INDUSTRIAL MICROBIOLOGY PRACTICUM REPORT

MODULE : Isolation of Microorganisms from a Mixture

GROUP : 4 /Monday
Practitioner Name : Kresna Ramadhani Fauzan
NRP : 5008201072

ASSISTANT
Assistant Name : Adidoyo Prakoso
NRP : 02211940000105

LECTURER
Lecturer Name : Dr. Ir. Sri Rachmania Juliastuti, M.Eng.
NIP : 195907301986032001

Tanggal Praktikum : 27/02/2023

Tanggal Pengumpulan Laporan : 06/03/2023

INDUSTRIAL CHEMICAL PHYSICS AND MICROBIOLOGY LABORATORY

DEPARTMENT OF CHEMICAL ENGINEERING
FACULTY OF INDUSTRIAL TECHNOLOGY AND SYSTEMS ENGINEERING
SEPULUH NOPEMBER INSTITUTE OF TECHNOLOGY
 I. Objectives
Studying the isolation of microorganisms from a mixture with the scratch plate
technique and a pouring cup.

II. Basic Theory

Microorganisms in a natural environment are mixed populations of various
types, both microorganisms in soil, water, air, food, and those found in the bodies of
animals and plants. Separation of bacteria is needed to determine the type, study culture,
morphology, physiology, and characteristics. The separation technique is called
isolation accompanied by purification. The definition of bacterial isolation is a process
of taking bacteria from the medium or from their environment of origin and then
growing them in an artificial medium so that a pure culture is obtained.
(Singleton & Sainsbury, 2006).

The principle of microbial isolation is to separate one type of microbe from

another which comes from a mixture of various microbes. This can be done by growing
it in solid media, microbial cells will form colonies of cells that remain in place. Several
ways or methods to obtain pure culture from a mixed culture. The two most frequently
used are the scratch cup method and the pour cup method. Which is based on the
principle of dilution with a view to obtaining individual species. Assuming that each
colony can be separated from one observable cell type.
(Afrianto, 2004)
Conventional bacterial identification processes based on bacterial phenotypic
characters such as Gram staining, colony morphology, and enzyme activity are often
not static and can change with evolution. Identification errors often occur due to the
presence of unusual bacterial phenotypic characteristics or lack of experience in
interpreting phenotypic character data. This gave rise to the presence of a molecular
identification method using the 16S rRNA gene. This also reduces interpretational bias
and eliminates the need for a possible “pretest” regarding the classification of
microorganisms for direct examination and database selection.
(Petti et al., 2005)

STREAK METHOD

III. Materials
1. Bacterial suspension/mixture (2 types) The medium is liquid (liquid NB) and
the suspension is made 1 day before the practicum; as well as enterincubator
2. NB agar media
3. Brown paper

IV. Tools
1. Petri Dishes (2 pieces)
2. Ose wire
3. Bunsen burner
4. Incase
5. Incubator
V. Procedure
1. Turn the petri dish upside down, and using a wax pencil or marker, divide the
entire bottom area of the cup as shown in Figure III.1. (Later, when you can
 master this etching technique well, then such division will not need to be
described again). Note that the O sector is smaller than the other sectors and the
inoculating strokes are not as well separated as they are in the following sectors.
2. Add NB Agar media to 2 petri dishes.
3. Guided by Figure 1, scratch the mixed culture provided on the petridish cup.
Caution: don't forget to turn on your loupe every time you switch sectors.
4. Do the same streaking using the same mixed culture on the second petri dish.
5. Place your petri dishes upside down in the basket provided for incubation at 30
oC for 24-48 hours.
6. Draw the shape of the observed colonies (assistant's choice). Give sufficient
explanation (colonial shape (from above), colony edge, colony surface (from
side), color, density, and colony diameter)!
7. Compare the data you got with the literature you read!

