LAPORAN PRAKTIKUM MIKROBIOLOGI INDUSTRI

MODUL : Sterilization Technique and Aseptic

KELOMPOK : (4 / 6 March 2023)

Nama Praktikan : Adlina Firza
NRP : 5008211142
Nama Praktikan : Rayhan Shahreza Ferdian Ferryanto
NRP : 5008211143
Nama Praktikan : Kresna Ramadhani Fauzan
NRP : 5008201072

ASISTEN
Nama Asisten : Adidoyo Prakoso
NRP : 02211940000105
Nama Asisten :
NRP :

DOSEN PENGAMPU
Nama Dosen Pengampu : Dr.Ir.Sri Rachmania Juliastuti,M.Eng
NIP : 195907301986032001

Tanggal Praktikum : 6 March 2023

Tanggal Pengumpulan Laporan : 13 March 2023

LABORATORIUM KIMIA FISIKA DAN MIKROBIOLOGI INDUSTRI

DEPARTEMEN TEKNIK KIMIA - FAKULTAS TEKNOLOGI INDUSTRI DAN
REKAYASA SISTEM INSTITUT TEKNOLOGI SEPULUH NOPEMBER
I. Objective

1. Recognize several ways of sterilization.

2. Understand the purpose of sterilization and disinfection.
3. Perform physical and chemical sterilization, and can perform aseptic work.
4. Obtain sterile tools and media.
5. Knowing the types of media and how to make them.
6. Recognize and know the function of microbial growth media.

II. Basic Theory

Disinfectants are chemicals used to inhibit or kill microorganisms on inanimate

surfaces. Disinfectants cannot be used on the skin or mucous membranes, because their
properties that damage tissues have the risk of irritating the skin and potentially triggering
cancer (Rutala, 2019).
Antiseptics are non-toxic disinfectants because they are applied to the skin, mucosa or
other living tissues. Antiseptics have several requirements, among others, have a broad
spectrum, do not stimulate the skin or mucosa, toxicity or absorption through the skin and
mucosa is low, the effect is fast and lasts a long time (Septiari, 2012).
The effectiveness or power of a disinfectant is influenced by concentration, pH,
contact time, age of bacteria, temperature, media, and solvent used. For example, at slightly
higher concentrations the fungicidal effect is stronger than the bactericidal effect. At pH 6 the
effect of chlorhexidine is 10 times stronger than at pH 9, also benzoic acid and its esters are
more active at acidic pH. Another factor that affects the effectiveness of antiseptics is the
number of microbes, where the more the more time it takes to kill them. The culture of
microorganisms used in the antiseptic-disinfectant test requires nutrients as a growth medium
that contains nutrients and a suitable growth environment for microorganisms (W. Lay,
1994).
Growth media is a nutrient medium that is prepared to grow bacteria. Some bacteria
can grow well on any medium and some bacteria require special media. The media must be
able to provide the energy needed for bacterial growth (Radji, 2010).

III. Tools & Material

3.1 Material

1. Media cair Nutrient Broth (NB) 6. Aquades

2. Media Nutrient Broth Agar (NBA) 7. Fatty cotton
3. Media Potato Dextrose Agar (PDA) 8. Brown wrapping paper (wrapper)
4. AAA Stem Agar 9. Aluminium foil
5. D-Glucose 10. Rubber band
11. Filter paper

3.2 Tools

1. Pipet taker 10. Autoclave

2. Pipette drops 11. Oven
3. Tweezers 12. Wire ose
4. Spatula 13. Bunsen
5. Glass stirrer 14. Analytical Balance
 6. Beaker glass 15. Watch Glass
7. Test tube 16. Hotplate stirrer
8. Petridish / petri dish 17. Magnetic Stirrer
9. Measuring cup

IV. Procedure:

4.1. Preparation Media

• Preparation of liquid media (NB)

