Staining techniques

What is the purpose of simple staining? Why stain bacteria?

To enhance visibility of bacteria under a microscope, making it easier to observe their shape and arrangement.

What is the purpose of heat fixing a smear?

To adhere the bacteria to the slide and kill them, preventing them from moving during staining.

Which type of dyes are used to perform a positive stain and a negative stain?

Positive stains use basic dyes (e.g., methylene blue), while negative stains use acidic dyes (e.g., India ink).

Why didn't you heat-fix the slide before adding the dye in a negative stain?

Heat-fixing can distort the bacteria and affect the results of the negative stain.

What would happen if the heat fixing step was not performed? What would you see under the microscope?

Without heat fixing, bacteria may wash off the slide during staining, leading to fewer or no visible cells.

Which dye was used to perform the negative stain?

An acidic dye, such as India ink.

Which dye was used to perform the positive stain?

A basic dye, such as methylene blue.

What is the purpose of the second slide in the negative stain?

To spread the dye evenly across the slide, creating a thin film for better observation.

Which of the three differential stains would most likely be the first stain to use in identifying an unknown bacterium?

The Gram stain.

How does a counterstain differ from a primary stain?

A counterstain is applied after the primary stain to provide contrast and visualize cells that did not retain the primary stain.

What differentiates a Gram-positive bacterium from a Gram-negative bacterium?

Gram-positive bacteria have a thick peptidoglycan layer and retain the primary stain, appearing purple, while Gram-negative bacteria have a thin peptidoglycan layer and do not retain the primary stain, appearing pink after counterstaining.

What is the most critical step in the Gram staining process? Why?

The decolorization step, as it determines whether bacteria retain the primary stain or take up the counterstain.

What is the purpose of the heat during the acid-fast and endospore staining procedures?

Heat acts as a mordant, helping the primary stain penetrate the bacterial cell walls.

What makes a bacterium acid-fast?

The presence of mycolic acid in the cell wall, which retains the primary stain even after decolorization.

Which stain is the primary stain for the Gram stain, and which one is the primary stain for the acid-fast stain?

The primary stain for the Gram stain is crystal violet, and for the acid-fast stain, it is carbol fuchsin.

Which stain is the counterstain for the Gram stain, and which one is the counterstain for the acid-fast stain?

The counterstain for the Gram stain is safranin, and for the acid-fast stain, it is methylene blue.

What color would Gram-negative cells be if the decolorizing step was not performed?

They would remain purple, as they would retain the primary stain.

What color would endospores be if heat was not used as a mordant?

They would not take up the primary stain and would appear colorless.

What is the function of an endospore?

To protect the bacterial DNA during unfavorable conditions, allowing the bacterium to survive extreme environments.