[Biology Class Notes] on Difference Between Gram positive and Gram negative Bacteria Pdf

In a typical bacterial cell, there are three layers :

• The innermost layer is the plasma membrane which directs what enters and exits the cell; phospholipid bilayer with some protein scattered between it

• The middle layer is the Cell wall that provides structural support to the cell and is made up of peptidoglycan or polysaccharide. The cell wall of bacteria Gram-positive and negative bacteria differ in their thickness. It is an important layer to understand the structure and difference between Gram-positive and negative bacteria, which we will understand later in this write-up. 

• The third layer is the Capsule which is the sticky outer layer for attachment and protection. 

The major differences between Gram-positive and Gram-negative bacteria can be understood with the structural differences and Gram staining differences between them.

Highlighting the Difference Between Gram-Positive and Gram-Negative

 

Gram Stain Process

It is a purpose that helps us to identify the type of infectious bacteria for proper diagnosis and treatment based upon outcome. It helps us to highlight the Gram positive and gram-negative bacteria differences.

Steps of Gram Staining

1. Take a glass slide and smear bacteria samples from the culture on it with the help of an inoculation loop. 

2. Fix the bacteria to the slide to avoid it getting washed away in the later steps, this is done by heat fixing the glass slide over a flame. 

3. Add a few drops of crystal violet to the bacteria sample.

4. Add iodine molecules to the sample; it is golden brownish in colour. 

5. Here, the crystal violet and iodine molecules bind together. 

6. Now, wash the bacteria with alcohol which is a de-staining chemical. A major happening in the Gram positive bacteria takes place which is when the capsule of the cell gets washed away and shrinks the cell. And in Gram-negative bacteria, the second plasma membrane layer gets washed away along with the capsule due to the dehydrative nature of the alcohol. As the Gram -ve bacteria have a thin cell wall left, the crystal violet-iodine molecules also get washed away, and hence the cell loses its colour. On the other hand, Gram +ve bacteria has retained the colour from crystal violet dye and therefore appears purple violet colour under the microscope.

7. The next step is to apply safranin to the bacterial samples; safranin is a biological stain, pinkish in colour. 

8. Results: The results for Gram positive vs. Gram-negative bacteria will be different as mentioned below. 

What are Gram-Positive Bacteria Result after Staining? 

Now under observation underneath the microscope, Gram-positive bacteria appear purple in appearance as the pinkish color of safranin is overshadowed by the deep color of crystal violet dye. 

Clinical Importance of Gram Staining

It is the first and foremost important step within the classification of bacteria. The morphology and arrangements of bacteria a
re often observed well once stained because it helps within the screening of pathogens in clinical specimens.  While Gram staining is used to detect the differentiate bacteria there are many other microscopic organisms that can be found on the smear of a gram stain, which is subjected to the test.

Disadvantages of Gram Staining

• Misinterpretation: Organisms that are gram-negative would appear red once they retain counterstain, even after getting washed out of the complex made of crystal violet and iodine in the heat fixed cell, by alcohol or acetone. Meanwhile, the gram-positive organisms, on over-decolourization after the loss of complex even though they retain the complex after decolourization and remain purple.

• Side Effects: It is noted that blood samples would lead to side effects such as bruising due to the blood draw,  even though some other types of samples would not entail certain side effects.

• Limitations Under Environmental Microbiology: It is observed that in most medical conditions, this method is not solely recommended for bacterial identification since; it will not be able to identify bacteria to its specific level. Therefore, it is used in a combination of other molecular techniques in identifying bacteria. 

 Mishaps during Gram Staining

Decolourization is one of the most prominent steps which is prone to be performed incorrectly, mishaps may occur more often.

• Unbridled Heat During the Time of Fixation: It makes it easier to decolourize the cell once the cell morphology is altered once heat fixation is done in an excessive amount.

• Overindulgent Washing between each step: CV stain is easily washable with water and it is recommended to not use more than 5 s water rinse during any stage of the procedure.

• Prolonged Decolorization: It is recommended to decolorise no more than 15 seconds,  and if the specimen is left for a longer period of time,  the possibility of the positive cell losing Crystal Violet and turning red is higher.

• Excessive Counterstaining: With overexposure, It is possible to replace the CV Iodine in the gram-positive cells with counterstain instead

• Misread Stains: where the negative strains were misread as positive and positive stains as negative.

• Mixed Culture

• A minimal number of bacterial cells.

Alternatives for Gram Stain

• KOH String test

• Aminopeptidase Test

• L-alanine aminopeptidase is the enzyme that is localized in bacterial cell walls that bisects amino acid L-alanine from different peptides. The test is proven to be more useful in non-fermenters and gram-variable organisms, with faster results in the short span of 5 minutes.

• Fluorescent Stains

• The test involves fluorescent nucleic acid binding dyes like hexidium iodide (HI)and SYTO 13. Gram-negative organisms turn green by SYTO 13 and gram-positive organisms are reddish-orange fluorescent by HI, overpowering the green colour of SYTO 13 when the dyes are used together in a single step.

• LAL-based Assay

• This type of test uses the same reaction which is being used for the chromogenic LAL test. In the LAL test, clotting is formed due to the enzymes cleaving a peptide from the horseshoe crab coagulant. Gram-positive organisms lacking endotoxin do not trigger the colour change in this method, but gram-negative on the other hand is triggered.

The provided pieces of information will be reliable for students and will provide a clearer understanding of the difference between gram-positive and gram-negative bacteria.

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