1. Gram Stain Watch the Gram Staining Video at
To see the process of Gram Staining.
You ARE responsible for everything in this power point as well as the video for the
upcoming test!
Principles of Biomedical Science
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2. Two Categories of Bacteria
A Gram stain is usually one of the first steps in identifying bacteria. In addition to the gram stain, the morphology, or structure, of the bacteria is also observed. You have learned that are three basic morphologies of bacteria (based on the shape of a single cell): bacillus (rod), coccus (sphere), and spirillum (spiral).
All bacteria are divided into two main groups, depending on how they react to a specific set of dyes called the Gram stain. A microbiologist named Hans Christian Gram developed the staining protocol in the 1880s, and it remains one of the first steps in classifying or identifying bacteria. If the bacteria appear purple after being treated with the stain, they are classified as Gram positive. The bacteria are considered to be Gram negative if they appear pink.
Gram Negative
Have a very thin cell wall
Antibiotics are NOT very effective against Gram Negative because there is not enough cell wall for the antibiotic to target
Gram Positive
Have a thick Cell Wall
Antibiotics ARE effective against Gram Positive because they target the cell wall and attack and destroy the bacteria

3. Gram Negative vs. Gram Positive Bacteria
Gram positive bacteria differ from Gram negative bacteria in the structure of their cell walls. The cell walls of Gram positive bacteria are made up of 20x more peptidoglycan, a polymer made of sugar and amino acids.

4. Gram Stain Process Gram Positive Bacteria Gram Negative Bacteria
Presentation Name
Gram Stain Process
Course Name
Unit # – Lesson #.# – Lesson Name
Gram Positive Bacteria
Step 1: Heat fix the slide so that that bacterial cells are fixed to the slide. (This means passing the slide over a flame to make the bacteria stick to the slide permanently.)
Step 2: Flood the slide with crystal violet dye.
The individual crystal violet ions penetrate the thick peptidoglycan layer of the cell as well as the plasma membrane.
Gram Negative Bacteria
Step 1: Heat fix the slide so that that bacterial cells are fixed to the slide. (This means passing the slide over a flame to make the bacteria stick to the slide permanently.)
Step 2: Flood the slide with crystal violet dye.
The individual crystal violet ions penetrate the thin peptidoglycan layer of the cell as well as the plasma membrane.
Note: the Gram staining procedure is the same for all cells, it is just broken down here to show you how it works in both cases.

5. Gram Stain Process Continued
Gram Positive Bacteria
Step 3: Rinse with water.
Step 4: Flood the slide with iodine.
The iodine ions penetrate the cell wall and bind with the crystal violet.
The iodine ions and crystal violet react, forming a crystal violet-iodine complex that is a very large molecule and is insoluble in water.
Gram Negative Bacteria
Step 3: Rinse with water.
Step 4: Flood the slide with iodine.
The iodine ions penetrate the cell wall and bind with the crystal violet.
The iodine ions and crystal violet react, forming a crystal violet-iodine complex that is a very large molecule and is insoluble in water.

6. Gram Stain Process Continued
Gram Positive Bacteria
Step 5: Rinse with water.
Step 6: Decolorize the slide with alcohol.
The alcohol causes water to leave the cell wall. Because of its larger size, the crystal violet-iodine complex is blocked from moving easily through the cell wall and thus is prevented from leaving the cell.
Gram Negative Bacteria
Step 5: Rinse with water.
Step 6: Decolorize the slide with alcohol.
The alcohol disrupts and dissolves the outer membrane. Therefore the big crystal violet-iodine complex is able to leave the cell.

7. Gram Stain Process Continued
Gram Negative Bacteria
Step 7: Rinse with water.
The crystal violet-iodine complex is washed away, leaving colorless, unstained cells.
Step 8: Flood the slide with Safranin dye.
Gram Positive Bacteria
Step 7: Rinse with water.
The crystal violet-iodine complex is still stuck inside the cell. The cell is therefore stained purple.
Step 8: Flood the slide with Safranin dye.

8. Gram Stain Process Continued
Gram Positive Bacteria
Safranin penetrates the cell, but since it is a lighter color than the crystal violet-iodine complex, it is not visualized over the purple color of the crystal violet-iodine.
The cells appear purple.
Antibiotics will work!
Gram Negative Bacteria
Safranin penetrates the cell and stains the cell a reddish-pink color.
The cells appear reddish-pink.
Antibiotics will be minimally effective.