Why does the gram positive bacteria stain purple while gram negative stains pink?
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The Gram stain procedure relies on structural differences of the cell walls between Gram-positive and Gram-negative bacteria that result in Gram-positive bacteria staining purple, and Gram-negative bacteria staining pink/red.
(All bacterial cells, like all cells, have a cytoplasmic membrane, but in addition to their cytoplasmic membrane, have additional structures that surround the cell, and these structures are what differs between Gram-positive and Gram-negative bacteria, and thus the Gram stain exploits these differences).
Bacteria that stain purple are known as Gram-positive, and their cytoplasmic membrane is surrounded by a thick cell wall composed of peptidoglycan, a molecule composed of amino acids (“peptido-”) and sugars (“-glycan”).
Bacteria that stain pink/red are known as Gram-negative, and their cytoplasmic membrane is surrounded by a very thin peptidoglycan layer, and surrounding that is another outer membrane.
Now that we’ve gone over the key structural differences, let’s take a look at how the Gram stain works:
The Gram stain involves a step-by-step procedure of applying dyes and washes to a glass slide of heat-fixed bacteria.
- Crystal violet dye is placed on the bacterial smear slide for one minute. At this stage, both Gram-positive and Gram-negative cells will stain purple.
- Wash/rinse to remove excess dye.
- Gram’s iodine is placed on the bacterial smear slide for one minute. Iodine acts as a mordant, a substance that helps to fix the dye to the thick peptidoglycan cell wall of Gram-positive bacteria.
- Wash/rinse to remove excess iodine.
- Alcohol, a decolorizer, is used to rinse the slide. Due to the iodine acting as a mordant for the crystal violet dye in Gram-positive bacteria, the Gram-positives will remain purple even after decolorizing. Gram-negative bacteria will become colorless.
- Wash/rinse to remove excess alcohol.
- Safranin dye is added to the slide for one minute. Gram-positive bacteria, already purple, will not show this pink/red stain. Gram-negative bacteria, colorless from the alcohol rinse, will show this pink/red stain.
(Pictures courtesy of Google)
Crystal violet stains peptidoglycan. Gram positive bacteria have a thick peptidoglycan layer and gram negative bacteria have a thin layer. Therefore, gram negative bacteria won’t be stained much when the dye is removed with the alcohol. So after adding iodine and alcohol you have stained, purple gram+ and unstained gram-
The counterstain, safranin is pink so it stains the unstained bacteria pink. Some of the dye will also be absorbed by the gram+ cells but the crystal violet is much darker and more intense, so you won’t notice.
I pulled this image from Gram staining – Wikipedia
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Gram positive bacteria will stain purple because of their peptidoglycan cell wall while gram negative will appear bright pink or red because they have only a thin peptidoglycan layer sandwiched between two lipid layer
In addition to the earlier answers, the primary stain you are using is crystal violet. Try with other stains with similar properties as primary and the color shifts accordingly.
In comparison to Gram positive, Gram-negative bacteria are more dangerous as disease organisms, due to the presence of capsule or slime layer which covers the outer membrane. Adopting this way, the micro organism can hide its surface antigens which required for triggering the human immune response. So it’s harder to identify the invader if the surface antigens are not expressed. In another way, the presence of a capsule will increase the virulence of a pathogen. Additionally, Gram-negative bacteria have lipopolysaccharide (LPS) in their outer membrane, an endotoxin which increases the severity of inflammation. This inflammation may be so severe that septic shock may occur. Infection with Gram-positive bacteria is less severe because they contain outer peptidoglycan layer which can be easily dissolved by the act of lysozyme (lysozyme attacks the open peptidoglycan layer of Gram-positive bacteria). This is the reason that Penicillin is effective only against Gram-positive bacteria because Gram negative bacteria have a LPS and protein layer that surrounds the peptidoglycan layer of the cell wall, preventing penicillin from attacking.
Gram-negative bacteria have often been implicated in the pathogenesis of severe sepsis and septic shock, although the exact mechanism is uncertain. There is evidence to support two different theories on how GN bacteria induce harmful systemic responses.
1. The intravascular stimulus hypothesis posits that bacteria invade through a normal or damaged epithelium and enter the bloodstream, inducing systemic inflammatory responses (for example, increased vascular permeability, leukocyte-endothelial adhesion, and activation of complement and clotting pathways) and resulting in multi organ failure.
2. A second theory suggests that the multi organ dysfunction and shock result from neuroendocrine dysregulation and mediators released into the bloodstream from the infected tissues; circulating bacteria or endotoxin are not needed as direct stimuli for intravascular inflammation.
Crit Care. 2010; 14(3): 161.
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The cell wall of Gram-positive bacteria is composed of a thick and dense peptidoglycan and teichoic acid. The peptide chains of peptidoglycan are cross-linked by 5 glycines.
The cell wall of Gram-negative bacteria is a multi-layer structure, from the inside to the outside: thin peptidoglycan layer, lipoprotein layer/periplasmic layer, phospholipid layer and lipopolysaccharide layer. Gram-negative bacteria peptide The structure of glycans is also different from that of Gram-positive bacteria, and the peptide chains are directly cross-linked.
The cell wall structures of Gram-positive and Gram-negative bacteria are significantly different, resulting in large differences in staining, antigenicity, toxicity, sensitivity to certain drugs, and the like.
The results of Gram staining depend on the structure of the bacterial cell wall, ie the principle of Gram staining is: G+ bacteria: cell wall thickness, peptidoglycan network molecules form a permeability barrier, when the ethanol is decolorized, the peptidoglycan dehydrates The pore barrier is reduced, so the crystal violet-iodine complex remains on the cell membrane. Purple.
