NAME : MANUEL, Arianne Nicole Rating : ________

EXERCISE NO . 5
THE STUDY OF BACTERIAL MORPHOLOGY USING GRAM’S STAIN

I. OBJECTIVE:
To be acquainted with gram’s stain as a staining method in the study of bacterial
morphology.

II. INTRODUCTION :

Gram’s stain is the most widely used staining technique in diagnostic microbiology .
The gram’s stain differentiates gram positive and gram-negative organisms, thus, it is
classified as a differential stain. The gram-positive organisms will appear deep purple
while the gram negative will appear red. The terms positive and negative have nothing to
do with electrical charge, but simply designate two distinct morphological groups of
bacteria.

Gram positive and gram negative bacteria stain differently because of fundamental
differences in the structure of their cell walls. The bacterial cell wall gives the organism
its size and shape and prevents osmotic lysis. The material in the bacterial cell wall
which confers rigidity is peptidoglycan.

In electron micrographs, the gram-positive cell wall appears as a broad, dense wall
20-80 nm thick and consisting of numerous interconnecting layers of peptidoglycan
which constitute 60-90% of the cell wall. Interwoven in the cell wall of gram positive are
teichoic acids which are peculiar to the gram-negative cell wall. These extend through
and beyond the rest of the cell wall and are composed of polymers of glycerol,
phosphates, and the sugar alcohol ribitol. Some have a lipid attached (lipoteichoic acid).
The outer surface of the peptidoglycan is studded with proteins that differ with the strain
and species of the bacterium.

The gram-negative cell wall, on the other hand, contains only 2-3 layers of
peptidoglycan which constitute only 10-20% of the cell wall. This is surrounded by an
outer membrane composed of phospholipids, lipopolysaccharide, lipoproteins.
Thephospholipids are located mainly in the inner layer of the outer membrane, as are the
lipoproteins that connect the outer membrane to the peptidoglycan. The
lipopolysaccharides, located in the outer layer of the outer membrane, consist of a lipid
portion called lipid A embedded in the membrane and a polysaccharide portion
extending outward from the bacterial surface. The outermembrane also contains a
number of proteins that differ with the strain and species of the bacterium.

The following rules will help in remembering the gram’s staining reaction of the more
important bacteria:

1. ALL bacilli are gram negative EXCEPT Corynebacterium, Mycobacterium,

aerobic and anaerobic spore formers
2. ALL cocci are gram positive EXCEPT Neisseria and Veilonella.
III. MATERIALS :
Glass slides
Wire loop
Alcohol lamp
Staphylococcus stock culture
Salmonella stock culture
Gram’s stain
Microscope
Oil immersion

IV. PROCEDURE :

How to prepare the bacterial smear :

1. Take a clean glass slide and gently heat one side to remove any grease that may
be present. Lay the slide on the table with the flame side up.
2. Sterile the wire loop.
3. If the source of the specimen is growing on a solid media, place a loopful of
sterile distilled water in the center of the slide. With the sterilized wire loop, pick a
small colony and emulsify in the drop of distilled water in the slide. Smears to be
made from a liquid media are made directly to the slide by taking one or two of
the culture and spread over the center of the slide over an area of 1 inch by ½
inch.
4. Lay the prepared slides on the table and allow it to dry in the air.
5. Fix the smear with the heat by passing the slide with the smear side up over a
flame 5 to 6 times.
6. Stain the preparation with the desired staining method.

Steps in Gram’s Staining:

1. Make a thin smear.

2. Cover the entire smear with some drops of crystal violet for 1 minute.
3. Wash off the excess stain with water until no more stain comes off.
4. Cover the smear with gram’s iodine for 1 minute.
5. Wash off the iodine with tap water.
6. Decolorize the smear with 95% alcohol. Flood the smear with alcohol and allow it
to stand for 15 to 30 seconds. Pour it off.
7. Wash again with tap water.
8. Counter stain with safranin for 30 seconds.
9. Wash off the excess stain with tap water.
10. Blot or air dry and examine under the oil immersion objective.
V. RESULTS :
Draw and identify the gram staining reaction, shape and arrangement .

