Simple vs. Differential Stain

Differential Staining

Simple Staining

Simple Staining vs Differential Staining

by Andrea Dougherty

Differential Stain

Simple Stain

Simple and Differential

1.

2. Air Dry. Do not blow or heat. This may

force bacteria into air and infect.

3. Heat fixate. This denatures bacterial proteins and causes cells to stick to slide. This also kills bacteria to make safe for handling.

4. Place in staining rack. Use stain of choice. Stain for 30 seconds then rinse with water and gently blot. Cover strip not necessary.

2. Basic Dye

3. Mordant

4. Decolourize

5.Counterstain

(From colony) Apply small drop of water

to slide. Sterilize loop and transfer

bacteria to water and mix.

(From broth) Make sure mixed well.

Use sterile loop to transfer 1-2

drops bacteria to slide.

Gram staining

Differential Staining

Simple Staining

Gram Staining

Acid-Fast Technique

Spore Staining

Crystal Violet

Staphylococcus aureus

Unidentified bacterium

Images of Simple Staining

Acid-fastness is a physical property of certain bacteria, specifically their resistance to decolorization by acids during gram staining procedures.

Once stained, these organisms resist the dilute acid and/or ethanol-based decolorization procedures common in other staining techniques, hence the name acid-fast.

Acid-fastness is due to the high mycolic acid (waxy) content of certain bacterial cell walls, like those of Mycobacteria.

Acid-fasting is usually used after Gram staining fails to identify the bacteria.

Separates spore-forming from non spore-forming bacterium.

During the primary stain, the smeared culture is heated over a steam bath to soften the resistant outer layer of the cell and allow the Primary stain to bind within the spore. The subject is then counterstained.

Schaeffer-Fulton method uses Malachite Green and Safranin. It produces a red/pink color on the vegetative cells. The Dorner method uses Carbolfuchsin and Nigrosin and Vegetative cells are colorless, endospores are red, and the background is black.

Differentiates between two groups, gram positive and gram negative.

Usually the first step in identifying bacterial organism.

Hans Christian Gram was the inventor of Gram staining in 1884.

Gram-positive bacteria have a thick cell wall made of peptidoglycan, which are stained purple by crystal violet, whereas Gram-negative bacteria have a thinner layer which are stained pink by the counter-stain.

Shows basic morphology of Bacterium.

Morphology is the structure of the microorganism. This includes the shape, size and arrangement.

Will not distinguish between thin or thick peptoglycan layers. They will stain the same with simple staining.

Positively charged dyes stick to negatively charged microorganisms.

Basic Fuchsin

Methylene Blue

Notice the stains to the left appear in three different colors.

Bacillus subtilis

Tuberculous infections

Spore Staining

Acid-Fast Technique

Spore Stain of Bacillusmegaterium

Summary

Simple Staining is 'simply' coloring the bacteria, which appears clear on the slide, so that it can be seen under the light microscope.

Differential Staining is used to 'differentiate' between different types of bactrium.

Gram staining, Acid-Fast Staining and

Spore Staining are all used to differentiate between types.