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

END