Exercise 6

Study of Cell Wall

Structure • Peptidoglycan (PG)- a.k.a murein; mucopeptide

~ unique component of bacterial cell wall

~ a polymer of disaccharides (2 sugar derivatives) linked by polypeptides (chain of different D amino acids)

- N-acetylglucosamine (NAG)

- N-acetylmuric acid (NAM); has attached side chain amino acid

~ cross bridge amino acids primarily contributes to the strength

Function

• Gives bacteria shape and rigidity

• Contributes to pathogenicity of pathogenic bacteria (due to enzymatic excretions) especially Gram- negative bacteria

• Protects bacteria from toxic substances

• Site of action of some antibiotics (due to the presence of the receptors; Ag-Ab reaction)

Gram- positive Bacteria

Gram- negative bacteria

Comparison on Cell Wall

Laboratory Procedures A. Difference in Thickness of Gram (-) and (+) walls

B. Gram Staining

C. Gregersen’s Method of Determining Gram Reaction

Difference in Thickness of Gram (-) and (+) walls

- Use of CEPACOL (for differential staining)

~ cationic mordant that coats the CW with positive charge

- Congo red (negatively charged)

~ stains the cell wall

- Methylene Blue (positively charged)

~ stains the cytoplasm

Result:

BACILLUS IS STAINED DARKER THAN E. COLI.

Gram positive and negative bacteria have

negative overall charge because of the Peptidoglycan. The interaction of Cepacol varies due to the difference in the thickness of the PG layer.

Cell wall of Gram- postive bacteria will be stained darker than that of the Gram- negative bacteria due to the GREATER interaction of congo red (negatively charged) with cepacol (cationic mordant) present on cell surface of the cells.

Gram Staining

- Use of crystal violet (primary stain)

- Gram’s iodine (mordant)

- 95% ethanol (decolorizer)

- Safranin (secondary stain)

Result:

E. coli is colored pinkish red while Bacillus became blue violet.

After applying crystal violet, both Gram positive and Gram negative will be stained blue to violet.

Result:

E. coli is colored pinkish red while Bacillus became blue violet.

Iodine increases the interaction between the bacterial cell and the dye so that the dye is more tightly bound or the cell is more strongly stained.

Result:

E. coli is colored pinkish red while Bacillus became blue violet.

Decolorization with ethanol or acetone removes crystal violet- iodine complex from gram-negative cells but not from gram-positive cells. The gram-negative cells then turn pink- red when counterstained with safranin.

Gram positive Gram negative

Crystal violet Blue to violet Blue to violet

Iodine Blue to violet

Blue to violet

Ethanol/ acetone Blue to violet

colorless

Safranin Blue to violet

Pinkish- red

Gregersen’s Method of Determining Gram Reaction

- Use of 3 % potassium hydroxide

Result:

Bacillus did not produce slimy thread compared to E. coli.

Potassium hydroxide(lyzing agent) have the capability to lyse thin cell wall(release of cellular constituents). This is the reason why gram-negative bacteria can be produce slimy thread after the procedure.

Gregersen’s Method of Determining Gram Reaction

Why use this method? Isn’t Gram staining enough?

1. Gram staining result depends on the age of the culture. Gram positive bacteria may turn gram negative if they are too old. It is best to do the procedure in the cultures where the organisms are vigorous rather than old ones.

2. Some bacterial species are gram variable. That is, some cells in the same culture will be gram positive and some, gram negative. Therefore, one should always be CERTAIN to run Gram stains on several cultures UNDER CAREFULLY CONTROLLED CONDITIONS in order to make certain that a given bacterial “strain” is TRULY GRAM POSITIVE OR GRAM NEGATIVE.

Answers to questions

1. Can you vary the primary stain and counterstain in the Gram- staining?

Yes. Gram reagents are not truly specific for the differentiation. Any dye or mordant can be used if the dye can deeply color the cell and if the precipitate of these reagents are soluble in alcohol and relatively soluble in the counterstain.

2. What would be the effect of replacing iodine with other oxidizing agents?

Oxidizing agents can shift the gram positive character of bacteria toward the gram negative state. Thus, there will be no formation of Crystal Violet complex and there will be reversal of charges.

3. What is the function of cepacol? KOH?

Cepacol is a cationic mordant that coats the cell wall with positive charges.

KOH is used in determining the reaction Gram reaction of the bacterial cell wall depending on their thickness. Gram positive has thicker wall than Gram negative and will not be able to be lyzed easily compared to Gram negative, thus, making the suspension viscous due to the release of cellular DNA/ constituents.

4. What is the influence of pH on Gram reaction?

1. The pH may alter staining effectiveness since the nature and degree of the charge on cell components change with pH. Thus anionic dyes stain best under acidic conditions when proteins and many other molecules carry a positive charge; basic dyes are most effective at higher pHs.

2. The Gram positive organism become Gram negative upon treatment with an oxidizing agent, whereas the Gram negative organism become Gram positive upon treatment with reducing or alkaline agents.