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Microbial Isolation Techniques Report

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FINAL REPORT

ENVIRONMENTAL MICROBIOLOGY PRACTICUM

MICROBIAL ISOLATION

BY:
NAME : HAIKAL RAMADHANA AKMAL
STUDENT ID NUMBER 2310942009
PRACTICUM DAY/DATE : TUESDAY / NOVEMBER 12th 2024
GROUP / SHIFT : 7 (SEVEN) / II (TWO)
GROUP MEMBERS : 1. SYFA ADI PUTRI (2310942013)
2. ARETA YASMINA LUTHI (2310942043)
3. AGUSTI NURUL RAHMI (2310943006)

ASSISTANT:
THARA ALIYAH EDIHARSI

ENVIRONMENTAL MICROBIOLOGY LABORATORY

DEPARTMENT OF ENVIRONMENTAL ENGINEERING
FACULTY OF ENGINEERING-UNIVERSITAS ANDALAS
PADANG
2024
LIST OF CONTENTS

LIST OF CONTENTS ........................................................................................ i

LIST OF TABLES ............................................................................................ iii

LIST OF FIGURES .......................................................................................... iv

CHAPTER I INTRODUCTION .................................................................... I-1

1.1 Background ...................................................................................... I-1
1.2 Aims and Objectives of the Experiment ............................................ I-2
1.2.1 Aims of the Experiment .......................................................... I-2
1.2.2 Objectives of the Experiment .................................................. I-2
1.3 Principle of the Experiment .............................................................. I-3
1.4 Scope of the Experiment ................................................................... I-3
1.5 Systematization of the Experiment .................................................... I-3
CHAPTER II LITERATURE REVIEW ...................................................... II-1
2.1 General ........................................................................................... II-1
2.2 Definition of Microbial Isolation .................................................... II-4
2.3 Microbial Isolation Methods ........................................................... II-6
2.4 Microbial Isolation Media............................................................... II-8
2.5 Applications of Microbial Isolation in Environmental Engineering..II-8
CHAPTER III EXPERIMENT PROCEDURE .......................................... III-1
3.1 Tools ............................................................................................. III-1
3.2 Materials ....................................................................................... III-4
3.3 Experimental Procedures ............................................................... III-6
CHAPTER IV RESULTS AND DISCUSSION ........................................... IV-1
4.1 Results Data .................................................................................. IV-1
4.2 Calculation .................................................................................... IV-2
4.2.1 Soil Sample .......................................................................... IV-2
4.2.1 Air Sample ........................................................................... IV-2
4.3 Discussion ..................................................................................... IV-3
CHAPTER V CLOSING ............................................................................... V-1
5.1 Conclusions .................................................................................... V-1
5.2 Suggestions .................................................................................... V-2

i
BIBLIOGRAPHY
ATTACHMENT

ii
LIST OF TABLES

Table 4.1 Data on the Number of Colonies in Soil Sample Experiments ..........IV-1
Table 4.2 Colony Physiology in Soil Samples ................................................ IV-1
Table 4.3 Data on the Number of Colonies in Air Sample Experiments ........... IV-1
Table 4.4 Colony Physiology in Air Samples ..................................................IV-2

iii
LIST OF FIGURES

Figure 3.1 Petri dish ........................................................................................ III-1

Figure 3.2 Measuring pipette........................................................................... III-1
Figure 3.3 Measuring cup................................................................................ III-1
Figure 3.4 Test tubes ....................................................................................... III-2
Figure 3.5 Spritus............................................................................................ III-2
Figure 3.6 Colony counter ............................................................................... III-2
Figure 3.7 Analytical balance .......................................................................... III-2
Figure 3.8 Erlenmeyer..................................................................................... III-3
Figure 3.9 Spatula ........................................................................................... III-3
Figure 3.10 Suction ball .................................................................................. III-3
Figure 3.11 Paralon pipe ................................................................................. III-3
Figure 3.12 Shaker .......................................................................................... III-4
Figure 3.13 NA media ..................................................................................... III-4
Figure 3.14 PCA media ................................................................................... III-4
Figure 3.15 SDA media .................................................................................. III-4
Figure 3.16 70% alcohol ................................................................................. III-5
Figure 3.17 0,1% BPW solution ...................................................................... III-5
Figure 3.18 Aquadest ...................................................................................... III-5
Figure 3.19 Sample ......................................................................................... III-5
Figure 3.20 Cups sterilized in an oven or autoclave ......................................... III-6
Figure 3.21 Workbench sprayed with alcohol.................................................. III-6
Figure 3.22 5 grams of soil taken .................................................................... III-6
Figure 3.23 Soil is put into an erlenmeyer and homogenized ........................... III-7
Figure 3.24 Samples put into test tubes ........................................................... III-7
Figure 3.25 Samples put into test tubes ........................................................... III-7
Figure 3.26 Samples put into a petri dish ......................................................... III-7
Figure 3.27 Samples put into a petri dish ......................................................... III-8
Figure 3.28 Sample homogenized ................................................................... III-8
Figure 3.29 Media allowed to solidify ............................................................. III-8
Figure 3.30 Incubation process........................................................................ III-8

iv
Figure 3.31 Colony counting ........................................................................... III-8
Figure 3.32 NA and SDA media poured into petri dishes (near the fire) .......... III-9
Figure 3.33 Media allowed to solidify ............................................................. III-9
Figure 3.34 The cups were left open................................................................ III-9
Figure 3.35 Incubation process...................................................................... III-10
Figure 3.36 Colony counting ......................................................................... III-10
Figure 4.1 PCA Soil ........................................................................................ IV-1
Figure 4.2 SDA Soil........................................................................................ IV-1
Figure 4.3 NA Air ........................................................................................... IV-2
Figure 4.4 SDA Air ......................................................................................... IV-2

v
INITIAL REPORT
ENVIRONMENTAL MICROBIOLOGY PRACTICUM

MICROBIAL ISOLATION

ASSISTANT:
THARA ALIYAH EDIHARSI

ENVIRONMENTAL MICROBIOLOGY LABORATORY

DEPARTMENT OF ENVIRONMENTAL ENGINEERING
FACULTY OF ENGINEERING-UNIVERSITAS ANDALAS
PADANG
2024
CHAPTER I

INTRODUCTION

1.1 Background

Microbes are defined as organisms that require a tool to be observed due to their
very small size. Known as microscopic organisms, they are often single-celled
(unicellular) or multicellular. Microbes are found in various environments, such as
air, water, and soil, where coexistence with other organisms occurs. In nature,
microbes exist in mixed populations, so isolation is conducted, usually initiated by
serial dilution, to obtain pure cultures. Microorganism isolation is understood as the
process in which microbes are extracted from their environment and then cultivated
in a laboratory medium. During this process, the target microbe must be separated
from other microbes with which it coexists. This isolation is considered essential
for studies on microbial identification, morphology, physiology, serology, and
culture characteristics (Rohmah, 2017).

The isolation process involves cultivating and propagating the microbe within a
culture that contains only the target bacteria, ensuring no contamination from other
microorganisms. Identifying the specific nutrients required by the bacteria and
understanding the ideal physical environment are essential. Performing laboratory
work accurately allows for a detailed examination of the microbes, not only by
observing their morphology and colony count but also by identifying their shape
and intricate structures under a microscope, ultimately determining the type of
isolated microbe (Sabbathini, 2017).

Achieving high efficiency in fermentation relies heavily on selecting and using the
appropriate microbes. Microbial isolation is performed to identify microbes that are
best suited to produce the desired product. This process can be carried out through
three main techniques, the streak method, the pour method, and the spread method.
The population of microorganisms in nature around us is huge and quite complex.
Hundreds of microbial species reside in every part of our body. One gram of feces
can contain millions of bacteria. The nature around us, be it soil, water, or air is also
inhabited by a collection of microorganisms (Istiamah et al., 2018).
MINISTRY OF HIGHER EDUCATION, SCIENCE, AND TECHNOLOGY
DEPARTEMENT OF ENVIRONMENTAL ENGINEERING
ENVIRONMENTAL MICROBIOLOGY LABORATORY
Kampus Unand Limau Manis Padang 25163
Telp. (0751) 7862901, Fax (0751 72566)

The majority of bacteria are single-celled and so tiny that they require magnification
to be seen. However, some single-celled bacteria and other larger organisms can be
visible to the naked eye. Typically, an object must be at least 100 micrometers (μm)
in size to be observable without a microscope (Mayasari, 2022).

Bacteria are difficult to identify and classify because they cannot be observed
directly and are not visible to the naked eye. To differentiate and categorize
bacteria, scientists must isolate, culture, and create methods to study their
biochemical traits. Despite these challenges, microbiologists consistently refine and
update guidelines to assist in the identification and classification of bacteria
(Mayasari, 2022).

1.2 Aim and Objective of the Experiment

1.2.1 Purpose of the Experiment

The goal of this practicum is to meet the requirements for completing the
environmental microbiology course and to identify a microorganism in order to
understand its characteristics.

1.2.2 Experiment Objective

The objectives of this practicum are:

1. For practitioners to know the type of equipment and culture media needed to
make pure cultures.
2. Understand the concept of sterilization and the procedures required for
successful subculture of microorganisms.
3. Inoculating streak plates, spread plates, and pour plates to separate
microorganisms from mixed cultures of microorganisms that will be used for
further isolation of pure cultures.
4. Knowing the morphological characteristics and colony shape of microorganism
cultures that grow.

Haikal Ramadhana Akmal (2310942009) I-2

MINISTRY OF HIGHER EDUCATION, SCIENCE, AND TECHNOLOGY
DEPARTEMENT OF ENVIRONMENTAL ENGINEERING
ENVIRONMENTAL MICROBIOLOGY LABORATORY
Kampus Unand Limau Manis Padang 25163
Telp. (0751) 7862901, Fax (0751 72566)

1.3 Principle of Experiment

The principle of this experiment is:

1. The technique of separating a particular species of microorganism from other
organisms is known as isolation technique, which is accompanied by
purification techniques.
2. Colonies are the result of the growth of microorganisms that can be seen
macroscopically on the surface of a solid medium. Each colony can be the result of
multiplication of one microorganism.

1.4 Scope of the Experiment

The scope of this experiment is to know how to isolate microbes to obtain pure
culture and study the properties of colonies using NA and PDA media. The samples
used in this experiment were soil from irrigation sediments and air under the stairs.

1.5 Systematization of Report Writing

The systematic writing of the report is as follows:

CHAPTER I INTRODUCTION

This section covers the background for writing the report, the objectives and goals
of the experiment, the principles underlying the experiment, the scope of the
experiment, and the structure of the report.
CHAPTER II LITERATURE REVIEW
This section provides an explanation of the references used in the creation of the
report, as well as other sources related to the microbial isolation experiment.

CHAPTER III EXPERIMENTAL PROCEDURE

This section details the equipment and materials used, as well as the procedural
steps followed during the experiment.

CHAPTER IV RESULTS AND DISCUSSION

Haikal Ramadhana Akmal (2310942009) I-3

CHAPTER V CLOSING

This section outlines the conclusions drawn from the experiment's results and
provides recommendations for improving the execution of the experiment.

LITERATURE

ATTACHMENT

Haikal Ramadhana Akmal (2310942009) I-4

CHAPTER II

LITERATURE REVIEW

2.1 General

Bacterial isolation or the cultivation of a single type of bacterium is referred to as a

pure or enriched culture, while a mixed culture is formed when more than one type
of bacterium is present. A biculture is formed when two types of bacteria are
combined. The isolation and cultivation of bacteriophages require the existence of
optimum conditions for the host organism's growth. The host habitat is considered
the best and most important source of bacteriophages. For example, coliphages are
found during digestion and can be isolated from waste or feces (Amelya, 2023).

