I. ABSTRACT
Culture media is essential to obtain
pure cultures, grow and count microbial
cells, and cultivate and choose
microorganisms in most microbiological
experiments. The preparation of culture
media requires a thorough understanding of
the various models or approaches used for
the preparation of any type of culture media.
Sterilization is essential to the preparation of
culture media. The sterility of the culture
media will determine whether microbes will
thrive on it. Accordingly, there is no chance
of microbes growing on sterile culture
media. The main objective of this task is to
prepare sterile culture media by practicing
the specific models or approaches used in
preparing culture media. Among the specific
approaches used in this task is the aseptic
technique, which involves handwashing and
aseptic transfer (from flask to plate).
Following the steps and procedures, the
aseptic media is created. Sterilization of the
media was done in an autoclave before it
was transferred to sterile plates. Although
the culture media was incubated, there were
no visible colonies of microbes on it. Even
in an inapt environment, students were able
to practice methodologies for preparing
culture media, and proved that microbes
cannot grow on sterile media.
II. INTRODUCTION
Culture media are essential for
obtaining pure cultures, growing and
counting microbial cells, and cultivating and
choosing microorganisms for the majority of
microbiological experiments. Accurate,
reproducible, and repeatable microbiological
test results are less likely without high-
quality media (Sandle, 2014). A substance
that promotes the growth, support, and
survival of microorganisms is known as a
microbiological culture medium. Nutrients,
growth-promoting substances, energy
sources, buffer salts, minerals, metals, and
gelling agents are all present in culture
media.
Figure 1. Simple Media—Nutrient Agar
(Shyamal, 2013).
The goal of the medium sterility test
is to find any microbial contamination that
occurred during production. All living
things in a pure culture are descended from
the same species. When working with
microorganisms, we also need a sterile
medium that contains nutrients so that the
organisms can thrive (Kaiser, 2021). A
medium is anything that we use to grow
microorganisms in or on. Any life forms are
absent from a sterile medium. Typically, it is
sterilized by being heated to a temperature at
which all potentially harmful germs are
eliminated. Last but not least, while working
with microorganisms, we need a way to
transfer developing organisms (referred to as
the inoculum) from a pure culture to a sterile
medium without adding any undesirable
external pollutants. Aseptic technique refers
to this approach to avoiding unwelcome
microbes from obtaining entry. To stop
pollutants from developing in microbial
growth media and other liquids needed for
working with bacteria, sterilization is
required. Heating liquids in an autoclave or
pressure cooker is the most effective
approach to sterilize them.

Figure 2. Sterilization process in an
Autoclave (Choudhary, 2020).
Figure 1 shows the sterilization
process of medium in an autoclave. When
employing aqueous preparations for
materials, this method of thermal sterilizing
using an autoclave should be employed
whenever practical. The idea behind this is
to replace the air in the sterilizing chamber
with saturated steam and keep the items
being sterilized exposed to saturated steam
for a long enough period of time to maintain
an efficient combination of time and
temperature to assure sterility. The objective
of this study is to produce a sterile media in
a sterile petri dish using a Nutrient agar.
III. MATERIALS AND METHODS
This chapter discusses the materials
and methods in this laboratory work. The
researchers describe the various processes in
a schematic diagram, which includes the
aseptic techniques, computations, and steps
in culturing a media and incubation. 
A. ASEPTIC TECHNIQUE
Microorganisms being present
everywhere is a hindrance in identifying
certain microbes in the laboratory. Isolating
certain microbes from the rest of unwanted
microbes is an obstacle, especially in a pure
and sterile media. The principle of aseptic
technique helps the researchers, especially
microbiologists, in maintaining a sterile
environment, isolating only the microbes of
interest.  The main defense against
unintentional exposure to harmful
substances or biological agents is proper
handwashing. Despite wearing gloves, it is a
must to always wash hands before and after
each procedure in the laboratory. Aside from
handwashing, proper transferring of media
was observed in this laboratory work.
A.1. Aseptic Transfer (flask to petri dish)
Avoiding contamination of both the
researchers and the culture being maintained
was the determination whether the aseptic
transfer in the microbiology laboratory was
a success or not.
Materials:
1. Alcohol lamp
2. Cotton plug
3. Erlenmeyer flask
4. Petri dish
Ingredients:
1. Distilled water
2. Diluted agar
Figure 1. Materials and Ingredients in
Aseptic Transfer
 Procedure:

