Colegio de San Juan de Letran

College of Business Administration and Accountancy

Food Technology Area

Gram Staining
(Experiment 4)

Submitted by:

ACUJEDA, Jerick James

BRUAN, Ma. Trixia Joyce

MAGBITANG, Francis John

MANALO, Aliyah Juliana

RANCES, Vincent

SACAYANAN, Katherine Dianne

SALVADOR, Kurt Danielle

Date Submitted:

October 13, 2022

Submitted to:

Ms. Sheena Marie Napata

Instructor
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I. ABSTRACT
Gram staining is a widely used technique in
microbiology that allows bacteria to be identified under the
microscope. This technique relies on the ability of bacterial
peptidoglycan cell walls to retain the crystal violet dye. Gram-
positive bacteria with thick peptidoglycan are stained blue or
purple due to the retention of crystal violet-iodine complex
within their cell walls, while Gram-negative bacteria with
thinner peptidoglycan are stained red or pink due to the
presence of safranin instead of crystal violet. Bacteria may be
obtained from various sources, such as surrounding surfaces,
the air, or even from human hands. Thus, it is necessary to
always practice aseptic techniques when handling
microorganisms to prevent cross-contamination.

II. KEYWORDS: Gram staining, crystal violet, safranin, peptidoglycan, smear

III. INTRODUCTION
Gram staining is a procedure that distinguishes between two large groups of
bacteria and can differentiate gram positive and gram negative by the cells red and
violet. Gram stain plays an important role in microbiology and it can help professionals
in the diagnosis of infectious diseases directly from the samples as the cell wall of gram
positive bacteria gets completely different from gram negative bacteria. To understand
the gram nature and to provide the appropriate treatment for the infection.

Schaeffer-Fulton method performs endospore staining malachite green and is

also used for simple stains in bacterial cells. By heating the bacterial emulsion,
malachite green is pushed into the spores. It relatively weakly binds dye to the cell wall
and spore wall, and this heating step colors the vegetative cells and the endospore.
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There are four different bacterial morphologies: coccus is any bacteria or

archaeon that has a spherical, ovoid, or generally round shape. They differ from other
bacteria and archaeons in that they can exist alone or in pairs. Bacilli are cylindrical or
rod-like bacteria. Spirilla are bacteria with a rigid spiral (helical) structure that is difficult

to bend, rigid, lengthy, and moves with flagella, measuring 6–15 μm in length. Vibrio
are comma-shaped.

This experiment aims to inoculate a smear made from solid media using a sterile
pipette, cells from a culture are applied in a thin layer to a tiny region of a microscope
slide, dried, and then fixed to the slide using heat or other chemical fixatives. A Gram
stain is a laboratory test that looks for bacteria in some physiological fluids or at the
probable location of an infection. This involves taking the sample, mounting it on a
microscope slide, staining it, and then examining it using a microscope.

IV. METHODOLOGY
Materials
The materials were used for the Gram staining of previously prepared samples,
as well as observation after staining. Microscope slides were used for the preparation of
smears. An alcohol lamp was also used to heat fix the sample. Solutions used for Gram
staining included crystal violet as primary stain, Gram’s iodine as mordant, acetone-
alcohol as decolorizer, and safranin as secondary stain. Plastic pipettes were used to
apply each solution, and a wash bottle containing distilled water was used in washing
off the slide in between the application of solutions. After staining, the samples were
observed through a compound light microscope, using immersion oil for viewing under
the oil immersion objective (100X).

