Ex 3

Gram-positive bacteria have a thick mesh-like cell wall made of peptidoglycan (50–90% (sa google) 60-80% (sa lec) of cell envelope), and as a result are stained purple by crystal violet, whereas gram-negative bacteria have a thinner layer (10% of cell envelope), so do not retain the purple stain and are counter-stained pink by safranin

Why do gram positive bacteria retain

the primary stain?

Gram-positive bacteria remain purple because they have a single thick cell wall that is not easily penetrated by the solvent; gram-negative bacteria, however, stain red because they have cell walls with much thinner layers that allow removal of the dye by the solvent, so it is colored only by the safranin counterstain.

Why do gram negative bacteria stain

red and not violet/blue like the gram

positive bacteria?

☞ Organisms such as Mycobacteria are extremely difficult to stain by ordinary methods like gram stain. Because of the high concentration of mycolic acid, a lipid in their cell wall.

☞ Phenolic compound carbol fuchsin is used as the primary stain because it is lipid soluble and penetrates the waxy cell wall. Staining by carbol fuchsin is further enhanced by heating the preparation to melt the wax and allow the stain to move into the cell

☞ Acid is used to decolorize non-acid fast cells because acid fast cells resist this decoloration. The ability of the bacteria to resist this decoloration with acid confers acid fastness to the bacterium. Following the colorization, the smear is counterstained with methylene blue which stains the background material, providing a contrast color against which the red AFB can be seen.

OR

The cell walls of mycobacteria contain high concentrations of lipid, making them waxy, hydrophobic and impermeable to routine stains such as the gram stain.

But stained with carbol fuchsin combined with phenol???
Ziehl-Neelsen staining technique has 3 main steps

1. Primary Staining

2. DE colorization

3. Counter Stain

The carbol fuchsin stain is heated to enable the dye to penetrate the waxy mycobacterial cell wall.
The stain binds to the mycolic acid in the mycobacterial cell wall.
After staining, decolorization is applied. This removes the red dye from the background cells, tissue fibers and any organisms in the smear except mycobacteria.
Finally apply the methylene blue which stains the background material, providing a contrast color against which the red acid fast bacilli can be seen.

Give the principle of acid fast staining

Blue

Color of bacteria in non-acid fast

To penetrate the cell wall, because it is lipid soluble

Why do we need to use carbol fuchsin

in acid fast

a.) Improperly heat fixed

b.) Too much acid alcohol

c.) Improper labelling

Common error in gram staining

a.) Mycobacterium Tuberculosis

b.) Bacillus Subtilis

 Bacili that are not gram(-)

a.) Neisseria

b.) Moraxella

c.) Catarrhalis

d.) Veillonella

Cocci that are note gram (+)

Red

Color of gram - in micro

Purple

Color of gram + in micro

Red

Color of bacteria in acid fast

Gram-positive bacteria have a thick mesh-like cell wall made of 60-80% peptidoglycan of cell envelope), and as a result are stained purple by crystal violet, whereas gram-negative bacteria have a thinner layer (10% of cell envelope), so do not retain the purple stain and are counter-stained pink by safranin

Why do gram positive bacteria retain the primary stain?

Gram-positive bacteria remain purple because they have a single thick cell wall that is not easily penetrated by the solvent; gram-negative bacteria, however, stain red because they have cell walls with much thinner layers that allow removal of the dye by the solvent, so it is colored only by the safranin counterstain.

Why do gram negative bacteria stain red and not violet/blue like the gram positive bacteria?

•  Organisms such as Mycobacteria are extremely difficult to stain by ordinary methods like gram stain. Because of the high concentration of mycolic

  acid, a lipid in their cell wall.

•  Phenolic compound carbol fuchsin is used as the primary stain because it is lipid soluble and penetrates the waxy cell wall. Staining by carbol fuchsin is further enhanced by heating the preparation to melt the wax and allow the stain to move into

  the cell

•  Acid alcohol is used to decolorize

  non-acid fast cells because acid fast cells resist this decoloration. The ability of the bacteria to resist this decoloration with acid confers acid fastness to the bacterium. Following the colorization, the smear is counterstained with methylene blue which stains the background material, providing a contrast color against which the red AFB can be seen.

Give the principle of acid-fast staining

○  Mycobacterium Tuberculosis

○  Bacillus Subtilis

Bacilli that are not gram (-)

○  Neisseria

○  Moraxella

○  Catarrhalis

○  Veillonella

Cocci that are not gram (+)

Red

Color of gram (-) in microscope

Purple

Color of gram (+) in microscope

Red

Color of an acid-fast bacteria

Blue

Color of a nonacid-fast bacteria

To penetrate the cell wall, because it is lipid soluble

Why do we need to use carbol fuchsin in acid-fast staining

○  Wrong labeling

○  Inadequate fixing

○  Too thick or too thin smear

○  Stain deposits

○  Over decolorizati

What are the common errors in gram staining?

○ Because they have different cell ends and the difference of how they multiply

Why do bacterias have different arrangement?