GRAM STAINING

OBJECTIVES
Explain the theory behind the Gram stain chemical
reaction.
Perform and interpret Gram stains.
Describe the consequences of specific mistakes in the
Gram stain technique, and be able to recognize likely
sources of error; recognize if and why a bacterial slide
was incorrectly Gram stained.
Properly use the oil immersion lens.
Characterize and identify bacteria based on their cell
morphology and arrangement.

GRAM STAIN

The gram stain is a differential stain that

classifies bacteria based on the physical and
chemical properties of their cell wall
structures.
The name comes from its inventor, Hans
Christian Gram.
Gram (+) Bacteria turn Purple.
Gram (-) Bacteria turn Red.

GRAM STAIN RESULTS

Gram
(+)

http://www.standardsingenomics.org/index.php/sigen/article/view
File/417/944/8518

Gram (-)

http://people.upei.ca/jlewis/html/lab_1.html

GRAM POSITIVE &

NEGATIVE STRUCTURES

http://www.pc.maricopa.edu/Biology/rcotter/BIO%20205/LessonBuilders/Chapter%204%20LB/Ch4Lessonbuilder7.html

GRAM NEGATIVE

Important Features:
Outer Membrane
Cell Wall: Thin Peptidoglycan
Periplasmic Space
Cell Membrane

http://classes.midlandstech.edu/carterp/Courses/bio225/chap04/ss4.htm

GRAM POSITIVE

Important Features:
Cell wall: Thick Peptidoglycan
Cell Membrane
No Outer Membrane
No Periplasmic Space

http://classes.midlandstech.edu/carterp/Courses/bio225/chap04/ss4.htm

GRAM STAINING PROCEDURE

1.Prepare Smear and Heat Fix

2.Basic Stain with Crystal Violet: 1 min
3.Wash: ~3 sec distilled water
4.Mordant - Grams Iodine: 1 min
5.Decolorizer: alcohol 6 sec (Colorless)
6.Wash: ~3 sec distilled water
7.Counterstain: Safranin 1 min
8.Wash: ~3 sec distilled water
9.Blot Dry: Bibulous paper, Air dry

GRAM STAINING PROCEDURE

http://biology200.gsu.edu/houghton/4595%20'04/lecture1.html

CRYSTAL VIOLET-IODINE
COMPLEX

In the gram staining procedure, Grams

Iodine acts as the mordant and forms a
complex with the crystal violet dye.
Gram (+): Holds the Crystal Violet-Iodine
complex (CV-I) during decolorization.
Gram (-): Does not hold the CV-I complex
during decolorization.

GRAM STAINING ERRORS

What if we forgot
Decolorizer?
Both gram positive and gram negative bacteria
will appear purple due to gram negative
bacteria not being decolorized.

What if we forgot Iodine?

Both gram positive and gram negative bacteria
will appear pink due to the crystal violet
complex not forming and crystal violet being
washed out.

GRAM STAINING ERRORS

What if we excessively
apply heat during fixation?
Heat fixing the cells, when done to excess,
alters the cell morphology and makes the cells
more easily decolorized.

What if we excessively
counterstain?
As the counterstain is also a basic dye, it is
possible to replace the crystal violetiodine
complex in gram- positive cells with an overexposure to the counterstain.

GRAM STAINING ERRORS

What if we excessively
decolorized?
If ethanol is applied excessively, all bacteria in
a sample will appear pink due to the counter
stain Safranin.