Exercise IV- Differential Stains

Bacterial smear

Primary material for morphology screening or evaluation

Simple stain

• uses one stain

• cannot differentiate different colors

Differential stain

• routinely used

• uses 2 or more stains

• can differentiate different colors

Special stain

• not routinely used

• used to demonstrate structures.

1. Primary staining

2. Mordant

3. Decolorization or Differentiation

4. Counterstaining

Four steps in Gram staining:

Crystal violet

• Used in primary staining

• violet/purple-colored solution hexamethyl-p-rosaniline chloride

• alternative – gentian violet

Gram’s Iodine

• Used in mordant step

• brown-colored solution

Acetone

• Used as a decolorizer

• colorless solution

Safranin

• Used as a counter sain

• red-colored solution

thick peptidoglycan layer with teichoic acid

What is the reason for the appearance of gram-positive organisms under the microscope which is purple/blue?

disaccharides and amino acids

teichoic acid is composed of?

Thin peptidoglycan layer with lipopolysaccharides

What is the reason for the appearance of gram-negative organisms under the microscope which is pink/red?

Polysaccharides and lipids

lipopolysaccharides is composed of?

it intensifies the color of the primary stain

What does the mordant do to the cell wall of a microorganism?

it is the only colored stain that has been employed yet to the microorganisms

In primary staining, why do both gram-positive and gram-negative organisms appear violet?

1. Air drying

2. Heat fixing

Before submitting the organisms for gram-staining, two prior steps needs to be completed first. What are these steps?

It kills microorganism and allows the smear to adhere to the slide

What does heat fixing do?

Crystal violet-Iodine complex

Once the iodine molecule in mordant step had entered the cell wall, the iodone molecule will form an attachment with the crystal violet molecule forming a complex called?

red hot

What is the indicator that the wire loop exposed to the fire of the alcohol lamp is already sterilized?

to remove grease and to avoid bacteria present in the dirty slide

What is the rationalization behind sterilizing the glass slide by wiping tissue and heating both sides three times?

to avoid contamination

Whhat is the rationalization behind sterilizing the wire loop?

to preserve the morphology of the organisms

What is the rationalization behind air drying the smear?