Lecture 4: Gram stain

Who developed the Gram stain and in what year?

H.C. Gram in 1884

How was gram staining developed?

By looking at pneumoniae and seeing that some bacteria took up the dye and some didn't

What is the main purpose of the Gram stain?

To distinguish between types of bacteria based on cell wall composition (positive or negative)

What cellular feature distinguishes Gram-positive from Gram-negative bacteria?

Cell wall composition (2 layers in gram-positive, and 3 layers in gram-negative) and thicker peptidoglycan in gram-positive

What are the two primary cells distinguished by the Gram stain?

Gram-positive and Gram-negative

What does PG stand for in bacterial cell wall descriptions?

Peptidoglycan layer

Which bacterial type features an outer membrane (OM)?

Gram-negative bacteria

What are the cell wall layers in gram-positive bacteria?

Inner plasma membrane, and thick PG layer

What are the cell wall layers in gram-negative bacteria?

Inner plasma membrane, thin PG layer, and outer membrane

Lipoteichoic acid

A molecule on gram-positive cell walls that are also connected to the membrane.

Teichoic acid

A molecule embedded in the cell wall (PG layer) of gram-positive cells

What do lipoteichoic acid and teichoic acid do?

Contribute to structure in cell wall and negative charge of gram-positive cells

LPS (lipopolysaccharide)

A molecule that coats the surface of the outer-membrane in gram-negative cells that has porins to let things go in and out of the cell

Step 1 of gram staining

Crystal violet is added to cells and dissociates into chloride ions and CV ions which associate/combine with the negative PG layer to stain all cells purple

Step 2 of gram staining

Iodine is added that makes the molecules get big so that CV ions get stuck in the PG/cell walls so the bacteria retain the purple color

Step 3 of gram staining

Alcohol is added and decolorizes gram-negative cell walls (since alcohol ruins outer membrane), while causing gram-positive cell walls to shrink and traps purple color even more

Step 4 of gram staining

Safranin (counterstain) is added and stains all cells (but is not seen in gram-positive cells since the purple dye is so dark) so gram-negative cells are now pink and gram-positive cells are purple