Procedure
1. Before Gram staining, the smear was prepared by placing a drop of distilled water
on a clean slide.
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2. An inoculating loop was flamed and cooled before obtaining a single colony from the
Petri dish containing the sample.
3. The inoculated colony was then placed on the slide with distilled water and spread
across the surface.
4. After smearing, the slide was set aside to allow the water to evaporate.
5. Once the slide had dried, it was passed over the alcohol lamp’s flame two to three
times to heat fix the sample.
6. After fixing, crystal violet was dropped onto the slide and allowed to stay for one
minute.
7. After applying crystal violet, it was washed off with distilled water by gently passing
the water onto the smear surface with the slide slightly tilted.
8. After rinsing, Gram’s iodine was then dropped onto the slide and allowed to stay for
one minute. It was again rinsed with distilled water after.
9. The decolorizer was dropped onto the slightly tilted slide until the blue color had
stopped running from the smear. The slide was immediately washed off with distilled
water after.
10. Lastly, safranin was applied onto the slide and allowed to stay for one minute before
a final rinse-off with distilled water.
11. The stained sample was set aside to allow it to dry, then placed under the
microscope for viewing under the oil immersion objective (100X). Observations were
noted.

V. RESULTS AND DISCUSSION

Gram Staining
Following the steps of the gram staining procedure correctly is to classify if it has
a bacterial infection, and it determines what type of bacteria are causing it. The initial
staining of the slide with crystal violet dye is the first stage in the gram staining
procedure. But first you need to do a heat fix in the slide. Heat fixation ensures the
elimination of contaminating organisms from the smear preparation. It adheres the cells
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to the slide and coagulates the bacterial proteins, effectively killing the bacteria and
necessary to provide contrast between the cells and the background.

The following stage, also known as fixing the dye, entails using iodine to create a
crystal violet-iodine combination in order to stop the dye from being removed easily. The
dye is then removed using a decolorizer, which is frequently a solvent made of ethanol
and acetone. The ability of the bacterial cell wall to hold onto the crystal violet dye after
solvent treatment is the fundamental tenet of gram staining. Gram-positive microbes
contain more peptidoglycan, while gram-negative organisms contain more lipids. All
bacteria initially absorb the crystal violet dye, however gram-negative bacteria's lipid
coating is eventually broken down by the use of a solvent.

Gram negatives lose the main stain when the lipid layer dissolves. As a result of
the pores being closed and the violet-iodine complex being unable to diffuse, solvent
dehydrates gram-positive cell walls, leaving bacteria marked. Gram staining depends on
how long the bacteria are exposed to the decolorizing chemical because lengthy
treatment can completely erase both types of bacteria's stains. The final step in gram
staining is to use safranin to give decolorized gram-negative bacteria pink color for
easier identification, also known as counterstain. Some laboratories use safranin as a
counterstain; however, there is something called basic fuchsin that stains gram-negative
organisms more intensely than safranin.

To avoid contamination at every stage, distilled water should be utilized. The

decolorizing agent can reduce the gaps through which the crystal violet-iodine
complexes might be able to pass by removing water from or dehydrating the
peptidoglycan. This makes it more challenging to get the purple stain off.

The two major groups of bacteria in gram stain can be divided into Gram Positive
and Gram Negative. The difference in the structure of the two groups' cell walls and
membranes serves as the foundation for the gram stain procedure. Gram-positive
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organisms have a thick layer of peptidoglycan that helps preserve the primary dye,
crystal violet, once iodine has been added as a mordant. Within the peptidoglycan, an
intricate mixture of iodine and crystal violet exists. When a decolorizer is used on the
cells, the color changes from dark purple to blue.

There is no significant peptidoglycan layer present in Gram-negative organisms.

Between the inner cell and outside cell membranes, the peptidoglycan was unevenly
distributed. The crystal violet complexes are not trapped inside of the peptidoglycan
after the crystal and iodine application. Applying the acid alcohol decolorizer causes the
outer cellular membrane to dry, causing pores in the membrane and effectively washing
or eliminating the crystal violet complex from the cells. The cells appear to be colorless.
Safranin, a secondary stain that turns gram-negative cells pink to red, is used to make
the colorless cells apparent.

Environmental Samples
There were five agar plates that were exposed in different areas: inside and
outside of the microbiology laboratory, preparation room, specimen room, and in the
laboratory office. Each of the agar plates were exposed for about 30 minutes, then
placed in the incubator for 24 hours.