GRAM-POSITIVE GRAM-NEGATIVE

Staphylococcus Salmonella

Staphylococcus appears Gram positive Salmonella is Gram-negative (pink to red)

(violet) and cocci with clustered or in Gram stain. It appears as solitary or
grape-like arrangement of cells. It also linear and short rod-shaped cells.
sometimes appears in diplococci or in
chains.

VI . QUESTIONS :

1. What is the purpose of fixing the smear ?

Fixing the smear allows for the attachment of the bacterial specimen to
the slide so that when staining with the reagents and when rinsing it off with
water, the smear will not be removed. Fixing with heat or fixing with chemicals
also kills the bacteria but preserves its cellular structures and enables the dye to
penetrate the cells better. Thus, stained fixed smears can be kept and stored.

2. What is the primary stain ?

The primary stain is crystal violet, which is the first dye applied during the
series of steps in Gram staining. This provides the initial blue to violet color to the
cells, whether gram-positive or gram-negative before counterstaining. It is also
the dye that is retained by Gram-positive cells. It is applied for 60 seconds before
carefully rinsing with water.
3. What is the role of iodine ?

Iodine is considered a fixing agent or mordant in Gram-stain. It reacts with

crystal violet and creates an insoluble crystal-violet iodine complex within the cell
wall. This prevents the removal of the primary stain from washing off with water
and by the decolorizer (Tripathi and Sapra, 2023).

4. Is there any counterstain that may be used ? If yes, name some.

Safranin is usually the counterstain, which is also the last dye applied for
60 seconds in the Gram-staining protocol. Another dye that can be used is basic
fuchsin which also gives a brighter pink to red color to the gram-negative cells.
This is also used over safranin in some laboratories because some gram
negative bacterial cells such as Haemophilus spp., Legionella spp., and some
anaerobes are poorly stained with safranin, but stains well with basic fuchsin
dyes (Tripathi and Sapra 2023).

5. State the principle why the gram positive organisms take up primary stain
while the gram negative organisms take up the counterstain ?

Gram positive and Gram negative cells differ in their cell wall composition.
Of importance is the thickness of the peptidoglycan layer which reacts with
crystal violet. Since the peptidoglycan layer in Gram positive cells is thicker, the
dye adheres more to this. Initially, all cells stain with the crystal violet and will
have a blue to violet color. But because of the thin peptidoglycan layer in
gram-negative cells, not much of the dye will be retained when washing off the
stain and especially when the decolorizer (ethanol) is applied. Ethanol also
dissolves the abundant lipid component of the gram-negative cell wall. On the
other hand, when washed with alcohol, the crystal violet-iodine complex is
retained in Gram-positive cells further because it causes dehydration of the cell
structure, trapping the crystal violet dye within the cell wall, while decolorizing the
Gram-negative cells (Tripathi and Sapra, 2023).

Counterstain is used to differentiate the gram-negative cells since these

cells show off the dye’s color after just being decolorized. Gram-positive cells
also take up the counterstain, however, the crystal violet-iodine complex that is
already within the cell membrane masks the pink color of the safranin or basic
fuchsin dye. Thus, only the decolorized cells with a thinner peptidoglycan layer
are able to show off the pink color of the counterstain.

6. What is a simple stain?

A simple stain is a staining technique which directly applies one dye into
bacterial cells to give contrast to colorless cells under a light microscope. It
enables the visualization of the cell’s morphology, size, and arrangement of the
cell that aids in its identification. The cells take up the color of the die as it is
 stained. This is effective in staining the vegetative cells, however, endospores are
not stained and requires a different method for visualization (Moyes et al., 2009).

Three dyes are commonly used in microorganisms - methylene blue,

crystal violet, and Ziehl’s carbol fuchsin which are all basic dyes (Moyes et al.,
2009). Basic dyes are cationic dyes which stain negatively-charged components
on the surface of the cell. Unlike acidic or anionic dyes which stain the
background of the cells to provide contrast.

