Colegio de San Juan de Letran

College of Business Administration and Accountancy

Food Technology Area

Isolation of Normal Flora

(Experiment 5)

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:
December 9, 2022

Submitted to:
Ms. Sheena Marie Napata
Instructor
 Colegio de San Juan de Letran
College of Business Administration and Accountancy
Food Technology Area

I. ABSTRACT
Normal flora are microbes that live in the human body
without causing diseases. They may become opportunistic
pathogens once an imbalance in population count occurs. In
this experiment, mannitol salt agar (MSA) was used to
selectively grow halophiles and differentiate mannitol
fermenters from non-fermenters with the help of the phenol
red indicator. Scalp and forearm swab samples were
inoculated to confirm the presence and identity of the
microbes in the skin’s normal flora. Normal scalp and
forearm flora were found to be dominated by
Staphylococcus aureus while Staphylococcus epidermidis
and Micrococcus luteus appeared occasionally. These
bacteria, as with other normal flora, are involved in the
body’s immune defense by providing competition against
foreign pathogens. However, imbalances in microbial count
could cause diseases. Because the experiment was
conducted without a uniform streaking technique, it is also
recommended that a uniform streak be established for later
experiments.

II. KEYWORDS: normal flora, opportunistic pathogen, mannitol salt agar (MSA),
halophile, phenol red

III. INTRODUCTION
Microorganisms classified as normal flora are those that thrive on a different
living thing (human or animal) or an inanimate object without spreading disease. The
human body is not sterile; from the moment we are born, we are surrounded by
microorganisms.
 Colegio de San Juan de Letran
College of Business Administration and Accountancy
Food Technology Area

In addition to the high salt concentrations that prevent the development of the
majority of gram-negative bacteria, mannitol salt agar (MSA) is a nutritional agar that
additionally includes the sugar mannitol and the pH indicator phenol red. Mannitol is
used to help identify an unidentified microorganism. Mannitol salt agar is a specialized
bacterial growth medium that is selective for halophiles and can distinguish between
pathogenic and non-pathogenic staphylococci.

To isolate pathogenic Staphylococci, mannitol salt agar is used as a selective

medium. It is advised for the identification and counting of coagulase-positive
Staphylococci in milk, food, and other specimens.

Mannitol salt phenol red agar is used for the isolation and putative identification
of Staphylococcus aureus in non-sterile pharmaceuticals and foods.

Selective isolation of presumptive pathogenic (pp) Staphylococcus species is

accomplished using mannitol and phenol red. For fermentation, mannitol serves as a
carbohydrate substrate, enabling a range of medium conditions. The pH indicator is
phenol red.

This experiment aims to isolate bacteria from normal flora using aseptic
techniques.

IV. METHODOLOGY
Materials
The materials were used for isolating the sample that is captured from the
forearm and head normal flora. Mannitol salt agar (MSA) plates were used to put the
sample from the scalp and forearm. Sterile saline used to wet sterile swabs. Sterile
swabs were used to get samples from skin and head.

Procedure
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College of Business Administration and Accountancy
Food Technology Area

1. Sterile swabs and sterile saline were prepared.

2. The agar plate was labeled with the participant’s last name and divided into two
portions, one for the scalp culture and another for the forearm culture.
3. A sterile swab was wet with the sterile saline and firmly rubbed onto the participant’s
scalp, ensuring no contact with other areas of the head.
4. The swab slowly rolled across the MSA plate’s surface allotted for the scalp culture.
5. The procedure was repeated once again on the student’s forearm using a new
swab.
6. The plate was incubated at 35–37°C for 24-48 hours.

V. RESULTS AND DISCUSSION

Scalp Culture
The scalp culture procedure used sterile water and swab cotton to expose it to
the person's scalp to determine the presence of microorganisms on their scalps. The
swab must be dipped in sterile water first because the swab must be moist or wet to
pick up microbes in the area (Landers, Hoet, & Wittum, 2010). After inoculation, the
dishes were incubated for 24 hours.

Two out of 12 MSA dishes had more presence of golden or yellow color, as
shown in Figure A1 of Appendix A. The presence of numerous yellow or golden color
colonies on the mannitol salt agar indicates that the person's scalp contains
Staphylococcus aureus, and the bright golden color found in the agar is the result of
Staphylococcal fermentation. They are the only ones that produce golden colors due to
the acidic by-product that changes the pH of the agar, which in turn changes into yellow.
And the lower the number of golden color colonies created, the fewer the
Staphylococcus aureus that live on the person's scalp.

Forearm Culture
 Colegio de San Juan de Letran
College of Business Administration and Accountancy
Food Technology Area

Aside from the scalp part of the human body, another part, the forearm was
cultured. In the first procedure of the experiment, the sterile swab was wet with sterile
saline. This was done to gather more microorganisms from the site, rather than the dry
swab (Landers, Hoet, & Wittum, 2010). After it was wet with the solution, the wet swab
was smeared onto the forearm. The procedure was done twice, then placed into the
incubator for two days, enough for the bacteria to grow.

Two out of 12 of the dishes were identified with yellow appearance, indicated as
pathogenic bacteria named as Micrococcus luteus, shown in Figure A1 in Appendix A. It
also produced a golden stain which only means that the bacteria produced
fermentation. The bacteria is called Staphylococcus aureus. Out of 12 dishes, two
dishes were revealed with no bacterial growth which only means that the source of the
sample is clean. The remaining dishes revealed the same white appearance which is
the Staphylococcus epidermidis. The table below shows the summary of the
experiment.

