Laboratory Activity 4

Gram Staining

Differential staining requires the use of at least three chemical reagents that are applied sequentially to a heat-
fixed smear. The first reagent is called primary stain. Its function is to import its color to all cells. In order to establish a
color contrast, decolorizing agent is used. Based on the composition of cellular components, the decolorizing may or
may not remove the primary stain. If the primary stain is removed, the decolorized cellular components will accept and
assume the color of the counterstain. In this way, cell types or their structures can be distinguished from each other on
the basis of the stain that is retained.

The most important differential stain used in bacteriology is the Gram stain, named after Dr. Christian Gram. It
divides the bacteria into two major groups: gram positive and gram negative, which makes it an essential tool for
classification and differentiation of microorganisms. The gram stain reaction is based on the difference in the chemical
composition of bacterial cell walls. Gram positive cells have thick peptidoglycan layer, whereas the peptidoglycan layer
in gram negative cells is much thinner and surrounded by outer lipid-containing layers.

Objectives: At the of this activity you should be able to:

1. Perform Gram staining of bacterial smears.
2. Differentiate gram negative from gram positive bacteria.
Materials:
Bacterial cultures of E. coli and S. aureus.
Reagents:
Crystal violet solution
Acetone / Acid Alcohol
Gram’s iodine
Safranin solution
Microscope

Procedure:

1. Obtain 3 clean glass slides

2. Using aseptic technique, prepare three smears for each organism.
3. Allow smears to air, then heat fix.
4. Gently flood smears with crystal violet and stand for 1 minute
5. Wash excess stain with water until no more stain comes off.
6. Put a few drops of Gram’s iodine over the smear and let it stand for 1 minute.
7. Wash off iodine with water.
8. Flood smear with 95% alcohol or acid-alcohol and let it stand for 15 -30 seconds. Repeat step until no more color
comes with the alcohol
9. Wash with water.
10. Counter-stain with safranin for 1 minute.
11. Air-dry and examine in the microscope using oil immersion objective.
 Laboratory Activity 4
GRAM STAINING
Name: _______________________________Yr& Section: ______Grp No. _________Date:__________ Score: ________
I. Microscopic observation.

II. indicate the Shape, Arrangement, Color, and Gram staining reaction of each bacteria.

III. Indicate the function of the different reagents used in Gram-Staining.

Expected Results
Reagent Function Gram-positive Gram-negative
Crystal Violet

Gram’s Iodine

Acid-alcohol

Safranin

IV. Differentiate Gram-positive cell wall from Gram-negative cell wall.

A. Peptidoglycan
B. Complexity
C. Teichoic acid
D. Lipopolysaccharide complexes
E. Endotoxin