Lab Report 1

Gram Staining Technique

- BIO 223 L -

Student Name: Sara Alabdulkarim

Student ID: 201136
Submission date: Sunday 19, 2021

Abstract:

Since microorganisms are known to be colorless and impossible to see

through the naked eye some techniques were developed to help
visualize their shape and cellular structure like the staining technique.
This technique allows the microorganisms to gain colors as well as
allowing us to study and see them. In this experiment, Our aim is to be
able to differentiate between two different bacteria’s (S. aureus and E.
coli) by using gram staining. To do this we started with creating and
preparing smears of the bacteria and fixing them in place by heating
them and adding chemicals (in the right order). If these steps are done
correctly, they will provide accurate results. Lastly, we will take the
stained bacteria and observe them under a compound light microscope.
The result we receive should match the world wide known information
about the bacteria we are using which are: the S. aureus is gram positive
and will show as a violet color while E.coli will be gram- and will show a
redish pink color.

Objective:

Remembering how to use the light microscope. Second, How to Prepare cultures and
explain the Gram stain reactions, cell shape, and arrangement in S. aureus and E.
coli.
Introduction:

Microbes are microscopic, single-celled organisms that are found all around us. They live
in water, soil, and in the air. The human body is home to millions of these microbes too,
also called microorganisms. Some may be harmful (pathogenic) and other are not
(nonpathogenic). On a microscope slide we fix, stain, and dry the bacterial smear of both
S. aureus and E. coli. A smear that is thin and even enables the shape and arrangement
of cells to be clearly seen and ensures that the staining procedure is applied uniformly
There are two types of staining methods:
o Simple Staining: Uses one basic dye and provides basic information about
the cell shape and structure.
o Differential Staining: Uses more than one dye. This procedure reacts with
different kinds of bacteria and helps distinguish between them by gram stain
and acid fast stain. Also helps visualize the structures with spore stain and
capsule stain. Most common type of differential stain is Gram staining.

Gram staining is what we’ll be doing in our lab. Gram staining is a differential stain that
distinguishes bacteria based on their cell wall structure. It is commonly used in
microbiology labs. Based on the composition of their cell walls, most bacteria can be
divided into two groups:
o Gram Positive: Beyond the plasma membrane, cell walls have a thick
peptidoglycan layer. The Gram stain turns violet when applied
o Gram Negative: Beyond the plasma membrane, cell walls have a thick
peptidoglycan layer. The Gram stain turns red.
In this experiment, we will differentiate between S. aureus (gram-positive) with a
purple color and E. coli (gram-negative) with a red/pink color. We’ll use a light
microscope after staining to observe them and see their colors shape and
arrangement.

Material:

 Bacterial broth culture of S. epidermidis and E. coli

 Microscopic Slides
 Disposable inoculating loops
 Compound light Microscope
 Flame (Bunsen Burner)
 Gram stain reagents
Procedure:

o Preparing the smear:

1) Pick up and Label a clean microscope slide with the group number using a marker
pen.
2) Light the Bunsen burner.
3) Using aseptic technique, remove the cap and flame the neck of the tube for few
seconds before inoculating the sample on the microscopic slide.
4) Transfer one or two loopfuls of bacterial culture on to the center of the
microscopic slide.
5) Swirl the loopful of bacteria on the slide area.
6) Dispose the inoculating loop and reflame the neck of the tube for few seconds.
7) Air-dry the slide until the liquid evaporates.
8) Heat-fix the smear by quickly passing the slide through the flame three times.

o Staining the smear:

1) Cover the smear area with the crystal violet stain and leave it for 1 minute, then
rinse the slide with a gentle stream of water.
2) Apply Gram's iodine solution covering the smear completely for 1 minute, then
rinse with water.
3) Using the Gram’s decolorizer, apply a drop at a time to the smear area until no
more color leaves the area. (This is the most crucial and difficult step in the
procedure. Apply it evenly to the entire smear.) Quickly rinse with water to stop
the decolorizing process.
4) Apply Safranin to the smear for 30 seconds, and then rinse with water.
5) Allow the smear to air dry.

o Observing and evaluating the smear:

The slide should appear only lightly colored to the naked eye. A good slide is evenly stained
and the bacteria are spread thinly enough that you can identify individual cells.

1) Observe the slides under low power lens to orient yourself, then move to the 40X
lens and finally the oil immersion lens.
2) Thoroughly clean the microscope. Discard the used slides in the proper container.
Place the cultures in the rack.
3) Clean your area, wipe down the desk and wash your hands before leaving.
 Results:

Figure 1: of S. epidermidis and E. coli Figure 2: Using the burner to create a smear
Figure 3: After adding crystal violet Figure 4: After adding iodine

Figure 5: After adding alcohol Figure 6: After putting safrannine

Figure 8: S. epidermidis (gram+) Figure 9: Same bacteria with a drop of immersion oil

Figure 10: E.coli (gram-) Figure 11: Sane bacteria with a drop of immersion oil
Discussion:

In this experiment, my part was fixated around S.epidermidis. First things first I
created a smear of the bacteria with an inoculating loop to spread on the slide and
fixed the smear in place with heating (type of fixation process). After the smear was
created and fixed in place I began the staining process, the type of staining was gram
staining (differential stain) to help classify the bacteria into gram positive or
negative. The chemicals we added were:

1) Crystal Violet (primary stain): stains cell wall of both gram+ and gram-.
2) Iodine (mordant): Combines with CV to form an insoluble complex that will
be trapped in thicker peptidoglycan layer (gram+)
3) Alcohol (decolorizing agent): it will remove the violet color from the gram-
bacteria but will not remove the color from the gram+ bacteria because
gram- has a thin peptidoglycan layer. If the alcohol is left for sometime it may
end up leaving both gram+ and gram- and –colorless. So to prevent this it
must be washed out immediately after its application.
4) Safranin (counter stain): Gram+ already stained violet and gram- is colorless.
So safranin will stain the gram- bacteria a redish pink color.

*Steps must be followed in this particular order and after every step the slide
containing the smear must be washed to remove the chemicals.

After finishing the staining process the bacteria is ready to be observed under the
microscope. The results I got were correct according to the instructions said in the
lab and showed the the E.coli did stain with a redish color and the S.epidermids was
stained with a violet color. After I observed the bacteria under the microscope for
some time I added a few drops of immersion oil on the slide to intensify the
resolution.
In conclusion, The results did affirm the real world facts about E.coli bacteria being
redish and being gram- and the S.epidermids being violet and also gram+.

Reference:

 Lab manual
 Lab ppt
 Post lab file
 http://www.diffen.com/difference/Gram-negative_Bacteria_vs_Gram-