Gram Staining of Bacteria Ex-4

Spring, 2024
North South University

Department of Pharmaceutical Sciences
Course: PHR124L (Pharmaceutical Microbiology-I Laboratory)
[EX-04] Title of Experiment: Gram Staining of Bacteria
Objective: The main objective of this experiment is to differentiate two large groups of bacteria based
on their different cell wall constituents.
Principle: The most important differential stain used in bacteriology is the gram stain. It is widely used
as differential staining technique which was developed by Dr. Christian Gram in 1884, and categorizes
bacteria according to their Gram character (Gram positive or Gram negative). In addition, this stain also
allows determination of cell morphology, size, and arrangement. It is typically the first differential test
run on a specimen brought into the laboratory for identification. In some cases, a rapid, presumptive
identification of the organism or elimination of a particular organism is possible.
The structure of the organism’s cell wall determines whether the organism is gram positive or negative.
The cell wall for gram positive bacteria has a higher peptidoglycan layer and lower lipid content than
gram negative bacteria. When stained with a primary stain and fixed by a mordant, some bacteria are
able to retain the primary stain by resisting decolorization while others get decolorized by a decolorizer.
Those bacteria which retain the primary stain are called Gram positive and those bacteria which get
decolorized and then get counterstained are called Gram negative.
Fig-1: The cell wall of gram Positive and Negative bacteria
Department of pharmaceutical Sciences, North South University

Spring, 2024

Materials:
1. Equipment: Compound Microscope and Slide

2. Stain:
➢ Crystal Violet: Act Primary a primary stain
➢ Grams Iodine: Used as a mordant
➢ Safranin: For Counter Satin
4. 95% Ethyl Alcohol (Decolorizing agent)

5. Microbial Culture
Procedure:
There are three major steps in procedure-
➢ Smear Preparation
➢ Staining of Cell (Gram Staining)
➢ Interpretation
Smear Preparation:
1. Take a grease free dry slide.

2. Sterilize the inoculating loop on a flame of a Bunsen burner.
3. Transfer a loopful of culture (or the specimen) by sterile loop and make a smear at the center.
Smear should not be very thin or very thick
4. Allow the smear to dry in the air.
5. Fix the dry smear by passing the slide 3-4 times through the flame quickly with the smear side facing
up.
Gram Staining:
1. Place the slides on the staining case.
2. Cover the smear with crystal violet stain and leave for 1 minute.
3. Wash carefully under running tap water.
4. Flood the smear with Gram’s iodine solution and leave for 1 minute.
5. Drain off the iodine Wash the slide for the again in a gentle stream of tap water.

Spring, 2024

6. Flood the slide with the decolorizing agent (95% Ethyl Alcohol) then wait for 10-15 seconds. This can
also be done by adding a drop by drop to the slide until the decolorizing agent (95% Ethyl Alcohol)
running from the slides runs clear.
7. Gently wash the slide under running tap water and drain completely.
8. Counterstain with safranin for and and wait for about 40 seconds.
9. Wash slide in a gentile and indirect stream of tap water until no color appears in the effluent and
then blot dry with absorbent paper.
10. Observe under microscope.
Fig-2: Gram Staining
How does the stain work on Cell:
➢ Crystal violet: Crystal violet is the primary stain. It is a positive charge dye which is attracted to
the negative charge in the cell and also the thicker peptidoglycan layer helps retain the dye
more.
➢ Grams Iodine: The function of iodine solution in the gram stain is to fix the dye on the slide in
order to form insoluble substance. It also forms a complex, (sometimes called CV-I Complex)
between the crystal violet and iodine. Gram's iodine serves as a mordant; a substance that
combines with stain to enhance the staining ability.

Spring, 2024

➢ 95% Ethyl Alcohol: Either acetone or 95% ethyl alcohol can be used as the decolorizing agent.
The alcohol dissolves lipids found in the outer cell membrane of Gram-negative bacteria,
allowing the crystal violet-iodine complex to leak out of the thinner peptidoglycan layer. The
alcohol is added for 10 to 20 seconds; it is poured on the slide until all the iodine is washed away
and the run-off is colorless. At this point in the Gram stain process, Gram-negative bacteria are
colorless while Gram-positive bacteria still retain the crystal violet. Once finished the slide needs
to be rinsed with water to stop the decolorizing effect.
➢ Safranin: The purpose of safranin in the Gram’s stain procedure is it directly stains the gram-
negative bacteria that became decolorized. The gram-positive bacteria are already stained and
not affected by the safranin.
Result/Observation:
The staining results of gram stain are as follows :
➢ Gram Positive : Dark purple

➢ Gram Negative : Light Pink to dark pink
Appearance Staining Result Name of the Bacteria

1. Shape:
2. Form:
3. Color:
Precaution:
1. Wear apron, gloves and mask.

2. Use only fresh cultures, not more than 48 hours old.
3. Don’t heat the slide more during heat fixation.
4. Don't despair if the stained smear is not visible to the naked eye; this may happen with a
Gram-negative reaction.
5. Take care to make an even smear otherwise alcohol will continue to wash the violet purple
color from thick parts of the smear while thin parts are being over-decolorized.

Spring, 2024

Report:
Write the laboratory report for this experiment and submit it one week after the experiment to
your instructor. Remember to include the following in your report:
1. Title of the experiment

2. Purpose/objective
3. Materials required
4. Procedure
5. Result/Observation
6. What you have learned from this experiment
7. Precaution

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Spring, 2024

North South University

Fig-1: The cell wall of gram Positive and Negative bacteria

Department of pharmaceutical Sciences, North South University

North South University

1. Equipment: Compound Microscope and Slide

4. 95% Ethyl Alcohol (Decolorizing agent)

There are three major steps in procedure-

1. Take a grease free dry slide.

Department of pharmaceutical Sciences, North South University

North South University

Fig-2: Gram Staining

How does the stain work on Cell:

Department of pharmaceutical Sciences, North South University

North South University

The staining results of gram stain are as follows :

➢ Gram Positive : Dark purple

Appearance Staining Result Name of the Bacteria

1. Wear apron, gloves and mask.

Department of pharmaceutical Sciences, North South University

North South University

1. Title of the experiment

Department of pharmaceutical Sciences, North South University

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