1. Introduction to Lab: Differential Stains
Gram Staining

2. Introduction to Lab Differential Stains – Gram Staining
Basic classification of bacteria is based on the cell wall structure.
There are 2 main groups: Gram positive and Gram negative.
Gram staining is a differential staining technique that provides an easy differentiation of bacteria into one of two groups.

3. Differential Stains – Gram Staining
The staining technique, developed in the late 1700’s by Christian Gram classifies the rigid cell walled bacteria into one of two groups.
based on whether they are able to resist the decolorizing action of an alcoholic solution.

4. Differential Stains – Gram Staining
Those that resist decolorization by 95% ethanol are arbitrarily termed Gram positive and those that do not are Gram negative
(the terms positive and negative have nothing to do with charges
of the cell but based on differences in the cell wall structure of
these two groups of bacteria).

5. Gram-positive cell walls Gram-negative cell walls
The characteristic compound found in all true bacterial cell walls
is peptidoglycan. The amount of PPG is among one of the
differences between the GP and GN cell walls.
Gram-positive cell walls Gram-negative cell walls
Thin peptidoglycan
5-10% peptidoglycan
No teichoic acids
3 layers
Outer membrane has lipids, polysaccharides
No acid- fast cells (mycolic acid)
Thick peptidoglycan
90% peptidoglycan
Teichoic acids
1 layer
Not many polysaccharides
In acid-fast cells, contains mycolic acid

6. Figure 4.13b, c

7. The process includes the use of:
a primary stain (crystal violet)
a mordant (helper) iodine solution,
a decolorizer (95% ethanol),
a counterstain (safranin).

8. The Gram stain Thin smear/heat fix Gram stain:
a. Flood slide with crystal violet and let stain for 1 minute.
b. Drain off crystal violet and rinse off with distilled water; flood slide with Gram's iodine for 1 minute. 
c. Rinse off Gram's iodine with distilled water.
d. Hold the slide on an angle (preferably with a clothes pin) and drop 95% ethyl alcohol onto it until the alcohol leaving the slide no longer has a purple tint; be sure to drop the alcohol onto the upper portion of the slide so that the smears are subjected to uniform decolorization.
e. Rinse with distilled water and flood the slide with safranin and let stain for minutes. 
f. Rinse with distilled water and blot dry with bibulous paper.
Gram
positive
Gram
negative

9. The crucial step in the staining process is the decolorizing step.
The most accepted theory relies on the fact that the PPG is found in layers and the stain molecules are trapped within the many layers of the GP CW when they form the complex with the mordant Iodine molecules.
Since the GN CWs lack much PPG the amount of stain captured in
those CWs is much lesser.
When the cells are treated with the decolorizer – the ethanol – this
causes denaturation of the proteins in the outer membrane of the
GN CWs resulting in gaping holes in these CWs that lead to the
removal of the crystal violet-iodine complexes easily, leaving these
cells unstained.
The counterstain -safranin- thus is used to make these cells visible.

10. There are 4 conditions to be followed for a valid Gram staining
procedure: 
Young cultures - must be young within 18-24hrs old
(older cultures lose their Gram staining properties
due to changes in the CWs as the cells get older)
Thin smear
thicker or uneven smears will result in uneven staining
and decolorization
Fresh reagents - of proper strength
Control cultures - for a known GP bacterium and GN culture (S.aureus & E.coli)

11. Demos: Gram stained slides of
Neisseria, Streptococcus, Pseudomonas, Actinomyces species.
Pseudomonas
Neisseria
Streptococcus