Microorganisms in the environment are found as a mixed population of various

microbial species, present in soil, water, air, and on the bodies of plants and animals.
The isolation of these microbes helps in identifying species and studying their
culture, morphology, physiology, and characteristics. This technique, known as
isolation with purification, is used to transfer bacteria from their original medium
to an artificial medium, allowing them to grow and establish a pure culture
(Sabbathini, 2017).

Various types of bacterial growth media are commonly used for purposes such as
isolation, transportation, inoculation, and differentiation. Bacterial culture media
generally consist of meat extract, yeast extract, peptone, and agar. Selective media
are employed in bacterial isolation to inhibit the growth of specific bacteria, and
several types of media are used for this purpose. Differential media are also used to
distinguish between various types of bacteria based on their metabolic
characteristics. Many media developed today possess both selective and differential
properties, allowing for more precise identification and isolation of bacteria.
Additionally, the composition of the media can be modified to suit the requirements
of particular bacteria, enabling the successful isolation of diverse microbial species
from mixed cultures Examples of such media include Nutrient Agar (NA) and Potato
Dextrose Agar (PDA) (Amelya, 2023).
MINISTRY OF HIGHER EDUCATION, SCIENCE, AND TECHNOLOGY
DEPARTEMENT OF ENVIRONMENTAL ENGINEERING
ENVIRONMENTAL MICROBIOLOGY LABORATORY
Kampus Unand Limau Manis Padang 25163
Telp. (0751) 7862901, Fax (0751 72566)

The principle of microbial isolation is to separate one type of bacteria from other
bacteria in a mixture of different microorganisms. This is achieved by cultivating
microbial cells on solid media, where permanent colonies are formed in place.
Several methods are used to obtain pure cultures from mixed cultures, with the
streak plate method and pour plate method being the most commonly employed
(Sabathini, 2017).

Microbial isolation is conducted to select the appropriate microbes for the

production of the desired product. The desired microbes may be in the form of a
pure culture or a mixed culture, depending on the required product. Isolation of
microbes is typically performed using three techniques: the streak plate method,
pour plate method, and spread plate method (Nur and Feronika, 2018).

Microbial isolation is performed by collecting microorganisms from the

environment and growing them in an artificial medium. The separation or
purification of other microorganisms is required, as every microbiological task,
such as the study and identification of microorganisms, demands a population
consisting of only one microbial species. The principle of microbial isolation is to
separate one type of bacteria from others in a mixture of different bacterial species.
This is achieved by growing them on solid media (Rohmah, 2017).

Microbial cells are formed into colonies that remain in place. When captured by
solid media at separate locations, each cell or group of cells will develop into a
distinct colony, allowing for easier separation. In liquid media, individual microbial
cells are difficult to separate because they are too small and do not stay in place.
When the cells are separated by dilution and then cultured on solid media, colonies
will form. These colonies can then be isolated into separate test tubes or Petri dishes
(Rohmah, 2017).

Microbial isolation is performed to select the appropriate microbes for the

production of the desired product. The desired microbes may be obtained in the
form of a pure culture or a mixed culture, depending on the product required. The
microorganisms are separated from others in the environment to ensure that only
the target species grow. Methods such as streak plate, pour plate, and spread plate
are used to isolate pure cultures. The microbes are analyzed for their morphological

Haikal Ramadhana Akmal (2310942009) II-2

Microorganisms are isolated either as pure cultures or mixed cultures, depending

on the desired product. Three techniques—streak plate, pour plate, and spread
plate—are used for microbial isolation (Nur and Feronika, 2018).

Microbial isolation is performed by growing microorganisms outside their natural

environment. The goal of separating the microbes is to obtain a pure culture, free
from other microbial species. The principle of microbial isolation is to separate one
microbe from others in a mixture. This is accomplished by growing them on solid
media, where colonies of microbial cells are formed and remain in place (Deny,
2020).

The importance of isolating microbes from environments such as food (solid

substrates), beverages (liquid substrates), and from oneself is recognized due to the
difficulty in observing or distinguishing many microbes directly through the senses.
Isolation makes it easier to observe microbial growth and examine their morphology
on various media. Inoculation is performed by transferring a specific culture from
an old medium to a new one to obtain a pure culture free from contamination by
unwanted microbes. Several methods are employed to obtain a pure culture from a
mixed one. The cup scratch and pour cup methods are commonly used, based on the
principle of dilution to obtain individual species, with the assumption that each
colony will be separable. Pure cultures are required for numerous microbiological
techniques, including microbial identification. The study of morphological,
physiological, and serological culture characteristics necessitates the use of
microbes from a single species (Deny, 2020).

Microbes are considered a group of living organisms found within ecosystems,

contributing to the biodiversity of those ecosystems. An exceptionally high species
diversity is observed in microbes. More than 60 percent of the ecosystem is
occupied by them, with critical roles played in processes such as nutrient cycling,
decomposition, and symbiosis with other organisms. Microbes are also vital for
various industries, medicine, and environmental management. Their small size and
diversity are regarded as essential for maintaining ecological balance. Microbes also
play a pivotal role in bioremediation, where they help in breaking down pollutants
and contaminants in the environment, they also have contribute to soil.

Haikal Ramadhana Akmal (2310942009) II-3

Microbes, which have evolved for at least 3.8 billion years, are considered a
significant part of biomass. To maintain their role in ecosystems, interactions with
their environment must be established. Beneficial and harmful microbes exist, and
distinguishing between them is made difficult due to their presence in mixed
populations. The identification and separation of beneficial microbes from harmful
ones are achieved through the process of microbial isolation from their environment.
This process is known as microbial isolation (Amelya, 2023).

The principle of microbial isolation is to separate a specific type of microbe from

others in a mixture of various microbes. This is achieved by cultivating the microbes
on solid media, where colonies of the microbes are formed and remain fixed in
place. The importance of isolating microbes from the environment, such as food
(solid substrates), beverages (liquid substrates), and even the human body, lies in
the fact that many microbes are difficult to observe or distinguish directly using the
five senses. Through isolation, it becomes easier to observe and study the growth
patterns of microbes in different media, and the morphology of the microbes can be
examined, which includes inoculation. Additionally, this process plays a critical
role in various microbiological methods, including identification and testing of
microbial species (Amelya, 2023).

The presence of microbes in the environment is evidence that nutrients present in

the surroundings are utilized by microbes. Energy sources for microbes are derived
from light (phototrophs) and inorganic carbon (chemotrophs), with carbon sources
coming from inorganic compounds such as potassium nitrate, and organic nitrogen
being supplied in the form of proteins and amino acids. Non-metals like sulfur and
phosphorus, metal elements including potassium, sodium, magnesium, iron, and
copper, as well as water for metabolic functions and growth, are also utilized by
microbes (Amelya, 2023).

2.2 Definition of Microbial Isolation

Microbial isolation is performed by extracting microorganisms from the

environment and cultivating them in an artificial medium. The separation or
purification from the purific of other microorganisms is required because all
activities related to microbiological research necessitate a pure population of

Haikal Ramadhana Akmal (2310942009) II-4

microbes. In microbiological studies, the examination and identification of

microorganisms require a population composed solely of a single microorganism
type. The principle behind microbial isolation is the separation of one type of
microbe from others within a mixture of various microbial species. This separation
is achieved by growing the microbes on a solid medium (Rohmah, 2017).

Colonies of microbial cells are formed and remain in fixed locations. The cells that
settle in various spots on a solid medium develop into individual colonies,
facilitating their subsequent separation. Isolation of single microbial cells in liquid
media is challenging due to their small size and tendency to move. When cells are
diluted, cultured on a solid medium, and permitted to form colonies, they can later
be isolated into separate test tubes or petri dishes (Rohmah, 2017).

Microbial isolation is conducted to separate microbes from their environment and

grow them as a pure culture on an artificial medium. Bacteria are stained with dyes
to increase contrast, facilitating easier observation under a microscope. This
bacterial staining is crucial for identification and classification, as it aids in
distinguishing between different types. This study was aimed at identifying airborne
microbes in a microbiology laboratory. Isolation and identification tests were
performed on nutrient agar (NA) media, followed by simple and Gram staining,
disinfection tests, and analyses of growth factors. Bacteria are stained with dyes to
increase contrast, facilitating easier observation under a microscope. This bacterial
staining is crucial for identification and classification (Amelya, 2023).

Microorganisms, given their widespread presence, play significant roles in both

beneficial and harmful ways. In areas like water closets, where they frequently
interact with human contact, microbial contamination poses potential health risks.
Proper sanitation and hygiene practices are essential to control harmful microbes,
especially in spaces prone to high human interaction. Through isolation and
identification, specific types of microbes can be studied to better understand their
impact on health and hygiene. Additionally, regular monitoring of microbial
populations in such environments can aid in preventing the spread of pathogens.

Haikal Ramadhana Akmal (2310942009) II-5

The water closet is known to harbor a high quantity of microbes due to

environmental factors that facilitate optimal microbial growth and reproduction
(Deny, 2020).

Some microbes are beneficial, while others are harmful and pathogenic.
Distinguishing between beneficial and harmful microbes is challenging, especially
as they often exist within mixed populations. This challenge can be addressed
through a process of identifying and separating the mixed microbial population
from its environment, a method known as microbial isolation. Microbes can only
be observed effectively if they are isolated in specific areas, which makes
observation easier. To study particular microbes, they must first be separated from
both the environment and other microorganisms, which can be achieved through
isolation techniques (Deny, 2020).

2.3 Microbial Isolation Methods

Microbial isolation or culture techniques for growing microbes must consider the
factors of nutrition and oxygen demand (gas, O2 or air). The way anaerobic
microbes grow is very different from aerobic ones. Microbial isolation is defined as
the process of separating a microbe from its environment in nature and growing it
as a pure culture in an artificial medium. What must be known from microbial
isolation is how to plant and grow microbes on culture media and other conditions
for growth. Microbes rarely occur purely in nature. Most are a mixture of diverse
microbial species (Solikah, 2019).

To cultivate microorganisms, it is essential to consider nutritional factors and

oxygen requirements (either oxygen gas or air). The growth methods for anaerobic
microbes differ significantly from those for aerobic microbes. Isolating a microbe
involves separating it from its natural environment and growing it as a pure culture
in an artificial medium. This isolation process requires knowledge of how to
cultivate bacteria in cultures as well as understanding the other conditions necessary
for their growth. Additional factors such as temperature and pH levels are also
critical in establishing an optimal environment for microbial growth (Microbiology
Laboratory Team, 2023).

Haikal Ramadhana Akmal (2310942009) II-6

Microbes rarely exist in nature in a pure state. Most are a mixture of various
microbial species. Various ways to isolate and grow microbes are (Microbiology
Laboratory Team, 2023):
1. Spread plate method;
2. Pour plate method;
3. Streak plate method.
a. Spread Plate Method
The spread plate technique is a microbial isolation technique by inoculating
microbial cultures by spreading on the surface of solidified agar media. This
method is done by diluting the culture of microbial cultures. Because the
concentration of microbial cells is generally unknown, dilution needs to be done
in several stages, so that at least one of the dilutions contains separate colonies
(30-300 colonies). Separate microbial colonies allow the colonies to be counted.
b. Pour Plate Method
This method is basically to inoculate the agar medium that is melting at a
temperature of 45-50 ° C with a suspension of material containing microbes,
and pour it into a sterile Petri dish. After incubation, colonies will be seen
scattered on the surface of the agar which may come from 1 bacterial cell, so
that it can be isolated further.
c. Streak Plate Method
The streaking method is commonly used to isolate microbial colonies on agar
plates to obtain separate colonies, which then form a pure culture. In this
method, a suspension containing microbes is streaked across the surface of an
appropriate agar medium in a petri dish. Following incubation, individual
colonies grow along the streak lines, each potentially originating from a single
microbial cell, allowing for further isolation. Proper streaking ensures the
formation of distinct colonies. Bacteria with flagella often form spreading
colonies, especially on moist plates. To prevent this, it is essential to use agar
plates with a thoroughly dry surface. This approach requires careful handling to
maintain sterility, as well as consistent environmental conditions to promote
optimal microbial growth.

