LAPORAN PRAKTIKUM MIKROBIOLOGI INDUSTRI

MODUL : Determination of Microorganism Cell Count

KELOMPOK :2

Nama Praktikan : Muhamad Farhan Randhie Hakim

NRP : 5008231191
Nama Praktikan : Afif Mughni
NRP : 5008231105
Nama Praktikan : Ardino Wibowo Purwoko
NRP : 5008231147

ASISTEN
Nama Asisten : Dian Maulidya Hanif
NRP : 5008201120

DOSEN PENGAMPU
Nama Dosen Pengampu : Dr. Ir. Sri Rachmania Juliastuti, M.Eng. IPM
NIP : 195907301986032001
Tanggal Praktikum : 9 October 2024
Tanggal Pengumpulan Laporan : 16 October 2024

LABORATORY OF PHYSICAL CHEMISTRY AND INDUSTRIAL

MICROBIOLOGY
DEPARTMENT OF CHEMICAL ENGINEERING - FACULTY OF INDUSTRIAL
TECHNOLOGY AND SYSTEMS ENGINEERING
INSTITUT TEKNOLOGI SEPULUH NOPEMBER
 I. OBJECTIVES

The objectives are to understand various sterilization methods, comprehend

the purpose of sterilization and disinfection, and effectively perform both physical and
chemical sterilization while maintaining aseptic techniques. Additionally, the aim is to
obtain sterile tools and media, become familiar with different types of media and their
preparation methods, and recognize the functions of microbial growth media.

II. BASIC THEORY

The purpose of the second practicum module on Sterilization Techniques and

Asepsis, along with Media Preparation, is to familiarize participants with various
methods of sterilization, understand the purposes of sterilization and disinfection,
perform both physical and chemical sterilization, and carry out aseptic procedures.
Participants are expected to obtain sterile equipment and media, learn about various
types of media and their preparation methods, and recognize and comprehend the
functions of microbial growth medium.

This practicum involves autoclave sterilization and aseptic work. Participants

must follow aseptic procedures from the start of the practicum until the end, which
includes washing their hands, removing unnecessary items from the workspace,
wearing masks and latex gloves, cleaning the workspace with 70% alcohol, and
wiping with dry tissue. Participants must also spray a disinfectant (70% alcohol) on
their gloved hands before and after each experimental procedure.

The next step after aseptic preparation is to prepare the media.

Microorganisms use media as a growth and propagation medium because it contains
the nutrients they require. As a result, the media must meet certain requirements, such
as maintaining a neutral pH, containing various nutrients required by microorganisms,
and being sterilized prior to use (Juariah, 2018). In microbiology, aseptic techniques
are used to reduce contamination from unwanted microorganisms. To achieve asepsis,
all equipment and culture media must be sterilized (Johnson, 2018). Sterilization is
the process of eliminating all microorganisms, including endospores. This can be
 accomplished using chemical, mechanical, thermal, or ultrasonic energy sources.
Disinfection is the process of chemically sterilizing inanimate objects' surfaces.
Mechanical sterilization is accomplished through filtration, which employs a special
filter designed to remove microorganisms. Thermal sterilization can be performed
with an autoclave (moist heat), an oven (dry heat), or a direct flame (Green, 2021).
The autoclave is the most effective sterilization tool, as it sterilizes equipment,
supplies, and media used in experiments to ensure aseptic results. The autoclave uses
steam heated to 15 psi and 121°C, with sterilization lasting 15 minutes (Hardono and
Supriyadi, 2020).

In addition to sterilizing the equipment to be used, the culture media must be

carefully examined. Culture media is required for culturing, transferring, and storing
microorganisms; thus, it must contain all nutrients appropriate for the
microorganisms. Culture media are classified based on their chemical composition,
physical properties, and functions. Culture media can be classified as synthetic or
complex based on their chemical composition. Culture media are classified into three
types based on their physical properties: liquid, semi-solid, and solid. Culture media
can also be classified as supportive, enriched, selective, or differential based on their
functions (Willey, 2020). This experiment used two types of media: nutrient broth
(NB) and nutrient broth agar (NBA). Nutrient Broth (NB) is a liquid medium that is
commonly used and contains carbon and nitrogen. Beef extract is one of the
components of NB and serves as a nutrient source for bacteria (Wahyuningsih, 2018).
Nutrient Broth Agar, also known as Nutrient Agar, is a culture medium used to isolate
bacteria. It is made by solidifying Nutrient Broth (1%) and adding agar (2%)
(Napitupulu et al., 2019).

