Introduction: 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's important to know the difference between gram positive and gram negative bacteria. It tells you one of the things you need to know to find out how to treat a patient or an animal. You do not to give the patient or the animal to wrong antibiotic because the bacteria could gain antibiotic resistance. You also need to know how long you need to give the antibiotic, so they do not get sick again. There are five things you will need which are crystal violet, Gram's iodine, 95% ethanol, safranin and distilled (D.I.) water.

Step 1: Crystal Violet

First, you get a slide with heat fixed bacteria. You have to heat-fix the bacteria, so the bacteria sticks to the slide, it preserves the slide, and it kills the bacteria. Crystal violet is the primary stain which is the first stain you apply to the slide.

Apply the crystal violet for 1 minute. This will stain the cells purple.

Wash with D.I. water.

Step 2: Gram's Iodine

This is the mordant. A mordant will intensify the stain on the bacteria that are gram positive.

Apply Gram's iodine for 1 minute.

Wash with D.I. water.

Step 3: 95% Ethanol

This is the decolorizer. A decolorizer determines if the bacteria will stay purple or the color will wash off the slide. You do not want to have this on the slide for too long otherwise bacteria that should remain purple will lose its' color as well. If you do not leave the ethanol on the slide long enough then all the bacteria will be purple.

Apply the 95% ethanol for 3-5 seconds.

Wash with D.I. water.When the slide does not look oily anymore then you rinse all the ethanol off the slide.

Step 4: Safranin

This is the counterstain. This will turn the colorless bacteria pink.

Apply the safranin for 1 minute.

Wash with D.I. water.

Step 5: Looking at the Results

Use the Bibulous Paper to dry the slide. Bibulous Paper is just a special type of paper that dries a slide without removing bacteria from the slide.

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