Haikal Ramadhana Akmal (2310942009) II-7

2.4 Microbial Isolation Media

There are 2 media in microbial isolation, namely:

1. Nutrient Agar (NA)
Nutrient agar is a medium for testing water and dairy products. Nutrient Agar (NA)
is also used for the growth of most non-selective microorganisms in the sense of
heterotrophic microorganisms. Based on its shape, this medium is solid because
agar is a compacted material. Nutrient Agar is a simple medium made from beef
extract, peptone and agar (Putri, 2017).
2. Potato Dextrose Agar
late count agar is presently the recommended medium for the standard bacterial
plate count (35 degrees C, 48-h incubation) of water and wastewater. However,
plate count agar does not permit the growth of many bacteria that may be
present in treated potable water supplies. A new medium was developed for use
in heterotrophic plate count analyses and for subculture of bacteria isolated
from potable water samples. The new medium, designated R2A, contains 0.5 g
of yeast extract, 0.5 g of Difco Proteose Peptone no. 3 (Difco Laboratories), 0.5
g of Casamino Acids (Difco), 0.5 g of glucose, 0.5 g of soluble starch, 0.3 g of
K2HPO4, 0.05 g of MgSO4 X 7H2O, 0.3 g of sodium pyruvate, and 15 g of
agar per liter of laboratory quality water. Adjust the pH to 7.2 with crystalline
K2HPO4 or KH2PO4 and sterilize at 121 degrees C for 15 min (Putri, 2017).

2.5 Factors Affecting Microbial Isolation

One crucial aspect to consider when culturing microbes is sterilization. Sterilization

is a process used to eliminate microorganisms from media and tools that will be
used for cultivation. There are various methods of sterilization, but the most
commonly used are heat sterilization, which includes dry heat sterilization (hot air)
and moist heat sterilization. Dry heat sterilization involves using hot air (typically
in an oven or sterilizer) at temperatures between 160°C and 180°C for 1.5 to 3 hours,
and is typically applied to sterilize empty glassware or glass pipettes. On the other
hand, moist heat sterilization employs steam that flows freely through the
equipment. This method is typically used for sterilizing items that need to be
exposed to higher temperatures for shorter durations, ensuring effective microbial

Haikal Ramadhana Akmal (2310942009) II-8

elimination. Sterilization must be conducted under controlled conditions to prevent

contamination during microbial cultivation temperature of 100°C and is usually
used for solutions that are thermolabile (tend to be damaged or changed)
(Nurmalasari, 2021).

The application of microbial isolation in the field of Environmental Engineering is

biotransformation. Biotransformation is chosen because its reactions are enzymatic,
making the biotransformation process selective and highly specific in altering
existing substrates. This specificity and selectivity are due to the chiral structure of
the enzyme proteins. When multiple functional groups are present, only specific
positions are affected. Biotransformation reactions can target functional groups that
cannot be efficiently activated or require several intermediary steps before they can
react chemically. Therefore, researchers are interested in studying the
biotransformation of catechins, a major secondary metabolite of the gambir plant,
using endophytic microbial isolates from leaves and soil microbes isolated from the
gambir plant’s growing environment, with the goal of producing other compounds
or new substances that may have different potential from catechins as the starting
compound(Sabbathini,2017).

Bacteria play many roles in human life and in environmental engineering.

Microorganisms found in the natural environment constitute a mixed population
from various ecosystems, including microorganisms in soil, water, air, food, and
those found in the bodies of animals and plants. With such microbial diversity,
bacterial isolation in a single colony must be carried out. The technique used for
this is microbial isolation. This isolation technique is performed by taking samples
from a mixture of microorganisms and cultivating them in controlled conditions to
separate them into pure cultures. By isolating bacteria in this way, it becomes
possible to study specific microbial characteristics and their potential applications
in environmental engineering. Bacteria from the medium or from environment for
then grow it in an artificial medium so that a pure culture is obtained. Microbial
isolation is important to know and determine the type, study the culture,
morphology, physiology, and characteristics of the microorganisms sampled. The

Haikal Ramadhana Akmal (2310942009) II-9

application of microbial isolation in the field of environmental engineering,

microbial isolation can be used to determine the presence of pollution involving
microorganisms in it, so that it can be known and solutions can be taken how to
minimize the source of pollution produced by these microorganisms to the
environment. Microorganisms have many benefits for the environment, for example
using microbes as environmentally friendly insecticides, microorganisms are used
in procces of Environmental Engineering (Sabbathini, 2017).

Haikal Ramadhana Akmal (2310942009) II-10

CHAPTER III
EXPERIMENT PROCEDURE

3.1 Tools
The tools used in microbial isolation practicum are:
1. Petri dish
Serves as a place to cultivate bacteria.

Figure 3.1 Petri dish

2. 1 mL measuring pipette
Serves to measure and move substances.

Figure 3.2 Measuring pipette

3. 10 mL measuring cup
Serves to measure the substance to be used.

Figure 3.3 Measuring cup

III-1
MINISTRY OF HIGHER EDUCATION, SCIENCE, AND TECHNOLOGY
DEPARTEMENT OF ENVIRONMENTAL ENGINEERING
ENVIRONMENTAL MICROBIOLOGY LABORATORY
Campus Unand Limau Manis Padang 25163
Telp. (0751) 7862901, Fax (0751 72566)

4. Test tubes and test tube racks

Serves as a container for dilution.

Figure 3.4 Test tubes

5. Spritus
Serves as a sterilization tool.

Figure 3.5 Spritus

6. Colony counter
Serves to count the number of bacterial colonies.

Figure 3.6 Colony counter

7. Analytical balance
Serves to weigh the soil sample.

Figure 3.7 Analytical balance

Haikal Ramadhana Akmal (2310942009) III-2

MINISTRY OF HIGHER EDUCATION, SCIENCE, AND TECHNOLOGY
DEPARTEMENT OF ENVIRONMENTAL ENGINEERING
ENVIRONMENTAL MICROBIOLOGY LABORATORY
Campus Unand Limau Manis Padang 25163
Telp. (0751) 7862901, Fax (0751 72566)

8. Erlenmeyer 250 mL
Serves as a container to dilute the sample.

Gambar 3.8 Erlenmeyer

9. Spatula
Serves as a tool for taking soil sample.

Figure 3.9 Spatula

10. Suction ball
Serves to suck the solution to be measured using a measuring pipette.

Figure 3.10 Suction Ball

11. Paralon pipe

Serves as a container for taking sample.

Figure 3.11 Paralon pipe

Haikal Ramadhana Akmal (2310942009) III-3

12. Shaker
Serves to homogenize the solution at a certain speed.

Figure 3.12 Shaker

3.2 Material

Materials used in microbial isolation practicum are:

1. NA media
Serves as a medium for growing bacteria.

Figure 3.13 NA media

2. PCA media
Serves as a medium for growing bacteria.

Figure 3.14 PCA media

3. SDA media
Serves as a medium for growing fungal.

Figure 3.15 SDA media

Haikal Ramadhana Akmal (2310942009) III-4

4. 70% alcohol
Serves to sterilize the workplace.

Figure 3.16 70% alcohol

5. Buffered Peptone Water (BPW) solution 0,1%

Serves to increase the growth of bacterial cultures that are below their optimum
conditions.

Figure 3.17 0,1% BPW solution

6. Aquadest
Serves to dilute the solution and sterilize the tool.

Figure 3.18 Aquadest

7. Sample
Serves as a test material.

Figure 3.19 Sample

Haikal Ramadhana Akmal (2310942009) III-5

3.3 Procedures

Procedures on microbial isolation practicum is:

1. All equipment is washed and dried;
2. petri dishes are wrapped in paper and sterilized in an oven or autoclave;

Figure 3.20 Cups sterilized in an oven or autoclave

3. the workplace is cleaned by spraying 70% alcohol, and allowing a few minutes
to dry;

Figure 3.21 Workbench sprayed with alcohol

MICROBES IN SOIL
4. soil weighed as much as 5,00 grams;

Figure 3.22 5,00 grams of soil taken

5. The soil was put into a 250 mL erlenmeyer with an additional 45 mL of 0,1%
BPW solution. The sample was homogenized using a shaker, at 160 rpm for 10
minutes (10-1);

Haikal Ramadhana Akmal (2310942009) III-6

Figure 3.23 Soil is put into an erlenmeyer and homogenized

6. 1 mL of sample was taken from the erlenmeyer and put into the first dilution
tube (10-2) aseptically (from suspension preparation) with 9 mL of 0,1% BPW
solution;

Figure 3.24 Samples put into test tubes

7. 1 mL was taken from tube 10-1 with a measuring pipette then transferred to tube
10-2 aseptically then homogenized. The transfer continued until the last dilution
tube in the same way until tube 10-4 ;

Figure 3.25 Samples put into test tubes

8. 1 mL of sample was taken from the 10-3 dilution, then put into 2 petri dishes
(1 mL of sample each) and 1 mL of sample was taken from the 10-4 dilution,
then put into 2 petri dishes (1 mL of sample each);

Figure 3.26 Samples put into a petri dish

9. PCA media was poured into 2 petri dishes that already contained samples and
SDA media, poured into 1 other petri dish;
Haikal Ramadhana Akmal (2310942009) III-7
MINISTRY OF HIGHER EDUCATION, SCIENCE, AND TECHNOLOGY
DEPARTEMENT OF ENVIRONMENTAL ENGINEERING
ENVIRONMENTAL MICROBIOLOGY LABORATORY
Campus Unand Limau Manis Padang 25163
Telp. (0751) 7862901, Fax (0751 72566)

Figure 3.27 Samples put into a petri dish

10. then put PCA and SDA media into a petri dish, then homogenized by rotating
the petri dish to form a figure 8;

Figure 3.28 Sample homogenized

11. petri dishes were covered and allowed to solidify;

Figure 3.29 Media allowed to solidify

12. then incubated for 24 hours at temperature 370 and then observed;

Figure 3.30 Incubation process

13. the shape of the colonies is noted and examined for fine structure;

Figure 3.31 Colony counting

Haikal Ramadhana Akmal (2310942009) III-8
MINISTRY OF HIGHER EDUCATION, SCIENCE, AND TECHNOLOGY
DEPARTEMENT OF ENVIRONMENTAL ENGINEERING
ENVIRONMENTAL MICROBIOLOGY LABORATORY
Campus Unand Limau Manis Padang 25163
Telp. (0751) 7862901, Fax (0751 72566)

14. record the results:

a. The number of growing colonies was counted using a colony counter.
b. Colonies that grow are observed and recorded.
1) Shape (whether point-shaped, round, regular, thread-shaped, thin, thick,
convex, and flat).
2) The fringe.
3) Surface (whether flat, shiny, rough, and irregular dull).
4) Color.

