Gram Stain
Abstract
Simple staining depends on the fact that bacteria di er chemically from their surroundings,
meaning that they can be stained to contrast with their environment. Also, bacteria di er
from one another chemically and physically and may react di erently to a given staining
procedure. Di erential staining can distinguish between the types of bacteria. The Gram
stain is the most useful di erential stain in bacteriology, which is named after Christian
Gram, the Danish scientist. The Gram stain divides most bacteria, except archaea, into two
groups: Gram-positive and Gram-negative.
Introduction
The foundation or basis of the Gram stain is that gram-positive bacteria retain the color of
the primary dye, but the gram-negative bacteria lose the primary dye (crystal violet) when
washed in a decolorizing solution and then take on the color of the second dye (the
counterstain safranin). In this experiment, the bacteria are stained with the basic dye
crystal violet and treated with the iodine solution that functions as a mordant. The mordant
increases the interaction between the bacterial cell and the dye that causes the dye to
form large complexes in the peptidoglycan meshwork of the bacterial wall. Due to the
peptidoglycan layer in gram-positive bacteria being thicker, the entrapment of the dye is far
more extensive in them than in gram-negative bacteria.
The Gram stain does not always yield clear results, so it is best to Gram stain young,
vigorous cultures rather than older ones. Also, some bacteria are gram variable.
Materials
Staphylococcus aureus Escherichia coli Crystal violet
Safranin Glass slides Gloves
Inoculating loop Bunsen burner Bibulous paper
Microscope Lens paper and lens cleaner Immersion oil
Staining rack Latex gloves
Procedure
1. Prepare heat-fixed smears of E. coli, S. aureus, and the mixture of E. coli and S. aureus.
2. Place the slides on the staining rack.
3. Pour crystal violet on the smears and let it stand for 30 seconds.
Abstract
Simple staining depends on the fact that bacteria di er chemically from their surroundings,
meaning that they can be stained to contrast with their environment. Also, bacteria di er
from one another chemically and physically and may react di erently to a given staining
procedure. Di erential staining can distinguish between the types of bacteria. The Gram
stain is the most useful di erential stain in bacteriology, which is named after Christian
Gram, the Danish scientist. The Gram stain divides most bacteria, except archaea, into two
groups: Gram-positive and Gram-negative.
Introduction
The foundation or basis of the Gram stain is that gram-positive bacteria retain the color of
the primary dye, but the gram-negative bacteria lose the primary dye (crystal violet) when
washed in a decolorizing solution and then take on the color of the second dye (the
counterstain safranin). In this experiment, the bacteria are stained with the basic dye
crystal violet and treated with the iodine solution that functions as a mordant. The mordant
increases the interaction between the bacterial cell and the dye that causes the dye to
form large complexes in the peptidoglycan meshwork of the bacterial wall. Due to the
peptidoglycan layer in gram-positive bacteria being thicker, the entrapment of the dye is far
more extensive in them than in gram-negative bacteria.
The Gram stain does not always yield clear results, so it is best to Gram stain young,
vigorous cultures rather than older ones. Also, some bacteria are gram variable.
Materials
Staphylococcus aureus Escherichia coli Crystal violet
Safranin Glass slides Gloves
Inoculating loop Bunsen burner Bibulous paper
Microscope Lens paper and lens cleaner Immersion oil
Staining rack Latex gloves
Procedure
1. Prepare heat-fixed smears of E. coli, S. aureus, and the mixture of E. coli and S. aureus.
2. Place the slides on the staining rack.
3. Pour crystal violet on the smears and let it stand for 30 seconds.
