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Questions and Answers
What is the primary purpose of Gram controls in the Gram staining procedure?
What is the primary purpose of Gram controls in the Gram staining procedure?
- To identify the specific species of bacteria present.
- To confirm the staining procedure is performed correctly and the unknown has stained correctly. (correct)
- To ensure the reagents are prepared correctly.
- To speed up the staining process for multiple samples.
Which component of Gram-negative bacteria is affected by ethanol during the decolorization step, leading to the loss of the crystal violet-iodine complex?
Which component of Gram-negative bacteria is affected by ethanol during the decolorization step, leading to the loss of the crystal violet-iodine complex?
- Lipopolysaccharide layer (correct)
- Peptidoglycan layer
- Cell membrane
- Teichoic acid
During Gram staining, if the decolorization step is skipped, what is the likely appearance of both Gram-positive and Gram-negative bacteria under the microscope?
During Gram staining, if the decolorization step is skipped, what is the likely appearance of both Gram-positive and Gram-negative bacteria under the microscope?
- Gram-positive will be purple, and Gram-negative will be pink.
- Both will appear purple. (correct)
- Gram-positive will be pink, and Gram-negative will be purple.
- Both will appear pink.
What color will Gram-positive bacteria appear after completing the Gram staining procedure?
What color will Gram-positive bacteria appear after completing the Gram staining procedure?
What is the function of Gram's iodine in the Gram staining procedure?
What is the function of Gram's iodine in the Gram staining procedure?
Which of the following is the most likely outcome of over-decolorizing a Gram stain?
Which of the following is the most likely outcome of over-decolorizing a Gram stain?
Why is it essential to use a very small amount of bacteria when preparing a smear for Gram staining?
Why is it essential to use a very small amount of bacteria when preparing a smear for Gram staining?
When examining a Gram-stained bacterial smear under a microscope, you observe multiple colors in a pure culture. What is the most likely cause of this?
When examining a Gram-stained bacterial smear under a microscope, you observe multiple colors in a pure culture. What is the most likely cause of this?
During a Gram stain, you heat-fix the bacterial smear, but do not do it properly. What is the likely consequence?
During a Gram stain, you heat-fix the bacterial smear, but do not do it properly. What is the likely consequence?
If Gram-negative bacteria appear purple after Gram staining, what procedural error most likely occurred?
If Gram-negative bacteria appear purple after Gram staining, what procedural error most likely occurred?
You are performing a Gram stain on a sample of bacteria. After adding crystal violet, you skip the iodine step. What is the likely appearance of Gram-positive bacteria after the final safranin step?
You are performing a Gram stain on a sample of bacteria. After adding crystal violet, you skip the iodine step. What is the likely appearance of Gram-positive bacteria after the final safranin step?
What is the primary reason for using Gram controls (positive and negative) when performing a Gram stain on an unknown organism?
What is the primary reason for using Gram controls (positive and negative) when performing a Gram stain on an unknown organism?
You examine a Gram-stained slide under the microscope, but cannot bring the bacteria into focus. What is the FIRST troubleshooting step you should consider?
You examine a Gram-stained slide under the microscope, but cannot bring the bacteria into focus. What is the FIRST troubleshooting step you should consider?
Which step in the Gram staining procedure is most critical in differentiating Gram-positive from Gram-negative bacteria?
Which step in the Gram staining procedure is most critical in differentiating Gram-positive from Gram-negative bacteria?
What is the purpose of safranin in the Gram staining procedure?
What is the purpose of safranin in the Gram staining procedure?
Which statement accurately describes the structural difference between Gram-positive and Gram-negative bacterial cell walls that contributes to their differential staining?
Which statement accurately describes the structural difference between Gram-positive and Gram-negative bacterial cell walls that contributes to their differential staining?
Ethanol and Gram's iodine are stored in apothecary bottles. What is the correct procedure for dispensing these reagents?