III. TOOLS AND MATERIALS

The equipments required for this practicum is as follows:
Table 1. Tools

No. Tools Quantity

 1. Weighing paper 1

2. Volumetric pipette 1

3. Beaker glass 2

4. Autoclave 1

5. Test tube 4

6. Washing bottle 1

7. Inoculating loop 1

8. Petridish 2

9. Analytical balance 1

10. Hotplate stirrer 1

11. Magnetic stirrer 1

12. Bunsen burner 1

13. Incubator 1

Table 2. Materials
 No. Materials Quantity

1. Nutrient Broth (NB) Liquid Media 8g

2. Nutrient Broth Agar (NBA) Media 20 g

3. Distilled Water (Aquades) 2L

4. Fatty Cotton 4

5. Brown Wrapping Paper 2

IV. METHODOLOGY
a. Preparing Liquid Media (NB):
1. Weigh 8 grams of Nutrient Broth.
2. Dissolve the Nutrient Broth in 1000 mL of distilled water (aquades).
3. Cover the beaker glass with aluminum foil or brown paper.
4. Sterilize in an autoclave at 121°C for 15 minutes.
5. Label the beaker glass.
6. The media is ready to use.

b. Preparing Solid Media (NBA):

1. Weigh 39 grams of PDA or 20 grams of NBA.
2. Dissolve the PDA in 1000 ml of distilled water (aquades) while heating.
3. Stir until fully mixed and homogeneous.
4. Cover the beaker glass with aluminum foil or brown paper.
5. Sterilize in an autoclave at 121°C for 15 minutes.
6. After sterilization, pour the media into petri dishes or test tubes. The volume
of media in the petri dish should not exceed half, and in the test tubes, it
should not exceed one-third of the tube.
 7. Each test tube should be covered with oil-free cotton. Then, tilt the test tubes
so that the solid media is slanted, but do not let it touch the cotton plug. Let it
cool.
8. Label each test tube and petri dish.
9. The media is ready to use.

c. Direct Flame Sterilization

1. Light the Bunsen burner.
2. Before use, heat the inoculating loop from the tip to the base of the wire until
it glows red.
3. Move it away from the flame.
4. Hold the inoculating loop until it cools, keeping it near the lit Bunsen burner.
5. The inoculating loop is now ready for use.
6. Reheat the inoculating loop after use, following step 2.
7. Do not place the inoculating loop on the workbench if it has not been heated
until it glows.

d. Autoclave Sterilization
1. Prepare the media to be sterilized. The volume of liquid and media placed in
the container should not exceed half of the container's capacity to prevent
spills. Ensure the media is covered and wrapped. Media in Erlenmeyer flasks
should be sealed with oil-soaked cotton stoppers and wrapped in brown paper.
2. Place all test tubes in a beaker glass and cover the top with brown paper or
aluminum foil.
3. Wrap all petri dishes in brown wrapping paper.
4. Place all the media into the autoclave. Arrange them carefully to prevent
spills. Sterilize in the autoclave at 121°C for 15 minutes.

e. Aseptic Work (Aseptic Technique)

1. Always wash your hands with soap before and after working.
2. Before starting, remove any unnecessary items from the workbench. Ensure
you are wearing a mask and disposable latex gloves. Disinfect the surface of
the work area by spraying disinfectant (70% alcohol). Let it sit for a few
seconds, then wipe it with a dry tissue.
 3. Use hand sanitizer or spray disinfectant on your palms, the back of your
hands, and your wrists (while still wearing gloves). Let it dry.
4. Turn on the Bunsen burner. Be cautious when working with alcohol near the
Bunsen flame.
5. Pass and rotate the edge of the petri dish near the Bunsen flame, then slightly
open the lid of the petri dish while working near the flame. Use your thumb
and index finger to open the petri dish lid, while the other three fingers hold
the base. Close the petri dish and pass and rotate its edge near the Bunsen
flame again.
6. Open the cotton stopper near the Bunsen flame. Pass and rotate the mouth of
the test tube/Erlenmeyer flask near the flame. Replace the cotton stopper.
7. Do not place stoppers, test tube caps, or any container lids on the workbench.
Hold the lid in your hand throughout the aseptic process.
8. After finishing, disinfect the workbench by spraying disinfectant (70%
alcohol). Let it sit for a few seconds, then wipe with a dry tissue.
9. Dispose of gloves in a designated waste bin for gloves and masks.
10. Wash your hands with Dettol soap.

