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Gram Stain for Identifying Bacteria - P2
SCIENCE PHOTOS
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
Escherichia coli @ 1000xTM
SPO VIRTUAL CLASSROOMS
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.
SPO VIDEO:
How to Do a Gram Stain
- 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.
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.
Page last updated: 11/2015
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- 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.
- Gently blot the slide dry. It is now ready to be viewed under oil immersion (1000x TM) with a bright-field compound microscope.
Photographic Guide to Completing Gram Stain
Double click on photo strip for a slideshow of larger images.
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