POURINGMETHOD

VI. Materials
1. Bacterial liquid suspension (mixture)
2. Aquades
3. NB Media
4. Brown paper

VII. Tools
1. Petridishes (5 pieces)
2. Test tubes (6 pieces)
3. Micropipette
4. Vortex
5. Bunsen burner
6. Incase
7. Incubator
VIII. Procedure
A. Series of Dilution
1. Take 6 test tubes and label them No. 1-6
2. Fill each test tube with 9 mL of distilled water
3. Take 1 mL of the bacterial suspension with a micropipette and put it in
test tube no.1 then shake it with a vortex
4. Take 1 mL of solution in Test Tube No. 1 and put it in test tube No. 2
then shake with vortex
5. Take 1 mL of solution in test tube No. 2 and put it in test tube No. 3 then
shake with vortex
6. Take 1 mL of solution in Test Tube No. 3 and put it in test tube No. 4
then shake with vortex
7. Take 1 mL of solution in test tube No. 4 and put it in test tube No. 5 then
shake with a vortex
8. Take 1 mL of solution in test tube No. 5 and put it in test tube No. 6 then
shake with vortex
B. Pour Method
1. Take 5 petri dishes and label them no. 1-5
2. Take 0.1 solution from test tube No. 2 and pour into petri dish No. 1
 3. Take 0.1 mL of solution from test tube No. 3 and pour into petridish No
2
4. Take 0.1 mL of solution from test tube No. 3 and pour into petridish No
3
5. Take 0.1 mL of solution from test tube No. 4 and pour into petridish No
4
6. Take 0.1 mL of solution from test tube No. 5 and pour into petridish No
5
7. Then pour the agar liquid into petridish No. 1
8. Rotate petridish No. 1 clockwise then rotate counterclockwise, then
rotate the petridish to form the number 8.
9. Perform Steps 7-8 for petridish No. 2-5.
10. After the agar media has frozen, pack the petridish No. 1-5 using brown
paper
11. Incubation for 24-48 hours at 37 oC (according to the type of microbe)
with the petridish position upside down
12. Make observations

IX. Results and Discussion

IX.I Results

Isolation Suspension Observation Reference with same

Method practicum
Streak Method Colonies of
Bacteria
 Pouring Method Colonies of 6th Dilution:
Fungi

7th Dilution:

8th Dilution:

IX.II Discussion
An experiment in industrial microbiology was conducted to study the isolation of
microorganisms from a mixture using the streak plate technique and the pouring cup. The
variables included variations in the isolation method, such as the streak method and the
pouring method, as well as the types of microorganism samples used, such as bacteria
suspension for the streak method and fung suspension for the pouring method.. Nutrient agar
was used as the culture medium since it is a non-selective medium suitable for the growth of
most microorganisms.The initial method of isolating bacteria is known as the streak method.
To begin the experiment, sterile petri dishes containing nutrient agar are prepared. The dishes
are then taken to a small laminar air flow to commence the streaking stage. Before
inoculating the bacteria, the wire loops used to scratch them are sterilized by heating them in
a Bunsen flame until they become warm or hot. This is to ensure that the wire loops are fully
sterile and prevent contamination. Additionally, the petri dishes containing agar media are
sterilized by evenly passing the edges of the dish over a Bunsen flame until they become
warm. The next step involves using a sterile loop to obtain the bacterial suspension and then
streaking it onto the agar medium with the same loop. The strokes made during the streaking
process must be consistent with the image provided in the reference. The scratches are made
in a zigzag pattern to ensure that the bacteria are widely spread during their growth, resulting
in a larger quantity of bacteria that are evenly distributed in the direction of the stroke.
Moreover, the pour plate method involves observing the variable of fungi. The experiment
begins by sterilizing all tools with alcohol and preparing three empty petri dishes and eight
test tubes. Dilution is necessary for this method as it enables fungal colonies to grow on
limited media where it is not possible to count thousands of fungi. Clean petri dishes are
prepared and taken to a laminar air flow, followed by adding 9 ml of distilled water into each
test tube. A micropipette is used to take 1 ml of the fungal suspension, which is then injected
into the first test tube. The test tube is then placed in a vortex to facilitate mixing between the
distilled water and fungi. Next, 1 ml of liquid from the first test tube is taken and injected into
the second test tube, and the process is repeated with the remaining test tubes. This dilution
process is carried out eight times to reduce the density of bacteria in the sample. The last
three dilutions are poured into each petri dish, and nutrient agar is poured on top of the liquid
resulting from the dilution. The petri dishes are then left to stand for a few minutes until the
agar hardens. Once the agar hardens, the petri dishes can be inverted and wrapped in brown
paper, then placed in an incubator for 24 hours. Upon observation, it appears that the streak
plate method performed in the experiment looks messy, which may have been caused by
carelessness during the streaking process. The outcome should resemble the examples
provided in the references. Another factor that may have contributed to the discrepancy is
using wire loops that were too forceful, resulting in the removal of a small amount of nutrient
agar. In the pour plate method, it is noticeable that the 7th dilution yields the most visible
results, but there is no visible fungal growth as compared to the reference. This lack of visible
growth could be due to insufficient dilution stages, resulting in unclear outcomes.