1. Alcohol lamp was prepared by the

researcher. The researchers used a match to
lit up the cotton wick of the alcohol lamp.
2. The cotton plug was pulled by the
researcher. The dominant was used by the
researchers in holding the flask and used
their left hand in pulling the cotton plug with
the cotton facing towards the ceiling to
ensure that the cotton would not touch any
surfaces. Figure 3. Flask to plate transfer
3. The tip or mouth of the flask was
heated by using the alcohol lamp. The
researchers made sure that the tip of the B. Sterile Medium in a Sterile Petri Dish
flask was not touching the cotton wick of the —Culture Media (Nutrient Agar)
alcohol lamp. An indication that it was Microorganisms cannot be cultured
properly heated was when a clear tip or with just one kind of culture medium
mouth of the flask was observed. 
because of their many natures, traits,
habitats, and even dietary needs. Culture
media are nutrient- and mineral-rich
mediums that facilitate the development of
microorganisms in the lab.
Materials:
1. Petri dish
2. Alcohol lamp
3. Erlenmeyer flask
Figure 2. Heating the tip of the flask 4. Graduated cylinder
5. Digital weighing scale
3. The agar was transferred from flask to 6. Stirring rod
petri dish by the researcher. A minimal 7. Cling wrap
opening was done in the petri dish by the 8. Paper box
researcher as the laboratory was not a closed 9. Paper
area. About 3 millimeters of agar was 10. Aluminum pot
poured on the petri dish. The researcher 11. Autoclave
slightly swirled the petri dish to make sure
that the agar was balanced.  Ingredients:
1. Distilled water
2. Nutrient agar
 Computation of the needed
ingredients was determined by 28g
of Nutrient agar as per the bottle
indication and 1000 mL of distilled
water. The researcher needed 1.15g
of Nutrient agar computed in figure
5.

28.0 g g
x =1.15 g
1000 mL 41mL

3. 41 mL of distilled water were

weighed by the researchers. After
computing the amounts needed for
the agar, the researchers weighed 41
mL of distilled water into a
graduated cylinder.

Figure 5. Measuring 41mL Distilled

Water

4. 1.15g of Nutrient Agar were

weighed by the researchers. Using
Figure 4. Schematic Diagram of Nutrient a digital weighing scale, the
Agar Preparation researchers were able to weigh an
exact amount of agar needed.
Procedure:
1. The ingredients and materials
were prepared by the researchers.
The researchers organized, washed,
and prepared the petri dish,
Erlenmeyer flask, graduated
cylinder, alcohol lamp, stirring rod,
and cling wrap. The digital weighing
scale and autoclave was provided by
the adviser.
2. Needed ingredients were Figure 6. 1.15g Nutrient Agar
calculated by the researchers.
5. Nutrient agar and distilled water 7. The researchers used a cling wrap to
were mixed into the Erlenmeyer flask seal the petri dish. The researcher used a
by the researchers. The laboratory small piece of petri dish and wrapped it
does not have any funnel to provide, so around both mouths of the petri dish to
the researcher poured the agar into the secure it.
flask with the improvised funnel paper
to make sure that every bit of the agar
was mixed into distilled water. After the
researchers poured all the ingredients,
they mixed it with a stirring rod.

Figure 9. Petri dish wrapped using cling

wrap

8. The researchers autoclaved the media

in Erlenmeyer flask and the petri dish. A
Figure 7. Mixing the ingredients
paper was used by the researchers to wrap
the petri dish and wrapped the mouth of the
6. The mixed ingredients in the flask
flask with foil and sealed it with a tape that
were heated using water bath by the
served as a protection of the media from
researchers. In heating, the researchers
contamination before putting it inside the
used an aluminum pot with water on it as
autoclave. The media was autoclaved at
an alternative for water bath. The media
121C for 15-20 minutes by the researchers
was lifted time to time and stirred in
with the supervise of their proctor.
circular motion by the researcher until all
particles were diluted. The agar to cool
down for a bit before transferring it to the
petri dish.

