Experiment 3-6 Gram Staining

Front 1

Who developed the Gram stain and the general info behind it?

Back 1

The Gram Stain was developed in the late 1800’s by Christian Gram, and it allows bacteria to be classified into two groups, ones that were Gram-positive (appear purple) and ones that
were Gram-negative (appears the color of the counterstain, in this case red). It is the most
important and widely used microbiological differential stain.

Front 2

What is the Primary Stain in this procedure

Back 2

Crystal Violet

Front 3

What is the mordant in this procedure

Back 3

Gram's iodine

Front 4

What is the decolorizing agent and give the purpose of decoloriztion

Back 4

95% Ethanol - washes away the dye in Gram-negative bacteria and does not touch the dye in Gram-positive bacteria

Front 5

What is the purpose of the counterstain? What is the counterstain?

Back 5

Safranin stains all the bacteria - Gram-negative bacteria appear red under the microscope

Front 6

What is the determining factor if something is Gram Positive or Gram Negative?

Back 6

The cell wall is the part of the bacteria that is the determining factor of whether the organism is Gram-positive or Gram-negative. The differentiation is based on the composition/ appearance of the cell wall.

Front 7

What is the major element of the cell wall of all bacteria?

Back 7

The major element of the cell wall of all bacteria is peptidoglycan. It is a rigid layer made up of two sugar derivatives - N-acetylglucosamine and N-acetylmuramic acid, and several amino acids. The sugars bind to each other in chains with glycosidic
linkages and cross link with the amino acids providing a strong, rigid structure.
- NOTE: The type of linkages vary due to the variety of amino acids.

Front 8

What is the breakdown of peptidoglycan is Gram Positive and Gram Negative (in percentages)?

Back 8

For Gram-positive bacteria, the cell wall is mostly (~90) peptidoglycan while for
Gram-negative bacteria, the cell wall is only ~10% peptidoglycan.

Front 9

What is responsible for the negative charges in the walls of Gram Positive bacteria and a general description of teichoic acids

Back 9

Many Gram-positive walls have teichoic acids attached to the cell wall. They are
polysaccharides connected to sugars and alanine bonded to phosphate through ester
linkages and are partly responsible for the negative charge of the cell. (See pg 77 in Brock for a picture of the cell wall)

Front 10

What is the other layer besides the peptidoglycan layer in Gram Negative bacteria?

Back 10

Gram-negative bacteria have a second layer besides the peptidoglycan layer called the
outer membrane or the lipopolysaccharide layer because it is made of lipids and a
variety of sugars/polysaccharides. (See pg 81 in Brock for a picture of the cell wall).

Front 11

Give a detailed description of how Gram Staining works (ie the mordant, decolorizer, and stain)?

Back 11

In both types of cell wall, the iodine forms an insoluble compound with crystal violet.
A thick peptidoglycan layer prevents decolorization because the alcohol causes
dehydration and shrinks the cell wall, so the dye becomes trapped. On the other hand,
alcohol can move through the lipid layer so dehydration does not occur, and the dye
can easily be washed away.

Front 12

What is the Gram Positive organism used in this experiment?

Back 12

S. epidermis

Front 13

What is the Gram Negative organism used in this experiment?

Back 13

P. Aeruginosa