Gram Staining and Bacterial Cell Structure

Hans Christian Joachim Gram was a Danish bacteriologist noted for his development of the Gram stain.

On the basis of composition of cell wall, the bacteria are classified into two groups, namely Gram positive and Gram negative bacteria. Cell wall of all bacteria is made up of peptidoglycan. The gram positive bacteria contains many layers of peptidoglycan. However, Gram negative bacteria contains single layer of peptidoglycan. Apart from this, it also contains an outer layer of lipopolysaccharide (LPS). 

Bacteria can be classified into two groups based upon reaction bacteria to Gram's stain (Christian Gram, 1884. Bacteria are first stained with weakly alkaline solution of crystal violet and then treated with 0.5% iodine solution. This is followed by washing with water and then with alcohol or acetone. Bacteria which retain blue or purple colour are known as Gram (+) bacteria (e.g Bacillus subtilis). Bacteria which do not retain stain and become colourless are called Gram (-) bacteria (e.g. Escherichia coli). Washing of the stain in Gram (- ve) bacteria is due to high lipid content of their cell wall which gets dissolved in organic solvents like acetone. So, the correct answer is '4 →1 →2 →3'.

Bacteria can be classified into two groups based upon reaction bacteria to Gram's stain (Christian Gram, 1884. Bacteria are first stained with weakly alkaline solution of crystal violet and then treated with 0.5% iodine solution. This is followed by washing with water and then with alcohol or acetone. Bacteria which retain blue or purple colour are known as Gram (+) bacteria (e.g Bacillus subtilis). Bacteria which do not retain stain and become colourless are called Gram (-) bacteria (e.g. Escherichia coli). Washing of the stain in Gram (- ve) bacteria is due to high lipid content of their cell wall which gets dissolved in organic solvents like acetone. So, the correct answer is '(i), (ii) and (iii)'.

Hans Christian Gram used carbolfuchsin, safranin, and crystal violet to classify bacteria as gram positive and gram negative.

1. Gram staining distinguishes between the Gram-positive and Gram-negative bacteria on the basis of the composition of the cell wall.
   
2. Gram-positive bacteria have a thick layer of peptidoglycan which helps to retain stain.
   
3. The Gram-positive bacteria is first treated with Gram stain which is retained by them. After staining they are treated with alcohol to decolourize. 
4.  Decolorizing the cell causes this thick cell wall to dehydrate and shrink, which closes the pores in the cell wall and prevents the stain from exiting the cell. 
   
5. When they are stained by saffranine, they are unable to take the stain and appears blue. 
6. Gram staining bacteria take up the Gram stain but they are unable to retain them due to the thin layer of peptidoglycan.
7. When they are exposed to alcohol, decolourizer dissolves the lipids in the cell walls, which allows the crystal violet-iodine complex to leach out of the cells.
   
8. When stained with safranin, they take the stain and appears red in colour.

Cell membrane invaginates and gets folded inside the cell forming mesosomes in bacillus because mesosomes are present only in the gram-positive organisms. All the other bacteria are gram-negative organism.

Gram-negative bacteria are bacteria that do not retain the crystal violet stain used in the gram-staining method of bacterial differentiation. They are characterized by their cell envelopes, which are composed of a thin peptidoglycan cell wall sandwiched between an inner cytoplasmic cell membrane and a bacterial outer membrane. They are an important medical challenge, as their outer membrane protects them from many antibiotics (including penicillin); detergents that would normally damage the peptidoglycans of the (inner) cell membrane; and lysozyme, an antimicrobial enzyme produced by animals that form part of the innate immune system. Additionally, the outer leaflet of this membrane comprises a complex lipopolysaccharide (LPS) whose lipid A component can cause a toxic reaction when these bacteria are lysed by immune cells. 

In the vesicle of mesosomes, the respiratory enzymes and the components of electron transport such as ATPase, dehydrogenase, cytochrome are present in low amount. So, they were considered anologous to mitochondria for their respiratory function. 

Teichoic acids are found within the cell wall of Gram-positive bacteria. The main function of teichoic acid is to provide rigidity to the cell wall by attracting cations, such as magnesium and sodium.

Crystal violet dye is used as a histological stain and in Gram's method of classifying bacteria.
Rest all other dyes in option as acetocarmine, haematoxylin and Schiff's reagent are used to stain chromosome.
Thus, the correct answer is option B.