Results were revealed after the plates were removed from the incubator. One out
of five of the agar plates, the one that was placed inside the laboratory, had resulted
with no growth of bacteria while the majority had. Two out five of the agar plates were
gone through a gram-staining method and studied through the use of a compound
microscope – the exposed agars in the specimen room and outside of the laboratory.
The two samples showed two types of morphology which are bacilli and spirilla.

The sample that was placed outside the laboratory after being stained and
looked through the microscope has bright violet bacilli, shown in Figure A1. The violet
color appears to be gram-positive which only means that the bacterias have thick
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peptidoglycan. This type of bacteria is prone to produce spores. By its ability to form
spores, it can survive in its environment for years (NCBI - WWW Error Blocked
Diagnostic, n.d.).

Shown in Figure A2 is the result of the agar that is exposed outside the
microbiology lab near the trash can. As seen here, spirilla are the one to live and
survive in the plate. The results have gone through exposure for a short period of time,
incubated after 24 hours and gone through Gram staining procedure and a microscope.
With the objective of microscope used the highest lens possible and the help of oil
immersion, it can be seen clearly in the naked eye and can be distinguished as a spirilla
bacteria that has gram-negative cell wall which can be identified by their shape, as
spirilla bacterias are long shaped worm-like appearance, and it can identified by their
color. The color of the microorganisms as positive cell walls are violet to blue colors,
and negative cell walls are pink to red color.

Mixed Culture Sample

The mixed culture sample contained strains of both Escherichia coli and
Staphylococcus capitis. Due to unsuccessful isolation during streaking, both types may
be seen under the microscope, as shown in Figure A3 in Appendix A.

The bacteria like Staphylococcus capitis is coagulase-negative. It is a natural

component of the skin's flora on the human scalp, face, neck, scrotum, and ears.
Lipopolysaccharides are present in these bacteria's outer membrane. The sphere-
shaped bacteria known as aerobic gram-negative cocci also require oxygen to survive.

Under the microscope, bacteria with two different morphologies are observable –
Gram-negative bacilli and Gram-positive cocci. Of the two species known to be in the
mixture, the E. coli species is known to be Gram-negative and rod-shaped (Lim, Yoon,
& Hovde, 2010). This description fits one of the two types observed.

Discussing it specifically, the next sample observed revealed that a variety of

ailments are caused by Gram-negative bacilli. Some of these species are common
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members of the typical gut flora. Disease can be caused by communal organisms as
well as others found in the environment or in animal reservoirs. The Bacillus species are
rod-shaped, endospore-forming, aerobic or facultatively anaerobic, Gram-positive
bacteria. In some species, cultures may eventually turn Gram-negative (S.H. Gillespie
MB, BCh, BAO, MRCP(UK), MRCPath, 1994).

On the same slide, Gram Positive Bacteria, another kind of bacteria, were visible.
They have uniformly thin, peptidoglycan-based walls. Because they maintain their blue-
black hue, they are referred to as gram positive bacteria. Gram-positive cocci are a
sizable collection of loose bacteria that have a common form. All of them are spherical
or almost so, but their sizes differ substantially. With 20% to 30% of the population
being regular carriers of this bacteria, these ubiquitous gram-positive cocci are
frequently detected on the skin and nasal mucosa.

Therefore, Gram-negative Escherichia coli, appearing as bacilli, and Gram-

positive Staphylococcus capitis, appearing as cocci, were both present in the smear
viewed under the microscope.

Finger and Swab Samples

In a swab sample, we looked for different things that can be swabbed and smear
the swab into nutrient agar. After taking the sample, the swab cotton must be
incinerated and then apply aseptic technique. And then, we put the samples or petri
dishes in the incubator, so that we are able to see if there are bacterias where we took a
sample. Incubators make the bacteria grow because it gives the right temperature
where bacterias grow.

