What are Gram-positive and Gram-negative bacteria?
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
Take a glass slide and smear bacteria samples from the culture on it with the help of an inoculation loop.
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.
Add a few drops of crystal violet to the bacteria sample.
Add iodine molecules to the sample; it is golden brownish in colour.
Here, the crystal violet and iodine molecules bind together.
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.
The next step is to apply safranin to the bacterial samples; safranin is a biological stain, pinkish in colour.
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 are 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
The test has proven effective for food microorganisms and Bacillus spp, with a bit of mishap for some anaerobes. It has the advantage of simplicity and is effective on older samples/cultures.
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.
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FAQs on Difference Between Gram positive and Gram negative Bacteria
1. Differentiate between gram positive and negative bacteria.
There are structural differences between Gram positive and Gram negative bacteria that makes them appear different after Gram staining experiment. The cell wall of the former kind is thicker peptidoglycan wall whereas that of latter is thinner. The former bacterial cell has a capsule after the cell wall whereas a second plasma membrane layer is present in the latter kind of bacterial cell. This makes it harder for antibiotics to get inside the Gram negative bacteria cell and kill it. So, it is difficult to treat if infected with Gram negative bacteria like Salmonella, Pseudomonas, Shigella, and other Enterobacteriaceae, Moraxella, Helicobacter, Stenotrophomonas, acetic acid bacteria, Legionella, etc. On the other hand, Gram positive bacteria like cocci, bacilli, Staphylococcus aureus, and Streptococcus pneumonia can be treated easily after their diagnosis.
2. What are the infections which are caused by gram-positive bacteria?
Some of the infections caused by Gram positive bacteria include Urinary Tract Infections and pneumococcal infections. These are caused commonly in people who are elderly or pregnant.
3. What are the infections which are caused by gram-negative bacteria?
Some of the infections caused by Gram negative bacteria include bloodstream infections, wound infections, and meningitis.
4. Which are easily treated, Gram positive or Gram negative bacterial infections?
Gram-positive bacterial infections can be treated with antibiotics easily as they possess cell walls that can absorb cleaning medications. They also do not have a second phospholipid plasma membrane before the capsule in their cellular structure as compared to the Gram-negative bacteria where it is present. This makes it easier for antibiotics to work on the former type.
5. What is the major difference between Gram positive and Gram negative bacteria?
The major difference can be found out after Gram staining with crystal violet and iodine and safranin at the end where Gram positive bacteria are purple in colour under the microscope and the latter are pinkish in appearance.
6. How can we distinguish gram-positive and negative bacteria?
Some of the key points are
In the case of Gram-negative bacteria, it has thinner peptidoglycan and has an outer lipid membrane
Gram-positive bacteria have a thicker peptidoglycan layer and have no outer lipid membrane.
Once stained by safranin, the gram-positive cells turn purple and gram-negative cells do not retain purple color.
Gram-positive bacteria make exotoxins
Gram-negative bacteria make endotoxins
7. What can cause gram-positive bacteria to appear gram-negative?
Some conditions in which the gram-positive bacteria appears gram-negative are listed below:
Over-decolorization
Exposure to lysozymes
Old cultures
Bacterias are phagocytosed
Over-decolorization- Gram-Positive Bacteria can appear Gram-Negative by using Acetone alone during the decolorization or over-decolorize by prolonged exposure to decoloriser.
Exposure to lysozymes - Gram-Positive Bacteria appears to be Gram-Negative when the cell walls get damaged by exposure to lysosomes or antibiotics such as penicillin.
Old Cultures- They can appear to be Gram-Negative bacteria when the cell wall is weakened or by the action of autolytic enzymes.
Bacterias are Phagocytosed - The cell wall is acted upon by lysosomal contents on bacterias that are phagocytic in nature.