An exotoxin is a toxin secreted by bacteria. An exotoxin can cause damage to the host by destroying cells or disrupting normal cellular metabolism. Exotoxin is secreted during lysis of the cell. 

The Gram-negative cell wall is characterized by presence of outer membrane, a thinner peptidoglycan layer and extensive periplasmic space surrounding the peptidoglycan. The uppermost layer of the outer membrane has lipopolysaccharide, while the innermost layer is a typical lipid layer. Presence of membrane channels formed by porin proteins in outer membrane impart make them less penetrable as compared to Gram positive cells. Gram positive cells are characterized by presence of single thick layer of cell wall which is made up of peptidogycan, teichoic acid and lipoteichoic acid. They lack outer membrane and porin and have narrow periplasmic space. Plasma membrane is the unit membrane in both, Gram positive and Gram negative bacteria, present beneath cell wall with primarily phospholipids (making up about 30–40% of the membrane mass) and proteins (60–70%). The dense gelatinous solution surrounded by plasma membrane is cytoplasm which has same composition in both, Gram positive and Gram negative cells. Cilia are the eukaryotic structures which are absent in bacteria as they are prokaryotic. 

A prokaryotic cell lacks a nucleolus. A prokaryote is a single-celled organism that lacks a membrane-bound nucleus, mitochondria, or any other membrane-bound organelles. All the intracellular water-soluble components are located together in the same volume enclosed by the cell membrane, rather than in separate cellular compartments.

The cell wall of certain bacteria shows a characteristic reaction to the stain devised by C. Gram. Those bacteria which retain the stain are known as Gram positive and those which do not retain the stain are called Gram negative.

• The cell wall in bacteria contains peptidoglycan, a polymer of N-acetyl glucosamine, N-acetyl muramic acid and amino acids.
• Gram-positive cell walls contain a thick layer of peptidoglycan that encircles the cell.
• Gram-negative cell walls contain a thin layer of peptidoglycan between the cytoplasmic membrane and the outer membrane.

The oligosaccharide component of bacterial cell wall consists of linear chains of alternating NAG and NAM linked by ${\beta}$ (1-4)linkage.

The peptidoglycan layer of the gram negative bacteria are thin and are also referred to as the cell envelope. The layer is wavy and provides resistance to antibiotics. They are more virulent as compared to Gram positive bacteria

Outer membrane and periplasmic space are absent in Gram positive bacteria but present in Gram negative bacteria. The outer membrane in Gram negative bacteria is made up of lipopolysaccharides.

Cell wall of Gram +ve bacteria is made up of peptidoglycans (mucopeptide). Mucopeptide form about 50% of component of cell wall. Cell wall of Gram -ve bacteria is made up of single layer of peptidoglycans.

The mucopeptide or murein is a polymer of two amino sugars namely N-acetylglucosamine (NAG) and N-acetyl muramic acid (NAM). These NAG and NAM are derivatives of Glucose molecules.

Exotoxin is produced by mostly gram positive bacteria, these are protenaceous and heat labile compound which are excreted by bacteria out side the cell.

Gram-negative bacteria have a unique outer membrane. This outer membrane prevents penetration of antibiotics within the cell. However, Gram-positive bacteria do not have an outer LPS membrane. This makes them more susceptible or less resistant to antibiotics than gram-positive bacteria.

Gram staining procedure is based on the principle of the ability of microorganisms to retain color of the stains used during the gram staining process. Gram-positive bacteria retain purple color after staining. They are not decolorized by treatment with alcohol. Whereas gram-negative bacteria are decolorized by the alcohol and lose purple color. When again stained with the second stain, they retain pink color. This major difference is due to the difference in the cell wall component of both the bacteria. Gram-negative bacteria contain additional peptidoglycan layer and a thin layer of LPS layer. 

All bacteria posses cell wall. However, the bacteria are classified as Gram positive bacteria and Gram negative bacteria on the basis of their cell wall. Cell wall of Gram positive bacteria consist of multiple layer of peptidoglycan. Cell wall of Gram negative bacteria is made up thin layer of peptidoglycan. In addition, they have one more outer layer of lipopolysaccharide (LPS).

