Lab Homework G: Lab Stain

Arrange the steps of the Gram staining procedure in their correct order. Do not overlap any steps.

1. Flood slide with violet crystals and rinse 2. Flood slide with iodine and rinse 3. Decolorize and rinse 4. Flood slide with safranin and rinse 5. Blot slide 6. View slide under microscope

After you add crystal violet and rinse the slide, what should be your next step?

Add Gram's iodine. Gram's iodine serves as a mordant. Notice in the image below how it forms an insoluble complex with crystal violet. This helps the crystal violet remain in the gram-positive cells during decolorization.

What is the best procedure for decolorization?

Add decolorizing agent until run-off is clear. This method allows the decolorizing agent to dissolve the outer membrane of Gram-negative cells and rinse out the crystal violet from the thin layers of peptidoglycan. This causes the run-off to be purple. When that purple color ceases to appear in the run-off, decolorization should be stopped so as not to also cause the Gram-positive cells to lose their purple color. Watch the video to review the process of decolorization.

After Gram's iodine is added and then rinsed, what is the next step in the Gram stain procedure?

Add decolorizing agent. The decolorizing step removes the crystal violet from the gram-negative cell walls, but not the gram-positive walls. Watch the video to review the addition of decolorizing agent.

If you FORGOT to do the decolorizing step, what colors would the Gram-positive and Gram-negative cells be when viewed at the end of the procedure?

All would be purple. If the decolorization step were omitted, both types of cell walls would retain the primary stain, which is crystal violet. This micrograph shows Gram-positive cocci and Gram-negative bacilli that were Gram stained with the decolorizing step omitted. Note that both are the same color.

Which of the following describes the correct cellular morphology and Gram reaction for Staphylococcus aureus?

Clusters of gram-positive cocci

Which of the following represents an incorrect association regarding the reagents used during the Gram Staining procedure?

Counterstain = Methylene blue

Which of the following basic stains is used in the Gram staining procedure?

Crystal violet and safranin

Which of the following organisms is gram-negative?

Escherichia coli

Which of the following substances is used as a decolorizing agent in the Gram stain?

Ethyl alcohol

What is the purpose of using Gram's iodine during the Gram staining procedure?

Gram's iodine is a killing agent, binds to crystal violet, and serves as a mordant.

Which of the following statements is INCORRECT regarding the Gram staining procedure?

Gram-negative cells carry a net negative charge in their cell walls; gram-positive cells carry a net positive charge. True: The primary stain, crystal violet, is able to stain all bacterial cells. Gram staining separates cells based on the composition of their cell walls. The decolorizing agent is designed to remove the primary stain from only gram-negative cells.

Sort each of these characteristics as either belonging to Gram-positive or Gram-negative bacteria.

Gram-positive bacteria Crystal violet retained during decolorization Many layers of peptidoglycan Stain purple by Gram stain Gram-negative bacteria One or two layers of peptidoglycan Crystal violet NOT retained during decolorization Stain pink/red by Gram stain

Which of the following describes the correct cellular morphology and Gram reaction for Bacillus cereus?

Gram-positive rods

If you were staining Gram-positive bacilli and Gram-negative cocci, which of these micrographs would correctly represent the appearance of these cells after staining with crystal violet, rinsing, and blotting?

If you view the slide after the crystal violet step, you will have performed a simple stain on this smear. All cells would stain purple, as you will see in this video. The decolorization step is the step that rinses the crystal violet from the Gram-negative cell wall, but not the Gram-positive. This allows the differential staining of the Gram-negative cell wall with safranin.

As bacterial cells age, their peptidoglycan begins to break apart. What would be the effect on decolorization?

Old Gram-positive cells will be decolorized. Normally, Gram-positive cells aren't decolorized because they have many layers of peptidoglycan that hold the crystal violet/iodine complex during decolorization. However, old Gram-positive cells have less intact peptidoglycan, and the crystal violet/iodine complex is washed out more easily. These cells are indicated by the arrow in this micrograph.

Why is it recommended that Gram staining be performed on young bacterial cultures (not older than 24 hours)?

Over time, gram-positive cells lose their ability to retain the crystal violet stain after decolorization.

