Final Instructable: How to Do a Gram Stain

In microbiology, Gram stains are a common procedure. It was developed in 1884 by Christian Gram. A Gram stain is used to differentiate between gram-positive and gram-negative bacteria. It is important to know the difference between gram-positive and gram-negative bacteria. It can tell you one of the many different things you need to know in order to find out how to treat your patient or your animal. You do not want to give your patient or animal the wrong antibiotic because the bacteria could gain antibiotic resistance. If the bacteria becomes resistant then it becomes harder to treat. You also need to know how long you have to give the antibiotic to your patient or animal, so they do not get sick again.

There are five things you will need in order to do a Gram stain which includes crystal violet (primary stain), Gram's iodine (mordant), 95% ethanol (decolorizer), safranin (secondary stain) and distilled (D.I) water. The primary stain, crystal violet, is the first dye that you use on your slide of bacteria and the cells turn purple. The mordant, Gram's iodine, is used to intensify the color of the cells by combining with the crystal violet. The decolorizer, 95% ethanol, determines what your slide looks like at the end of the Gram stain. If you leave it on too long, all the cells lose their color. If you don't leave it on long enough then all the cells remain purple. The secondary stain, safranin, is the counterstain that will stain the colorless cells pink. Distilled water is water that doesn't have any extra ions in it. You use the D.I. water to rinse off each of the different dyes you put on your slide.

Step 1: Crystal Violet

First, you get a slide with heat-fixed bacteria. You heat-fix the bacteria by taking a sample of the bacteria you have and mixing it with a droplet of D.I. water on a slide. Let the slide dry and then heat the slide for about 20-30 seconds, so the bacteria will stick to the slide, you can preserve the slide to look at it later and it kills that bacteria. Crystal violet, the primary stain, is applied to your slide first.

Apply enough crystal violet to cover the bacterial smear on your slide for 1 minute. This will stain the cells purple.

Rinse your slide with D.I. water.

Step 2: Gram's Iodine

Gram's iodine is the mordant in the Gram staining process. The mordant will intensify the stain on the bacteria.

Apply enough Gram's iodine to cover the bacterial smear on your slide for 1 minute.

Rinse your slide with D.I. water.

Step 3: 95% Ethanol

95% ethanol is the decolorizer in the Gram staining process. The decolorizer determines if the bacteria will remain purple or if the color will wash off your slide.

Apply just enough of the 95% ethanol over the entire bacterial smear on your slide for 3-5 seconds.

Rinse your slide with D.I. water. When the slide doesn't look oily anymore then you have rinsed all the ethanol off your slide.

Step 4: Safranin

Safranin is the secondary stain in the Gram staining process. This is the counterstain that will turn the colorless bacteria cells pink.

Apply just enough safranin to cover the bacterial smear for 1 minute.

Rinse your slide with D.I. water

Step 5: Looking at the Results

Use the Bibulous Paper to dry the slide. Open the booklet and place your slide inside. Then close the booklet and lightly press down on it to dry the slide. Bibulous Paper is just a special type of paper that dries your slide without removing your bacteria from the slide like a paper towel would.

After you dry the slide, you are now ready to look at your slide under the microscope.

Step 6: Interpreting Your Results

The purple colored bacteria are gram-positive bacteria. Gram-positive bacteria have a thick peptidoglycan layer than absorbs the crystal violet. Peptidoglycan is a phospholipid bilayer with sugars and amino acids that forms a cell wall to protect the bacteria. The Gram's iodine makes the peptidoglycan layer thicker, so that ethanol has a hard time removing the color from the bacteria. This means the bacteria stays purple.

The pink colored bacteria are gram-negative. Gram-negative bacteria have a outer membrane that surrounds the thin peptidoglycan layer. When crystal violet is added, it doesn't have the peptidogylcan layer to absorb it. The Gram's iodine has nothing to make the cell wall thicker, so when the ethanol is added the outer membrane and the crystal violet with Gram's iodine is washed off your slide. The cell is now open to absorb a stain and when you use the safranin, it turns the bacteria pink.