Questions and Answers
What is Gram stain used for?
Identifying and classifying bacteria.
What classification does a Gram stain allow for bacteria?
What are the steps in the staining technique?
Apply the primary stain (crystal violet), apply mordant (grams iodine), apply decolorizing agent (ethanol or acetone), apply secondary stain (safranin).
What is the primary stain in Gram staining and what color does it stain the bacteria?
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What does the mordant (grams iodine) combine with in Gram staining?
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What is the decolorizing agent in Gram staining?
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What does the secondary stain or counterstain do in Gram staining?
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What are those bacteria that decolorize easily known as?
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What are those bacteria that decolorize slowly (aren't washed out) known as?
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Why do bacteria stain differently?
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What is bacterial cell walls primarily composed of?
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How many layers of peptidoglycan do Gram-positive cell walls contain?
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How many layers of peptidoglycan do Gram-negative cell walls contain?
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What does the alcohol decolorizing agent do to the cell wall of gram-positive bacteria?
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What cannot be washed out of Gram-positive cells?
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In Gram-negative cells, what does the decolorizing agent dissolve?
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What color does safranin stain the decolorized bacteria?
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How old should the cultures be for the Gram stain to be most consistent?
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What color will a Gram-negative cell stain?
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What color will a Gram-positive cell stain?
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Which organism is categorized as a coccus?
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Which of the following organisms is the largest among staphylococcus, bacillus, and escherichia?
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Which of the given bacterial cultures were Gram-positive?
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Why will Gram-positive cells older than 24 hours stain as Gram-negative?
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Can iodine be added before the primary stain in a Gram stain?
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In Gram-positive cells, what color does the crystal violet turn the cells?
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In Gram-positive cells, what color does the iodine turn the cells?
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In Gram-positive cells, what color does alcohol-acetone turn the cells?
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In Gram-positive cells, what color does safranin turn the cells?
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In Gram-negative cells, what color does crystal violet turn the cells?
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In Gram-negative cells, what color does the iodine turn the cells?
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In Gram-negative cells, what color does alcohol-acetone turn the cells?
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In Gram-negative cells, what color does safranin turn the cells?
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Which step can you omit without affecting determination of Gram reaction?
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Study Notes
Gram Staining Overview
- Gram stain is essential for identifying and classifying bacteria.
- Bacteria are classified as gram-positive or gram-negative based on their cell wall composition.
Staining Technique Steps
- Primary stain: Crystal violet is applied, staining bacteria purple.
- Mordant: Gram's iodine forms a crystal violet-iodine complex (CV-I), enhancing stain retention by bonding to peptidoglycan in cell walls.
- Decolorizing agent: Ethanol or acetone washes out primary stain from some bacteria, while others remain unaffected.
- Secondary stain: Safranin is used to stain decolorized bacteria red.
Cell Wall Differences
- Gram-negative bacteria have a thin peptidoglycan layer and an outer membrane of lipoproteins and lipopolysaccharides.
- Gram-positive bacteria possess multiple layers of peptidoglycan, making them more resilient to decolorization.
- Chemical and structural variations in cell walls lead to differences in staining outcomes.
Reaction to Staining
- CV-I cannot be washed out of gram-positive cells, allowing them to maintain the purple color after the decolorization step.
- In gram-negative bacteria, the decolorizing agent dissolves the outer membrane, leading to color loss and staining red with safranin.
Bacterial Cultures and Staining Results
- Gram-negative cells stain pink/red, while gram-positive cells retain a purple/blue hue.
- The Gram stain is most reliable on young cultures, ideally 24 hours old, as older cells may show degeneration and appear gram-negative.
Specific Organisms and Characteristics
- Staphylococcus is identified as a coccus.
- Bacillus subtilis is the largest among the mentioned bacteria: Staphylococcus, Bacillus, and Escherichia.
- Bacillus subtilis is classified as gram-positive.
Staining Process Observations
- Staining colors for gram-positive cells: Purple for crystal violet, iodine, alcohol-acetone, and safranin.
- For gram-negative cells: Initially purple with crystal violet and iodine, but becomes colorless after alcohol-acetone and turns red with safranin.
Important Notes
- Omitting the safranin step does not affect the determination of Gram reaction.
- Crystal violet and iodine influence the staining of gram-negative cells, where alcohol-acetone results in a colorless state before applying safranin.
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Description
This quiz covers the essential aspects of the Gram staining technique, including its importance in identifying bacteria. Explore the steps involved in the staining process and understand the differences between gram-positive and gram-negative bacteria based on their cell wall structures.
