Exp#5 Gram Staining

EXPERIMENT 10
GRAM STAIN TECHNIQUE
A. PURPOSE
• To determine bacterial morphology and chemical structure of bacterial cell wall

• To examine chemical and structural differences of cell walls of Gram positive and Gram
negative bacteria, in other words, to understand how the Gram stain reaction influences
Gram positive and Gram negative bacteria based on the biochemical and structural
differences of their cell walls.
B. THEORY
Staining is a supplementary technique used in microscopic techniques used to improve the

clarity of the microscopic image. Stains and dyes widely used in the scientific field to highlight
the structure of the biological specimens, cells, tissues etc. The most widely used staining
procedure in microbiology is the Gram stain, discovered by the Danish scientist and physician
Hans Christian Joachim Gram in 1884. Gram staining is a differential staining technique that
differentiates bacteria into two groups: gram-positives and gram-negatives. The procedure is
based on the ability of microorganisms to retain color of the stains used during the gram stain
reaction. Gram-negative bacteria are decolorized by the alcohol, losing the color of the primary
stain, purple. Gram-positive bacteria are not decolorized by alcohol and will remain as purple.
As mentioned above, the Gram stain procedure enables bacteria to retain color of the stains,
based on the differences in the chemical and physical properties of the cell wall.
Gram positive bacteria: Stain dark purple due to retaining the primary dye called Crystal Violet
in the cell wall, i.e., Staphylococcus aureus.
Gram negative bacteria: Stain red or pink due to retaining the counter staining dye called
Safranin or Basic Fuschin, i.e., Escherichia coli.
Stain Reaction
There are four basic steps in Gram Stain. These are:
1) Application of the primary stain Crystal Violet (CV) to a heat-fixed smear of bacteria
culture: CV dissociates in aqueous solutions into CV+ and Cl- ions. These two ions then
penetrate through the cell wall and cell membrane of both Gram-positive and Gram-negative
cells. The CV+ ions later interacts with negatively charged bacterial components and stains
the bacterial cells purple.
2) Addition of Gram’s Iodine: Iodine (I- or I3-) acts as a mordant and as a trapping agent. A
mordant is a substance that increases the affinity of the cell wall for a stain by binding to
the primary stain, thus forming an insoluble complex which gets trapped in the cell wall. In
the Gram stain reaction, the crystal violet and iodine form an insoluble complex (CV-I)
which serves to turn the smear a dark purple color. At this stage, all cells will turn purple.
In this experiment, the name of the source of Iodine is Lugol.
3) Decolorization with 95% ethyl alcohol: Alcohol or acetone dissolves the lipid outer
membrane of Gram negative bacteria, thus leaving the peptidoglycan layer exposed and
increases the porosity of the cell wall. The CV-I complex is then washed away from the thin
peptidoglycan layer, leaving Gram negative bacteria colorless. On the other hand, alcohol
has a dehydrating effect on the cell walls of Gram positive bacteria which causes the pores
of the cell wall to shrink. The CV-I complex gets tightly bound into the multi-layered, highly
cross-linked Gram positive cell wall thus staining the cells purple.
The decolorization step must be performed carefully, otherwise over-decolorization may

occur. This step is critical and must be timed correctly otherwise the crystal violet stain will
be removed from the Gram-positive cells. If the decolorizing agent is applied on the cell for
too long time, the Gram-positive organisms to appear Gram-negative. Under-decolorization
occurs when the alcohol is not left on long enough to wash out the CV-I complex from the
Gram-negative cells, resulting in Gram-negative bacteria to appear Gram-positive.
4) Counterstain with Safranin: The decolorized Gram negative cells can be rendered visible
with a suitable counterstain, which is usually positively charged safranin, which them pink.
Pink color which adheres to the Gram positive bacteria is masked by the purple of the crystal
violet (Basic fuschin is sometimes used instead of safranin in rare situations). In this
experiment, Basic fuschin will be used.
Figure 1: Color changes occurring at each step in the staining process
C. APPARATUS AND MATERIALS

