1. Generally, how does the peptidoglycan differ between a Gram negative and Gram
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Question
1. Generally, how does the peptidoglycan differ between a Gram negative and Gram Positive cell? In the specific case of Cyanobacteria, what effect might the 'increase in peptidoglycan chain cross-linking' have on the process of Gram staining?
Based on the presence of peptidoglycan, Cyanobacteria are considered prokaryotes. But, due to the likely endosymbiotic relationship that has evolved with algae and plants they also possess chloroplast. Cyanobacteria are very difficult to classify as gram positive or gram negative. This is normally due to the fact that although there is an overall gram negative structure, the peptidoglycan is thicker in Cyanobacteria than what is typically seen in gram negati bacteria. Additionally, it has been found that there is an increase in the peptidoglycan chain cross-linking of Cyanobacteria (Hoiczyk et al. 2000). ve Below is an electron microscopy comparison of the gram-negative cell envelopes of (A) cyanobacterium (Phormidium uncinatum) and (B) Escherichia coli Mp (Hoiczyk E, Hansel A J. Bacteriol. 2000;182:1191-1199) he above comparison, EL represents a serrated External Layer, characteristic of the specific type of Cyanobacteria pictured. OM represents the Outer Membrane, P is the Peptidoglycan layer, and CM is the Cytoplasmic Membrane. You not concerned with the EL for staining purposes; focus on the three primary structures that help distinguish a gram from a gram negative cell n order to answer why Cyanobacteria (A), given the characteristics as shown above, are make the cell challenging to gram stain correctly as GRAM NEGAT VE.Explanation / Answer
1.In the Gram-positive Bacteria, the cell wall is thick (15-80 nanometers), consisting of several layers of peptidoglycan. In the Gram-negative Bacteria the cell wall is relatively thin (10 nanometers) and is composed of a single layer of peptidoglycan surrounded by an outer membrane.
The peptidoglycan layer found in cyanobacteria is considerably thicker than that of most gram-negative bacteria. In unicellular strains such as Synechococcus, its thickness is about 10 nm reaching 15 to 35 nm in filamentous species like Phormidium uncinatum and more than 700 nm in large cyanobacteria like Oscillatoria princeps. Chemical analysis of cyanobacterial peptidoglycans has revealed further unusual characteristics. The degree of cross-linking between the peptidoglycan chains within the murein of Synechocystis sp. strain PCC 6714 is far higher than the usual 20 to 33% found in most gram-negative bacterial peptidoglycans and in fact, the extent of cross-linking (56 to 63%) is more similar to the values reported for gram-positive bacteria.On the other hand, most pentapeptides involved in cross-linkage contained only the typical gram-negative bacterial diamino acid meso-diaminopimelic acid, in contrast to l-diaminopimelic acid or l-lysin in gram-positive peptidoglycan tetrapeptides, although l-lysin has been found in the pentapeptides of the cyanobacterium Anabaena cylindrica. Clearly, more studies are needed to establish the general composition of cyanobacterial peptidoglycans. Another typical constituent of gram-positive peptidoglycans, teichoic acid, is also missing in cyanobacterial cell walls. Nevertheless, the cyanobacterial peptidoglycan is complexed with specific polysaccharides in a fashion very similar to that of gram-positive bacterial peptidoglycan; this feature is also found in the peptidoglycan of the closely related prochlorophytes.
Cyanobacteria has many layers outside the cell membrane as seen in the figure. Outermost layer is the slime coat, followed by capsule, mucoid sheath, outer membrane and then peptidoglycan layer.
So the same principle applies even for cyanobacteria.
Another point I would like to make is that, capsule layer has low affinity for stains. So when we add the primary stain, crystal violet and mordant, cyanobacterial cell doesn’t take up the stain. In the next step we add alcohol (i.e. the decolourizer). This alcohol increases the porosity of cyanobacterial outer membranes. Therefore, when we add the counter stain i.e. saffranine. It takes up that colour and appear pink.
3. E.coli is a gram negative bacteria showing pink colour.
Gram staining procedure and mechanism:
Gram Staining is the common, important, and most used differential staining techniques in microbiology, which was introduced by Danish Bacteriologist Hans Christian Gram in 1884. This test differentiate the bacteria into Gram Positive and Gram Negative Bacteria, which helps in the classification and differentiations of microorganisms.
When the bacteria is stained with primary stain Crystal Violet and fixed by the mordant, some of the bacteria are able to retain the primary stain and some are decolorized by alcohol. The cell walls of gram positive bacteria have a thick layer of protein-sugar complexes called peptidoglycan and lipid content is low. 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. So the ethanol cannot remove the Crystal Violet-Iodine complex that is bound to the thick layer of peptidoglycan of gram positive bacteria and appears blue or purple in colour.
In case of gram negative bacteria, cell wall also takes up the CV-Iodine complex but due to the thin layer of peptidoglycan and thick outer layer which is formed of lipids, CV-Iodine complex gets washed off. When they are exposed to alcohol, decolorizer dissolves the lipids in the cell walls, which allows the crystal violet-iodine complex to leach out of the cells. Then when again stained with safranin, they take the stain and appears red in color.
Gram Positive: Blue/Purple Color
Gram Negative: Red Color
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