Gram staining procedure is based on the principle of the ability of microorganisms to retain the color of the stains used during the gram staining process. Gram-positive bacteria retain purple color after staining with Crystal Violet stain. Iodine is added as a mordant to form the crystal violet-iodine complex. They are not decolorized by treatment with alcohol. Whereas gram-negative bacteria are decolorized by the alcohol and lose the purple color of Crystal Violet stain. When again stained with second stain Saffranin, they retain pink color. This major difference is due to the difference in the cell wall component of both the bacteria. Gram-negative bacteria contain additional peptidoglycan layer and a thin layer of LPS layer. 

Streptomycin is an antibiotic which is used in the treatment of bacterial infections. It belongs to the Aminoglycoside antibacterial class. It is a broad-spectrum antibiotic. It is effective against both Gram-positive and Gram-negative bacteria. It is used in the treatment of tuberculosis, endocarditis, plague, rat bite fever. 

Both Gram negative and Gram positive bacteria have a peptidoglycan layer in the cell wall. The peptidoglycan in Gram negative bacteria is present in the periplasmic space. Apart from this, Gram negative bacteria have an outer wall made up of lipopolysaccharide. Peptidoglycan is also called as mucopeptide. It is a polymer that is made up of sugars and amino acids. 

Gram staining procedure is based on the principle of the ability of microorganisms to retain color of the stains used during the gram staining process. Gram-positive bacteria retain purple color after staining with crystal violet stain. They are not decolorized by treatment with alcohol. Gram-negative bacteria also get stained with crystal violet stain. However, they are decolorized by the alcohol and lose purple color. When again stained with the second stain safranin, they retain pink color. This major difference is due to the difference in the cell wall component of both the bacteria. 

Both Gram negative and Gram positive bacteria have a peptidoglycan layer in the cell wall. It is also called as mucopeptide or murein. However, Gram positive bacteria have thick layer of peptidoglycan. Hence, mucopeptide is abundant in the cell wall of gram positive bacteria. Gram negative bacteria have thin layer of mucopeptide. Apart from this, Gram negative bacteria have outer wall made up of lipopolysaccharide. 

Both Escherichia coli and Rhizobium japonicum are Gram-negative bacteria, meaning they have a thin peptidoglycan layer and an outer membrane.

The mucopeptide is a peptidoglycan that is insoluble in nature. It is also called as murein. It is present in almost all bacterial cell walls. However, its percentage is higher in cell wall of gram positive bacteria than in cell wall of gram negative bacteria. 

It holds receptor molecules that help bacteria detect and respond to chemicals in their surroundings

Gram positive bacteria differs from Gram negative bacteria in having multiple layers of peptidoglycan. They have 20–30 nm thick and smooth cell wall. They contain 70-80% murein or peptidoglycan in cell wall. They do not have outer membrane like Gram negative bacteria, which have an outer LPS layer. Due to absence of outer LPS layer, Gram positive bacteria has two rings in basal body of flagellum, one attached to the plasma membrane and one attached to the peptidoglycan wall. They have prominent mesosomes. Mesosomes are folded invaginations of the plasma membrane.

Teichoic acids are found within the cell wall of most Gram-positive bacteria such as species in the genera Staphylococcus, Streptococcus, Bacillus, Clostridium, Corynebacterium, and Listeria, and appear to extend to the surface of the peptidoglycan layer.

Gram-negative bacteria are bacteria that do not retain the crystal violet stain during gram-staining procedure. Gram positive bacteria are bacteria that retain the crystal violet stain during gram staining procedure. Examples of Gram negative bacteria include Escherichia coli, Helicobacter pylori. Examples of Gram positive bacteria include Bacillus subtilis, Streptomyces coelicolor, Streptomyces orientalis (Ampycolatopsis orientalis).

• Glycocalyx or pericellular matrix is a carbohydrate-enriched coating of glycoprotein and a glycolipid that covers the cell membranes of few bacteria, epithelial cells and other cells. 
• It is mainly used in the recognition of the cell (distinguish between healthy and diseased cells) and also regulates the endothelial vascular tissues.
  
• So, the correct answer is 'Oligosaccharide part of glycolipids and glycoproteins'.