1. Weight 8-gram Nutrient Broth.
2. Dissolve Nutrient Broth in 1000 ml of distilled water.
3. Cover the beaker glass with aluminum foil / brown paper.
4. Sterilize in autoclave at 121OC for 15 minutes.
5. Label the beaker glass.
6. The media is ready to use.
• Solid media manufacturing (PDA/NBA)
1. Weigh 39-gram PDA / 20-gram NBA.
2. Dissolve PDA in 1000 ml of distilled water while heating.
3. Stir until mixed homogeneously.
4. Cover the beaker glass with aluminum foil / brown paper.
5. Sterilize in autoclave at 121OC for 15 minutes.
6. Once sterile, pour the media into the petridish or test tube. The media volume in the
petridish should not be more than ½ petridish and in the test tube it should not be more
than 1/3 of the test tube.
7. Each test tube is covered with fat-free cotton. Kemudain tilt the test tube so that the
solid media is tilted. But don't get it on the cotton plug. Let it cool.
8. Label each test tube and petridish.
9. The media is ready to use.

4.2. Direct fire heat sterilization

1. Turn on the Bunsen.

2. Before use, burn the loop wire from the end to the base of the wire until it glows
red.
3. Keep away from fire.
4. Hold the loop until it cools while still holding it around the lit Bunsen.
5. Wire loop is ready to use.
6. Burn the loop wire again after use, like step no. 2.
7. Don't put the ose wire on the work table if it hasn't been heated until it's red hot.

4.3. Sterilization by autoclave.

1. Prepare the media to be sterilized.The volume of liquid and media filled into in the
container do not exceed half the volume of the container, so that the liquid and media not
spilled.Make sure the media is covered & wrapped. Media in erlenmeyer was covered in
fat cotton plugs and wrapped in brown paper covers.
2. Put all the test tubes in a beaker glass and cover it with brown paper/aluminum foil
on top.
 3. All petridishes are wrapped in brown cover paper.
4. Put all the media in the autoclave. Place in such a way that the media does not spill.
Sterilize in an autoclave at a temperature of 121OC for 15 minutes.

4.4 Work aseptically (technically aseptic)

1. Before and after work, always wash your hands with soap.
2. Before work, remove items that are not needed from the work table. Make sure
you are wearing a disposable latex mask and gloves. Disinfect the surface of the
workbench area by spraying disinfectant liquid (70% alcohol). Allow a few
seconds. Wipe with dry tissue.
3. Use a hand sanitizer or spray disinfectant liquid on the palms and backs of the
palms and wrists (still wearing gloves). Let it dry.
4. Turn on the burner. Be careful working with alcohol around a Bunsen flame. 5.
Pass and rotate the lips of the petridish in the Bunsen flame, then slightly open the
lid of the petridish while still doing it around the Bunsen flame. Use your thumb
and forefinger to open the lid of the petridish, while the other 3 fingers hold the
base of the petridish. Close the petridish and pass & turn the lip of the petridish near
the Bunsen flame.
6. Open the cotton plug around the Bunsen flame. Pass and rotate the lip of the test
tube/Erlen Meyer around the Bunsen flame. Close the cotton plug again. 7. Do not
place cork caps, test tube caps or any container lids on the work table. Hold the lid
with your finger during the aseptic process. 8. After work, disinfect the workbench
by spraying disinfectant liquid (70% alcohol). Allow a few seconds. Wipe with dry
tissue. 9. Dispose of gloves in a special waste bin for gloves and masks. 10. Wash
your hands with Dettol laundry soap.

