BIO 3305 Fall 2021
Lab 7 Part 2: Gram Stain
The Gram stain is a differential stain in which a decolorization step occurs between the application of
two basic stains. The primary stain is Crystal Violet. This is followed by Iodine, which acts as a mordant. A
mordant is a chemical that is added to a stain to help fix the stain within the organism. In this case, Iodine
enhances the effect of the Crystal Violet dye by forming a crystal violet-iodine complex. Decolorization
follows and is the most critical step in the procedure. Gram-negative cells are decolorized by the alcohol
solution whereas Gram-positive cells are not. Gram-negative cells are thus able to be colorized by the
counterstain safranin. Upon successful completion of the Gram Stain procedure, Gram-Positive cells appear
purple and Gram-Negative cells appear pink/red.
It is possible to over-decolorize by leaving the alcohol on too long, causing Gram-positive cells to
appear reddish. You can also under-decolorize and produce purple Gram-negative cells. Neither of these
changes the actual gram reaction for the organism being stained they are simply false results due to poor
technique.
The difference in results of the Gram Stain is due to the differences of the cell wall of these two types
of bacteria. The lipopolysaccharide layer in Gram-negative organisms is partially dissolved by the alcohol,
making this cell wall more porous and incapable of hanging on to the violet-iodine complex. Thus is it
decolorized. In the Gram-positive organisms, the alcohol actually makes the cell wall close up, allowing the
Gram-positive organisms to keep the violet-iodine complex within. A picture of a gram stain of both types of
organisms is shown.
APPLICATION:
In this exercise, the Gram stain will be performed on two different organisms. One will be Gram-positive and
the other will be Gram-negative. This is typically the first test performed on unknown organisms to help
differentiate and identify the specimen. So, if you get mixed results, you may want to redo the test until you
are confident that you have the procedure down. This will be an important first step when you begin
identification of your unknowns.
MATERIALS:
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Gram Staining Kit (Gram crystal violet, Gram iodine, ethanol, Gram safranin)
Squirt bottle with DI water
Staining tray
Inoculating loop
Bunsen burner
Bibulous paper
Microscope slides
Disposable gloves
Compound light microscope
Immersion oil
Lens paper
BIO 3305 Fall 2021
Organisms:
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Environmental isolate
Bacillus cereus (Gram + rod)
 Serratia marcescens (Gram – rod)
PROCEDURE:
(Gram Stain Procedure – Simplified)
1) Begin with a heat-fixed emulsion of the organism
2) Cover dried emulsion with Crystal Violet stain. Let stand 1 minute.
a. Use the staining tray to catch excess stain.
b. Wear gloves.
3) Rinse with DI water.
4) Cover dried emulsion with Iodine. Let stand 1 minute.
5) Decolorize by rinsing with ETOH (ethyl alcohol)
6) Counterstain with Safranin. Let stand 1 minute.
7) Rinse with DI water.
8) Gently Blot dry with bibulous paper.
9) When dry observe under oil immersion with the microscope.
RESULTS:
1) Record the Gram stain and morphology of the organisms
a. Gram negative = pink/red or Gram positive = purple
b. Gram bacilli = rods or cocci = spheres
c. Also note if the organisms are in chains or clusters.