On the basis of composition of cell wall, the bacteria are classified into two groups, namely Gram positive and Gram negative bacteria. Cell wall of all bacteria is made up of peptidoglycan. The gram positive bacteria contains thick layer of peptidoglycan. However, Gram negative bacteria contains thin layer of peptidoglycan. Apart from this, it also contains an outer layer of lipopolysaccharide (LPS). Hence, the wall of Gram negative bacteria is two-layered.

• Cell recognition and adhesion occur due to biochemicals of cell membranes name glycoproteins and glycolipids.
  
• Glycocalyx or pericellular matrix is a carbohydrate-enriched coating of glycoprotein and a glycolipid that covers the cell membranes of few bacteria, epithelial cells and other cells. 
  
• It is mainly used in the recognition of the cell (distinguish between healthy and diseased cells) and also regulates the endothelial vascular tissues.
• So, the correct answer is 'Glycoproteins and glycolipids'.

The mucopeptide is a peptidoglycan that is insoluble in nature. It is also called as murein. It is present in almost all bacterial cell walls.

Glycocalyx in eukaryotes (often as part of the cell coat) is involved in cell-cell recognition, immune response, and receptor function. While the term is used differently in prokaryotes, in the context of broader biological questions, it covers these functions.

Gram-positive organisms have two of these basal body rings, one in the peptidoglycan layer and one in the plasma membrane.

So, the correct answer is 'Microtubular organisation and type of movement'.

Statement (b) is wrong because a capsule is a thick, tough layer, not a loose sheath. Statement (c) is wrong because a slime layer is the loose sheath, not the thick/tough one.

• Glycocalyx or pericellular matrix is a carbohydrate-enriched coating of glycoprotein and a glycolipid that covers the cell membranes of few bacteria, epithelial cells and other cells. 
  
• It is mainly used in the recognition of the cell (distinguish between healthy and diseased cells) and also regulates the endothelial vascular tissues.
  
• So, the correct answer is 'Plasmalemma'.

Both the slime layer and the capsule are variations of the glycocalyx, which is the outermost layer of the bacterial cell envelope.

Teichoic acid is a characteristic component of the cell wall of Gram-positive bacteria, providing structural support and helping in ion regulation.

After two divisions, four bacterial cells are formed in which two daughter bacterial cells have normal DNA and other two have both normal and heavy DNA because DNA replicates by semiconservative method in bacteria.

• Glycocalyx or pericellular matrix is a carbohydrate-enriched coating of glycoprotein and a glycolipid that covers the cell membranes of few bacteria, epithelial cells and other cells. 
  
• It is mainly used in the recognition of the cell (distinguish between healthy and diseased cells) and also regulates the endothelial vascular tissues.
• So, the correct answer is 'A layer present between cell wall and membrane of bacteria'.

• The peptidoglycan layer in Gram-positive bacteria is made up of with anionic glycopolymers called wall teichoic acids.
•  It plays an important role in determining the cell shape, regulation of cell division etc.
• Teichoic acids are found within the cell wall of Staphylococcus, Streptococcus, Bacillus, Clostridium, Corynebacterium etc.
  
• So, the correct answer is 'Cell wall'.

The cell envelope of Gram-negative bacteria consists of three layers: the inner cytoplasmic membrane, a thin peptidoglycan layer, and an outer membrane.

Gram staining differentiates bacteria based on the structure and composition of their cell wall, specifically the thickness of the peptidoglycan layer.

Eubacterial cell walls are composed of peptidoglycan and a sticky mesh of polysaccharides, a glycocalyx often surrounds the wall.

Gram-positive bacteria are stained dark blue or violet by Gram staining. This is in contrast to gram-negative bacteria, which take up the counterstain and appear red or pink. 

Gram stain is the most important staining procedure. Gram-positive bacteria stain purple, whereas gram-negative bacteria stain pink. 

Bacteria can be classified into two groups based upon reaction of bacteria to Gram's stain (Christian Gram, $1884$). Bacteria are first stained with weakly alkaline solution of crystal violet and then treated with $0.5$% iodine solution. This is followed by washing with water and then with alcohol or acetone. Bacteria which retain blue or purple colour are known as Gram $(+)$ bacteria (e.g., Bacillus subtilis). Bacteria which do not retain stain and become colourless are called Gram $(-)$ bacteria (e.g., Escherichia coli). Washing of the stain in Gram $(-ve)$ bacteria is due to high lipid content of their cell wall which gets dissolved in organic solvents like acetone.