Table 1. Forearm results, with (-) indicating no growth and (+) indicating bacterial growth.

Participants Trial 1 Trial 2

S. M. luteus S. aureus S. M. luteus S. aureus
epidermidis (yellow) (golden) epidermidis (yellow) (golden)
(white) (white)

P1 + - - + - -

P2 - + + - + +

P3 - - - - - -

P4 + - - + - -

P5 + - - + - -

P6 + - - + - -

Benefits and Threats of Normal Flora

 Colegio de San Juan de Letran
College of Business Administration and Accountancy
Food Technology Area

Normal flora are found on the skin and in various mucous membranes in the
human body (Davis, 1996). These membranes include the oral and upper respiratory
tract, the gastrointestinal tract, the urogenital tract, and the conjunctiva. Because these
membranes exist in different parts of the body, the microorganisms present in each also
vary.

On the skin, the most common microorganisms are Gram-positive bacteria such
as Staphylococcus spp., Micrococcus spp., and diphtheroids, with Gram-negative bacilli
and Streptococcus spp. appearing in small amounts. Streptococci are also found on the
oral and upper respiratory tract, Helicobacter pylori and Escherichia coli in the
gastrointestinal tract, E. coli in the urogenital tract, and Haemophilus and
Staphylococcus spp. in the conjunctiva (Davis, 1996).

Normal flora is often overlooked when non-medical professionals consider

potential causes of diseases. However, they are intricately connected with our health
status.

Normal flora on the skin contribute to the body’s first line of defense by acting as
competition against the invading pathogens (Sommer, Reboli, & Heymann, 2018).
Normal flora present in the body’s mucous membranes are also involved in the body’s
immune response by providing competition against foreign pathogens, similar to the
normal flora on the skin (“Normal flora of human body”, n.d.). In the gastrointestinal
tract, the normal flora has an additional role involving the production of vitamin K, which
cannot be synthesized by the body itself, as well as the fermentation of dietary fibers
(Valdes, Walter, Segal, & Spector, 2018).

Although normal flora are largely associated with good health, they may also
cause harm under certain circumstances. When imbalances occur in their environment,
causing a drastic increase or decrease in population count, they may lead to various
diseases.
 Colegio de San Juan de Letran
College of Business Administration and Accountancy
Food Technology Area

On the skin, an infection may occur once the skin stops acting as a barrier, as in
the case of a wound. As for the mucous membranes, imbalances may lead to infection
as well, manifesting as colds in the respiratory tract, genital infection in the genitalia,
and ulcers in the gastrointestinal tract.

VI. CONCLUSION
The results indicated that the amount of normal flora may differ from person to
person. The presence of microorganisms is largely dependent on the person’s hygiene,
as shown by two trials presenting an outlier case for both trials. A variety of microbes
were observed, with Staphylococcus aureus, Staphylococcus epidermidis, and
Micrococcus luteus appearing in both forearm and scalp cultures.

Normal flora may present both benefits and threats to the body, dependent on
their balance and the environment they are in. Benefits include enhancement of immune
defense, as well as the production of vitamin K for gastrointestinal flora specifically.
Threats include infection when microbial imbalances occur.

When conducting the inoculation of normal flora, it is recommended that samples

continue to be obtained using wet swabs. Further experiments may also be performed
with other skin portions as sample source for further comparison with the obtained
results from this experiment.

Additionally, streaking of swabs must be uniformly performed for all trials and
participants, and considered as a controlled variable to reduce the factors that may
affect the results.
 Colegio de San Juan de Letran
College of Business Administration and Accountancy
Food Technology Area

REFERENCES

Davis, C. (1996). Normal flora. In: T. Albrecht & S. Baron (Authors), Medical
microbiology (4th ed.). Galveston, Texas: University of Texas Medical Branch at
Galveston, Dept. of Microbiology & Immunology. Retrieved from
https://www.ncbi.nlm.nih.gov/books/NBK7617/.

Landers, T. F., Hoet, A., & Wittum, T. E. (2010). Swab type, moistening, and
preenrichment for Staphylococcus aureus on environmental surfaces. Journal of
Clinical Microbiology, 48(6), 2235-2236. doi:10.1128/jcm.01958-09.

Normal flora of human body (n.d.). Retrieved from

https://www.nios.ac.in/media/documents/dmlt/Microbiology/Lesson-07.pdf.

Sommer, L. L., Reboli, A. C., & Heymann, W. R. (2018). Bacterial diseases. In: J. L.
Bolognia, J. V. Schaffer, & L. Cerroni (Authors), Dermatology (4th ed., pp. 1259-
1295). Elsevier Limited. Retrieved from
https://www.clinicalkey.com/#!/content/book/3-s2.0-B978070206275900074X.

Valdes, A., Walter, J., Segal, E., & Spector, T. (2018). Role of the gut microbiota in
nutrition and health. BMJ. doi:10.1136/bmj.k2179.
 Colegio de San Juan de Letran
College of Business Administration and Accountancy
Food Technology Area

APPENDIX A
Documented Images

Figure A1. Scalp and forearm cultures for both trials of one participant, showing S.
aureus (golden colonies) and M. luteus (yellow colonies)