Cell Wall and Gram Staining Flashcards
What is the cell wall made of and describe the material.
Made of a macromolecule called peptidoglycan aka Murein which is a vast polymer consisting of interlocking chains of identical peptidoglycan monomers.
Describe a peptidoglycan monomer.
A peptidoglycan monomer consists of two joined amino sugars, N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM), with a tetra- or pentapeptide as a functionality of the NAM.
Describe the 5 properties of the bacterial cell wall.
- Peptidoglycan polymer system prevents osmotic lysis.
- Allows for rigidity and provides shape/structure (main function)
- Maintains a high water potential in the cytoplasm and prevents entry of excess water.
- The monomer is made in the cytosol and transported to the cell wall.
- Enzymes insert peptidoglycan monomers into the cell wall to allow for growth and division.
Explain how the peptidoglycan cross-linkage occurs.
Cross-linkage occurs between the COOH group of the terminal D-Ala residue and either a free amine of the glycan pentapeptide or tetrapeptide
= condensation reaction forming peptide bond.
Describe the structure of Gram-positive cell wall.
The penta/tetra peptide dangles down from monomer NAM and is cross-linked to an adjacent penta/tetra peptide with an interbridge. This allows the backbone of peptidoglycan to lie closely together. This creates a dense network which is tight and rigid.
Describe the structure of Gram-negative cell wall.
Much less thicker than Gram-positive cell wall due to lack of interbridges and only direct linkages between the tetrapeptides.
Explain the mechanism of cell wall synthesis.
- Peptidoglycan monomers synthesised in the cytosol which then attached to membrane carrier molecules - bactoprenols.
- Autolysins break glycosidic bonds at the point of growth along the existing peptidoglycan wall - allows insertion of monomers.
- Transglycosidase enzymes catalyse glycosidic bond formation.
- Transpeptidase enzymes stitches peptides together, crosslinking the wall.
Which 3 components found in bacterial cell wall are not found in eukaryotic cells?
D-amino acids, Muramic acid, Diaminopimelic acid (DAP)
Which bacteria is peptidoglycan cell wall not present?
Mycoplasmas and halobacteria.
What is LPS endotoxins? What do they do? (8)
Found in G- cells only. Endotoxins = cell-bound, heat-resistant toxins - released on cell death. These can
- cause endotoxic shock.
- are pyrogenic (induce fever).
- can initiate immune response.
- can induce interferon production causing inflammation.
- changes in white blood cell counts
- changes in vascular permeability = leaking blood vessels
- BP drop leading to vascular collapse and eventually shock
- death
Describe gram staining technique and results.
- Both Gram+/- are treated with crystal violet dye followed by iodine solution.
- G+ cells retain the dye (crystal-violet iodine complex) whereby G- lose the colour. 3. Both are stained again using safranin. 4. Under light microscope, G- cells appear red and G+ cells are seen as purple.
What shape and colour are G+ cells after staining? Likewise with G-?
G+ are seen as purple and are spherical/cocci. G- are seen as red/pink and are rod-shaped/bacilli.
What occurs in G+ cells when crystal-violet iodine complex forms?
When these combine, it forms a large insoluble complex which precipitates within the cell resulting in colour change.
Explain purpose of alcohol in Gram staining process for G+.
Alcohol causes dehydration of the multilayered peptidoglycan, thus decreasing the space between the molecules and causing cell wall to trap the crystal violet-iodine complex within cell.
Alcohol also aids in fixing the colours.
What happens to G- cell when treated with alcohol?
Alcohol dissolves the outer membrane of cell wall and may damage the cytoplasmic membrane to which the peptidoglycan layer is attached. Alcohol can dehydrate peptidoglycan layer.
