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Questions and Answers
What distinguishes gram-positive bacteria from gram-negative bacteria in a Gram stain?
What distinguishes gram-positive bacteria from gram-negative bacteria in a Gram stain?
- Gram-positive bacteria have a higher lipid concentration.
- Gram-negative bacteria retain the primary dye.
- Gram-negative bacteria have more crystal violet.
- Gram-positive bacteria have a thick peptidoglycan layer. (correct)
The Gram stain uses a single dye to differentiate between bacterial types.
The Gram stain uses a single dye to differentiate between bacterial types.
False (B)
What is the role of iodine in the Gram stain procedure?
What is the role of iodine in the Gram stain procedure?
Iodine acts as a mordant, fixing the crystal violet dye complex in the cell wall.
Gram-negative bacteria take on a _______ color after the counter stain is applied.
Gram-negative bacteria take on a _______ color after the counter stain is applied.
Which antibiotic is more effective against gram-positive bacteria and why?
Which antibiotic is more effective against gram-positive bacteria and why?
The Gram stain is advantageous over a simple stain because it provides more specific information about bacterial types.
The Gram stain is advantageous over a simple stain because it provides more specific information about bacterial types.
What happens to gram-negative bacteria during the decolorization step?
What happens to gram-negative bacteria during the decolorization step?
Match the components of the Gram stain with their functions:
Match the components of the Gram stain with their functions:
What color do gram-positive organisms appear after decolorization and counterstaining?
What color do gram-positive organisms appear after decolorization and counterstaining?
Acetone is the slowest decolorizing agent used in Gram staining.
Acetone is the slowest decolorizing agent used in Gram staining.
What may cause false Gram-stain readings in aged bacterial cultures?
What may cause false Gram-stain readings in aged bacterial cultures?
The Gram stain is particularly useful in the _________ laboratory.
The Gram stain is particularly useful in the _________ laboratory.
Match the following decolorizing agents with their speed:
Match the following decolorizing agents with their speed:
What term is used to describe gram-positive cells that appear falsely gram-negative due to aging?
What term is used to describe gram-positive cells that appear falsely gram-negative due to aging?
What is the purpose of using Gram's iodine in the staining procedure?
What is the purpose of using Gram's iodine in the staining procedure?
Over-decolorization leads to a potentially false Gram-stain reading.
Over-decolorization leads to a potentially false Gram-stain reading.
Flashcards
Gram Stain
Gram Stain
A differential stain that uses four reagents to categorize bacteria into two groups based on cell wall composition.
Gram-Positive Bacteria
Gram-Positive Bacteria
Bacteria that retain the primary dye (crystal violet) during the Gram stain procedure, appearing purple.
Gram-Negative Bacteria
Gram-Negative Bacteria
Bacteria that lose the primary dye (crystal violet) during the Gram stain procedure and take up the counterstain (safranin), appearing pink.
Peptidoglycan
Peptidoglycan
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Mordant
Mordant
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Decolorizer
Decolorizer
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Counterstain
Counterstain
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Why is Gram staining important in clinical diagnosis?
Why is Gram staining important in clinical diagnosis?
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Mordant (in Gram staining)
Mordant (in Gram staining)
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Decolorization (in Gram staining)
Decolorization (in Gram staining)
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Counterstain (in Gram staining)
Counterstain (in Gram staining)
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Gram-variable bacteria
Gram-variable bacteria
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Over-decolorization
Over-decolorization
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Study Notes
Gram Stain
- The Gram stain is a differential stain, using four reagents, based on differences in bacterial cell walls.
- Gram-positive bacteria retain crystal violet, appearing purple.
- Gram-negative bacteria lose the crystal violet, taking up a counterstain (like safranin), appearing pink.
Learning Objectives
- Explain two key differences between the cell walls of gram-positive and gram-negative bacteria.
- Recognize the Gram stain's importance in clinical diagnosis.
- Identify a step in the Gram stain procedure that can be omitted and why.
- Explain why some antibiotics (e.g., penicillin) are more effective against gram-positive bacteria.
- Compare the Gram stain's advantages to a simple stain.
Gram Stain Procedure
- Stain a smear with crystal violet, allow to stand for 1 minute (check with instructor).
- Cover the slide with Gram's iodine (a mordant). Leave for 1 minute.
- Wash off with tap water.
- Decolorize with 95% ethyl alcohol, dropwise until no more color washes off.
- Wash off with tap water.
- Apply safranin (the counterstain). Let sit for 1 minute.
- Wash off with tap water.
- Drain and blot with bibulous paper, air dry the slide.
- Examine the preparation under the microscope.
Gram Stain Variations
- The concentration of dyes, staining time, and decolorizer composition affect the method.
- 95% ethyl alcohol is commonly used in staining. Sometimes acetone or an acetone-alcohol combination are used for faster decolorization.
- Variations in the decolorization process are a crucial part of successful staining.
Gram Stain Results
- Gram staining analyzes and determines the colour, shape, and type of microscopic organisms.
Gram Cell Wall Differences
- Gram-positive cell walls have a thick peptidoglycan layer which traps the crystal violet-iodine complex.
- Gram-negative cell walls have a thin peptidoglycan layer and an outer membrane with high lipid content. The lipids dissolve in the decolorizer, removing the crystal violet-iodine complex.
Gram Variable Bacteria
- Bacteria that change Gram stain reactions as they age or are exposed to antimicrobial agents.
- Age, incubation time, and specimen processing are factors that can affect the outcome of a Gram stain. A bacterial culture from a collection longer than 24-48 hours may lead to inaccurate readings.
Gram Stain Materials
- 24-hour agar cultures (Staphylococcus epidermidis, Enterococcus faecalis, Neisseria sicca, Saccharomyces cerevisiae, Bacillus subtilis, Escherichia coli, Proteus vulgaris)
- Light suspension of 1 or more species.
- Simulated pus/clinical specimen from a wound.
- Microscopes
- Stains and Solutions: Crystal Violet, Gram's Iodine, 95% Ethyl alcohol, Safranin, Hucker's crystal violet
- Slides, slide labels
- Staining racks
- Bibulous paper
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