V. Result and Discussion

Figure 5.1 Result of Nutrient Broth Figure 5.2 Result of Nutrient Broth Agar

This practicum aims to physical and chemical sterilization, and can perform aseptic
work and recognizing and know the function of microbial growth media . Medium is a
material used to grow microorganisms on or in it, the medium must meet the requirements,
among others, must contain all nutrients that are easily used by microbes, must have osmotic
pressure, surface tension and pH according to the needs of the microbes to be grown, not
contain substances that can inhibit microbial growth, must be in a sterile condition before
use, so that the microbes that are grown can grow properly. The practicum this time is to NB
(Nutrient Broth) and.NBA (nutrient broth agar) was used as a medium for bacterial growth.
The medium for this experiment was prepared using NBA (nutrient broth agar), where in the
manufacture of instant NBA first by weighing the ingredients that are already available and
produced patently into an analytical balance in accordance with the required amount then put
the ingredients into a 500 ml Erlenmeyer, where the ingredients are 250 ml of aquades water,
5 gram NBA using a autoclave and occasionally magnetic stirring, the purpose of this
heating and stirring is to homogenize NBA with aquades, whereby heating can speed up the
dissolution of NBA and aquadest. After being heated for a few minutes, the solution changed
color from cloudy to brownish yellow, indicating that the solution was homogeneous.

The same is true process for NB (Nutrient Broth) is also the same, which is used in
this practicum with the composition of 0,8 g of NB and 100 ml of aquades, which is already
in a dosage form that has been made by the factory. And it was put in the hotplate magnetic
stirrer to homogenize and heat a solution when preparing the media which is heated at the
temperature of 200 celsius with an rpm above 250. It usually takes around 1 hour because NB
is easier to blend.Then it is put into the autoclave with the beaker mouth covered with
aluminum foil on the outside. After heating for a some minutes the solution changed color
from cloudy to dark yellow. After that it was put into the autoclave but before being put in
the Erlenmeyer mouth was closed with cotton and then wrapped in paper, this aims to
minimize the hazard. If an error occurs, the factor that causes an error in this practicum is that
when pouring the extract is not careful it will cause the extract to spill, not weighing the
composition of the media correctly will cause the media to be inhomogeneous. Autoclaves
use high temperatures and pressures that provide greater power to kill cells than ordinary hot
air. The autoclave has the advantage of being a high-pressure boiler. This tool is often used in
sterilization techniques because of the coefficient level and the nature of the tool that does not
damage the content of the growth media used, namely NBA, NB,.

Sterilization is carried out aims to avoid contamination, namely the entry of

unwanted microorganisms. Sterilization is a process (chemical and physical) that kills all
forms of life, especially microorganisms. The sterilization used in this experiment is physical,
namely using heat, where the heat used is with water vapor which is usually called wet
sterilization. In practicum, it is necessary to sterilize before using tools and materials.
Equipment such as mortar and pestle, petri dishes, media, test tubes, tools used in bacterial
casings are first sterilized using an autoclave. The proper method of sterilization depends on
the type and nature of the material being sterilized and what we practice this time is the
method of heating with water vapor and the effect of pressure (autoclave). The object to be
disinfected is placed on the filter plate and does not directly touch the water below. Heating is
carried out until the water boils (estimated at a temperature of 100˚C) at a pressure of 15 lb at
a temperature of 121˚ C

V.Conclusion

From this practicum with can conclude that the sterilization method used is to use an
autoclave to remove microorganisms from the tools and materials to be used in the next
practicum. And as the practicum conducted it can be conclude that Nutrient Broth and
Nutrient Broth Agar as an growth media were its provides a good surface and space to
aerobic microorganisms, especially bacteria, to grow well. NB are a type of liquid media used
to cultivate and maintain microorganisms. NB media remain as liquids even at the room
temperature were homogenous happen when physical change occurred during the heated and
stirred process, as the solution changed color from cloudy to yellow. NBA media functions as
the growth of bacteria/microbes in solid form as the media were supposed to be put in Petri
dish.
6. References

Donnenberg. 2013. Escherichia Coli Pathotypes and Principles of Pathogenesis. Baltimore:

University of Maryland School of Medicine Academic Press

Nasution,M. 2012. Pengantar Mikrobiologi. Medan : USU. 76-77

Radji, Maksum. 2010. Buku Ajar Mikrobiologi Panduan Mahasiswa Farmasi dan
Kedokteran. Jakarta: EGC.