Exercise 3-7 Gram Stain

Gram's iodine is used as the _______in the Gram stain procedure.

MORDANT

What factors can cause a gram-positive bacterium to appear gram-negative?

Overdecolarization: result in the loss of primary stain (if age of culture is over 24 hrs-or you can say age of culture) Under Decolorization: will not completely remove the crystal violet-iodine complex

Crystal violet is used as the ______in the Gram stain procedure.

PRIMARY STAIN

A gram-positive bacterium stains what color?

PURPLE

After decolorization, properly conducted, Gram-positive cells will be _______in color and Gram-negative cells will be ________?

Purple pink

A gram-negative bacterium stains what color?

RED or PINK (but I think she prefers PINK as the answer)

Describe a gram-positive cell wall?

*It has many sheaths of peptidoglycan

Describe a gram-negative cell wall?

*Outer membrane is composed of many types of lipids

What is the decolorization agent in the Gram stain procedure?

95% ethanol

Why is the decolorization step so crucial in the Gram stain procedure?

Because decolorization dehydrates the peptidoglycan making it shrink and tight, therefore the violet-iodine complex cannot get into the the tighten layers therefore trapping the cell in Gram positive bacteria

The purpose of the Gram stain procedure is to ______________________________?

Differentiate between the 2 large groups of bacteria. Which are: [GRAM+] and [GRAM-] MOs Violet (Gram+): due to the presence of a thick layer of peptidoglycan in their cell walls, which retains the crystal violet these cells are stained with red/pink (Gram-): attributed to a thinner peptidoglycan wall, which does not retain the crystal violet during the decoloring process

What are the four main steps of the Gram stain procedure and what are their functions?

CRYSTAL VIOLET (primary stain)- stains all the cells purple GRAM'S IODINE (mordant)-forms complex gram + cells 95% ETHANOL(counterstain) -removes the color of gram - SAFRANIN (secondary stain)- colorizes the gram - cells pink

What is the primary stain in the gram stain procedure?

Crystal violet

Give an example of a gram-negative microorganism

Escherichia coli

After adding the primary stain to the bacterial smear for the appropriate amount of time, Gram-positive cells will be what color? What color will Gram-negative cells be?

GRAM POSITIVE → will be a violet color (purple) GRAM NEGATIVE→ will be red-pink color

What would the color of a gram-variable bacterium be?

Gram variable: cells will have cells that stain pink and cells that stain purple in the same sample. So the color of these would be BOTH pink/red and purple bc its mixed.

What is the mordant in the Gram-stain procedure?

Gram's iodine Counterstain : safranin

Gram-positive bacteria stain one color, while Gram-negative bacteria stain another color. Why is this so?

Gram+ retains the crystal violet stain better because it is made up of more peptidoglycan layers in their cell wall MEANWHILE in Gram- thinner peptidoglycan wall, which does not retain the crystal violet during the decoloring process which causes different colors

What is the counterstain in the Gram stain technique?

SAFRANIN

Safranin is used as the _________in the Gram stain procedure.

SECONDARY STAIN

Give an example of a gram-positive microorganism.

Staphylococcus aureus

Why do we Gram stain bacterial cultures after we have determined that they are pure cultures?

To determine if they are [GRAM+] or [GRAM-]

The term gram-variable means___

When the bacterium is neither positive nor negative.__