1. Clean glass slides
2. Inoculating loop
3. Bunsen burner
4. Microscope
5. Immersion oil
6. Distilled water
7. Bibulous paper
8. 18 to 24 hour cultures of organisms
9. Crystal Violet (primary stain)
10. Lugol (mordant)
11. Ethyl Alcohol 95% (decolorizer)
12. Basic Fuschin (secondary stain)
D. PROCEDURE
Preparation of dyes used in gram staining method: Crystal Violet, Lugol, and Basic Fuschin
are used in Gram Stain Technique.
• Preparation of Crystal Violet: 0,5 gram of Crystal Violet and 100 mL of distilled water
are mixed.
• Preparation of Lugol: 1 gram of Iodine, 2 grams of potassium iodide and 300 mL of
distilled water are mixed.
• Preparation of Basic Fuschin: 3 grams of basic fuschin and 100 mL of ethyl alchol
are mixed.
Procedure of gram staining method:
1. Type the name of the bacterial stain on the back of the glass slide.
2. Drop a drop of water in front of the slide.
3. Place a loopful of culture on the slide with a sterile cooled loop. Spread by means of circular
motion of the inoculating loop to about one centimeter in diameter. Excessive spreading
may result in disruption of cellular arrangement. A satisfactory smear will allow
exanimation of the typical cellular arrangement and isolated cells. Here, it is very important
to prevent preparing thick, dense smears which contain on excess of the bacterial sample. A
very thick smear decreases the amount of light that can pass through, thus making it difficult
to visualize the morphology of single cells. Smears typically require only a small amount
of bacterial culture. An effective smear appears as a thin whitish layer on film after heat-
fixing.
4. Allow the smear to air dry for heat fixing. After the smear has air-dried, hold the slide at
one end and pass the entire slide through the flame of a Bunsen burner two to three times
with smear-side up. Now, the smear is ready to be stained. (Heat fixing kills the bacteria in
the smear, firmly adheres the smear to the slide, and allows the sample to more readily take
up stains.)
5. Gently flood smear with Crystal Violet and let stand for 1 minute.
6. Tilt the slide slightly and gently rinse with tap water or distilled water using a wash bottle.
7. Gently flood the smear with Lugol and let stand for 1 minute.
The smear will appear as a purple circle on the slide.
9. Decolorize using 95% ethyl alcohol. Tilt the slide slightly and apply the alcohol drop by
drop for 15 seconds until alcohol runs almost clear. Be careful not to over-decolorize.
10. Immediately rinse with water.
11. Gently flood with Basic Fuschin and let stand for 30 seconds.
13. Blot dry the slide with bibulous paper.
14. View the smear using light-microscope under oil-immersion.
D. OBSERVATIONS
What different types of bacteria did you see in light-microscope? Classify them.
E. DISCUSSION
1. Which step is the most crucial to bring about poor outcomes in the Gram stain? Why?
2. What is the aim of the alcohol solution in Gram stain?
F. REFERENCES
1. Brown E. Alfered, Benson’s Microbiological Applications, Nineth Edition, McGraw Hill
Publication.
2. Prescott M., Lansing, Harley P. John, Klein A., Donald, Microbiology, Sixth Edition,
McGraw Hill Publication.
3. Yalcınkaya, Y., Seyis, I., Unal, A., Basiacik, S., Candan, D., Cicek, H., Sam, M., Tugrul, T.
Hacettepe University, Department of Biology, General Microbiology Laboratory Manual.

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EXPERIMENT 10

GRAM STAIN TECHNIQUE

• To determine bacterial morphology and chemical structure of bacterial cell wall

Staining is a supplementary technique used in microscopic techniques used to improve the

The decolorization step must be performed carefully, otherwise over-decolorization may

Figure 1: Color changes occurring at each step in the staining process

C. APPARATUS AND MATERIALS

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