Lecture 3: Bacterial cell surface Flashcards
Learning Objectives
To understand and be able to describe:
- the procedure for the Gram staining
- The structure of the bacterial cell envelope
- The differences between gram-positive and gram-negative bacteria
Describe the method of simple staining.
1) Spread culture in thin film over slide (fewer cells for better stain)
2) Dry in air
3) Pass over flame/heat with hairdryer to fix
4) Flood slide with stain, rinse and dry
5) Place drop of oil on slide and examine under microscope with 100x objective
Stains which can be used: carbol fushin, safranin (negatively charged, bind to cell surface)
What is the Gram-staining procedure?
1) Flood the heat-fixed smear with crystal violet for 1 min (all cells purple)
2) Add iodine solution (all cells remain purple - iodine and crystal violet form an insoluble complex on cell walls)
3) Decolourise with alcohol briefly for about 20 seconds. The alcohol dehydrates the peptidoglycan layer, tightening it and trapping the crystal violet-iodine complex (Gram +ve cells are purple, gram -ve cells are colourless) (complex released from gram -ve cells, but stays associated with gram +ve cells)
4) Counterstain with safranin for 1 - 2 min (Gram +ve cells are purple, Gram -ve cells are pink to red) (safranin enters bacteria, gram -ve cells stain pink)
Compare the structures of Gram positive and Gram negative cell envelopes?
- Gram positive bacteria have a much thicker peptidoglycan layer than gram negative bacteria.
- Gram positive bacteria have one membrane with a thick layer of peptidoglycan on the outside, whereas Gram negative bacteria have two membranes with a thin layer of peptidoglycan between them.
- Gram negative bacteria also have an extra lipopolysaccharide and protein layer outside of the outer membrane.
What is the structure of peptidoglycan?
Peptidoglycan is composed of two sugar derivatives and a small number of amino acids.
The sugar derivatives:
- N-acetyl glucosamine (NAcGlc)
- N-acetyl muramic acid (NAcMur)
The amino acids:
- L- and D-alanine
- D-glutamic acid
- L-lysine (Gram positive) (NH2 on side chain allows formation of peptide bonds)
- Diaminopemelic acid (Gram negative) (NH2 on side chain allows formation of peptide bonds)
The sugar derivatives form a glycan backbone (repeating sequence) using beta 1,4 linkages. Strands of 3 to 5 amino acids branch off of this glycan backbone from the N-acetyl muramic acid (NAcMur). Cross-bridging can be direct (Gram -ve) or composed of penta-glycine (some Gram +ves). The peptide strands in E. coli are L-Ala, D-Glu, DAP, D-Ala. Branched aas, aromatic aas and sulphur containing aas are never involved in cross linkages.
Which microorganisms is peptidoglycan present in?
Bacteria. Archaea have a similar structure called pseudopeptidoglycan. Pseudopeptiglycan has different sugars (N-acetylalosaminuronic acid), beta 1,3 linkage (lysosome insensitive) and different amino acid crosslinkages (all L isomers).
What is peptidoglycan destroyed by?
Lysozyme and phage enzymes
What is another name for peptidoglycan?
Murein
