Questions and Answers
What color is Escherichia coli under a gram stain? Why?
Red/pink because it is gram negative. It does not have a thick peptidoglycan cell wall therefore, does not retain the CV-1 stain.
What color is Mycobacterium smegmatis under a gram stain? Why?
Purple because it is gram positive. It does have a thick peptidoglycan cell wall and therefore retains the crystal violet stain.
What color is Staphylococcus epidermidis under a gram stain? Why?
Purple because it is gram positive. It does have a thick peptidoglycan cell wall and therefore retains the crystal violet stain.
What color is Bacillus megaterium under a gram stain? Why?
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What color is Escherichia coli under an acid fast stain? Why?
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What color is Mycobacterium smegmatis in an acid fast stain? Why?
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What color is Staphylococcus epidermidis in an acid fast stain? Why?
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What color is Bacillus megaterium in an acid fast stain? Why?
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Which of the four samples from lab is the only acid fast positive bacteria?
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Which of the four samples from lab is the only gram negative bacteria?
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What is the primary stain in the gram stain?
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What is the mordant in the gram stain?
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What is the decolorizer in the gram stain?
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What is the secondary stain in the gram stain?
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What is the primary stain in the acid fast stain?
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What is the decolorizer in the acid fast stain?
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What is the counterstain in the acid fast stain?
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What is the primary stain in the spore stain?
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What is the counterstain in the spore stain?
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Is there a decolorizer in the spore stain?
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Which of the four samples from lab is positive for the spore stain?
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What are the results of Escherichia coli if you apply a gram stain, acid fast, and spore stain?
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What are the results of Mycobacterium smegmatis if you apply a gram stain, acid fast, and spore stain?
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What are the results of Staphylococcus epidermidis if you apply a gram stain, acid fast, and spore stain?
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What are the results of Bacillus megaterium if you apply a gram stain, acid fast, and spore stain?
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Name 4 gram positive bacteria.
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Name 4 gram negative bacteria.
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Name 4 acid fast positive bacteria.
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Name 4 acid fast negative bacteria.
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Name 4 spore positive (spore forming) bacteria.
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Name 4 spore negative (non-spore forming) bacteria.
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What is the purpose of a negative stain?
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What is total magnification?
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What is a petri sample for?
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What is a slant for?
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What is the dip sample for?
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What is the broth for?
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Why do you store the petri dish upside down?
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What temperature is a petri dish stored at? Why?
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What is a basic dye?
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What is an acidic dye?
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Why does basic dye attach to bacteria?
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What are chromophores?
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What is the purpose of a simple stain?
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What is a simple stain?
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What does the condenser do?
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What does the diaphragm do?
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What does coccus mean?
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What does bacillus mean?
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What does diplo mean?
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What does strepto mean?
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What does tetrads mean?
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What does sarcinae mean?
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What does staphy mean?
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What does chemotaxis mean?
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What does phototaxis mean?
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What happens to the gram positive bacteria cell when the decolorizer (alcohol acetone) is added?
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What happens to the gram negative bacteria cell when the decolorizer (alcohol acetone) is added?
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What are 5 considerations when using the oil immersion objective?
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Study Notes
Gram Staining
- Escherichia coli: Red/pink (gram-negative); does not retain crystal violet due to a thin peptidoglycan wall.
- Mycobacterium smegmatis: Purple (gram-positive); retains crystal violet due to a thick peptidoglycan wall.
- Staphylococcus epidermidis: Purple (gram-positive); thick peptidoglycan retains crystal violet.
- Bacillus megaterium: Purple (gram-positive); retains crystal violet due to a thick peptidoglycan wall.
Acid-Fast Staining
- Escherichia coli: Blue (acid-fast negative); carbolfuchsin washes out, leaving methylene blue visible.
- Mycobacterium smegmatis: Red (acid-fast positive); retains red from carbolfuchsin due to lipid-enriched cell wall.
