Bio 16 Gram 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.

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.

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.

Gram - Negitive

Crystal violet NOT retained, few layers of peptidoglycan stained pink by gram stain

Gram - Postitive

Crystal violet retained, many layers of Peptidoglycan, stained purple by Gram Stain

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. 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.

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. 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 not enough of it is retained to cause a color change......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. although safranin may occupy some of the remaining spaces, the overall effect is still purple.

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.

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.

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.

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, 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.