MICROBES IN THE AIR

15. NA media and SDA media is poured into petri dishes (work near the fire);

Figure 3.32 NA and SDA media poured into petri dishes (near the fire)

16. Petri dishes were covered and allowed to solidify;

Figure 3.33 Media allowed to solidify

17. petri dishes were left open for 10 minutes;

Figure 3.34 The cup is left open

18. then wrapped in paper and incubated for 24 hours at temperature 370 and
observed.;

Haikal Ramadhana Akmal (2310942009) III-9

Figure 3.35 Incubation process

19. the shape of the colonies is noted and examined for fine structure;

Figure 3.36 Colony counting

20. record the results:
a. The number of growing colonies was counted using a colony counter.
b. Colonies that grow are observed and recorded.
1) Shape (whether point-shaped, round, regular, thread-shaped, thin, thick,
convex, and flat).
2) The fringe.
3) Surface (whether flat, shiny, rough, and irregular dull).
4) Color.

Haikal Ramadhana Akmal (2310942009) III-10

CHAPTER IV
RESULTS AND DISCUSSION

4.1 Results Data

The following is the data on the results of this practical experiment:

Table 4.1 Data on the Number of Colonies in Soil Sample Experiments

Media Number of Colonies Characteristic Features
PCA 57 Colony Bacterial Thick round shape, irregular
edges, flat surface, and white in
color.
SDA 2 Colony Fungi Thick round, irregular edges, and
flat surface.
Source: Environmental Microbiology Practicum Result Data 2024

Table 4.2 Colony Physiology in Soil Samples

NA PCA

Figure

Figure 4.1 NA Soil Figure 4.2 PDA Soil

Source: Environmental Microbiology Practicum Result Data 2024

Table 4.3 Data on the Number of Colonies in Air Sample Experiments.

Media Number of Colonies Characteristic Features
Thick round shape, irregular edges,
NA 27 Bacterial Colonies
flat surface, and cream color
Thick round shape, irregular edges,
SDA 16 Fungal Colonies
flat surface, and cream color
Source: Environmental Microbiology Practicum Result Data 2024

IV-1
MINISTRY OF HIGHER EDUCATION, SCIENCE, AND TECHNOLOGY
DEPARTEMENT OF ENVIRONMENTAL ENGINEERING
ENVIRONMENTAL MICROBIOLOGY LABORATORY
Campus Unand Limau Manis Padang 25163
Telp. (0751) 7862901, Fax (0751 72566)

Table 4.4 Colony Physiology in Air Samples

NA SDA

Figure

Figure 4.3 NA Air Figure 4.4 SDA Air

Source: Environmental Microbiology Practicum Result Data 2024

4.2 Calculation

The following is data for calculating the number of microbial colonies by diluting
soil microbes 3 times.

4.2.1 Soil Sample

The total number of colonies on each soil sample medium are:

N total PCA soil = 1/fp x n colony counter
= 1/10-3 x 57 colonies
= 57.000 colonies
N total SDA soil = 1/fp x n colony counter
= 1/10-3 x 2 colonies
= 2.000 colonies
4.2.2 Air Sample
The total number of colonies on each air sample medium are:
5a × 104
N total NA air = b.t

5 (27) × 104
=
65,61 cm2 × 10 minutes

= 2.057,61 CFU/m3

5a × 104
N total SDA air = b.t

5 (16) × 104
=
65,61 cm2 × 10 minutes
= 1.219,32 CFU/m3

Haikal Ramadhana Akmal (2310942009) IV-2

4.3 Discussion
In the microbial isolation practicum, two types of samples were used: soil and air.
The soil sample was obtained from the Edufarm plantation area at Andalas
University, with sampling carried out on Monday, November 11 th, 2024, at 2:05
PM. The recorded coordinates for the sampling location were 0054'39.2112" South
Latitude and 100027'54.615" East Longitude, at an elevation of 291 meters above
sea level. The weather was clear at the time, with a temperature of 28°C. The air
sample was collected from the stairwell area of the Civil Engineering Department
at the Faculty of Engineering, Andalas University, on Tuesday, November 12 th,
2024. Bacterial cultures in this practicum were observed as pure cultures on NA,
SDA, and PCA media.

Before starting the practicum, the workspace was sterilized using 70% alcohol,
followed by preparation of the NA, SDA, and PCA media. The NA, SDA, and PCA
media were poured into petri dishes near a flame. Five grams of soil sample were
weighed using an analytical balance, dissolved in 45 mL of 0.1% Buffered Peptone
Water (BPW) in an Erlenmeyer flask, and homogenized for 10 minutes at a speed
of 160 rpm using a shaker. The soil sample was then diluted by transferring 1 mL
into the first dilution tube (10⁻²), adding 9 mL of BPW, and homogenizing. The
dilution process was repeated three times, after which 1 mL from the third dilution
(10⁻4 tube) was transferred to petri dishes containing NA, SDA, and PCA media
and spread in a figure-eight motion for even distribution. The media were then
incubated for 24 hours, and colonies were counted using a colony counter. For the
air sample, petri dishes with NA and SDA media were opened at the sampling site
for 10 minutes. After exposure, the media were incubated for 24 hours, and colonies
were counted using a colony counter.

The air sample microbes were incubated in an incubator for 24 hours. After colony
counting using a colony counter, the microbial count on NA media was found to be
2.057,61 CFU/m³, with characteristics of an irregular round shape, smooth and
uneven edges, a slightly raised flat surface, and a pale cream/white color.
Meanwhile, microbes on SDA media showed a count of 1.219,32 CFU/m³, with
similar characteristics: an irregular round shape, smooth edges, a flat surface, and a

Haikal Ramadhana Akmal (2310942009) IV-3

pale cream/white color. The experimental results indicated that the microbial
concentration in the air sample on NA media was higher than on SDA media.

The soil sample microbes were incubated in an incubator for 24 hours. After
counting, the results showed that bacterial growth on NA media reached 57.000
colonies per 5 grams of soil, while fungal growth on PDA media was recorded at
2.000 colonies per 5 grams of soil. These results indicate that the microbial content
in the soil sample was higher on PDA media than on NA media, suggesting that
microbes reproduce more readily in soil when more complete nutrients are
available.

Errors made by the practitioner during the microbial isolation practicum included
insufficient sterilization of the equipment used. Cleanliness and sterility are crucial
aspects in microbial isolation practices. Additionally, the practitioner demonstrated
a lack of precision in observing the weight of the sample and the amount introduced.
Practitioners must ensure they work in a clean environment and apply aseptic
techniques. Poor storage of microbial cultures can also lead to contamination or the
death of the isolated microorganisms. It is essential that practitioners use
appropriate containers, maintain the correct storage temperature, and follow proper
conservation methods.

The role of an Environmental Engineering graduate in microbial isolation includes

applying sterilization principles to eliminate all forms of life, such as microbes,
from materials or objects. Reducing harmful microbes involves managing waste
generation, which helps to decrease or prevent the growth of bacteria and fungi that
can negatively impact soil and air quality. An Environmental Engineering graduate
is expected to apply their knowledge and develop new innovations to address soil
and air pollution, thereby contributing to a cleaner environment.

Haikal Ramadhana Akmal (2310942009) IV-4

CHAPTER V
CLOSING

5.1 Conclusion

From the soil and air microbial isolation experiment, it can be concluded that:
1. In this practicum using soil samples located in Edufarm, Limau manis, Padang
City, West Sumatra on Monday, November 11th, 2024. Air samples were taken
at the Civil Engineering stairs on Tuesday, November 12th, 2024.
2. The practicum was carried out by sterilizing the working media first, then the
samples were treated and put into Petri dishes. Agar media is inserted then
incubated for 24 hours. Microbes were observed with a microscope and the
number of colonies was counted with a colony counter.
3. On NA air media there were 2.057,61 CFU/m3. While microbes on SDA media
were 1.219,32 CFU/m3, with characteristics of irregular round shape, smooth
and irregular edges with flat embossed surfaces, and creamy/pale white color..
4. After calculating the results of this practicum, it was found that the number of
bacteria growing on NA media was bacterial colonies per 5 grams of soil, while
on PDA media there were fungal colonies per 5 grams of soil.
5. Mistakes that are usually made in microbial isolation practicum are the
inaccuracy of practitioners in weighing samples, entering the number of
samples, and the lack of sterile tools used.
6. Bachelor of Environmental Engineering is expected to be able to apply the
knowledge that has been learned about the principles of sterilization so that the
environment remains safe and find various innovations to overcome
environmental pollution, one of which is environmental pollution in soil and
air.
MINISTRY OF HIGHER EDUCATION, SCIENCE, AND TECHNOLOGY
DEPARTEMENT OF ENVIRONMENTAL ENGINEERING
ENVIRONMENTAL MICROBIOLOGY LABORATORY
Kampus Unand Limau Manis Padang 25163
Telp. (0751) 7862901, Fax (0751 72566)

5.2 Advice

For future practicum, the following are suggested:

1. Practitioners are expected to understand everything related to practicum, be
careful when doing practicum, and be careful in using tools and materials.
2. People should be more aware of the benefits and harms of microorganisms, and
be aware of the importance of protecting the environment.
3. The government is expected to better supervise and pay attention to the state of
the environment.
4. Bachelor of Environmental Engineering is expected to prevent and overcome
environmental problems caused by microorganisms with appropriate
technology.

Haikal Ramadhana Akmal (2310942009) V-2

DAFTAR PUSTAKA

Deny. 2020. Identifikasi Morfologi Mikroba Pada Ruangan Water Closet Jurusan
Biologi. Makassar: Universitas Negeri Makassar.
Istiamah, dkk. 2018. Teknologi Bioproses. Malang: Universitas Brawijaya: Press.
Mayasari, Ulfayani. 2022. Mikrobiologi. CV Media Sains
Indonesia: Bandung.
Nurmalasari. 2021. Dasar-Dasar Mikrobiologi dan Penerapannya. CV Widiana
Media Utama: Bandung.

Putri. 2017. Mikrobiologi. BPPSDM Kementrian Kesehatan.

Putri Amelya. 2023. Identifikaasi Mikroba Udara Isolat Pink di Laboraturium

Mikrobiologi. Padang: Universitas Negeri Padang.

Rohmah, N. S. (2017). Isolasi dan identifikasi bakteri yang berpotensi sebagai agen
bioremediasi timbal (Pb) dari Lumpur Lapindo (Doctoral dissertation,
Universitas Islam Negeri Maulana Malik Ibrahim).

Sabbathini. 2017. Isolasi dan Identifikasi Bakteri Genus Sphingomonas dari Daun
Padi (Oryza Sativa) di Area Persawahan Cibinong. Semarang: Universitas
Diponegoro.

Solikah, Ed Lusi. 2019. Teknik-teknik Isolasi atau Penanaman Mikroba. Jurnal

Farmasi: UNIDA.

