Introduction to Lab: Differential Stains

Gram Staining

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.

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.

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).

• Thick peptidoglycan• 90% peptidoglycan• Teichoic acids• 1 layer• Not many

polysaccharides• In acid-fast cells,

contains mycolic acid

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)

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.

Figure 4.13b, c

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

The Gram stainThe Gram stainThin 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 2-3 minutes. 

f. Rinse with distilled water and blot dry with bibulous paper.

Gram positive

Gramnegative

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.

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 smearthicker or uneven smears will result in uneven stainingand decolorization

Fresh reagents - of proper strengthControl cultures - for a known GP bacterium and GN culture

(S.aureus & E.coli)

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

Neisseria

Pseudomonas

Streptococcus