Ethanol and Gram's iodine are stored in apothecary bottles. What is the correct procedure for dispensing these reagents?
In the Gram staining procedure, which of the following reagents is NOT a stain?
In the Gram staining procedure, which of the following reagents is NOT a stain?
Which characteristic of crystal violet and safranin is essential for their function in Gram staining?
Which characteristic of crystal violet and safranin is essential for their function in Gram staining?
You are observing Gram-stained bacteria under a microscope and notice that both the Gram-positive and Gram-negative bacteria appear dark purple. Which of the following is the MOST likely explanation for this result?
You are observing Gram-stained bacteria under a microscope and notice that both the Gram-positive and Gram-negative bacteria appear dark purple. Which of the following is the MOST likely explanation for this result?
Which of the following is NOT a step in the Gram staining procedure?
Which of the following is NOT a step in the Gram staining procedure?
Under-decolorizing during Gram staining will result in which outcome?
Under-decolorizing during Gram staining will result in which outcome?
What is the primary reason for counterstaining with safranin in Gram staining?
What is the primary reason for counterstaining with safranin in Gram staining?
You perform a gram stain and observe pink cocci and purple bacilli. What is the correct interpretation?
You perform a gram stain and observe pink cocci and purple bacilli. What is the correct interpretation?
A student performs a Gram stain, but forgets to rinse with water after applying crystal violet. How is this LIKELY to affect the final results.
A student performs a Gram stain, but forgets to rinse with water after applying crystal violet. How is this LIKELY to affect the final results.
What is the scientific basis for Gram-positive bacteria retaining crystal violet stain, while Gram-negative bacteria do not?
What is the scientific basis for Gram-positive bacteria retaining crystal violet stain, while Gram-negative bacteria do not?
Which of the following describes the correct order of reagents in the Gram staining procedure?
Which of the following describes the correct order of reagents in the Gram staining procedure?
What is the expected color of a Bacillus subtilis smear after properly performing a Gram stain?
What is the expected color of a Bacillus subtilis smear after properly performing a Gram stain?
What is the expected color of an Escherichia coli smear after properly performing a Gram stain?
What is the expected color of an Escherichia coli smear after properly performing a Gram stain?
What is the main difference between Gram-positive and Gram-negative cell walls that influence how crystal violet interacts with them?
What is the main difference between Gram-positive and Gram-negative cell walls that influence how crystal violet interacts with them?
After Gram-staining, you observe a field of bacteria that are a mix of both purple cocci and pink cocci. Assuming that all the steps of the procedure have been completed carefully, you can conclude that:
After Gram-staining, you observe a field of bacteria that are a mix of both purple cocci and pink cocci. Assuming that all the steps of the procedure have been completed carefully, you can conclude that:
Flashcards
What is Gram staining?
What is Gram staining?
A differential staining technique that uses two basic stains along with other reagents to distinguish between different types of bacteria.
What is Crystal violet?
What is Crystal violet?
Crystal violet is the primary stain used in Gram staining that colors all cells initially.
What is Safranin?
What is Safranin?
Safranin is the secondary stain (or counterstain) used in Gram staining, which stains Gram-negative bacteria pink or red.
What is Gram's Iodine?
What is Gram's Iodine?
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What is Ethanol's role?
What is Ethanol's role?
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What is Gram-positive?
What is Gram-positive?
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What is Gram-negative?
What is Gram-negative?
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What effect does Over-decolorizing have?
What effect does Over-decolorizing have?
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What effect does Under-decolorizing have?
What effect does Under-decolorizing have?
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What are Gram controls?
What are Gram controls?