V. RESULTS AND DISCUSSION

The practicum on sterilization techniques and media preparation aims to

familiarize participants with various methods of sterilization, understand the purposes
of sterilization and disinfection, perform physical and chemical sterilization, and carry
out aseptic work. Participants are expected to obtain sterile equipment and media,
learn about different types of media and their preparation methods, and recognize the
functions of microbial growth media. Aseptic techniques are crucial for preventing
contamination by unwanted microorganisms. To achieve asepsis, equipment and
culture media must be sterilized (Johnson, 2018). This practicum focuses on aseptic
work and sterilization techniques using an autoclave. Aseptic practices are followed
from the preparation phase before the practicum begins to its conclusion. This
includes washing your hands, cleaning the workspace with 70% alcohol, and properly
disposing of waste. Additionally, participants must spray 70% alcohol on their gloved
hands before and after each experimental procedure. Following aseptic preparation,
media preparation is the next step. Because of the composition of nutrients required
for microorganism growth and propagation, media serves as a growth and propagation
medium. As a result, certain requirements for the media must be met, such as
maintaining a neutral pH, containing a variety of microorganism-friendly nutrients,
and being sterilized before use (Juariah, 2018).

Picture 1. Prepared Media

This practicum involved preparing two types of media: nutrient broth (NB) in
test tubes and nutrient broth agar (NBA) in petri dishes and inclined test tubes. To
prepare NB, weigh 8 grams of NB powder with an analytical balance, then mix it with
1000 mL of distilled water in a beaker and stir until the powder dissolved, yielding a
clear yellow-orange solution. To prevent contamination by airborne microorganisms,
the beaker was covered with aluminum foil. For the NBA media, 20 grams of NBA
powder were mixed with 1000 mL of distilled water in a beaker and homogenized
with a magnetic stirrer. When the solution appeared clear and yellow-orange,
indicating complete dissolution, it was sterilized with an autoclave.

Picture 2. NBA (left) and NB (right) before sterilized

Other equipment, such as petri dishes and test tubes, were sterilized in a
separate autoclave based on its capacity. Before placing the petri dishes in the
autoclave, they were wrapped in brown wrapping paper, lids facing upward, and
placed on the paper. The glossy side of the wrapping paper faced out, and the test
tubes were filled with mineral oil-soaked cotton. This wrapping and plugging was
done to prevent water from entering the petri dishes and test tubes, thereby avoiding
contamination.
The autoclave uses moist heat (steam) to sterilize (Green, 2021). Initially, the
tank is filled with water up to the indicated level. The media and equipment to be
sterilized are then neatly placed inside the autoclave. The lid is securely fastened, and
the preheat mode is activated at 121°C. The autoclave makes a sound to indicate that
the temperature has reached 121°C. A timer is then set to 15 minutes for the
sterilization process. After this time, the autoclave will sound again, indicating that
the sterilization is complete. The steam valve is opened to lower the pressure inside
the autoclave, allowing it to be opened to retrieve the sterilized media and equipment
(Wati, 2021).
 After sterilization, pour the sterile media into the sterilized petri dishes and
test tubes. This step is carried out within a laminar air flow hood, which filters
external air in several stages to provide sterile airflow (Amrulloh et al., 2021). The
NBA is poured into petri dishes until they are half full, and then closed. NBA is
poured into the test tubes until one-third full, then plugged with mineral oil-soaked
cotton. The test tubes are tilted without touching the cotton plug to allow the media to
solidify. This inclination increases surface area, making it easier to inoculate or plant
microorganisms. To minimize microbial contamination, the pouring process must
strictly adhere to aseptic principles, such as flaming the edges of the petri dishes and
the mouths of the test tubes prior to and after pouring the media. To prevent spillage,
care is taken not to overfill the media. Prior to proceeding, the media is allowed to
solidify. Once solidified, the test tubes are sealed with mineral oil-soaked cotton and
placed in a beaker. The media-containing petri dishes are wrapped in plastic wrap and
placed in an incubator, along with the test tubes. The incubator includes temperature
and time controls to ensure that the incubation conditions are optimal for the
microorganisms being cultured (Halla et al., 2019).

