Microlab Tutor Video Transcript

This is a transcript of the video on the Educator Features page.

The Gram stain is used to stain bacteria as well as distinguish gram-positive from gram-negative cells.

This stain distinguishes bacteria based on their cell wall.

Bacterial cells are stained in the Gram stain and then ethanol is used to attempt to decolorize or remove the stain from the cells.

Gram staining is most consistent on cells less than 24 hours old, because young cells are less likely to have cell wall damage.

Remember to follow all safety instructions provided by your instructor while you're in the lab.

Draw two dime sized circles on the back of the slide and label each circle.

Prepare a smear.

Flame your loop until it is red-hot and let it cool.

Pass the tube through the flame and use the loop to obtain the broth culture.

Pass the tube through the flame again before recapping the tube.

Then spread the culture on the first circle on the slide.

Repeat this process so you have two or three loops of bacteria on the slide.

We'll make two smears on the slide.

Your instructor may have different procedures regarding the number of smears on a slide.

Follow the procedures used in your lab.

Repeat the process to make a smear in the second circle on the slide.

Don't forget to flame the loop and the mouth of the tube between steps.

Let the smear air dry.

Your instructor may have you use a slide warmer to speed the process.

Make sure the smear is completely dry then heat fix the smear by passing the slide through the flame two or three times.

Cover the smear with crystal violet for 30 seconds.

The exact time for each step will vary with the reagents used in your lab.

Wash the slide carefully with distilled water.

Tilt the slide and aim the water above the smear.

Let the water wash over the smear.

Let's look at what happens at the molecular level when crystal violet is added to the smear.

Crystal violet penetrates the cell wall and cytoplasm of both gram-positive and gram-negative bacteria.

Both types of bacteria stained purple at this stage.

Cover the smear with Grams iodine for 10 seconds to 1 minute.

Your instructor will advise you on the required time based on the reagents used in your lab.

Wash the slide by letting distilled water wash over the smear.

Wash the slide until the water runs clear.

Let's look at what happens at the molecular level when Graham's iodine is added to the smear.

Graham's iodine is a mordant that forms a larger complex with the crystal violet making it more difficult to wash out.

At this stage the gram-positive cell would still appear purple under the microscope.

When Graham's iodine is added to the gram-negative cell, it also forms a larger complex with the crystal violet resulting in Graham's iodine having the same effect in the gram-negative cell.

At the end of this step both cell types still appear purple.

To decolorize let ethanol run through the smear until no large amounts of purple wash out.

Be careful not to over decolorize as this can rinse the crystal violet from gram-positive bacteria giving false results.

Wash the slide by letting distilled water wash over the smear.

wash the slide until the water runs clear.

Let's see what happens to the cell wall during decolorization.

In the gram-positive cell the decolorizing agent causes the peptidoglycan molecules to be more tightly connected making it more difficult for the crystal Violet iodine complexes to move through.

The decolorizing agent dissolves the cytoplasmic membrane causing the dye to wash out of the cytoplasm toward the wall.

However, the dye cannot easily pass through the smaller spaces in the multiple layers of peptidoglycan and gets further trapped by two types of connecting amino acid chains in the gram-positive wall.

After decolorization the gram-positive cell remains purple.

In the gram-negative cell the decolorizing agent dissolves the outer membrane, passes through and dehydrates the thin peptidoglycan layer, and begins to dissolve the cytoplasmic membrane.

Because the gram-negative cell has little peptidoglycan and fewer connecting amino acids to trap the crystal violet complexes, the D colorizer easily washes the complexes away.

Thus the gram-negative cell is no longer dyed purple.

After decolorization gram positive cells retain the crystal violet, while gram-negative cells lose the die and become colorless.

Cover the smear with safranin for 30 seconds.

Wash the slide with distilled water until the water runs clear.

When added to the gram-positive cell, which is already dyed a dark purple, the light red saffron and dye has no effect on the visible color. Thus at the end of the Gram stain, the gram positive cells are purple.

This is not the case with gram negatives.

because the purple dye has been removed by decolorization the red safranin colors the wall and cytoplasm.

The thin peptidoglycan of gram-negative cells allows them to be decolorized and counterstained red to pink with safranin.

The gram-positive cell walls have many layers of peptidoglycan tightly linked by connecting chains of amino acids which help them to retain the crystal violet color after decolorization.

Thus the Gram stain procedure makes it easy to distinguish gram positive cells from gram negative cells.

Use a kim wipe or bibulous paper to block the slide dry.

Do not wipe.

Just gently blot the slide.

As you observe this slide under the microscope note the purple appearance of these gram-positive cells.

You can also determine cell morphology and arrangement by observing the stained cells.

These are gram-positive rods in chains.

In this photo micrograph note the red appearance of the gram-negative cocci in pairs.

The gram-negative cells have been decolorized and counterstained by the safranin.

When you finished observing your results gently blot oil from the slide and store it in a slide box.