X. Conclusion

Concluding on the experiments that were conducted, several points can be inferred, as
follows:
1. When culturing bacteria using the streaking method, it is crucial to spread
them in a zigzag pattern both horizontally and vertically, while being cautious
not to apply excessive pressure to avoid harming the nutrient agar.
2. For fungal cultures using the pouring method, it is necessary to dilute them
more extensively to obtain clearer observations, as dilution is a critical factor
that can facilitate more precise visualization of the fungi.

XI. Bibliography
[1] Yabuuchi, E., dan Y. Kosako. 2005. Order IV. Sphingomonadales ord. nov. In
: Garrity, G.M., Brenner, D.J., Krieg, N.R., Staley, J.T (Eds). Bergeys Manual Of
Systematic Bacteriology Second Edition. Department of Microbiology and Molecular
Genetics, Michigan State University. USASingleton, P., dan Sainsbury, D. 2006.
Dictionary of Microbiology and Molecular Biology. Edisi ke-3.John Wiley & Sons Ltd.
UK
[2] Afrianto, L., 2004, Menghitung Mikroba Pada Bahan Makanan, Cakrawala
(Suplemen Pikiran Rakyat untuk Iptek), Farmasi FMIPA ITB. Bandung
[3] Petti, C.A., C.R. Polage dan P. Schreckenberger. 2005. The Role of 16S rRNA Gene
Sequencing in Identification of Microorganisms Misidentified by Conventional
Methods. Journal of Clinical Microbiology 12 (43) : 6123 6125
 ATTACHMENTS

LAPORAN SEMENTARA
PRAKTIKUM MIKROBIOLOGI INDUSTRI
SEMESTER GENAP 2022/2023
Kelas : IUP
Kelompok : 4
Nama Mahasiswa 1 : Adlina Firza NRP : 5008211142

Nama Mahasiswa 2 : Rayhan S F Ferryanto NRP : 5008211143

Nama Mahasiswa 3 : Kresna Ramadhan Fauzan NRP : 5008201072

Judul Modul : Isolation of Microorganisms from a Mixture

Hasil Praktikum

A. streak method
Using bacteria as our materials.
1. Firstly take a sterile petri dish which already have the NBA in it from the
previous practicum
2. Next, draw a line that can divide into 4 parts in the petri dish. For this
method, we have to streak it in different zigzag movements.
3. Place your petri dishes upside down in the basket provided for incubation at
30 oC for 24-48 hours.

Observation
Pictures Description

In petridish observation, the streak

method does not look neat. This can be
caused by being careless in scraping
and even tearing the NBA. However, it
can still be seen that there is bacterial
growth in the petridish.
B. Pouring Method
Using fungi as our materials. In this step there needs to be a series of dilution. The
more the dilution the better because it can help to see the fungi clearer.

1. Firstly take 3 petri dish that has already from autoclave and is in sterile condition
2. Next, prepare 10 ml measurement cup, 8 test tube, micropipette, and a vortex
3. Next, insert 9 ml of aquades into each test tube and then label it 1-8. Then for the first
dilution, insert 1ml of fungi into the first test tube then shake in on the vortex.
4. Repeat this action until 8 times for the 8th test tube.
5. Next we’re going to use the last three dilution and pour them into each 3 petri dish.
Then, those petridish will be added an NBA to cover them up.
6. Lastly, wait for them to harden and then wrap them with brown paper. Additional
information, the lid of the petri dish have to be at the bottol (upside down)

Step Pictures Description

6th From the picture, it can be

dilution seen that the fungi liquid is
not evenly pured therefore
it curled up at the edge.
There is no contamination.

7th For the 7th dilution, we can

dilution see clearer regarding the
fungi because it is almost
evenly poured. There is no
contamination.
8th For the last dilution, we
dilution can’t really see the fungi
but it succeeds due to the
changing color just like the
previous dilution.

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