Bacteria gram stain

Question 1

What does the gram stain classify based on?

Answer 1

Cell wall structure and composition

Question 2

Gram positive structure

Answer 2

Single membrane and a relatively exposed peptidoglycan cell wall

Question 3

Gram negative structure

Answer 3

Two membranes (inner and outer membrane), lipopolysaccharide (LPS), and a relatively thin peptidoglycan in the periplasm between the two membranes

Question 4

Why cant a gram stain be used on mycoplasma?

Answer 4

Lack a cell wall

Question 5

Why cant a gram stain be used on intracellular pathogens?

Answer 5

Cannot be stained as inside cells

Question 6

Why cant a gram stain be used on spirochetes?

Answer 6

Too thin to be visualised

Question 7

Why cant a gram stain be used on mycobacterium tuberculosis?

Answer 7

Bacteria is ‘acid fast’ and cannot be identified with a Gram stain because the first stains don’t get through the mycobacterial outer membrane.

Question 8

How are bacteria fixed to a slide?

Answer 8

With heat

Question 9

Gram stain steps

Answer 9

1. Primary staining with Crystal Violet (originally Gentian Violet) This dark purple stain penetrates the cell wall of organisms irrespective of their structure, and stains them purple.
2. Staining with Gram’s Solution. This is a mixture of iodine and potassium iodide. It reacts with the Crystal Violet forming large complexes within the cells.
3. Decolourisation. This is a critical step. Gentle treatment with alcohol leads to loss of the Gram-negative outer membrane and the cells become leaky; as a result the large crystal violet-iodine complexes are washed out and cells with this wall structure become colourless. In Gram positive organisms, the thicker peptidoglycan retains the Crystal-violet complexes within the cell and they remain purple.
4. Counter stain. The final step is addition of a pink counter stain (Saffranin or Carbol Fuschin) which enables Gram negative organisms to be visualised.

Question 10

Gram positive bacteria stain…

Answer 10

Purple

Question 11

Gram negative bacteria stain…

Answer 11

Pink

Question 12

Peptidoglycan

Answer 12

Peptidoglycan or murein is a polymer consisting of sugars and amino acids that forms a mesh-like layer outside the plasma membrane of most bacteria, forming the cell wall.

Question 13

PG structure

Answer 13

Polymer of (1‐4)‐linked N‐acetyl glucosamine (GlcNAc/NAG) and N‐acetyl muramic acid (MurNAc/NAM)

NAM is substituted with peptides, typically 3‐5 alternating L‐ and D‐amino acids.

Peptides from adjacent strands are often cross linked, directly or through short peptides. Covalent linkages are formed.

Question 14

Gram negative PG peptides

Answer 14

Each NAM is attached to a short (4- to 5-residue) amino acid chain, containing L-alanine, D-glutamic acid, meso-diaminopimelic acid (mesoDAP), and D-alanine

Question 15

Gram positive PG peptides

Answer 15

Each NAM is attached to chain of L-alanine, D-glutamine, L-lysine, and D-alanine with a 5-glycine interbridge between tetrapeptides.

Question 16

Teichoic acids are a feature of…

Answer 16

Gram positive bacteria

Question 17

Teichoic acids structure, role and types

Answer 17

Fibres located in the outer layer of the gram-positive cell wall. Composed of polymers of glycerol phosphate or ribitol phosphate.

Wall teichoic acids: anchor to the muramic acid of the peptidoglycan (NAM).

Lipoteichoic acids: penetrate the peptidoglycan layer and are covalently linked to the lipid in the cytoplasmic membrane.

Involved in cell shape, growth and division, resistant to microbial peptides.

Question 18

Three units of lipopolysaccharides

Answer 18

Lipid A (endotoxin): Lipid extensions allow lipid A to integrate with the membrane. Endotoxic portion of LPS

Core polysaccharides (conserved within a species)

O-antigen (repeating polysaccharide chain of varying length ~20): Involved in virulence and pathogenesis. Allows Molecular mimicry (H pylori can mimic a Lewis blood group)