So, the correct answer is '$4\rightarrow 1 \rightarrow 2\rightarrow 3$'.

Bacteria can be classified into two groups based upon reaction of bacteria to Gram's stain (Christian Gram, $1884$). Bacteria are first stained with weakly alkaline solution of crystal violet and then treated with $0.5$% iodine solution. This is followed by washing with water and then with alcohol or acetone. 

Glycocalyx (mucilage sheath) is the outermost layer of the bacterial cell envelope which consists of non-cellulosic polysaccharides with or without proteins. It may occur in the form of loose sheath, called slime layer. If it is a thick covering, it is called capsule. Glycocalyx gives sticky character to the cell and is not absolutely essential for survival of bacteria. It prevents desiccating, protects from phagocytes, toxic chemicals and viruses and serves in attachment. It may give selective advantage through in certain situations.

a quick version of buttermilk, known as acidified buttermilk, is used to make paneer. The tartness of buttermilk is due to acid in milk, where iodine is suitable.

The glycocalyx is a glycoprotein-polysaccharide covering that surrounds the cell membranes of some bacteria, epithelia and other cells. The glycocalyx is a type of identifier that the body uses to distinguish between its own healthy cells and transplanted tissues, diseased cells, or invading organisms. 
Thus, the correct answer is option C.

When a decolourizer such as alcohol or acetone is added, it interacts with the lipids of the cell membrane. A Gram-negative cell loses its outer lipopolysaccharide membrane and the inner peptidoglycan layer is left exposed. The crystal violet complexes are washed from the Gram-negative cell along with the outer membrane.

Pyrogens can be the so called endotoxins or lipopolysaccharides, cell wall components of Gram negative bacteria such as E. coli, Salmonella or Bordetella, but also of the cell walls from Gram positive bacteria, e.g., so called lipoteichoic acids (LTA). When bacteria or viruses invade the body and cause tissue injury, one of the immune systems responses is to produce pyrogens. These chemicals are carried by the blood to the brain, where they disturb the functioning of the hypothalamus, the part of the brain, that regulates body temperature.

Gram staining differentiates bacteria by the chemical and physical properties of their cell walls by detecting peptidoglycan, which is present in a thick layer in gram-positive bacteria. In a gram stain test, gram-positive bacteria retain the crystal violet dye, while a counterstain (safranin or fuchsine) added after the crystal violet gives all gram-negative bacteria a red or pink colouring.

Gram - negative bacteria display the following characteristics: outer membrane containing lipopolysaccharide (which consists of lipid A, core polysaccharide, and O antigen) in its outer leaflet and phospholipids in the inner leaflet; porins exist in the outer membrane, which act like pores for particular molecules; lipoproteins are attached to the polysaccharide membrane.

Gram positive cells are characterized by presence of single thick layer of cell wall which is made up of peptidogycan, teichoic acid and lipoteichoic acid. Teichoic acid is acidic polymer of ribitol phosphate /glycerol phosphate and is covalently linked to peptidoglycan layer. Acetic acid is organic compound and is the main component of vinegar. Glutamic acid is one of the 20 standard amino acids and also serves as neurotransmitter. Malic acid is organic compound and serves as important intermediate in number of biochemical pathways; Kreb cycle and Glyoxalate cycle, etc. Thus, the correct answer is option B.

Eosin is an acidic dye with negatively charged chromophore and stains positively charged molecules such as certain WBCs; presence of acidic substances in large quantity with negatively charged surfaces on bacterial cells inhibit their staining with eosin. Option A is incorrect. Haematoxylin is positive/basic dye which stains acidic substances carrying negative charge and is used with eosin in histology; option C is incorrect. Carmine is a natural dye produced by insect Dactylopius coccus and is used food and cosmetics; option D is incorrect. Gram staining includes staining of both Gram positive and Gram negative bacterial cell purple with crystal violet followed by stabilization of dye in peptidoglyacan layer with mordant—Gram’s iodine and application of alcohol to dissolve outer membrane and dye from peptidoglycan layer. Presence of thick peptidogycan layer in Gram positive cells make them retain the dye after alcohol treatment while Gram negative bacteria become colourless. Thus, option B is the correct answer.