Ch. 3 - Observing Microorganisms Through a Microscope

Tortora - Microbiology: An Introduction 14th Edition

Tortora14th EditionMicrobiology: An IntroductionISBN: 9780138200398Not the one you use?Change textbook

Tortora 14th Edition

Ch. 3 - Observing Microorganisms Through a Microscope

Problem 8

Chapter 3, Problem 8

What is the purpose of a decolorizer in the Gram stain? In the acid-fast stain?

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Understand that both the Gram stain and the acid-fast stain are differential staining techniques used to classify bacteria based on their cell wall properties.

In the Gram stain, the decolorizer (usually alcohol or acetone-alcohol) is applied after the primary stain (crystal violet) and the mordant (iodine). Its purpose is to selectively remove the crystal violet-iodine complex from Gram-negative bacteria, which have a thinner peptidoglycan layer, while Gram-positive bacteria retain the stain due to their thicker peptidoglycan layer.

In the acid-fast stain, the decolorizer (usually acid-alcohol) is used after the primary stain (carbol fuchsin). Its role is to remove the stain from non–acid-fast bacteria, which lack the waxy mycolic acid in their cell walls, while acid-fast bacteria retain the stain because their cell walls resist decolorization.

Recognize that the decolorizer is a critical step in both stains because it differentiates bacteria based on their cell wall composition by selectively removing the primary stain from certain groups.

Summarize that the decolorizer's purpose is to act as a selective agent that differentiates bacterial types by their ability to retain or lose the primary stain during the staining process.

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Key Concepts

Here are the essential concepts you must grasp in order to answer the question correctly.

Gram Stain Procedure

The Gram stain is a differential staining technique used to classify bacteria into Gram-positive and Gram-negative based on their cell wall structure. It involves applying a primary stain, a mordant, a decolorizer, and a counterstain to distinguish bacterial types by color.

Role of Decolorizer in Gram Stain

In the Gram stain, the decolorizer (usually alcohol or acetone-alcohol) selectively removes the primary stain from Gram-negative bacteria by dissolving their outer membrane and washing out the crystal violet-iodine complex. This step differentiates Gram-negative (colorless after decolorization) from Gram-positive bacteria (retain purple color).

Decolorizer Function in Acid-Fast Stain

In the acid-fast stain, the decolorizer (acid-alcohol) removes the primary stain from non–acid-fast cells but not from acid-fast bacteria, which have waxy mycolic acid in their cell walls. This selective decolorization allows acid-fast bacteria to retain the red stain, distinguishing them from non–acid-fast cells.

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Textbook Question

What is the purpose of a counterstain in the acid-fast stain?

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Textbook Question

Which of the following is not a modification of a compound light microscope?

a. Brightfield microscopy

b. Darkfield microscopy

c. Electron microscopy

d. Phase-contrast microscopy

e. Fluorescence microscopy

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Textbook Question

Assume that you are viewing a Gram-stained field of red cocci and blue rods through the microscope. You can safely conclude that you have

a. Made a mistake in staining

b. Two different species

c. Old bacterial cells

d. Young bacterial cells

e. None of the above

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Textbook Question

Assume you stain Clostridium by applying a basic stain, carbolfuchsin, with heat, decolorizing with acid-alcohol, and counterstaining with an acidic stain, nigrosin. Through the microscope, the endospores are 1, and the cells are stained 2.

a. 1—red; 2—black

b. 1—black; 2—colorless

c. 1—colorless; 2—black

d. 1—red; 2—colorless

e. 1—black; 2—red

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Textbook Question

In 1996, scientists described a new tapeworm parasite that had killed at least one person. The initial examination of the patient’s abdominal mass was most likely made using:

a. Brightfield microscopy

b. Darkfield microscopy

c. Electron microscopy

d. Phase-contrast microscopy

e. Fluorescence microscopy

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Textbook Question

Fill in the following table regarding the Gram stain:

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