If you forget to add the decolorizing agent when performing a Gram stain, what color will the gram-negative cells be?

Purple

After you add crystal violet to the slide, what should be your next step?

Rinse with distilled water. For each reagent to work effectively, it is important to rinse the slide with water after each reagent has been applied. Watch the video to see the effect of rinsing the slide with water. If there is a large pool of excess crystal violet on the slide, then the molecules of the next reagent (Gram's iodine) will encounter and adhere to those crystal violet molecules and be less likely to reach the crystal violet within the cell.

How does safranin affect Gram-positive cells?

Safranin penetrates the cell wall, but is masked by the darker crystal violet stain. In the Gram-positive cell walls, most of the spaces between the molecules that make up peptidoglycan are already occupied by crystal violet/iodine complexes.

Assuming the Gram stain smear was blotted and observed following the decolorization step, which micrograph correctly illustrates how Gram-positive cocci and Gram-negative bacilli would appear?

The Gram-positive cells have thick walls, and they will retain the purple crystal violet stain if decolorization was carried out correctly. The Gram-negative cells will be decolorized and are colorless. Watch the video to review the effect of decolorization on both types of cells.

Why is it important that crystal violet be a contrasting color to safranin?

The contrast allows you to differentiate between gram-negative and gram-positive cells by observing the color of the organism.

What is the most critical step in the Gram staining procedure?

The decolorization step

What happens to the Gram-positive cell wall during decolorization?

The decolorizing agent dehydrates the peptidoglycan. Removing water from (or dehydrating) the peptidoglycan allows the decolorizing agent to shrink the spaces through which the crystal violet-iodine complexes might be able to pass. This makes it more difficult for the purple stain to be removed. Watch the video to view the effect of decolorization on the Gram-positive wall below.

What happens to the Gram-negative cell wall during decolorization?

The decolorizing agent dissolves the outer membrane. The decolorizing agent dissolves the outer membrane of Gram-negative cells. This allows the decolorizing agent to penetrate the thin layers of peptidoglycan and rinse away the crystal violet/iodine complexes. Watch the video to review the effect of decolorization on the Gram-negative cell wall.

What is the difference between doing a simple stain versus a differential stain on a bacterial slide?

The simple will only tell you morphological shape and size, while the differential stain will allow for determination of size, shape, and type of the cell wall.

True or false: The Gram stain differentiates between bacteria based on the thickness of their peptidoglycan layer.

True

A fellow student is unsure why his gram-negative Escherichia coli is purple. Upon examining the Gram stain kit, you notice it didn't have alcohol. What caused the bacteria to be purple instead of pink?

Without alcohol during the decolorizing step, the bacterial cell wall was not altered, which prevented the Gram stain's iodine and crystal violet from being rinsed away.

You performed the Gram stain. You are expecting to find purple Gram-positive cocci and pink Gram-negative bacilli. Instead, you observe purple cocci but don't seem to see any bacilli. How could you explain this?

You forgot the safranin step. Safranin is used to color the Gram-negative bacteria that have been decolorized in the previous step. At the beginning of this video, you will note that the Gram-negative cells have no color. If the safranin step is omitted, then the Gram-negative cells will be colorless and difficult to see.

You performed the Gram stain on a smear. You are expecting to find purple Gram-positive bacilli and pink Gram-negative cocci. Instead, you observe pink bacilli and pink cocci. Which of the following is NOT a possible explanation for this unusual result?

You skipped the safranin step. If the safranin step were skipped, none of the cells would take up a pink color. This would not explain why the Gram-positive cells were pink instead of purple.

If you forgot to add the counterstain to the mixed culture sample at the end of the staining procedure, gram-negative cells would appear __________ and gram-positive cells would appear __________.

colorless; purple

The Gram stain is an example of a __________ stain, because the process uses two contrasting stains to separate bacteria into groups based on cell wall composition.

differential

While gram-positive cells have a thick layer of __________ in their cell walls, gram-negative cells have a thin layer, surrounded by an additional lipopolysaccharide layer.

peptidoglycan

After performing a Gram stain, gram-negative cells appear __________, while gram-positive cells appear __________.

pink or red; purple