Picture 3. Contaminated Solidified NBA

 According to the practicum result, as shown in Picture 3., after 24 hours of
incubation, one NBA media sample was contaminated, while the other NBA and NB
media samples were uncontaminated. The contamination was identified by a cloudy
appearance and the presence of white spots, indicating that the media preparation was
not aseptic. Several factors contributed to these aseptic technique errors during the
practicum.

Picture 4. Uncontaminated NBA (left) and Uncontaminated NB (right)

The results on Picture 4. show that both Nutrient Broth (NB) and Nutrient
Broth Agar (NBA) remained uncontaminated after 24 hours of incubation. There was
no microbial growth on the Nutrient Broth surface in the test tube. The transfer of
Nutrient Broth into the test tube was done under strict aseptic conditions, effectively
eliminating the possibility of microbial contamination in the media. This ensured that
sterile conditions persisted throughout the experiment.

VI. CONCLUSION

Based on the results of the experiment, it is possible to conclude that

sterilization can be achieved effectively using an autoclave set to 121°C for 15
minutes. The primary goal of sterilization is to destroy all microorganisms and avoid
contamination. Disinfection, a chemical method of sterilization, focuses on the
surfaces of inanimate objects. Throughout the practicum, aseptic techniques were
used alongside the sterilization process. Physical sterilization was performed on both
media and equipment using an autoclave, whereas chemical sterilization involved
 disinfecting the work surface and hands with 70% alcohol during the experiment. As a
result, sterile equipment was obtained via autoclave.

However, while Nutrient Broth (NB) was successfully sterilized, one of the
Nutrient Broth Agar (NBA) remained contaminated despite being autoclaved under
the same conditions. This contamination was caused by lapses in aseptic technique
during practicum. The media perform the critical functions of cultivating, transferring,
and storing microorganisms by supplying the necessary nutrients.

BIBLIOGRAPHY

Amrulloh, M. F., Purnama, H., & Margana, A. S. (2021). Sistem Monitoring Kecepatan
Aliran Udara Dan Suhu Pada Laminar Air Flow Cabinet Menggunakan Hmi
Berbasis Mikrokontroller. SEMNASTERA (Seminar Nasional Teknologi dan Riset
Terapan, 3, 144-150.
Green, Lorrence H., and Emanuel Goldman. (2021). Practical Handbook of Microbiology
Fourth Edition. Florida: CRC Press.
Halla, S., Rohmi, R., & Agrijanti, A. (2019). Efektivitas Inkubator Portable Sebagai Alat
Inovasi Penunjang Laboratorium Mikrobiologi. Jurnal Analis Medika Biosains
(JAMBS), 6(1), 66-72.
Johnson, Ted R., and Christine L. Case. (2018). Laboratory Experiments in Microbiology
Twelfth Edition. Hoboken: Pearson Education, Inc.
Napitupulu G. Hatopan, dkk. (2019). Bacillus sp. SEBAGAI AGENSIA PENGURAI
DALAM PEMELIHARAAN Brachionus rotundiformis YANG
MENGGUNAKAN IKAN MENTAH SEBAGAI SUMBER NUTRISI. Manado:
Program Studi Ilmu Kelatuan, Universitas Sam Ratulangi.
Wati, R. Y. (2021). Perbandingan Performa Autoclave Manual Sebagai Alat Killing
Mikroba Di Laboratorium Mikrobiologi Teknologi Hasil Pertanian Unand. Jurnal
Temapela, 4(2), 56-60.