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

Gram Stain for Identifying Bacteria - P2
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.
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. More Gram stain photos.

Gram Negative Bacteria, E. coli @ 1000xTM
Escherichia coli @ 1000xTM
Gram Stain Procedure
The steps of Gram staining are 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.

How to Do a Gram Stain

Back to PAGE 1
  • Secondary stain (counterstain): The safrinin counterstain imparts pink color to the colorless Gram- bacteria, but doesn't change the color of Gram+ cells.​

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

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

Page last updated: 11/2015

PAGE 2                                                <  Back to Page 1
End of Article    
How to Make a Bacterial Smear 
<  Back to Page 1
  • 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.

Instructors Corner
 of Science Prof Online

You have access to a large collection of free science teaching materials used in high school and college-level introductory science courses. The SPO Instructors Corner has resources for teaching general science, anatomy & physiology, chemistry, cell biology genetics, immunology and microbiology.
Instructors Corner of Science Prof Online
from the free STEM 
education site 
Science Prof Online
SPO is a FREE science education website. Donations are key in helping us provide this resource with fewer ads. 
Please help!

(This donation link uses PayPal on a secure connection.)