Tim Laboratorium Mikrobiologi Lingkungan. 2023. Modul Praktikum

Mikrobiologi Lingkungan. Padang : Universiita Andalas.
ATTACHMENT
MINISTRY OF HIGHER EDUCATION, SCIENCE, AND TECHNOLOGY
DEPARTEMENT OF ENVIRONMENTAL ENGINEERING
ENVIRONMENTAL MICROBIOLOGY LABORATORY
Campus Unand Limau Manis Padang 25163
Telp. (0751) 7862901, Fax (0751 72566)

SAMPLING DOCUMENTATION

Group/Shift : 7 (SEVEN)/II (TWO)

Day/Date : Monday/November 11th 2024
Time : 2.05 PM
Location : Corn Garden Edufarm, Universitas Andalas, Limau Manis,
Pauh Sub-district, Padang City, West Sumatera
Coordinate : 0054’39,2112”SL 100027’54,615”EL
Elevation : 291 masl
Temperature : 280 C
Weather : Sunny

Figure 1. Existing Condition of Sampling Area

Group 7 Shift II
MINISTRY OF HIGHER EDUCATION, SCIENCE, AND TECHNOLOGY
DEPARTEMENT OF ENVIRONMENTAL ENGINEERING
ENVIRONMENTAL MICROBIOLOGY LABORATORY
Campus Unand Limau Manis Padang 25163
Telp. (0751) 7862901, Fax (0751 72566)

Figure 2. Sampling Method

Figure 3. Sample

Group 7 Shift II
Isolasi dan Seleksi Bakteri Penghasil..., Amilia, Mardya, Any, Andik

ISOLASI DAN SELEKSI BAKTERI PENGHASIL

BIOSURFAKTAN YANG TERDAPAT DI DALAM DEPOSIT
LILIN PADA PIPA TRANSMISI MINYAK MENTAH

Amilia1), Mardya Ning Tyas2), Any Juliani, Andik Yulianto

Jurusan Teknik Lingkungan, FTSP
Universitas Islam Indonesia
Email: 1)amilia.envir08server@[Link]; 2)Mardyaningtyaz@[Link]

ABSTRAK
Salah satu komponen berat di dalam minyak bumi yaitu parafin/wax akan
mengendap pada dinding pipa transmisi apabila temperatur berkurang seiring
minyak yang ditransmisikan mendekati permukaan bumi. Selain penanganan
secara mekanis dan isolasi termal juga terdapat penangan dengan penambahan
bahan kimia sejenis surfaktan untuk menurunkan tegangan permukaan kemudian
membentuk mikroemulsi sehingga minyak dapat larut di dalam air begitu
sebaliknya. Biosurfaktan menjadi alternatif menjanjikan yang ramah lingkungan,
relatif mudah untuk dikembangkan dengan biaya yang relatif lebih murah. Tujuan
dari penelitian ini yaitu menemukan isolat bakteri penghasil biosurfaktan
menggunakan metode isolasi bakteri dari deposit lilin di dalam pipa transmisi
minyak mentah kemudian diseleksi bakteri penghasil biosurfaktan. Didapatkan
isolat bakteriEnterobacter gergoviaedanBurkholderia cepacia. Bakteri yang
berkaitan dengan penghasil biosurfaktan disini yaitu bakteri Burkholderia cepacia
yang memiliki waktu generasi 68,4 menit dengan konstanta laju pertumbuhan
0,61 per jam.
Kata Kunci : Deposit Lilin, Biosurfaktan, Bakteri Indigen

ABSTRACT
When transmitting crude oil, paraffin wax contained in the crude oil will be
precipitated on the walls of the pipe as the crude closer to the earth’s surface.
Been applied mechanically handling, thermal insulation and with the addition of
chemical inhibitors. The workings of a chemical inhibitor in addition to preventing
the formation of wax crystals also lowers the temperature at which crude oil stopped
flowing (crude pour point). As the working principle of the surfactant will reduce
the surface tension so that then formed microemulsion oil soluble in water and oil
soluble in water. From this surfactant can be applied to prevent wax precipitation.
biosurfactant a promising alternative environmentally friendly, relatively easy to
49
KHAZANAH, Vol. 5 No.2 Januari 2012

develop a relatively cheaper cost. The study was conducted to obtain biosurfactant-
producing bacterial indigen isolates of wax deposition in crude oil transmission
pipelines. Bacteria isolated from deposition wax then selected biosurfactant-pro-
ducing bacteria by oil spreading technique and hemolysis [Link]-pro-
ducing bacteria Burkholderia cepacia bacteria are [Link] cepacia
obtained with a generation time of 68.4 minutes and a constant growth rate of
0.61 per hour.
Keywords: Wax Deposition, Biosurfactants, Bacteria Indigen

PENDAHULUAN
Endapan dalam pipa tersebut Bahan surfaktan telah lama dipakai
biasanya diatasi dengan berbagai oleh industri minyak untuk membantu
metode termasuk pembersihan secara proses bioremediasi limbah minyak
mekanis, pengaplikasian panas dan uap serta digunakan dalam proses En-
ataupun penginjeksian bahan-bahan hanced Oil Recovery (EOR). Dalam
kimia termasuk jenis surfaktan yang proses bioremediasi, biosurfaktan
dalam prosesnya membutuhkan biaya digunakan untuk meningkatkan kela-
yang tidak sedikit. Salah satu alternatif rutan polutan sehingga lebih mudah
pemecahan masalah yang dapat untuk didegradasi oleh mikroba. Dalam
dikembangkan adalah dengan aplikasi proses EOR, biosurfaktan dapat
proses biologis yang dalam hal ini mengekstrak kembali minyak yang
dilakukan oleh mikroba jenis bakteri. terjebak dalam substansi lain sehingga
Mikroba dapat dikembangkan untuk dapat meningkatkan produksi minyak
dapat mendegradasi endapan dalam dalam sumur minyak.
pipa yang pada dasarnya adalah senya- Biosurfaktan memiliki keunggulan
wa organik. Sementara itu, senyawa dibandingkan dengan surfaktan yang
organik merupakan sumber karbon dan disintesis secara kimiawi karena mem-
energi yang penting untuk mikroba. punyai kadar toksisitas yang rendah,
Namun aplikasi langsung tersebut dapat didegradasi secara biologis,
terkendala kondisi lingkungan dalam efektif terhadap suhu, pH dan salinitas
pipa yang memiliki temperatur dan ekstrim, serta mudah disintesis.
tekanan yang tinggi. Oleh karena itu Biosurfaktan merupakan calon poten-
proses biologis dapat diaplikasikan sial untuk aplikasi komersial dalam
secara tidak langsung untuk mengatasi industri obat–obatan dan makanan
masalah ini melalui produksi biosur- serta dalam industri pengambilan
faktan. minyak. (Banat, 1995).

50
LAPORAN AKHIR PRAKTIKUM
MIKROBIOLOGI PERAIRAN
(M10A104)

Disusun Oleh:
KELOMPOK 10/PERIKANAN C
Rizal Firdaus 230110130162
M. Salsabil 230110130198
Jumaidi Efendi 230110130200
Ruth Maria 230110130174
Christoper 230110130199
Rury Ratnafuri 230110130228

UNIVERSITAS PADJADJARAN
FAKULTAS PERIKANAN DAN ILMU KELAUTAN
PROGRAM STUDI PERIKANAN
JATINANGOR

2014
9

(Suriawiria, 2005). Beberapa indikasi pembiakan pada laboratorium

mikrobiologi meliputi:
1. Pengasingan (isolasi) mikroba pada biakan bakteri
2. Menunjukan sifat khas mikroba.
3. Untuk menentukan jenis mikroba yang diisolasi dengan cara-cara
tertentu.
4. Untuk mendapatkan bahan biakan yang cukup untuk membuatantigen
dan percobaan serologi lainnya.
5. Menentukan kepekaan kuman terhadap antibiotik.
6. Menghitung jumlah kuman
7. Mempertahankan biakan mikroba.
Mikroorganisme tidak memerlukan banyak ruangan untuk
perkembangannya, sebab itu media buatan (agar) dapat dimasukkan ke dalam
sebuah tabung percobaan labu atau cawan Petri. Pada permulaannya tabung atau
cawan Petri harus dalam keadaan steril (bebas dari setiap mikroorganisme
hidup) lalu setelah itu dimasukkan mikrobia yang diinginkan, tabung atau cawan
harus dilindungi terhadap kontaminasi dari luar. Sumber utama pencemaran dari
luar adalah udara, yang banyak mengandung mikroorganisme yang
berterbangan. Bentuk cawan petri, dengan tutup yang saling menyelubungi,
dirancang untuk mencegah pencemaran udara. Pencemaran tabung atau labu
dihindari dengan cara menyumbat mulutnya dengan penutup yang cocok,
biasanya dengan kapas.
Permukaan luar cawan biakan yang menjadi sasaran pencemaran, dan
bagian dalam labu atau tabung akan tercemar bila dibuka untuk memasukkan
atau mengeluarkan bahan. Bahaya ini dapat dihindari dengan cara membakar
bibir atau pinggiran cawan, tabung atau labu dalam api, segera setelah penutup
dibuka dan dibakar sekali lagi pada waktu akan ditutup.
Dalam mengisolasi bekteri dikenal empat cara, cara isolasi bakteri
tersebut yaitu :
a. Pour plate atau shake culture
10

Beberapa ml suspensi bakteri dicampur dengan mediaum yang masih cair

(belum membeku) dengan demikian akan diperoleh piaraan adukan. Digunakan
untuk mengencerkan atau mengisolasi yang terdapat pada contoh. Setelah
inkubasi pada suhu dan waktu tertentu, koloni akan tumbuh pada permukaan dan
bagian bawah agar.
b. Streak Plate atau culture
Ujung kawat imokulasi yang membawa bakteri digesekkan atau
digoreskan dengan bentuk zig-zag pada permukaan agar-agar dalam cawan Petri
sampai meliputi seluruh permukaan. Untuk memperoleh hasil yang baik
diperlukan keterampilan, yang biasanya diperoleh dari pengalaman. Metode
cawan gores yang dilakukan dengan baik kebanyakan akan menyebabkan
terisolasinya mikroorganisme yang diinginkan. Dua macam kesalahan yang
umum sekali dilakukan adalah tidak memanfaatkan permukaan medium dengan
sebaik- baiknya untuk digores sehingga pengenceran mikroorganisme menjadi
kurang lanjut dan cenderung untuk menggunakan inokulum terlalu banyak
sehingga menyulitkan pemisahan sel - sel yang digores.
c. Slant culture
Ujung kawat yang membawakan bakteri digesekkan pada permukaan
agar-agar miring dalam tabung reaksi. Dapat dilakukan dengan cara
menggoreskan secaa zig-zag pada permukaan agar miring menggunakan
jarum ose yang bagian atasnya dilengkungkan. Cara ini juga dilakukan pada agar
tegak untuk meminimalisir pertumbuhan mikroba dalam keadaan kekurangan
oksigen. (Rusdimin, 2003)
d. Stab culture
Ujung kawat yang membawakan bakteri ditusukkan pada media padat
(agar-agar) dalam tabung reaksi, berbeda dengan slant culture permukaan agar-
agar ini tidak miring. Media agar setengah padat dalam tabung reaksi, digunakan
untuk menguji gerak bakteri secara makroskopis
ISOLASI DAN IDENTIFIKASI BAKTERI YANG BERPOTENSI
SEBAGAI AGEN BIOREMEDIASI TIMBAL (Pb) DARI LUMPUR
LAPINDO

SKRIPSI

Oleh :
NITA SHILFIANI ROHMAH
NIM.12620101

JURUSAN BIOLOGI
FAKULTAS SAINS DAN TEKNOLOGI
UNIVERSITAS ISLAM NEGERI MAULANA MALIK IBRAHIM
MALANG
2017
39

PbrA yang diketahui dapat mentranspor Pb ke periplasma. Selain transporter

tersebut, terdapat pula dari famili CBA dan CDF (Arguello, 2003). CDF

merupakan transporter yang membawa ion dari sitoplasma ke periplasma,

sedangkan CBA berperan dalam mengeluarkan Pb dari sel (Hynninen, 2010).