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Study Notes
Gram Staining
- A differential staining technique is used in gram staining
- Stains used are crystal violet and safranin
- Other reagents used include iodine and ethanol
Stains Used
- Crystal violet and safranin are basic stains
- Have positively charged chromophores
- This attracts them to the negative charge of bacterial cell walls
- Crystal violet is the primary stain
- Safranin is the counterstain or secondary stain
Reagents Used
- Gram's iodine is a mordant that reacts with the crystal violet to form the crystal violet-iodine complex
- Ethanol is a decolorizing agent
- Ethanol dehydrates the thick peptidoglycan of gram-positive bacteria
- Ethanol dissolves the lipopolysaccharide layer of gram-negative bacteria
Gram Staining Procedure
- Cover the smear with primary stain (crystal violet) for 30 seconds
- Rinse gently with DI water
- Cover smear with Gram's iodine for 10 seconds
- Rinse gently with DI water
- Decolorize with alcohol
- Rinse gently with DI water
- Cover smear with secondary stain (safranin) for 30 seconds
- Rinse gently with DI water and blot dry
Gram Reaction
- Gram-positive cells stain purple
- Gram-negative cells stain red or pink
Bacterial Cells
- Gram-positive bacteria have a thick peptidoglycan layer that can absorb surrounding materials well
- Gram-negative bacteria have a thin peptidoglycan layer with multiple thin layers of membrane
- The lipopolysaccharide layer of gram-negative bacteria can be dissolved when ethanol is used
Gram Stain Mechanism
- Bacteria starts out colorless
- Crystal violet is attracted to the cell wall and the bacteria turns purple
- Iodine then combines with the crystal violet, forming the crystal violet-iodine complex
- The ethanol dehydrates the peptidoglycan layer, trapping the crystal violet iodine complex inside
- The bacteria is already colored with the crystal violet iodine complex, so pink color makes no difference
- For Gram negative bacteria, the bacteria starts out colorless
- Crystal violet is attracted to the cell wall, turning the bacteria purple
- Iodine combines with the crystal violet forming the crystal violet-iodine complex
- Ethanol dissolves the lipopolysaccharide layer, releasing the crystal violet iodine complex
- The safranin is now attracted to the cell wall and the bacteria turns pink
Gram Controls
- Gram controls should be used when gram staining unknown organisms
- Use gram controls whenever an unknown organism is being stained
Gram Positive Control
- A known Gram positive organism that will stain purple
Gram Negative Control
- A known Gram negative organism that will stain pink/red
Purpose
- Gram controls help confirm whether the unknown has stained correctly
Troubleshooting for Bacterial Smears
- Using a smear that is too thick makes staining more difficult and could produce confusing results
- Same mistakes and outcomes as before
- Not heat fixing correctly, means there will be no bactera to begin with
Troubleshooting for Decolorizing Step
- Over-decolorizing can cause gram-positive cells to lose their purple color and appear gram-negative
- Under-decolorizing can result in gram-negative cells retaining the purple color and appearing gram-positive
Apothecary Bottles
- Ethanol and Gram's Iodine will be stored in apothecary bottles
- Do NOT take the lid off
- Turn the spout to align with the bumps on the neck of the bottle
- Hold onto the lid with a finger to make sure it does not come off while pouring
Troubleshooting Staining
- If a pure bacterial smear has multiple colors when viewed under a microscope, one might have made the smear too thick, over-decolorized gram-positive cells, or under-decolorized gram-negative cells
Troubleshooting Microscope
- If you cannot get the bacteria into focus under the microscope, ensure the slide is not upside down, and that the side of the slide with the smear is facing up
Advice for Studying
- Describe the GRAM reaction including results and interpretations
- Write out process to have a clear understanding of stains and staining procedures
- Draw out bacteria step by step and color accordingly without looking at the lab manual
- Know consequences of making a mistake at any stage of gram-staining procedure for both positive and negative cells
Procedures
- Follow procedure on page 65
- Work with solid cultures ONLY
- Use a very small amount of bacteria
- Do not use tap water, use small dropper bottle at table
- Store slide inside class slide box if you are not able to complete procedure
- Place used slides inside the disinfectant pan, once viewing is complete
- Pour staining tray down the sink and rinse with water after used
- Properly store the microscope
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