STUDY OF CELL WALL
CELL WALL
contains peptidoglycan, a polymer of N-
acetyl glucosamine, N-acetyl muramic acid and amino acids. FUNCTIONS: major function of the cell wall is to act as a pressure vessel gives shape and rigidity to cell
Cell Wall differences
Divides bacteria into 2 major groups:
Gram Positive Bacteria
Gram Negative Bacteria
CELL WALL COMPONENTS
1. PEPTIDOGLYCAN
a thick rigid layer composed of an overlapping lattice of two sugars, N-acetyl glucosamine (NAG) and N-acetyl muramic acid (NAM), that are cross-linked by amino acid bridges
90 % of Gram Positive Cell Wall
10 % Gram Negative Cell Wall
2. TEICHOIC ACID
polymer of ribitol or glycerol phosphate with additional compounds such as glucose linked to the backbone of the polymer; found in the cell walls of some bacteria
Found in Gram Positives May function to effect passage of ions
through cell wall
Outer membrane
another lipid bilayer similar to the cytoplasmic membrane, and contains lipids, proteins, and also lipopolysaccharides (LPS)
OTHER CELL WALL COMPONENTS:
Brauns proteins Periplasmic space
Properties of cell walls
PROPERTY GRAM POSITIVE 20-80 nm 1 90% + 0-3 % 0 GRAM NEGATIVE 10 nm 2 10% 58% 9%
Thickness of wall
Number of layers in wall Peptidoglycan content Teichoic acid in wall Lipid and lipoprotein content Protein content
Lipopolysaccharide Sensitive to penicillin
Digested by lysozyme
0 Yes
Yes
13% Less sensitive
Weakly
GRAM POSITIVE BACTERIA
The cell wall is made mostly of
peptidoglycan, interspersed with teichoic acid which knits the different layers together. The amount of crosslinking is higher and the wall is thicker than in gram-negative cell walls.
Gram Negative Bacteria
have a more complicated structure than do
those of gram-positive organisms. Outside the cytoplasmic membrane is the periplasm, which contains the thin layer of peptidoglycan. The peptidoglycan in gram-negative cells contains less cross-linking than in grampositive cells with no peptide linker.
Methods of demonstrating the cell wall
Cepacol staining Gram Staining Gregersens method
Cepacol Staining
Differential staining Shows thickness of Cell Wall (CW)
serves as cationic mordant which coats the CW with (+) charge
Upon addition of congo red (-), it will stain
the CW red
methylene blue(+) stains the cytoplasm blue
Gram Staining
empirical method of differentiating bacterial
species into two large groups (Gram-positive and Gram-negative) based on the chemical and physical properties of their cell walls Developed by Danish scientist Hans Christian Gram (1853-1938)
Gram staining consists of four components:
Primary stain (Crystal violet, methyl violet
or Gentian violet) Mordant (Gram's Iodine) Decolorizer (ethyl alcohol, acetone or 1:1 ethanol-acetone mixture) Counterstain (Dilute carbol fuchsin, safranin or neutral red)
The Gram Stain Mechanism
RECALL:
Gram-positive bacteria have a thick mesh-like cell wall made of peptidoglycan (50-90% of cell wall), which stain purple and Gramnegative bacteria have a thinner layer (10% of cell wall), which stain pink.
Gram-negative bacteria also have an
additional outer membrane which contains lipids, and is separated from the cell wall by the periplasmic space.
Crystal violet (CV) dissociates in aqueous solutions into CV+ and chloride (Cl ) ions. These ions penetrate through the cell wall and cell
membrane of both Grampositive and Gramnegative cells. The CV+ ion interacts with negatively charged components of bacterial cells and stains the cells purple.
iodine treatment
Iodine (I or I3 ) interacts with
CV+ and forms large complexes of crystal violet and iodine (CV I) within the inner and outer layers of the cell.
Decolorization
When a decolorizer such as alcohol or acetone is added, it interacts with the lipids of the cell
membrane. A Gram-negative cell will lose its outer membrane and the peptidoglycan layer is left exposed. The CV I complexes are washed from the Gram-negative cell along with the outer membrane. In contrast, a Gram-positive cell becomes dehydrated from an ethanol treatment. The large CV I complexes become trapped within the Gram-positive cell due to the multilayered nature of its peptidoglycan.
!!!!
The decolorization step is critical and must be
timed correctly; the crystal violet stain will be removed from both Gram-positive and negative cells if the decolorizing agent is left on too long (a matter of seconds).
Counterstaining
After decolorization, the Grampositive cell remains purple and the Gram-negative cell loses its purple color. Counterstain, which is usually positively-charged safranin or basic fuchsin, is applied last to give decolorized Gram-negative bacteria a pink or red color.
NOTE:
G+ retain CV-I complex due to high cross
linking in the PG=blue-violet
G- lose CV-I complex due to low cross
linking=red to pink
E. coli
Bacillus subtilis
Gregersens method
Used to confirm gram reaction
3% KOH (lyzing agent)
disoolves thin CW G- more susceptible due to thin CW Formation of slimy thread due to release of cellular components
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