2.6 Isolasi dan Identifikasi Bakteri

2.6.1 Isolasi Bakteri

Isolasi adalah mengambil mikroorganisme yang terdapat di alam dan

menumbuhkannya dalam suatu medium buatan. Proses pemisahan atau pemurnian

dari mikroorganisme lain perlu dilakukan karena semua pekerjaan mikrobiologis,

misalnya telaah dan identifikasi mikroorganisme, memerlukan suatu populasi

yang hanya terdiri dari satu macam mikroorganisme saja. Prinsip dari isolasi

mikroba adalah memisahkan satu jenis mikroba dengan mikroba lainnya yang

berasal dari campuran bermacam-macam mikroba. Hal ini dapat dilakukan dengan

menumbuhkannya dalam media padat (Sutedjo, 1996).

Sel-sel mikroba akan membentuk suatu koloni sel yang tetap pada

tempatnya. Jika sel-sel tersebut tertangkap oleh media padat pada beberapa tempat

yang terpisah, maka setiap sel atau kumpulan sel yang hidup akan berkembang

menjadi suatu koloni yang terpisah, sehingga memudahkan pemisahan

selanjutnya. Bila digunakan media cair, sel-sel mikroba sulit dipisahkan secara

individu karena terlalu kecil dan tidak tetap tinggal di tempatnya. Akan tetapi bila

sel-sel itu dipisahkan dengancara pengenceran, kemudian ditumbuhkan dalam

media padat dan dibiarkan membentuk koloni, maka sel-sel tersebut selanjutnya
40

dapat diisolasi dalam tabung-tabung reaksi atau cawan petri-cawan petri yang

terpisah (Sutedjo, 1996).

Ada beberapa teknik isolasi bakteri menurut Hadioetomo (1993) yaitu:

a. Metode Cawan Gores (Streak Plate)

Prinsip metode ini yaitu mendapatkan koloni yang benar-benar terpisah

dari koloni yang lain, sehingga mempermudah proses isolasi. Cara ini dilakukan

dengan menggoreskan ose pada cawan petri berisi media steril

b. Metode Cawan Sebar (Spread Plate)

Teknik spread plate (cawan sebar) adalah suatu teknik di dalam

menumbuhkan mikroorganisme di dalam media agar dengan cara menuangkan

stok kultur bakteri atau menghapuskannya di atas media agar yang telah memadat,

sedangkan pour plate kultur dicampurkan ketika media masih cair (belum

memadat). Kelebihan teknik ini adalah mikroorganisme yang tumbuh dapat

tersebar merata pada bagian permukaan agar.

c. Teknik Pengenceran (Dilution Plate)

Tujuan dari teknik ini adalah melarutkan atau melepaskan mikroba dari

substratnya ke dalam air sehingga lebih mudah penanganannya (pemindahannya

ke tabung atau cawan). Sampel yang telah diambil kemudian disuspensikan dalam

aquades steril. Teknik dilusi sangat penting dalam analisa mikrobiologi karena

hampir semua metode penelitian dan perhitungan jumlah sel mikroba

menggunakan teknik ini, seperti TPC (Total Plate Counter) (Waluyo, 2005).
■ Mikrobiologi Keperawatan Gigi ■

2) Media sintetik yaitu media yang disusun oleh senyawa kimia. Contohnya media
untuk pertumbuhan dan perkembangbiakan bakteri Clostridium tersusun oleh :

K2HPO4 : 0,5 g
KH2PO4 : 0,5 g
MgSO4, 7H2O : 0,1 g
NaCl : 0,1 g
FeSO4, 7H2O : 0,01 g
MnSO4, 7H2O : 0,01 g
CaCO3 : seangin

3) Media semi sintetik yaitu media yang tersusun oleh campuran bahan-bahan
alami dan bahan-bahan sintetis.

Berikut ini beberapa media yang sering digunakan secara umum dalam mikrobiologi

Lactose Broth
Lactose broth digunakan sebagai media untuk mendeteksi kehadiran koliform dalam
air, makanan, dan produk susu, sebagai kaldu pemerkaya (pre-enrichment broth) untuk
Salmonellae dan dalam mempelajari fermentasi laktosa oleh bakteri pada umumnya. Pepton
dan ekstrak beef menyediakan nutrien esensial untuk memetabolisme bakteri. Laktosa
menyediakan sumber karbohidrat yang dapat difermentasi untuk organisme koliform.

EMBA (Eosin Methylene Blue Agar)

Media Eosin Methylene Blue mempunyai keistimewaan mengandung laktosa dan
berfungsi untuk memilah mikroba yang memfermentasikan laktosa seperti S. aureus,
P. aerugenosa, dan Salmonella. Mikroba yang memfermentasi laktosa menghasilkan koloni
dengan inti berwarna gelap dengan kilap logam. Sedangkan mikroba lain yang dapat tumbuh
koloninya tidak berwarna.

Nutrient Agar
Nutrien agar adalah medium umum untuk uji air dan produk dairy. NA juga digunakan
untuk pertumbuhan mayoritas dari mikroorganisme yang tidak selektif, dalam arti
mikroorganisme heterotrof. Media ini merupakan media sederhana yang dibuat dari ekstrak
beef, pepton, dan agar. NA merupakan salah satu media yang umum digunakan dalam
prosedur bakteriologi seperti uji air biasa, uji air limbah, produk pangan, untuk membawa
stok kultur, untuk pertumbuhan sampel pada uji bakteri, dan untuk mengisolasi organisme
dalam kultur murni.

Nutrient Broth
Nutrient broth merupakan media untuk mikroorganisme yang berbentuk cair. Intinya
sama dengan nutrient agar.

36
■ Mikrobiologi Keperawatan Gigi ■

MRSA (de Mann Rogosa Sharpe Agar)

MRSA merupakan media yang diperkenalkan oleh De Mann, Rogosa, dan Shape (1960)
untuk memperkaya, menumbuhkan, dan mengisolasi jenis Lactobacillus dari seluruh jenis
bahan. MRS agar mengandung polysorbat, asetat, magnesium, dan mangan yang diketahui
untuk beraksi/bertindak sebagai faktor pertumbuhan bagi Lactobacillus, sebaik nutrien
diperkaya.

Trypticase Soy Broth (TSB)

TSB adalah media broth diperkaya untuk tujuan umum, untuk isolasi, dan penumbuhan
bermacam mikroorganisme. Media ini banyak digunakan untuk isolasi bakteri dari spesimen
laboratorium dan akan mendukung pertumbuhan mayoritas bakteri patogen. Media TSB
mengandung kasein dan pepton kedelai yang menyediakan asam amino dan substansi
nitrogen lainnya yang membuatnya menjadi media bernutrisi untuk bermacam
mikroorganisme.

Plate Count Agar (PCA)

PCA digunakan sebagai medium untuk mikroba aerobik dengan inokulasi di atas
permukaan. Media PCA ini baik untuk pertumbuhan total mikroba (semua jenis mikroba)
karena di dalamnya mengandung komposisi casein enzymic hydrolisate yang menyediakan
asam amino dan substansi nitrogen kompleks lainnya serta ekstrak yeast mensuplai vitamin
B kompleks.

Potato Dextrose Agar (PDA)

PDA digunakan untuk menumbuhkan atau mengidentifikasi ragi dan kapang. Dapat
juga digunakan untuk enumerasi ragi dan kapang dalam suatu sampel atau produk makanan.
PDA cocok untuk pertumbuhan jamur. PDA mengandung sumber karbohidrat dalam jumlah
cukup yaitu terdiri dari 20% ekstrak kentang dan 2% glukosa sehingga baik untuk
pertumbuhan kapang dan khamir tetapi kurang baik untuk pertumbuhan bakteri.

2. Jenis-Jenis Media Berdasarkan Fungsinya

Berdasarkan fungsinya, media dapat dibedakan menjadi enam yaitu:
1. Media Basal (media dasar) adalah media yang digunakan sebagai bahan dasar untuk
membuat media lain yang lebih kompleks. Media ini dapat mendukung pertumbuhan
hampir semua jenis mikrobia, contohnya adalah nutrient broth, kaldu pepton, dsb.

2. Media Diferensial adalah media yang bila ditumbuhi oleh mikroba yang berbeda,
mikroba tersebut akan tumbuh dengan ciri khusus sehingga dapat dibedakan.
Contohnya: Media Triple Sugar Iron Agar (TSIA), Media Sulfit Indol Motility (SIM), dan
sebagainya. Pada media diferensial ditambahkan bahan-bahan kimia atau reagensia
tertentu yang menyebabkan mikroba yang tumbuh memperlihatkan perubahan-
perubahan spesifik sehingga dapat dibedakan dengan jenis lainnya.

37
Jurnal Biologi, Volume 6 No 1, Januari 2017
Hal. 59-64

ISOLASI DAN IDENTIFIKASI BAKTERI GENUS Sphingomonas DARI

DAUN PADI (Oryza sativa) DI AREA PERSAWAHAN CIBINONG

Gabriela Christy Sabbathini 1)*, Sri Pujiyanto 1)*, Wijanarka 1)* dan Puspita Lisdiyanti 2)*

1DepartemenBiologi, Universitas Diponegoro

2
Laboratorium Mikrobiologi Terapan, LIPI, Cibinong

ABSTRAK

Kemampuan unik bakteri dengan genus Sphingomonas seperti mendegradasi kontaminan yang tahan terhadap
panas, sebagai bakteri antagonis terhadap fungi fitopatogenik, dan dapat mensekresi eksopolisakarida gellan yang
sangat berguna membuat bakteri ini dapat berperan penting dalam berbagai bidang industri. Eksploitasi
kemampuam metabolism dari bakteri genus Sphingomonas dapat menyediakan keuntungan komersial yang
penting bagi bioteknologi. Spesies dari Sphingomonas sering ditemukan berasosiasi dengan tanaman padi sebagai
salah satu bakteri endofitik yang dapat dikultur. Penelitian ini bertujuan untuk mengisolasi bakteri lokal yang dapat
menghasilkan gellan gum dari daun tanaman padi (Oryza sativa). Proses isolasi dilakukan dengan metode cawan
sebar suspensi dari daun padi pada media Nutrient Dextrose Agar (NDA). Koloni tunggal bakteri yang dapat
diisolasi kemudian diidentifikasi dengan metode PCR koloni untuk dilanjutkan pada proses sekuensing. Hasil
sekuensing yang dilanjutkan dengan penyetaraan sekuens pada program BLAST menunjukkan empat isolat
dengan genus Sphingomonas yaitu isolat XA1, XA2, XA6, XA12 dengan hasil Sphingomonas sp. Fse41,
Sphingomonas sp. Fse41, Sphingomonas sanguinis strain L4-317 dan Sphingomonas sp. MLB01

Kata kunci : bakteri endofit, padi, Sphingomonas

ABSTRACT

The unique ability of the genus Sphingomonas bacteria as degrade the contaminants refractory contaminants, to
serve as the antagonists bacteria to phytopathogenic fungi, and capable to secrete hidhly useful exopolysaccharide
gellan make these bacteria may play an important role in various industrial fields. Exploitation of the metabolic
capabilities by genus Sphingomonas bacteria can provide significant commercial advantages for
[Link] species of Sphingomonas are often found associated with the rice plant as one of the endophytic
bacteria that can be cultured. This study aims to isolate the local bacteria that can produce gellan gum from the
leaves of the rice plant (Oryza sativa). The isolation process is done with a spread plate method suspension of rice
leaves on Nutrient Dextrose Agar (NDA) media. Single colonies of bacteria that can be isolated then identified by
colony PCR method to proceed at sequencing process. Sequencing followed by equalization sequences on the
BLAST program shows four isolates of the genus Sphingomonas which isolates XA1, XA2, XA6, XA12 with the
results are Sphingomonas sp. Fse41, Sphingomonas sp. Fse41, Sphingomonas sanguinis L4-317 strain and
Sphingomonas sp. MLB01