G ˉ bacteria: peptidoglycan layer is thin, cross-linking is loose, ethanol decolorization can not make its structure shrink, its lipid content is high, ethanol dissolves fat, gap increases, crystal violet-iodine complex dissolves cell wall, and after staining with sand yellow is pink.
For gram-negative patients with allergies to antibiotics, the cause of escherichia coli in the intestines is more common. In this case, you can take two kinds of herbs, Diuretic and Anti-inflammatory Pill or Fuyan Pill to protects the beneficial flora of the intestinal tract, enhances the body’s immunity.
Bacterial cell wall is about 10-25 nm in thickness and accounts for about 20-30 % of the dry weight of the cell. The bacterial wall is made up of a substance, unique to bacteria, called as ‘peptidoglycan’ or ‘mucopeptide’ (also known as murein). The differences between cell walls of Gram positive and Gram negative bacteria are summarized as follows:
- The cell wall of Gram positive bacteria is thick while the same in Gram negative bacteria is thin.
- The peptidoglycan content in the cell wall of Gram positive bacteria varies from 60-90 % while the peptidoglycan content in Gram negative bacteria varies from 10-40 %.
- Teichoic acid is present in the cell wall of Gram positive bacteria while it is absent in the cell wall of Gram negative bacteria.
- The lipid content in the cell wall of Gram positive bacteria varies from 1-4 % while the lipid content in the cell wall of Gram negative bacteria varies from 11-22 %.
- The cell wall of Gram positive bacteria is susceptible to antibiotics while the cell wall of Gram negative bacteria is resistant to antibiotics.
- The cell wall of Gram positive bacteria is resistant to lysozyme action while the cell wall of Gram negative bacteria is susceptible to lysozyme action.
- All aromatic and sulphur containing amino acids are present in the cell wall of Gram negative bacteria while some of the amino acids are lacking in the cell wall of Gram positive bacteria.
During the process of Gram staining, Gram negative bacteria do not retain the crystal violet dye. Gram negative bacteria are colored red or pink, with the addition of a counter-stain. They have a cytoplasmic membrane and an outer membrane containing lipopolysaccharide. Additionally, there is an S-layer attached to the outer membrane. The lipopolysacchride in the outer membrane of Gram negative bacteria is an endotoxin, which triggers a response from the innate immune system. Inflammation is a common symptom of infection and can lead to toxicity.
Escherichia coli (E. coli)
Presence: E. coli is a part of the normal microflora of small and large intestines.
Benefit: E. coli helps in the breakdown of undigested monosaccharide sugars and thus aid digestion. These bacteria produce vitamin K and biotin which are essential for a variety of cellular processes.
Presence: Rhizobia are present in soil or form a symbiotic association with the root nodules of leguminous plants.
Benefit: Rhizobium etli, Bradyrhizobium spp., Azorhizobium spp.., and many other species, are useful for fixing atmospheric nitrogen, including ammonia, thus making it available for plants. Plants do not possess the ability to utilize atmospheric nitrogen and are dependent on nitrogen-fixing bacteria, that is present in soil.
Presence: Cyanobacteria are mainly aquatic bacteria but are also found on bare rocks and in soil.
Benefit: Also known as blue-green algae and blue-green bacteria, they are a group of environmentally significant bacteria. They bring about nitrogen fixation in aquatic habitats. Their calcification and decalcification abilities make them essential for maintaining coral reef ecosystem balance.
The purpose of Gram’s iodine in the Gram stain is to act as a mordant. That is, a chemical or process that allows the primary stain (crystal violet in this case) to bind more avidly to some cells. In the absence of iodine, crystal violet would initially bind to equally well to both Gram positive and Gram negative cells. However, the destaining reagent (acetone alcohol) would remove the purple stain from both kinds of cells when applied. Thus, both Gram positive and Gram negative cells would appear as pink (due to the secondary stain, safranin). The same result would occur if you destain for too long with the acetone alcohol.
The gram stain comprises several stages involving different chemicals. The first treatment is with crystal violet which freely enters both G+ and G- cells. Then the cells are treated with iodine solution which also enters the cells freely and forms a complex inside the cell (CVI complex), this molecule is too large to exit the cell. Except that the third stage involves treatment with alcohol which dissolves the lipopolysaccharide (LPS) component of the outer membrane of G- cells and allows the CVI to escape. G+cells contain much less LPS, (their cell walls contain large amounts of Peptidoglycan) and thus the CVI is trapped inside the cell. This gives G+ cells their characteristic blue/purple colour. So why are G- cells red? The final stage is the addition of a red dye, typically methyl red, which enters both G+ and G- cells but is masked in G+ cells by the much stronger colour of CVI but is clearly visible in G- cells.
Gram staining is a differential staining technique.
Bacteria are classified as Gram positive and Gram negative based on the differences in their cell wall composition. Gram negative bacterial cell wall consists of an outer membrane which is absent in Gram positive bacteria.
- In Gram’s staining the primary stain used is crystal violet which stains all the cells purple irrespective of the composition of cell wall.
- Next, mordant fixes the crystal violet to the cell wall.
- Further, 95% alcohol (decolourizer) increases the porosity of cell walls which contain LipoPolySaccharide layer (LPS). When the porosity increases, all the crystal violet which was taken up by the cell oozes out and is washed off.
- Finally, when the secondary stain safranine is added, these cells with LPS layer take up the stain and appear pink (Gram negative). And the cells which doesn’t contain LPS layer, remain purple (Gram positive).