study of cell wall

8/4/2019 Study of Cell Wall

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STUDY OF CELL WALL


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CELL WALL

contains peptidoglycan, a polymer of N-acetyl glucosamine, N-acetyl muramic acidand amino acids.

FUNCTIONS:

major function of the cell wall is to act as apressure vessel

gives shape and rigidity to cell


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Cell Wall differences

Divides bacteria into 2 major groups:

Gram Positive Bacteria

Gram Negative Bacteria


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CELL WALL COMPONENTS

1. PEPTIDOGLYCAN

a thick rigid layer composed of an overlappinglattice of two sugars, N-acetyl glucosamine(NAG) and N-acetyl muramic acid (NAM),that are cross-linked by amino acid bridges


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90 % of Gram Positive Cell Wall

10 % Gram Negative Cell Wall


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2. TEICHOIC ACID

polymer of ribitol or glycerol phosphate withadditional compounds such as glucose linkedto the backbone of the polymer; found in thecell walls of some bacteria


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Found in Gram Positives

May function to effect passage of ionsthrough cell wall


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Outer membrane

another lipid bilayer similar to the cytoplasmic

membrane, and contains lipids, proteins, andalso lipopolysaccharides (LPS)


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OTHER CELL WALL COMPONENTS:

Brauns proteins

Periplasmic space


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Properties of cell walls

PROPERTY GRAM POSITIVE GRAM NEGATIVE

Thickness of wall 20-80 nm 10 nm

Number of layers in wall 1 2

Peptidoglycan content 90% 10%

Teichoic acid in wall + -

Lipid and lipoproteincontent

0-3 % 58%

Protein content 0 9%

Lipopolysaccharide 0 13%

Sensitive to penicillin Yes Less sensitive

Digested by lysozyme Yes Weakly


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GRAM POSITIVE BACTERIA

The cell wall is made mostly ofpeptidoglycan, interspersed with teichoicacid which knits the different layers together.The amount of crosslinking is higher and thewall is thicker than in gram-negative cellwalls.


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Gram Negative Bacteria

have a more complicated structure than dothose of gram-positive organisms.

Outside the cytoplasmic membrane is theperiplasm, which contains the thin layer ofpeptidoglycan.

The peptidoglycan in gram-negative cells

contains less cross-linking than in gram-positive cells with no peptide linker.


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Methods of demonstrating the

cell wall Cepacol staining

Gram Staining

Gregersens method


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Cepacol Staining

Differential staining

Shows thickness of Cell Wall (CW)


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serves as cationic

mordant which coatsthe CW with (+) charge


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Upon addition of congo red (-), it will stainthe CW red


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methylene blue(+) stains the

cytoplasm blue


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Gram Staining

empirical method of differentiating bacterialspecies into two large groups (Gram-positiveand Gram-negative) based on the chemicaland physical properties of their cell walls

Developed by Danish scientist Hans ChristianGram (1853-1938)


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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, safraninor neutral red)


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The Gram Stain Mechanism

RECALL:

Gram-positive bacteria have a thick mesh-like

cell wall made of peptidoglycan (50-90% ofcell wall), which stain purple and Gram-negative bacteria have a thinner layer (10% ofcell wall), which stain pink.


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Gram-negative bacteria also have anadditional outer membrane which containslipids, and is separated from the cell wall bythe periplasmic space.


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Crystal violet (CV)dissociates in aqueoussolutions into CV+ andchloride (Cl ) ions. Theseions penetrate through thecell wall and cellmembrane of both Gram-positive and Gram-negative cells. The CV+ ion

interacts with negativelycharged components ofbacterial cells and stainsthe cells purple.


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iodine treatment

Iodine (I or I3 ) interacts withCV+ and forms

large complexesof crystal violetand iodine (CV I) within theinner and outerlayers of the cell.


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Decolorization

When a decolorizer such as alcohol or acetone isadded, it interacts with the lipids of the cellmembrane. A Gram-negative cell will lose its

outer membrane and the peptidoglycan layer isleft exposed. The CV I complexes are washedfrom the Gram-negative cell along with the outermembrane. In contrast, a Gram-positive cell

becomes dehydrated from an ethanoltreatment. The large CV I complexes becometrapped within the Gram-positive cell due to themultilayered nature of its peptidoglycan.


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!!!!

The decolorization step is critical and must betimed correctly; the crystal violet stain will be

removed from both Gram-positive andnegative cells if the decolorizing agent is lefton too long (a matter of seconds).


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Counterstaining

After decolorization, the Gram-positive cell remains purpleand the Gram-negative cellloses its purple color.

Counterstain, which isusually positively-chargedsafranin or basic fuchsin, is

applied last to givedecolorized Gram-negativebacteria a pink or red color.


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NOTE:

G+ retain CV-I complex due to high crosslinking in the PG=blue-violet

G- lose CV-I complex due to low crosslinking=red to pink


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E. coli


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Bacillus subtilis


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Gregersens method

Used to confirm gram reaction


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3% KOH (lyzing agent)disoolves thin CW

G- more susceptibledue to thin CW

Formation of slimythread due to release

of cellular components


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END

study of cell wall

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