Keywords: endophytic bacteria, padi, Sphingomonas

PENDAHULUAN cukup beragam dalam hal filogenetik, ekologi,

dan sifat fisiologis. Sphingomonas dibagi
Genus Sphingomonas didefinisikan menjadi empat genera: Sphingomonas,
oleh Yabuuchi et al. (1990) sebagai kelompok Sphingobium, Novosphingobium, Sphingopyxis
Gram negatif, berbentuk batang, dan genera yang disebut secara kolektif sebagai
chemoheterotrophic, aerobic, serta "sphingomonads" (Balkwill et al, 2006).
mengandung glikosphingolipid (GSLs) Sphingomonads dapat menggunakan bahan
lipopolisakarida dalam amplop sel mereka, dan organic yang terdapat di alam termasuk
biasanya menghasilkan koloni berpigmen kontaminan lingkungan yang tahan terhadap
kuning. Terdapat lebih dari 20 spesies yang panas. Kemampuan Sphingomonads dalam
Jurnal Biologi, Volume 6 No 1, Januari 2017
Hal. 59-64

biodregradatif dan biosintetik membuat bakteri pertanian padi sebagai mata pencaharian
genus ini dapat digunakan dalam aplikasi (Craswell et al, 1981; Yoshida, 1981 dalam
bioteknologi secara luas. Mulai dari Hongrittipun et al, 2014). Padi (Oryza sativa)
bioremidiasi dari kontaminan lingkungan juga merupakan pangan biji padi-padian utama
sampai produksi ektraseluler polimer yang yang dikonsumsi oleh hampir setengah dari
dapat digunakan secara luas dalam industri populasi dunia, menjadikannya tanaman paling
makanan dan lainnya. penting untuk diproduksi. Sawah tropis
Sphingomonas tersebar secara luas di menawarkan lingkungan biologis beragam dan
alam dan dapat diisolasi dari banyak lingkungan dinamis untuk populasi mikroba dan bunga,
perairan maupun daratan termasuk dalam invertebrata dan vertebrata (Ramakrishnan,
jaringan tanaman , specimen klinik, dan sumber 2005). Studi oleh Mano et al (2007)
lainnya (Balkwill, 2006). Spesies dari menunjukkan bakteri endofit yang dapat
Sphingomonas sering ditemukan berasosiasi dikultur pada tanaman padi. Terdapat bakteri
dengan tanaman (White et al, 1996). Spesies endofitik terkait dengan Bacillus,
Sphingomonas dalam jaringan tanaman (biji, Curtobacterium, Methylobacterium,
daun dan bunga) dapat terdeteksi dan terinci Sphingomonas dan Pantoea terisolasi dari
dengan amplifikasi dari bagian spesifik subunit daun, dan Bacillus, Brevibacillus,
kecil gen 16S rRNA dan ditemukan di 26 Mycobacterium, Enterobacter, dan
spesies tumbuhan yang termasuk dalam 11 Chryseobacterium terisolasi dari akar .
famili (Kim et al, 1998). Salah satunya adalah
ditemukannya spesies Sphingomonas pada 2. Genus Sphingomonas
tanaman padi sebagai salah satu bakteri Genus Sphingomonas pertama kali
endofitik yang dapat dikultur (Mano et al, diusulkan oleh Yabuuchi et al. dan terdiri dari 5
2007). Oleh karena itu penelitian ini bertujuan spesies termasuk S. paucimobilis sebagai tipe
untuk mendapatkan isolat bakteri genus spesies. Sebagian besar strain yang digunakan
Sphingomonas dari daun padi (Oryza sativa). dalam penelitian mereka diisolasi dari pasien
atau dari bahan klinis. Namun, S. paucimobilis,
sebelumnya diklasifikasikan sebagai
REFERENSI Pseudomonas paucimobilis, diketahui dapat
diisolasi dari lingkungan non-klinis dan juga
1. Bakteri Endofit Tanaman Padi dari rizosfir tanaman padi (Kawahara et al,
Bakteri endofit dapat diartikan sebagai 1994). Genus Sphingomonas memiliki kriteria
bakteri yang berkolonisasi di dalam jaringan bakteri Gram negatif dengan sel rod/curved rod
internal tanaman tanpa menunjukkan tanda , 0,5µm-4,0µm, motil dan tidak motil, serta
eksternal dari infeksi atau pengaruh negatif memiliki varian koloni dari tidak berwarna
lainya pada inangnya (Holliday, 1989; Schulz sampai berwana jingga (Yabuuchi & Kosako,
& Boyle, 2006 dalam Ryan et al, 2007), dan 2005). Sphingomonas tersebar secara luas di
hampir 300.000 spesies tanaman yang ada di alam dan dapat diisolasi dari banyak lingkungan
bumi, setiap individu tanaman adalah inang perairan maupun daratan termasuk dalam
untuk satu atau lebih mikroorganisme endofitik jaringan tanaman , specimen klinik, dan sumber
(Strobel et al, 2004 dalam Ryan et al, 2007). lainnya. Strain Sphingomonas dikenal dapat
Bakteri endofit dapat diartikan sebagai bakteri menghasilkan biopolimer ekstraselular,
yang berkolonisasi di dalam jaringan internal termasuk eksopolisakarida terkait gellan
tanaman tanpa menunjukkan tanda eksternal (Balkwill, 2006).
dari infeksi atau pengaruh negatif lainya pada
inangnya (Holliday, 1989; Schulz & Boyle, 3. Isolasi
2006 dalam Ryan et al, 2007), dan hampir Mikroorganisme pada suatu
300.000 spesies tanaman yang ada di bumi, lingkungan alami merupakan populasi
setiap individu tanaman adalah inang untuk satu campuran dari berbagai jenis, baik
atau lebih mikroorganisme endofitik (Strobel et mikroorganisme pada tanah, air, udara,
al, 2004 dalam Ryan et al, 2007). makanan, maupun yang terdapat pada tubuh
Padi adalah tanaman yang paling hewan maupun tumbuhan. Pemisahan bakteri
penting di Asia sebagai sumber pangan utama diperlukan untuk mengetahui jenis,
dan bagi mereka yang bergantung pada mempelajari kultural, morfologi, fisiologi, dan
Jurnal Biologi, Volume 6 No 1, Januari 2017
Hal. 59-64

karakteristik. Teknik pemisahan tersebut secara molekuler menggunakan gen 16S rRNA.
disebut isolasi yang disertai dengan pemurnian. Hal ini juga mengurangi prasangka penafsiran
Pengertian isolasi bakteri yaitu suatu proses dan menyingkirkan kebutuhan untuk
mengambil bakteri dari medium atau dari kemungkinan "pretest" mengenai klasifikasi
lingkungan asalnya lalu menumbuhkannya di mikroorganisme untuk pemeriksaan langsung
medium buatan sehingga diperoleh biakan yang dan pemilihan database (Petti et al., 2005).
murni (Singleton & Sainsbury, 2006). Gen 16S rRNA adalah gen yang
Prinsip dari isolasi mikroba adalah digunakan dalam menentukan filogenetik dan
memisahkan satu jenis mikroba dengan taksonomi dari bakteri secara molekuler.
mikroba lain yang berasal dari campuran Penggunaan 16S rRNA sekuensing gen di
bermacam-macam mikroba. Hal ini dapat laboratorium klinis menjadi umum untuk
dilakukan dengan menumbuhkannya dalam mengidentifikasi bakteri biokimia tak dikenal
media padat, sel-sel mikroba akan membentuk atau menyediakan referensi identifikasi untuk
koloni sel yang tetap pada tempatnya. Beberapa strain yang tidak biasa. (Janda & Abbott, 2007).
cara atau metode untuk memperoleh biakan Penggunaan gen 16S rRNA dengan teknik
murni dari suatu biakan campuran. Dua Polymerase Chain Reaction (PCR)
diantaranya yang paling sering digunakan memungkinkan untuk mengidentifikasi bakteri
adalah metode cawan gores dan metode cawan amilolitik dengan cepat dan spesifik. PCR
tuang. Yang didasarkan pada prinsip merupakan suatu metode enzimatis untuk
pengenceran dengan maksud untuk melipat gandakan secara eksponensial suatu
memperoleh spesies individu. Dengan sekuens nukleotida tertentu dengan cara in vitro
anggapan bahwa setiap koloni dapat terpisah (Yuwono, 2006).
dari satu jenis sel yang dapat diamati (Afrianto,
2004). METODELOGI

4. Identifikasi [Link] Bakteri dari Daun Padi

Proses identifikasi bakteri secara Daun padi diambil dari persawahan
konvensional berdasarkan karakter fenotip Cibinong. Sampel daun kemudian dicuci
bakteri seperti pewarnaan Gram, morfologi dengan aquades steril. Daun dipotong menjadi
koloni, dan aktivitas enzim seringkali tidak bagian yang lebih kecil dan disterilisasi
bersifat statis dan dapat berubah seiring dengan menggunakan ethanol 70% selama 30 detik
adanya evolusi. Kesalahan identifikasi
dan di bilas dua kali menggunakan aquades
seringkali terjadi dikarenakan hadirnya
karakteristik fenotip bakteri yang tidak biasa steril. Daun yang telah steril tersebut kemudian
ataupun kurangnya pengalaman dalam dipotong dengan gunting steril secara aseptik
menginterpretasikan data karakter fenotip. Hal menjadi bagian yang lebih kecil dan direndam
ini menimbulkan hadirnya metode identifikasi dalam larutan garam fisiologis 0,85%. Suspensi
secara molekuler menggunakan gen 16S rRNA. bakteri diencerkan hingga 10-3 dan disebar
Hal ini juga mengurangi prasangka penafsiran mengunakan metode cawan sebar pada media
dan menyingkirkan kebutuhan untuk
kemungkinan "pretest" mengenai klasifikasi NDA (Peptone 5 g/L, dextrose 20 g/L, yeast
mikroorganisme untuk pemeriksaan langsung extract 1,5g/L, NaCl 5 g/L, beef extract 1,5 g/L,
dan pemilihan database (Petti et al., 2005). dan agar 15 g/L ). Langkah pemurnian koloni
Proses identifikasi bakteri secara dilakukan dengan metode gores kuadran hingga
konvensional berdasarkan karakter fenotip diperoleh koloni tunggal, diinkubasi selama 48
bakteri seperti pewarnaan Gram, morfologi jam pada suhu 30°C serta dilakukan KOH test.
koloni, dan aktivitas enzim seringkali tidak
Koloni tunggal yang telah diperoleh disimpan
bersifat statis dan dapat berubah seiring dengan
adanya evolusi. Kesalahan identifikasi dalam gliserol stock 10%.
seringkali terjadi dikarenakan hadirnya
karakteristik fenotip bakteri yang tidak biasa [Link] Bakteri
ataupun kurangnya pengalaman dalam Identifikasi isolat bakteri dilakukan
menginterpretasikan data karakter fenotip. Hal secara molekuler dengan analisis gen 16S
ini menimbulkan hadirnya metode identifikasi rRNA. Satu koloni tunggal pada setiap isolat
◼ Mikrobiologi dan Parasitologi Keperawatan ◼

2. Koloni kapang/mold
• membentuk koloni kering dan padat.
• tekstur menyerupai beludru atau kapas.
• Contoh: Aspergillus, Trichophyton, Epidermophyton.

