of decolorization. Then the cells are washed with water to stop the further decolorization (Hands-on-Labs, 2015).

Step 4: Safranin

The counterstain is added in this step. The specimen is counterstained with safranin for 30 to 60 seconds. Then, an ultimate rinse and dry is provided. The gram-positive cell wall which has not lost its primary stain of crystal-violet will remain purple. Whereas, the decolorized Gram-negative cell wall obtains a new pink color of safranin. This makes the decolorized Gram-negative cells easily evident and identifiable. Thus, gram stain is a visual stain which visually differentiates the bacteria (Hands-on-Labs, 2015).

A3. Results

Escherichia coli

The cells appeared to be rod shaped and pink. They acquired pink dye from counterstain of safranin due to their thin layered cell wall. Here, some cells are in the chain form (streptobacilli), some are attached end-to-end in pair (diplobacilli) and some are lined up side-by-side at angles in an arrangement or in stacks known as palisades. Thus, due to the above feature Escherichia coli is Gram-negative rod(bacillus) (Hands-on-Labs, 2015).

Staphylococcus epidermidis

These cells appeared to be spherical and purple in color. They retained the primary stain crystal-violet due to their thick cell wall. During the division of coccus bacterial cell if the daughter cells fail to separate completely after cell division, a duo of cocci known as diplococci and groups of four in form of squares called tetrads are formed that can be seen in the image. They can also be formed in cube like packets of eight, sixteen, and even larger numbers of cells, which are all named as sarcina and that is also apparent in the image. Cocci can also combine into hundreds of unceasing cells, bead-like chains known as streptococci and into grape-like clusters and asymmetrical bunches called staphylococci. In the image, we can see many bunches of spherical purple cells. Hence, Staphylococcus epidermidis is Gram-positive cocci (Hands-on-Labs, 2015).

A4. Discussion

The Gram-negative’s cell wall is thinner and less compact than that of Gram-positive bacteria, but remains strong, tough, and elastic to give them shape and protect them against extreme environmental settings. The outer membrane of Gram-negative bacteria invariably contains a specific molecules or proteins exclusive to Gram-negative cells called lipopolysaccharides (LPS) in addition to proteins and phospholipids. LPS consists of polysaccharides and lipid A. The type of chains made by polysaccharides and Lipid A are used to detect different Gram-negative bacteria. Here, the lipid A portion is responsible for the toxic properties that potentiates serious medical problem via any Gram-negative infection (Hands-on-Labs, 2015).

The LPS molecule is toxic and is classified as an endotoxin that elicits a strong immune response when the bacteria infect animals. Endotoxins produce reactions such as fever and expands blood vessels, so the blood pressure drops swiftly leading to shock in Gram-negative infections. Endotoxin is known for killing people in severe infections. Lot of endotoxins is released even when they are dying. Thus, the concentration of very toxic substance rapidly increases by killing them. Therefore, providing the antibiotics tardily in an infection may also worsen the symptoms, or even may cause death of the patient. Hence, the Gram-negative infections are a challenge to healthcare professionals due to the high morbidity and mortality associated with these infections (Hands-on-Labs, 2015).


Hands-on-Labs. (2015). Bacterial Morphology and Staining Techniques. Englewood, CO. Available from

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