7. Illustrate and label the gram positive and gram negative cell walls.

Figure 1. Composition of cell wall of Gram positive and Gram-negative bacteria (a) Gram-positive
(b) Gram-negative. Image source: Madigan et al., (2021). Brock Biology of Microorganisms. 16th
ed.

8. Differentiate the gram positive from gram negative cell wall. Enumerate the
components of the cell wall which are unique for each.

Gram-negative bacterial cell wall is more complex and has a thinner

peptidoglycan layer than Gram-positive cell wall. Peptidoglycan is a rigid
polysaccharide which is unique to bacterial cells, containing repeats of
N-acetylglucosamine and N-acetylmuramic acid in alternating fashion. However,
its thickness can differentiate the two types of cells.

Gram positive cell wall has a thick peptidoglycan layer which comprise
most of its cell wall, as much as 90%. Peptidoglycan can measure 20 to 35 mm
thick and can be up to 15 layers thick, stabilized by cross-links between adjacent
strands of peptidoglycan, both horizontally and vertically (Madigan et al., 2021).
The thickness allows the gram positive cell wall to retain the crystal violet dye
even when a decolorizer is applied. One unique feature of gram-positive cell
walls not found in gram-negative cell walls are teichoic acid and lipoteichoic
acid. Teichoic acids are composed of glycerophosphate or ribitol phosphate
 attached to glucose or D-alanine and are embedded in the cell wall. These are
antigenic molecules in Gram-positive bacteria. Some teichoic acids are bound to
lipoteichoic acids, a type of membrane teichoic acid bound to lipids (Riedel et al.,
2018).

Gram-negative cell wall is more complex and has many layers which
include a periplasmic space after the cytoplasmic membrane which contains
porins, followed by the thin peptidoglycan layer, and an outer membrane which
contains lipopolysaccharides. The cell wall has a single layer of peptidoglycan,
2-7 mm thick located in between the cytoplasmic membrane and outer
membrane (Madigan et al., 2021). For this reason, Gram staining differentiates
cell walls on the peptidoglycan layer. The peptidoglycan meshwork is pliable and
porous because it has less cross-linking than gram-positive cell walls. But it is still
rigid enough to resist pressure from the environment and rupture the cell
(Madigan et al., 2021). The unique features of the gram-negative cell wall are the
periplasm which contains porins, and an outer membrane with
lipopolysaccharide. The presence of porins extending to the outer membrane
allows the passive diffusion of low-molecular weight molecules to and hydrophilic
compounds to protect the cell. Lipopolysaccharide is an important virulence
factor which allows surface recognition and provides additional mechanical
strength to the cell wall. It is mainly composed of lipid A embedded on the outer
leaflet of the outer membrane (Riedel et al., 2018). The presence of the lipid
components and a thin peptidoglycan layer of the Gram-negative bacterial cell
wall is the reason for the easy decolorization in Gram-staining and why it is easily
counterstained with safranin.

REFERENCES:

Madigan, M. T., Bender, K. S., Buckley, D. H., Sattley, W. M., & Stahl, D. A. (2021).
Brock Biology of Microorganisms (16th ed.). Pearson Education.

Moyes, R. B., Reynolds, J., & Breakwell, D. P. (2009). Preliminary staining of bacteria:
Simple stains. Current Protocols in Microbiology, 15(1).
https://doi.org/10.1002/9780471729259.mca03es15

Riedel, S., Morse, S. A., Mietzner, T. A., & Miller, S. (2019). Jawetz, Melnick &
Adelberg’s Medical Microbiology (28th ed.). McGraw-Hill Education.

Tripathi, N. & Sapra, A. (2023, August 14). Gram Staining. StatPearls. National Library
of Medicine. StatPearls Publishing. Retrieved
https://www.ncbi.nlm.nih.gov/books/NBK562156/