Heat Fixed and Gram Stained Bacterial Smear with Positive and Negative Controls. Unknown in Center.

How to Do a Gram Stain 
Test for Gram+ & Gram- Bacteria Identification

LAB NOTES from Science Prof Online


Bacterial Cell Wall: Peptidoglycan Structure and Function
This rigid structure of peptidoglycan gives the bacterial cell shape, surrounds the plasma membrane and provides prokaryotes with protection from their environment. Peptidoglycan is a huge polymer of interlocking chains of identical monomers connected by interpeptide bridges.

From the peptidoglycan inwards all bacterial cells are very similar. Going further out, the bacterial world divides into two major classes: Gram positive (Gram+) and Gram negative (Gram-).

Gram-positive Cells: In Gram-positive bacterial cells, peptidoglycan makes up as much as 90% of the thick, compact cell wall, which is the outermost cell wall structure of Gram+ cells.

Gram-negative Cells: The cell walls of Gram-negative bacteria are more chemically complex, thinner and less compact. Peptidoglycan makes up only 5 – 20% of the cell wall, and is not the outermost layer, but lies between the plasma membrane and an outer membrane. This outer membrane is similar to the plasma membrane, but is less permeable and composed of lipopolysaccharides (LPS), a harmful substance classified as an endotoxin.

Gram Staining Procedure
Because most bacteria have one of these two types of cell walls, we can use this difference as a feature that can be identified using the Gram stain. The Gram stain is a differential stain that uses two dyes to differentiate between the two basic bacterial cell wall types.

First a bacterial smear must be heat fixed to a microscope slide. A smear is a sample of bacteria suspended in a small amount of water on a slide. That sample is then dried using heat. The heat kills the bacteria and attaches the sample to the slide so that it does not easily wash away.

Bacterial Smear and Gram Stain Videos
Article Summary: Gram staining involves the application of a series of dyes that leaves some bacteria purple (Gram +) and others pink (Gram -). Here's how the Gram stain works.
Gram Stain for Identifying Gram +/- Bacteria

​Page last updated: 9/2014
More Images of Gram-stained Bacteria Button
Gram Stain Reaction Explained
  • Primary stain: The blue-violet appearance of Gram-positive cells is caused by crystal violet, the primary (first) stain. 
  • Iodine mordant: Crystal violet binds to the iodine mordant causing this large molecule to become trapped in the layers of peptidoglycan of Gram+ cells. 
  • Decolorizer: This step does not remove the crystal-violet stain trapped within the Gram-positive cell wall, but does remove the stain from the thin layer of peptidoglycan of Gram-negative bacteria.
  • Secondary stain (counterstain): The safrinin counterstain imparts pink color to the colorless Gram- bacteria, but doesn't change the color of Gram+ cells.​

Gram Positive Bacteria, Staph. epidermidis @1000xTM
Gram-positive Staphylococcus epidermidis @ 1000xTM+
Comparison of Gram-positive and Gram-negative Cell Wall,  by Graevemoore on Wikipedia
Application of Primary Stain: 1. Heat fixed bacterial smear, Gram-positive control on left, Gram-negative control on right, and unknown bacteria in center; 2 & 3. Flood the slide with primary stain, crystal violet stain; 4. Rinse after 1 min.

Application of Mordant: 1 & 2. Next apply the iodine mordant to the slide; 3. Leave on for 1 minute; 4.Rinse.

Application of Decolorizer:  
1. Decolorizer, Acetone Alcohol, used in Gram-stain; 2. Hold slide at angle and run a stream of decolorizer over slide for 10 to 15 seconds, until negative control loses color; 3. Rinse. 

Application of Counterstain: 1. Safranin secondary stain; 2 & 3. Apply safranin counterstain to slide and leave on for 1 minute; 4. Rinse; 5. Gram-stained slide with positive control on left, negative control on right and unknown bacteria in center. Click here for more Gramstain photos.

Gram Negative Bacteria, E. coli @ 1000xTM
Gram-negative Escherichia coli @ 1000xTM
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How to Prepare a Bacterial Smear for Gram Staining
Virtual Microbiology

You have free access to a large collection of materials used in a college-level introductory microbiology course. The Virtual Microbiology Classroom provides a wide range of free educational resources including PowerPoint Lectures, Study Guides, Review Questions and Practice Test Questions.
Prokaryotic Cell, Mariana Ruiz
Steps of Gram Stain Procedure
The Gram staining procedure is as follows (see photos of steps below):
  • Start with heat-fixed bacterial smear
  • Apply primary stain: Flood slide with crystal violet stain.
  • Rinse: After 1 minute, rinse the slide with water.
  • Apply mordant: Flood the slide with iodine.
  • Rinse: After 1 minute, rinse the slide with water.
  • Apply decolorizer: Flood slide with acetone alcohol.
  • After 10 or 15 seconds, rinse the slide with water. (Do not leave the decolorizer on too long or it may remove stain from the Gram-positive cells as well.)
  • Apply secondary stain (counterstain): Flood slide with safrinin.
  • Rinse: After 1 minute, rinse the slide with water.

Photographic Guide to Completing Gram Stain
Double click on photo strip for a slideshow of larger images.

How to Do a Gram Stain
Instructor's Corner
In the 1800’s, Christian Gram, a Danish bacteriologist, developed a technique for staining bacteria that is still widely used today.

The Gram stain protocol involves the application of a series of dyes that results in some bacteria staining purple and others pink. Bacteria that stain purple are considered Gram-positive, and those that stain pink, Gram-negative. The specific stain reaction of a bacterium results from the structure of the bacterial cell wall.

Additional Gram Staining Resources
  • Gram Stain Bite Sized Tutorial: This is an extremely useful tutorial that shows, step-by-step, the Gram-staining procedure and the appearance of Gram+ and Gram- bacterial cells.

Photographic guides to differential stains 
now available!
1. Gram
2. Acid-fast
3. Endopsore

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