Figure 10. Autoclaved media and petri dish

9. The media was transferred from flask
to petri dish by the researcher. Using the Ta
Figure 8. Water bathing of Media aseptic transfer procedure in A.1., the PR
researchers transferred the media from flask that autoclaves are mostly used in sterilizing
to petri dish with minimal opening to avoid lab equipment. Autoclaves are essential
contamination in the laboratory as it was not laboratory equipment for the disinfection
a closed area. and sterilization of tools and biohazard
Figure 11. Flask to plate media transfer products. However, sterilizing at higher
altitude geographical locations with an
10. The culture media was incubated for autoclave presents special issues. Due to the
one (1) day. After autoclaving the media, decrease in atmospheric pressure and lower
the researchers let the media in the boiling points of higher altitude areas, a
incubator for one (1) day. The result of the high-altitude autoclave must be used to
media was provided in ensure proper sterilization. These autoclaves
figure…………………………. are specifically designed to compensate for
the decrease in pressure and lower boiling
points, allowing for the same level of
IV. RESULTS AND DISCUSSION sterilization as at lower altitudes.
Additionally, these autoclaves are equipped
This chapter presents, with a pressure gauge to monitor the
comprehensively discussed, and interpret the pressure inside the chamber, ensuring that
result observed by the researchers of their the correct pressure is maintained for proper
study. sterilization. High-altitude reprogramming is
essential for all autoclaves when operating at
1,200m or more above sea level. Without
FACTORS this reprogramming, autoclaves may not be
AREAS able to reach the necessary temperatures and
Altitud Climate Average
pressures for proper sterilization, leading to
e (m) Temperatu
potential safety risks. Additionally, improper
re (celcius)
sterilization can lead to the spread of
UP 1,480 Tempera 27-30 infectious diseases and other health hazards.
BAGUI te Therefore, it is critical to ensure that
O autoclaves are properly reprogrammed for
high-altitude operation in order to ensure the
PRMSU 11 Tropical 28-31 safety of patients and healthcare workers
IBA Monsoo WHO Laboratory Safety Manual (3rd
n Edition, p27). From table 1, it is clear that
there is a vast difference between the
Table 1 shows data regarding the
altitude of UP Baguio and PRMSU Iba, this
environmental factors surrounding President
entails that there will be a difference in their
Ramon Magsayday State University
sterilization parameters in order for them to
(PRMSU) Iba Campus and University of
achieve full sterilization. This problem can
The Philippines (UP) Baguio. Altitude refers
be solved by UP Baguio purchasing
to the area’s distance from sea level.
autoclaves designed for high-altitude use, a
Altitude determines the possible temperature
more practical way to solve this is
of an area. The higher the altitude the lower
autoclaving the instruments for a little
the temperature (Costa et al., 2022). 
longer to achieve full sterilization, if the
autoclave device they have has a fixed
The reason behind why determining
pressure. If the autoclave has an adjustable
altitude is important before sterilization is
pressure, then it is recommended that the
scientists increase the pressure to match the
altitude and to achieve sterilization time
equal to autoclaving at low altitudes.
A. Microbial Growth Prospects in
Various Set-Ups:
A.1. Set-up A: Non-sterile medium in a
sterile plate
In this setup, the growth of microbial
colonies was expected. The reason for this
expectation is that the medium is not sterile.
If the students conducting this set-up did not
use aseptic techniques when preparing the
medium, microbial growth in the medium is
unavoidable. In addition to that, the
environment where the set-up is placed is
not optimal for such laboratory exercises.
With these factors, the most plausible
expectation is that there will be microbial
growth in the said setup.
A.2. Set-up B: Sterile medium in a sterile
plate
In this setup, no growth of a colony
of any microbe is expected. The reason
behind this assumption is the conditions in
the given setup. The media used is sterilized,
similar to the plate into which the nutrient
agar was poured. However, accounting for
the environment where the set-up was done,
which is not suitable for the laboratory
activity assigned, the students initially
thought that there was a high chance that
microbe colonies would grow in the culture
media.
A.3. Set-up C: Non-sterile medium in a
non-sterile plate.
In this setup, the growth of microbes
is expected. Given the conditions of the
variables involved, not to mention the
environment in which this set-up is being
done, microbial growth is anticipated. Not
just one type, but a variety of colonies will
grow in this setup.
A.4. Set-up D: Sterile medium in a non-
sterile plate
In this setup, the growth of microbes
is expected. Even though the medium used
is sterilized, the plate where the medium is
going to be poured is not. Remember that in
this laboratory activity, if students aim to
produce a culture media with no microbial
growth both the medium and the plate must
be sterile. If those conditions are not met,
then microbial growth is unavoidable. The
development of microbes in this set-up will
be from the plate which was not sterilized
A.5. Set-up E: Swab Inoculation Method
(Skin)
Microbial growth is expected in this
setup, where human skin is swabbed to
obtain a sample. Microbial presence is the
result the students are aiming for. The main
expectation is what kind of bacteria is going
to grow. According to Grice and Segre
(2011), the primary bacteria that colonize
the human skin are Staphylococcus
epidermidis and other coagulase-negative
staphylococci. With this, one of the colonies
that will grow in the culture media is those
that primarily colonize it.
A.6. Set-up F: Swab Inoculation Method
(Paper Bill)
In this setup, where a paper bill is
swabbed to obtain a sample, microbial
growth is expected. There will be variations
in the microbial colony due to the following
conditions: the environment where the set-
up is located, the set-up's nature, and the
paper bill and the microbes that got stuck to
it from being handed from person to person.
The reason behind the assumption of various
microbial colony formation is due to the
experiences the paper bill has gone through
and the microbes from the hands of people
that handled the bill, therefore making the
bill a bacterial playground.

