Gram Stain - Online Tutor, Practice Problems & Exam Prep

The Gram stain is a differential staining technique that categorizes bacteria into gram-positive and gram-negative groups based on cell wall composition. The procedure involves four steps: staining with crystal violet, applying iodine as a mordant, decolorizing with alcohol or acetone, and counterstaining with Safranin. Gram-positive bacteria retain the purple color throughout, while gram-negative bacteria initially turn colorless and then pink. This method is crucial for identifying bacterial types and guiding antibiotic treatment, highlighting the importance of cell wall structure in microbial classification.

Gram Stain

This video, we're going to begin our lesson on the Gram stain. And so recall from our previous lesson videos that the Gram stain is an example of a differential stain. It can be used to differentiate groups of bacteria. And in fact, the Gram stain separates bacteria into 2 major groups based on cell wall differences. And those 2 major groups are the gram positive bacteria and the gram negative bacteria. Now the gram stain itself actually consists of a series of 4 steps that we have numbered down below in our table 1, 2, 3, and 4. And so notice that this table is outlining the overall procedure for gram staining, and in the far left it has a description of the specific steps. And this column here it has this a description of the state of the bacteria. And here what we have are the gram positive cells after each step, and the gram negative cells after each step as well. And so in the very first step of the gram staining procedure the, sample is gonna be stained with a particular dye called crystal violet dye. And the crystal violet dye is going to stain all of the cells a purple color. Regardless if they are gram positive or gram negative, all of the cells will turn purple. And so notice that before the gram staining procedure even begins, the gram positive and the gram negative cells are unstained. And so you can see that these cells are all white, which represents that they are unstained. They are not stained. But after the very first step of the gram staining procedure, staining them with crystal violet dye, all of the cells take on that crystal violet dye. So the gram positive and the gram negative cells are all going to be purple. And so in step number 2, the sample is actually gonna be treated with an iodine solution. And the iodine serves as what is known as a mordant. Okay? And the mordant is really just, going to help ensure that the crystal violet dye remains affixed to the gram positive cells. It basically helps the gram positive cells to hold on to that crystal violet dye, more strongly. And so all of the cells are still going to remain purple after step number 2. And so really all of these cells are gonna remain purple. And nothing really has changed other than the fact that the gram positive cells are going to retain and, have a stronger affinity to that crystal violet dye. And so in step number 3, the sample is gonna be treated with a decolorizer. And that decolorizer is usually going to be alcohol or acetone. And so after this step, the decolorizer, the gram positive cells are going to remain purple, whereas the gram negative cells become colorless, and they actually lose that crystal violet dye. And so notice that the gram positive cells once again are remaining purple, whereas the gram negative cells have lost the crystal violet dye and so they are unstained once again as they were originally. And so in the 5th, I'm sorry, in the 4th and final step, because these colorless gram negative cells are somewhat difficult to visualize, usually the sample is gonna be stained with a counterstain called Safranin. And so Safranin is just a counterstain that comes and stains the gram negative cells this pinkish color. And so once again the gram positive cells are gonna still remain purple, whereas the gram negative cells have changed from being colorless to taking on this pinkish color. And so that's exactly what we see is that the gram positive cells pretty much remain purple throughout the process. Whereas the gram negative cells, start off being purple and then they become colorless, and then they become pink with this counterstain safranin. And so, ultimately what happens at the very end of the Gram stain is that all of the Gram positive cells are going to be purple, whereas all of the Gram negative cells are going to be pink. And so you're able to visually distinguish these different groups of bacteria. And so this here concludes our introduction to the Gram stain, and we'll we'll be able to get some practice applying these concepts as we move forward. So I'll see you all in our next video.

Which of the following answers lists the steps of gram-staining in the correct order?

• A. Stain with primary stain (crystal violet), add iodine, add decolorizer, stain with counterstain (safranin).
• B. Add iodine, add decolorizer, stain with primary stain (crystal violet), stain with counterstain (safranin).
• C. Stain with primary stain (crystal violet), add decolorizer, stain with counterstain (safranin), add iodine.
• D. None of the above are correct.

