NAME: BOYE GLENDA NAA AYORKOR

PROGRAM: BSC BIOCHEMISTRY 2

INDEX NUMBER: 8363721
GROUP NO: FOUR
BENCH NO: 2
REF. NO: 20857805
DATE: 16/03/2023

TITLE: PREPARATION AND EXAMINATION OF STAINED SMEARS

AIMS
1. To determine the difference between gram-positive and gram-negative bacteria using staining
techniques.
2. To prepare and observe bacteria smears on a microscope using white oil immersion

INTRODUCTION

Microorganisms are everywhere in our surroundings and are vital to human survival. Microbes,
such as bacteria, fungi, and viruses, can be found in everything from the air we breathe to the
food we eat. In the study of microbiology, microorganisms are investigated in order to
comprehend their function in our environment and to create cures and defenses against illnesses
brought on by bacteria. Bacteriology is a complicated and fascinating subject. They are highly
diverse and can be found practically anywhere, including the interior of the human body and the
deepest parts of the ocean. Bacteria can be helpful, doing necessary tasks like breaking down
organic materials, or they can be dangerous, spreading diseases like leprosy, for example.

Gram staining is a common technique used to differentiate two large groups of bacteria based on
their different cell wall constituents. The Gram stain procedure distinguishes between Gram
positive and Gram negative groups by coloring these cells red or violet. Gram-positive bacteria
stain violet due to the presence of a thick layer of peptidoglycan in their cell walls, which retains
the crystal violet these cells are stained with. Alternatively, Gram-negative bacteria stain red,
which is attributed to a thinner peptidoglycan wall that does not retain the crystal violet during
the decoloring process.

The color of the Gram stain and the morphology of the bacteria provide hints as to what bacteria
may be the source of the infection. The bacteria responsible for staph infections, Staphylococcus
aureus, is an illustration of a gram-positive cocci. Escherichia coli, a gram-negative bacteria that
frequently causes urinary tract infections, is an illustration. The Gram stain can also be used to
initially identify fungi, such as yeasts or molds, however it cannot detect viruses.

Results from Gram stains are typically regarded as preliminary, despite the fact that they are
effective as early tests for locating and classifying common species of bacteria or fungi. To
confirm a diagnosis, results from a culture and/or other tests are required, such as antigen,
antibody, or molecular testing for specific species of bacteria. To decide which antibiotic will be
the most successful in treating the infection, susceptibility testing may occasionally be required.

A sterile surface or object is entirely devoid of viruses and live microbes. All microbes are
destroyed during sterilization treatments. Heat, ethylene oxide gas, hydrogen peroxide gas,
plasma, ozone, and radiation are some of the techniques utilized in sterilizing treatments.
MATERIALS
Filter paper, staining rack, forceps, jar, dropper, masking tape, sterile/distilled water, sterile loop,
Bunsen burner, Gram stain (0.5% crystal violet, iodine, absolute alcohol, safranin), and grease-
free slides are also required.
METHODS
Before the experiment, the grease-free slides and forceps was kept in an 80% ethanol solution in
a jar. Then the grease-free slides was then removed with forceps and cleaned with a tissue paper.
Two slides A and B were labelled with a masking tape at the posterior end of the grease-free
slide. With the use of a dropper, a drop of distilled water was pipetted onto the surface of the
slide and a flamed loop was used to pick a little portion of a bacterial colony which was smeared
on the surface of the slide containing the water. The labelled slides A and B was left to dry. After
sometime both slide A and B was passed through a Bunsen burner flame for about two to three
times. The slides which was placed on the staining rack were then flooded with 0.5% crystal
violet and was left to stand for two minutes.
The stain was removed with water and drained onto a tissue paper after two minutes. The slides
were also flooded with diluted iodine and left to stand for an additional two minutes. The stain
was removed with water and then poured onto a tissue paper after two minutes. The main stain
was gently removed with water after three carefully placed drops of absolute ethanol were
applied to the smears on slides A and B. Safranin dye was used to counterstain Slides A and B,
and the slides were then allowed to stand for a minute. A mild stream of water was used to
remove the discoloration, and any extra water was wiped with filter paper.
Slides A and B, on the other hand, were set in place and examined under a low power objective
before a drop of immersion oil was pipetted directly onto the surface of the smear and the area
was then examined via an oil immersion lens.
RESULTS
TABLE: 1
BACTERIUM GRAM REACTION SHAPE POSSIBLE NAME
 Bacterium A Positive Rod-like Bacillus spp
Bacterium B Negative spherical Staphylococcus spp

DISCUSSION AND CONCLUSION

The purpose of the smear preparation is to adhere the bacteria to the surface and stop the sample
from evaporating during the staining process. A solid or broth medium can be used to prepare a
smear. The forceps and slides were intentionally submerged in ethanol for sterilizing. The
concern was to take great precautions to prevent the smears from running into each other since
this could result in the indirect observation of a different bacteria on the slide. A small amount of
bacteria was picked up with the inoculation loop or needle and was gently spread.
The purpose of passing the slides through the flames twice or three times after they had dried
completely on the air was primarily to kill the cells and stick them to the slide; if the heat
fixation wasn't done properly, the cells might also come off. All of the cells on the smear are
colored using the first primary stain, crystal violet, while the second stain, diluted iodine, is
employed as a mordant to confirm the color of the first stain. Moreover, pure alcohol, which is
used to remove the principal stains from the smears, is non-corrosive to plastics.
Safranin is employed as a decolorizer. The most important phase in determining how a stained
smear will turn out is decolorization, which has the potential to provide inaccurate results. It is
recommended to employ a brief decolorization period when staining a thinly prepared slide. A
gram negative result may be obtained in error if gram-positive organisms stain pink to red due to
excessive decolorization. Gram negative microbes may stain blue to purple if the pigment is not
sufficiently removed, giving rise to an incorrect gram positive result.
According to the results, the dye produced two distinct color compositions: blue for gram
positive bacteria and pink for gram negative bacteria. Being a basic dye, it has a colored anion
and a colorless cation that can adhere to the bacteria's cell walls.
As a result, bacteria A and B may have had the designations Bacillus spp and Staphylococcus,
respectively.

REFERENCES
 https://serc.carleton.edu/microbelife/research_methods/microscopy/gramstain.html
 https://asm.org/Protocols/Gram-Stain-Protocols
 https://www.mountsinai.org/health-library/tests/gram-stain
 https://www.testing.com/tests/gram-stain/
 https://www.vet.cornell.edu/animal-health-diagnostic-center/testing/protocols/gram-stain