Lab Report Microbiology
Lab Report Microbiology
Lab Report Microbiology
Laboratory Report
Gram Staining
School of Food Science and Environmental Health Laboratory Report.
Olga Ciudin
Student name(s) and number: DT425
C19757639
AIMS AND OBJECTIVES
1.1 Aim: The aim of the experiment was to become familiar with method of staining used to
distinguish and classify bacterial species into two large groups (Gram-positive and Gram-
negative).
1.2 Objective: The objective of the experiment was to use standard microbiological method
of gram staining, in order to examine and compare E.coli and Staphylococcus Aureus
according to their morphology and biochemical characteristics.
INTRODUCTION
Being able to distinguish bacterial species is very significant for many reasons, from
diagnosing infection or checking food safety. Bacteria can include characteristics like their
shape, growth in particular nutrients and preference for high or low oxygen environments.
Depending on the feature being studied, microorganisms may be broken down into large
groups, but taken together this data can contracted the possible identities significantly.
Staining is an auxiliary technique used in microscopic techniques which are used to
enhance the clarity of the microscopic image. Gram-staining was first developed in 1883 by
Danish bacteriologist Hans Christian Gram, and according to him, this is a standard technique
to classify bacteria and make them more visible under a microscope. Gram staining was used
to distinguish between Gram-positive microorganisms and Gram negative microorganisms,
therefore these microorganisms being separated into 2 large categories. The aims of this
procedure consist to determine on the ability of microorganisms to retain colour of the stains
used during the experiment of gram stain reaction. Gram stain allows determining which of
the bacteria are retaining primary dye, and these are called gram-positive bacteria. There have
been done observations that the second group take the colour of the counterstain, are
therefore are called gram-negative microorganisms. The primary dye which is used for gram
staining experiment crystal violet and the secondary dye is usually either safranin O or basic
fuchsin. Some of the more common formulations include: saturated crystal violet
(approximately 1%), Hucker’s crystal violet, and 2% alcoholic crystal violet.
The Gram stain has many uses: principally, it classifies bacteria on the foundation of
its cell wall structure and allows inspection of their size and cellular morphology as well. It
can also be utilized to assess the quality of clinical specimens and as a significant test for
rapid and presumptive analysis of infective agents directly from specimens (W.Chan, 2016)
Gram-positive bacteria are classified by the color they turn after a chemical called
Gram stain is applied to them. Gram-positive bacteria stain blue when this stain is put to
them. Other bacteria stain red. They are called gram-negative. Gram-positive and gram-
negative bacteria stain in a different way because their cell walls are different. They also
cause different types of infections, and diverse types of antibiotics are effective against
them. (M.Bush, 2019)
All bacteria are differentiated as one of three basic profiles: spheres (cocci), rods (bacilli),
and spirals or helixes (spirochetes). Gram-positive microbes may be cocci or bacilli. Gram
positive bacteria contain a continuous wall, which is called sacculus, which is 20 to 80 nm
in thickness. The cell wall is composed of peptidoglycan knows as murein. (Lakna, 2017)
There is strong evidence that some Gram-positive bacteria cause infections. Others
normally occupy a specific site in the body, and mainly in the skin. These bacteria,
called resident flora, do not usually cause disease. A lot of investigations that took place in
the last years, shows that Gram-positive bacteria are more and more are becoming resistant
to antibiotics. For example, methicillin-resistant Staphylococcus aureus (MRSA) bacteria
are resistant to most antibiotics that are related to penicillin. Methicillin is a type
of penicillin. MRSA strains are normally implicated in infections obtained in health care
facilities and can cause infections acquire outside health care facilities (community-
acquired infections).
Bacteria which do not retain the crystal violet stain during gram staining are being
represented by gram negative microorganisms. The peptidoglycan layer, which is responsible
for retaining the crystal violet stain, is much thinner in gram negative bacteria and it is
sandwiched between the inner cytoplasmatic membrane and the outer membrane of the
microorganism. Therefore, gram negative bacteria can be stained using safranin, giving in the
end of the test, a red pink colour.
E.coli is an example of gram negative bacteria and therefore is being used is a model
organism in most of bacterial studies. There is strong evidence that Gram-negative
microorganisms are more pathogenic than gram positive bacteria, due to their less suscebility
to antibiotics. The cell wall of the gram negative bacteria is 5 to 10 nm in thickness, and
contains one layer of peptidoglycan.
The 2 main features that lead to the differing visualization properties of Gram
positive and Gram negative types of bacteria are the thickness of the peptodoglycan coat and
presence or lack of the outer lipid membrane. This is because the wall configuration affects
the cell’s ability to retain the crystal violet stain, which is being used in Gram staining
method which then can be observed using a light microscope.
List of materials
Methods: For this experiment, we used gram staining method. The procedure as per
manual.
RESULTS
Table 1 Gram Stain Results
In this experiment we stained 2 smears of E.coli and S.aureus, with crystal violet
dyes. There was observed that because of the Gram stain reaction, the crystal violet, and the
next agent used, which was Gram’s iodine, formed and insoluble complex which to turn the
smear into a dark purple colour. The acetone alcohol was used as decolorizing agent, and
dissolved the lipid outer membrane of the Gram negative bacteria, and therefore making the
bacteria colourless. For Gram positive bacteria, the alcohol dehydrated the layer of
peptydoglycan and it appeared in purple colour. In the end, after using suitable counterstain,
named safranin. This enabled Gram negative bacteria to be seen clearly under the
microscope. This appeared in pink colour stains. Gram-positive bacteria, which in this
experiment is represented by S.aureus did not turn in pink after using Safranin, because the
peptidoglycan layer already had crystal violet-iodine complex.
Therefore, it can be concluded that as we can see in the Table 1, S.aureus is a Gram-
positive microorganism, because it apperead in a purple colour. In the same same, the other
bacteria, which is E.coli, is a Gram-negative one, and it appeared in pink-red colour. There
was observed that the cell shape of S.aureus showed a grape like cocci, and E.coli came up as
small rods. The arrangement of the Gram-positive bacteria in this experiment appeared in
cluster. On other hand, Gram-negative bacteria shower up as rod shaped, and tending to occur
individually and in large clumps.
Fig. 1 Procedure of Gram Staining; note the colour change after each step
CONCLUSION
From this experiment, we can conclude that its aim was successfully achieved, making
differentiations between Gram-positive and Gram-negative bacteria through gram staining.
The proper use of materials, had a positive impact to reach the aims and objective of this
research. In conclusion, there was distinguished between Gram-positive and Gram-negative
bacteria, which has different level of thickness in cell wall, as well different arrangement and
morphology
.
Bibliography
Lakna. (2017, April 3). www.researchgate.net . Retrieved November 12, 2019, from
www.researchgate.net : www.researchgate.net/oublication/315