Which of the following reagents is used to stain gram negative cells pink (or red) in the gram stain?

• A. Iodine.
• B. Safranin.
• C. Crystal violet.
• D. Decolorizing agent.

Which of the following statements is false?

• A. Crystal violet is used to stain the cells purple.
• B. Alcohol is used to kill the bacteria that are not stained.
• C. Gram positive cells end up being stained purple.
• D. Iodine is used to affix the crystal violet dye to gram positive cells.

Results from a gram stain show that these bacteria are:

• A. Gram negative.
• B. Gram positive.
• C. Gram neutral.

A scientists has a sample with two different species of bacteria. The first species is Staphylococcus aureus, a gram-positive bacterium. The second species is Escherichia coli, a gram-negative bacterium. The scientist gram-stains his sample of bacteria. What colors will the two species of bacteria be after staining?

• A. Staphylococcus aureus:pink; Escherichia coli:purple.
• B. Staphylococcus aureus:purple; Escherichia coli:pink.
• C. Both Staphylococcus aureus and Escherichia coli will be purple.

Here’s what students ask on this topic:

What is the purpose of the Gram stain in microbiology?

The Gram stain is a differential staining technique used in microbiology to categorize bacteria into two major groups: gram-positive and gram-negative. This classification is based on differences in the bacterial cell wall composition. The Gram stain involves four steps: staining with crystal violet, applying iodine as a mordant, decolorizing with alcohol or acetone, and counterstaining with Safranin. Gram-positive bacteria retain the purple color throughout the process, while gram-negative bacteria initially turn colorless and then pink. This method is crucial for identifying bacterial types and guiding antibiotic treatment, as it highlights the importance of cell wall structure in microbial classification.

What are the four steps involved in the Gram staining procedure?

The Gram staining procedure involves four key steps: 1) Staining with crystal violet dye, which turns all cells purple. 2) Applying iodine solution as a mordant, which helps the crystal violet dye remain affixed to gram-positive cells. 3) Decolorizing with alcohol or acetone, which causes gram-negative cells to lose the crystal violet dye and become colorless, while gram-positive cells remain purple. 4) Counterstaining with Safranin, which stains the gram-negative cells pink, while gram-positive cells remain purple. This process allows for the differentiation of bacteria based on their cell wall composition.

How do gram-positive and gram-negative bacteria differ in their response to the Gram stain?

Gram-positive and gram-negative bacteria differ in their response to the Gram stain due to differences in their cell wall structure. Gram-positive bacteria have a thick peptidoglycan layer that retains the crystal violet dye, causing them to remain purple throughout the staining process. In contrast, gram-negative bacteria have a thinner peptidoglycan layer and an outer membrane that does not retain the crystal violet dye after decolorization with alcohol or acetone. As a result, gram-negative bacteria become colorless and are then counterstained with Safranin, turning pink. This differential staining allows for the visual distinction between the two groups.

Why is iodine used as a mordant in the Gram staining process?

Iodine is used as a mordant in the Gram staining process to enhance the retention of the crystal violet dye by gram-positive bacteria. When iodine is applied after the initial crystal violet staining, it forms a complex with the dye, making it less soluble and more difficult to wash out. This helps ensure that the crystal violet dye remains affixed to the thick peptidoglycan layer of gram-positive cells. As a result, gram-positive bacteria retain the purple color even after the decolorization step, while gram-negative bacteria, which have a thinner peptidoglycan layer, do not retain the dye and become colorless.

What role does the decolorizer play in the Gram staining procedure?

The decolorizer, typically alcohol or acetone, plays a crucial role in the Gram staining procedure by differentiating between gram-positive and gram-negative bacteria. During the decolorization step, the decolorizer disrupts the outer membrane of gram-negative bacteria and extracts the crystal violet-iodine complex from their thinner peptidoglycan layer, causing them to become colorless. In contrast, gram-positive bacteria, with their thicker peptidoglycan layer, retain the crystal violet-iodine complex and remain purple. This step is essential for distinguishing the two groups of bacteria based on their cell wall composition.