BIO461 Practical 2

BIO461
LABORATORY REPORT EXPERIMENT 2
DIFFERENTIAL STAINING &

STRUCTURAL STAINING
PREPARED FOR:
NUR HANIS BINTI MOHD YASIM
PREPARED BY:
NURUL AIHAN BINTI AHMAD HILMI
202269743
AS2011B
PRACTICAL 2
A. DIFFERENTIAL STAINING - THE GRAM STAIN
Result
a) bacillus subtilis b) Escherichia coli
Discussion
The basic principle of gram staining involves the ability of the bacterial cell wall to retain the
crystal violet dye during solvent treatment. Gram-positive cell walls have a thick peptidoglycan
layer beyond the plasma membrane. Gram-positive cell walls stain blue/purple with the Gram
stain. Gram-negative cell walls are more complex. They have a thin peptidoglycan layer and an
outer membrane beyond the plasma membrane. Gram-negative cells will stain pink with the Gram
stain.
The first step in gram staining is the use of crystal violet dye for the slide's initial staining. The
next step, also known as fixing the dye, involves using iodine to form crystal violet- iodine
complex to prevent easy removal of dye. Subsequently, a decolorizer, often solvent of ethanol and
acetone, is used to remove the dye. The basic principle of gram staining involves the ability of the
bacterial cell wall to retain the crystal violet dye during solvent treatment. Gram-positive
microorganisms have higher peptidoglycan content, whereas gram-negative organisms have
higher lipid content.
Initially, all bacteria take up crystal violet dye; however, with the use of solvent, the lipid layer
from gram-negative organisms is dissolved. With the dissolution of the lipid layer, gram negatives
lose the primary stain. In contrast, solvent dehydrates the gram-positive cell walls with the closure
of pores preventing diffusion of violet-iodine complex, and thus, bacteria remain stained. The
length of decolorization is a critical step in gram staining as prolonged exposure to a decolorizing
agent can remove all the stains from both types of bacteria.
The final step in gram staining is to use basic fuchsin stain to give decolorized gram-negative
bacteria pink color for easier identification. It is also known as counterstain. Some laboratories use
safranin as a counterstain; however, basic fuchsin stains gram-negative organisms more intensely
than safranin.
Bacillus subtilis is a rod-shaped bacterium, the bacterial rod is a symmetrical cylinder with
rounded ends. A significant difference in pressure across the cytoplasmic membrane pushes the
cell wall into a specific shape. Bacillus subtilis retain the crystal violet dye, appearing purplish-
blue under a microscope. This is due to the presence of a thick peptidoglycan layer. Bacillus
subtilis is a Gram-positive bacterium.
E. coli is a rod-shaped bacterium that under the microscope is shaped like a rod with a small
tail and a facultative anaerobic bacterium. E. coli appear pink in color. These bacteria have a thin
layer of peptidoglycan, as well as an outer membrane that prevents them from retaining the crystal
violet stain used in Gram's method. Use of a counterstain (e.g., safranin) stains such cells pink.
Escherichia coli is a Gram-negative bacterium.
B. STRUCTURAL STAINING
I. THE ENDOSPORE STAIN
Result
Bacillus subtilis
Discussion
For the Endospore staining technique, we will stain the smear by using the Schaeffer-
Fulton's method. The glass slide containing bacteria Bacillus cereus smear was used. Theoretically,
the bacteria with genera such as Bacillus have the ability to produce endospores. These endospores
can resist to heat, drying, radiation, and various disinfectants. The spore is usually produced within
each of the vegetative bacterium. Once the endospore is formed, the vegetative portion of the
bacterium is reduced and the dormant endospore is released.
First, the smear is filled with primary dye which is malachite green. The primary dye malachite
green is a weaker binding dye to the cell wall and spore wall. If washed well with tap water, the
dye comes right out of the cell wall, however the spore wall will lock in the dye. That is why there
is no need for a decolorizer in this staining technique because the binding of the malachite green
and the permeability of the spore and cell wall.
When smear was heated by a Bunsen burner to catalyze the malachite green to permeate the
low-permeability spore wall. A variety of chemicals exist within the spore wall such as keratin
protein and calcium but deeper in the spore wall is peptidoglycan. The keratin forming on the outer
portion of the endospore wall resists dye. The heating of the bacteria will make the spore wall
becoming more permeable to the malachite green dye, and it will then attach to the peptidoglycan.
Once the malachite green goes inside, the malachite green will not come out because the
overlapping spore wall becomes less permeable when the smear cools down. Vegetative cells may
be decolorized with tap water. This is because the vegetative cells have been disrupted by high
temperature.
Then the safranin is then applied, which stains the vegetative cells. The addition of a
counterstain is used to stain the decolorized vegetative cells to make it more visible. On the last
step, we observe our specimen using oil immersion microscope objective lenses. This lens has
more mirror inside and it requires the use of oil to refract light rays towards the center of the lenses.
As the results, the spore is stained green and the vegetative cell is stained a pinkish-red colour.
II. THE CAPSULE STAIN
Result
Escherichia coli
Discussion
For the capsule stain technique. The slide containing bacteria Klebsiella pneumonia smear was
used. Based on theory, a capsule is a gelatinous outer layer secreted by bacterial cell and that
envelope and sticks really fast to the cell wall. Most capsules are mainly composed of
polysaccharides, but some are mainly composed of polypeptides. The capsule is different from the
slime outer layer that most bacterial cells produce in that it is a thick layer outside the cell wall.
The capsule stain takes on an acidic stain and a basic stain to detect capsule production. The smear
is flooded with Indian ink.
The purpose of Indian ink is to provide a background color. The ink will stain the
background with cells but the capsule will be unstained. Then the cover slip was placed on the top
of the slide. Finally, the specimen is observed using microscope under oil immersion objective
lenses (100x). At the end of the staining process, the background of the cell will appear as black
while the capsule will be unstained and colorless. Based on observation the capsule is there.
In the motility test the microscope-based method for determining motility, it is called
hanging drop preparation method. In this method we will see if the bacterial culture, in this case
Escherichia coli is having a true motility movement or Brownian movement. The Petroleum jelly
(in this case Vaseline) are used to prevent the broth from evaporating. Then we use the microscope
with high light intensity to view the microbe and it turns out that Escherichia coli is a motile
microbe. The background will be dark (color of Indian ink). The bacterial cells will be stained
purple (bacterial cells take crystal violet-basic dyes as they are negatively charged). The capsule
(if present) will appear clear against the dark background (the capsule does not take any stain)

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BIO461

LABORATORY REPORT EXPERIMENT 2

DIFFERENTIAL STAINING &

NUR HANIS BINTI MOHD YASIM

NURUL AIHAN BINTI AHMAD HILMI

a) bacillus subtilis b) Escherichia coli

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