- Staphylococcus epidermidis: Blue (acid-fast negative); similar to E. coli.
- Bacillus megaterium: Blue (acid-fast negative); similar to E. coli.
Unique Bacterial Characteristics
- Only acid-fast positive bacteria in lab: Mycobacterium smegmatis.
- Only gram-negative bacteria in lab: Escherichia coli.
Staining Components
- Primary stain in gram stain: Crystal violet.
- Mordant in gram stain: Iodine.
- Decolorizer in gram stain: Alcohol-acetone.
- Secondary stain in gram stain: Safranin.
- Primary stain in acid-fast stain: Carbolfuchsin.
- Decolorizer in acid-fast stain: Alcohol-hydrochloric acid.
- Counterstain in acid-fast stain: Methylene blue.
- Primary stain in spore stain: Malachite green.
- Counterstain in spore stain: Safranin.
- Decolorizer in spore stain: None; only a water rinse.
Staining Results for Specific Bacteria
- Escherichia coli: Gram negative (red), acid-fast negative (blue), spore negative (red).
- Mycobacterium smegmatis: Gram positive (purple), acid-fast positive (red), spore negative (red).
- Staphylococcus epidermidis: Gram positive (purple), acid-fast negative (blue), spore negative (red).
- Bacillus megaterium: Gram positive (purple), acid-fast negative (blue), spore positive (red and green).
Bacterial Classification
- Gram Positive Bacteria: Mycobacterium smegmatis, Staphylococcus epidermidis, Bacillus megaterium, Streptococcus species.
- Gram Negative Bacteria: Escherichia coli, Neisseria species, Vibrio cholera.
- Acid-Fast Positive Bacteria: Mycobacterium species, Nocardia asteroides.
- Acid-Fast Negative Bacteria: Escherichia coli, Staphylococcus species, Bacillus megaterium.
- Spore-Forming Bacteria: Bacillus anthracis, Clostridium species.
- Non-Spore Forming Bacteria: Escherichia coli, Mycobacterium smegmatis.
Staining Techniques and Purposes
- Negative stain: Used to see if bacteria have a capsule.
- Petri dish: Isolation of bacteria.
- Slant: Preservation of cultures.
- Dip sample: Examination for flagella presence.
- Broth culture: Unrestricted growth of bacteria.
Storage and Maintenance
- Petri dishes stored upside down to prevent condensation.
- Incubation temperature for petri dishes: 38°C, optimal for most bacteria.
Staining Characteristics
- Basic dye: Positive ions (cations), attract negatively charged bacteria.
- Acidic dye: Negative ions (anions).
- Chromophores: Colored ions in stains.
Microscope Adjustments
- Condenser: Focuses light.
- Diaphragm: Controls light amount.
Bacterial Morphology Terms
- Coccus: Round shape.
- Bacillus: Rod shape.
- Diplo: Pairs.
- Strepto: Chains.
- Tetrads: Groups of four.
- Sarcinae: Groups of eight.
- Staphy: Grapelike clusters.
Bacterial Movement Responses
- Chemotaxis: Movement toward or away from chemicals.
- Phototaxis: Movement toward or away from light.
Gram Staining Mechanism
- Gram-positive bacteria retain crystal violet after decolorization due to dehydrated peptidoglycan.
- Gram-negative bacteria lose crystal violet due to dissolved outer membrane and porous peptidoglycan layer.
Oil Immersion Microscope Usage
- Apply oil between 40x and 100x magnification.
- Use one drop of oil; clean before and after use.
- Ensure 100x objective touches oil.
- Use fine focus adjustments for clarity.
- Oil maximizes numerical aperture and resolution; avoid using cloudy oil.
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Description
Test your knowledge of microbiology concepts with this lab review focused on Gram staining. Learn the differences between Gram-positive and Gram-negative bacteria, including Escherichia coli and Mycobacterium smegmatis. Use these flashcards to prepare for your microbiology exams effectively.