A.7. Set-up G: Capture Plate Method

(Sterile Media in a Sterile Plate)

This setup included 30 minutes of

holding the sterile culture media in the air.
All types of bacteria are known to be present Figure 1. Sterile media and sterile plate set-
in air, therefore depending on the up after 24 hours being incubated (bottom
environment the culture media is exposed to, view)
different microbial colonies will form. The
assumption from this arrangement is that In figure one (1), it is clear that there
there will be a variety of bacterial colonies is no visible growth of microbes’ colony in
to form, taking into account the length of the nutrient agar media. It shows that the
time the culture material was exposed. researchers were able to execute the
methodologies properly. In terms of set-up,
they were able to accomplish the given task
Incubation plays a vital role in to them despite the inapt environment where
catalyzing the growth of microbes. The the set-up was located. To further support
optimal temperature range for microbial this result here is another figure of the petri
growth is between 4.4 to 60 degrees Celcius dish.
(Danger Zone (40 °F - 140 °F), 2018). This
temperature range is often referred to as the
"Danger Zone" due to the fact that microbes  
can double in number in a short period of
time. It is important to ensure that
incubation temperatures remain within this
range to prevent the rapid growth of
microbes. Here is a figure for the nutrient
agar after being subjected to an incubator for
boosting the microorganisms’ growth. 
 

 

Figure 2. Sterile media and sterile plate set-
up after 24 hours being incubated (bottom
view against a light source)
Figure 2 shows the sterile plate
against the light to see if there is anything
that was missed in the first figure (figure 1).
This figure shows that the media is clear and
that there is indeed no microbial colony that
grew in the sterile media and sterile plate
set-up.  Culture-Media-in-Pharmaceutical-
V. CONCLUSIONS AND
RECOMMENDATION
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1. http://www.mmc.gov.bd/downloadable
%20file/Culture%20media%20by
%20Shyamal.pdf