After getting the sample or the petri dish, we are able to see if there is bacteria
growth. As for the results, the cotton swab in the shoes had microbial growth. In order to
see the bacteria, the bacteria was transferred on the slide while applying the aseptic
technique. Then, Gram staining was done. After applying the gram staining, we viewed
it in the optical microscope.
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For the swab sample shown in Figure A4, we saw Gram-positive bacilli and
Gram-positive spirilla while for the fingerprint, there were no results because acetone-
alcohol was not rinsed before applying safranin, destroying the sample.

VI. CONCLUSION
Gram staining is the most widely used staining technique in microbiology. It
involves the application of a purple stain (crystal violet), as well as a decolorizer and a
red counterstain (safranin). Gram-positive bacteria are stained blue or purple while
Gram-negative ones are stained pink or red.

Bacteria from natural sources such as the environmental samples may be varied
depending on the source and region. However, some species are expected to appear in
certain sources, such as Escherichia coli in fecal matter and Salmonella spp. in raw
chicken.

Gram staining is a resource-intensive procedure that requires sufficient time and

stain solutions to conduct. It is thus recommended that adequate time and materials be
allotted to participants, especially for beginners who are bound to make mistakes.

Additionally, the importance of following the correct order of solutions during

Gram staining must be emphasized. Each step performed is necessary and may affect
the quality of the smear.

Lastly, practice of aseptic techniques must be ensured to lessen the risk of cross-
contamination. Petri dishes already inoculated with samples must be adequately
protected from contamination to maximize the results and ensure their accuracy.
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REFERENCES

According to Baker H, Bloom WL. Further Studies on the Gram Stain. J Bacteriol;
56(4):387–390. Retrieved Oct 1948, from
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC518598/.

According to Bruner DW. Differentiation between Gram-positive and Gram-negative

Microorganisms by the use of Enzymes. J Bacteriol; 26(4):361-371. Retrieved Oct
1933, from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC533568/.

NCBI - WWW Error Blocked Diagnostic (n.d.). Retrieved from

https://www.ncbi.nlm.nih.gov/books/NBK470553/.

According to Bobby L. BoyantonJr., James Versalovic, in Feigin and Cherry's Textbook

of Pediatric Infectious Diseases (Sixth Edition), 2009 from
https://www.sciencedirect.com/topics/medicine-and-dentistry/microbial-morphology

According to Richard Coico on Gram staining on from

https://currentprotocols.onlinelibrary.wiley.com/doi/abs/

10.1002/9780471729259.mca03cs00

Lim, J., Yoon, J., & Hovde, C. (2010, January). A brief overview of escherichia coli

O157:H7 and its plasmid O157. Retrieved from

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3645889/.

Rupp, M. E., & Fey, P. D. (2015). Staphylococcus epidermidis and Other Coagulase-

Negative Staphylococci. In J. E. Bennett, R. Dolin, & M. J. Blaser (Authors),

Mandell, Douglas, and Bennett's principles and practice of infectious diseases

(9th ed., pp. 2432-2443). Philadelphia, Pennsylvania: Elsevier.

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S.H. Gillespie MB, BCh, BAO, MRCP(UK), MRCPath, S. H. G. M., BCh, BAO,

MRCP(UK), MRCPath. (1994). Gram-negative cocci (the Neisseriaceae).

ScienceDirect. https://www.sciencedirect.com/topics/immunology-and-

microbiology/gram-negative-cocci.

K A Bettelheim, S M Lennox-King (n.d.). The acquisition of Escherichia coli by new-born

babies. National Library of Medicine. https://pubmed.ncbi.nlm.nih.gov/789255/.

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APPENDIX A
Documented Images

Figure A1. Gram-positive bacilli under OIO (100X), environmental sample

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Figure A2. Gram-negative spirilla under OIO (100X), environmental sample

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Figure A3. Gram-negative bacilli and Gram-positive cocci under OIO (100X), mixed
culture sample
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Figure A4. Gram-positive bacilli and Gram-positive spirilla under OIO (100X), swab
sample