a. Candida albicans b. Koloni ragi bulat c. Koloni ragi pink

Gambar 5.7. Candida albicans, ragi bulat, dan koloni ragi pink
(Rahardjo, 2010)

C. PEMBIAKAN, PERTUMBUHAN BAKTERI, DAN JAMUR

1. Pembiakan dan Pertumbuhan Bakteri

Berbagai jenis media pertumbuhan bakteri lazim digunakan untuk tujuan isolasi,
transportasi, persemaian. dan diferensiasi. Untuk menunjang pertumbuhan yang optimal,
bakteri membutuhkan nutrisi yang amat beragam, sebagian bakteri dapat tumbuh dalam
medium yang hanya mengandung zat anorganik, sedangkan bakteri tertentu memerlukan
tambahan asam amino, vitamin, dan zat organik lain untuk pertumbuhannya. Media
pembiakan bakteri umumnya terdiri atas ekstrak daging, ekstrak ragi, pepton, dan agar.
Berdasarkan konsistensinya, media pembiakan bakteri dapat dibagi menjadi media
cair, media padat, dan semi solid. Kaldu nutrisi (pepton dan ekstrak daging) dan agar nutrisi
(pepton, ekstrak daging, dan agar) merupakan contoh medium cair dan padat yang sering
digunakan. Bakteri komponen dapat ditambahkan ke dalam medium untuk menghasilkan
medium dengan sifat tertentu sebagai contoh, penambahan zat warna dapat digunakan
untuk indikator aktivitas metabolisme bakteri.
Untuk menumbuhkan bakteri yang memerlukan nutrisi tinggi (fastidious
microorganism), medium dapat diperkaya dengan menambahkan darah, serum, vitamin,
dan komponen lain. Medium tersebut termasuk dalam medium diperkaya, contoh medium
tersebut adalah medium agar darah.
Isolasi bakteri menggunakan medium selektif yang dapat menghambat pertumbuhan
bakteri secara selektif, dan medium yang termasuk dalam medium selektif adalah medium
Salmonelle Shigella (SS).
Medium lain yang digunakan untuk membedakan beberapa jenis bakteri adalah
medium diferensial, medium yang banyak dikembangkan pada saat ini memiliki sifat selektif
dan diferensial, contoh medium tersebut adalah medium Agar Eosin Methylene Blue (EMB).

218
◼ Mikrobiologi dan Parasitologi Keperawatan ◼

Bila pengambilan spesimen di luar laboratorium, maka untuk mencegah kematian

bakteri, spesimen dapat ditanam pada medium transpor sebelum dipindahkan pada
medium pertumbuhan yang diperlukan. Contoh medium transpor yang sering digunakan di
laboratorium antara lain medium Carry Blair, Amies, dan Stuart.

Gambar 5.8. Bentuk media pembiakan bakteri (CDC, 1994)

2. Pembiakan Dan Pertumbuhan Jamur

Spesimen untuk pemerisaan jamur harus diinokulasikan pada medium yang dapat
menunjang pertumbuhan optimal. Berbagai macam media dapat digunakan, dapat berupa
medium dengan/atau tanpa antibiotika. Penambahan Chylohexamide dalam medium
diharapkan dapat menghambat pertumbuhan jamur saprofit yang tumbuh cepat sehingga
prtumbuhan jamur patogen yang lambat tidak terhalangi. Sedangkan Chloramfhenicol
dan/atau Gentamisin dapat menghambat kontaminasi bakteri.
Medium kultur jamur yang dianjurkan antara lain:
a. Sabouraud Dextrose Agar (SDA)
Medium standar yang mengandung mycological pepton, gula dekstrosa, dan agar.
b. SDA+Chylohexamide+Chloramfhenikol (agar mycobiotic atau agar mycosel)
Medium selektif untuk jamur Dermatofita dan Candida albicans.
c. Inhibitory Mold Agar (IMA).
Medium ini merupakan medium diperkaya yang mengandung Chloraamfenikol,
menunjang hampir semua pertumbuhan kapang dan khamir dan menghambat
pertumbuhan bakteri.
d. Brain-Heart Infusion Agar
Medium yang berfungsi sebagai penunjang pertumbuhan jamur dimorfik.

Temperatur optimal untuk pertumbuhan adalah 30oC, tetapi jika inkubator yang
diharapkan tidak tersedia, kultur harus diinkubasi pada suhu kamar (25oC) dengan
kelembaban yang harus tetap terjaga. Sebagian kultur jaringan diinkubasi selama 4 minggu
sebelum dinyatakan negatif. Kultur yeast dari tersangka oral trush atau vaginitis hanya
membutuhkan waktu 5 hari, sedangkan kultur dari tersangka jamur dimorfik (25 oC dan 37oC)
harus diinkubasi 8 minggu sebelum dinyatakan negatif.

219
INITIAL REPORT
ENVIRONMENTAL MICROBIOLOGY PRACTICUM

MICROBIAL ISOLATION

ASSISTANT:
THARA ALIYAH EDIHARSI

ENVIRONMENTAL MICROBIOLOGY LABORATORY

DEPARTMENT OF ENVIRONMENTAL ENGINEERING
FACULTY OF ENGINEERING-UNIVERSITAS ANDALAS
PADANG
2024
CHAPTER I

INTRODUCTION

1.1 Background

1.2 Aim and Objective of the Experiment

1.2.1 Purpose of the Experiment

The goal of this practicum is to meet the requirements for completing the
environmental microbiology course and to identify a microorganism in order to
understand its characteristics.

1.2.2 Experiment Objective

The objectives of this practicum are:

1. To familiarize participants with the equipment and media needed to create pure
cultures Participants will learn to use tools and media such as petri dishes and
agar for cultivating microorganisms.
2. To understand sterilization concepts and procedures for successful subculturing.
The practicum will cover sterilization techniques like autoclaving and flame
sterilization to prevent contamination.
3. To inoculate using streak plate, spread plate, and pour plate techniques for
isolating pure cultures. Participants will practice isolating microorganisms from
mixed cultures to obtain pure samples.
4. To recognize the morphological characteristics and colony shapes of microbial
cultures. Participants will learn to identify and describe colony features like
size, shape, and color to classify microorganisms.

Haikal Ramadhana Akmal (2310942009) I-2

1.3 Principle of Experiment

The principle of this experiment is:

1. The process of isolating a specific microorganism species from others is called
isolation, often combined with purification techniques.
2. A colony is a visible cluster of microorganisms that grow on a solid medium.
Each colony typically originates from the replication of a single microorganism.

1.4 Scope of the Experiment

The purpose of this experiment is to learn how to isolate microbes in order to obtain
a pure culture and examine the properties of colonies using NA and PDA media.
The samples used in this experiment were soil from irrigation sediments and air
collected from beneath the stairs.

1.5 Systematization of Report Writing

The systematic writing of the report is as follows:

CHAPTER I INTRODUCTION

CHAPTER III EXPERIMENTAL PROCEDURE

This section details the equipment and materials used, as well as the procedural
steps followed during the experiment.

CHAPTER IV RESULTS AND DISCUSSION

This section presents the data and results obtained during the experiment, along
with an analysis of how these results were achieved. It includes a detailed
interpretation of the findings and compares them with the expected outcomes.
Additionally, the implications of these results for future experiments and research
will be highlighted.

Haikal Ramadhana Akmal (2310942009) I-3

CHAPTER V CLOSING

This section outlines the conclusions drawn from the experiment's results and
provides recommendations for improving the execution of the experiment.

LITERATURE

Haikal Ramadhana Akmal (2310942009) I-4

CHAPTER II

LITERATURE REVIEW

2.1 General

Bacterial isolation or the cultivation of a single type of bacterium is referred to as a

Microorganisms in the environment are found as a mixed population of various

arious types of bacterial growth media are commonly used for purposes such as
isolation, transportation, inoculation, and differentiation. Bacterial culture media
generally consist of meat extract, yeast extract, peptone, and agar. Selective media
are employed in bacterial isolation to inhibit the growth of specific bacteria, and
several types of media are used for this purpose. Differential media are also used to
distinguish between various types of bacteria based on their metabolic
characteristics. Many media developed today possess both selective and differential
properties, allowing for more precise identification and isolation of bacteria.
Additionally, the composition of the media can be modified to suit the requirements
of particular bacteria, enabling the successful isolation of diverse microbial species
from mixed cultures Examples of such media include nutrient agar (NA) and potato
dextrose agar (PDA) (Padoli, 2016).
MINISTRY OF HIGHER EDUCATION, SCIENCE, AND TECHNOLOGY
DEPARTEMENT OF ENVIRONMENTAL ENGINEERING
ENVIRONMENTAL MICROBIOLOGY LABORATORY
Kampus Unand Limau Manis Padang 25163
Telp. (0751) 7862901, Fax (0751 72566)

CHAPTER II

LITERATURE REVIEW

2.1 General

Bacterial isolation or the cultivation of a single type of bacterium is referred to as a

Microorganisms in the environment are found as a mixed population of various

Haikal Ramadhana Akmal (2310942009) II-2

Microbial isolation is conducted to select the appropriate microbes for the

Microbial isolation is performed by collecting microorganisms from the

Microbial isolation is performed to select the appropriate microbes for the

Haikal Ramadhana Akmal (2310942009) II-2

Microorganisms are isolated either as pure cultures or mixed cultures, depending

on the desired product. Three techniques—streak plate, pour plate, and spread
plate—are used for microbial isolation (Nur and Feronika, 2018).

Microbial isolation is performed by growing microorganisms outside their natural

The importance of isolating microbes from environments such as food (solid

Microbes are considered a group of living organisms found within ecosystems,

Haikal Ramadhana Akmal (2310942009) II-3

The principle of microbial isolation is to separate a specific type of microbe from

The presence of microbes in the environment is evidence that nutrients present in

2.2 Definition of Microbial Isolation

Microbial isolation is performed by extracting microorganisms from the

environment and cultivating them in an artificial medium. The separation or
purification from the purific other microorganisms is required because all activities
related to microbiological research necessitate a pure population of microbes.

Haikal Ramadhana Akmal (2310942009) II-4

In microbiological studies, the examination and identification of microorganisms

require a population composed solely of a single microorganism type. The principle
behind microbial isolation is the separation of one type of microbe from others
within a mixture of various microbial species. This separation is achieved by
growing the microbes on a solid medium (Rohmah, 2017).

Microbial isolation is conducted to separate microbes from their environment and

grow them as a pure culture on an artificial medium. Bacteria are stained with dyes
to increase contrast, facilitating easier observation under a microscope. This
bacterial staining is crucial for identification and classification, as it aids in
distinguishing between different types. This study was aimed at identifying airborne
microbes in a microbiology laboratory. Isolation and identification tests were
performed on nutrient agar (NA) media, followed by simple and Gram staining,
disinfection tests, and analyses of growth factors. Observations revealed that, after
simple staining, the bacterial morphology appeared as a dense shell structure, and
the red Gram stain confirmed it as a Gram-negative bacterium (Putri Amelya, 2023).

Microorganisms, given their widespread presence, play significant roles in both

Haikal Ramadhana Akmal (2310942009) II-5

The water closet is known to harbor a high quantity of microbes due to

environmental factors that facilitate optimal microbial growth and reproduction
(Deny, 2020).

2.3 Microbial Isolation Methods

To cultivate microorganisms, it is essential to consider nutritional factors and

Haikal Ramadhana Akmal (2310942009) II-6

Mikroba jarang terdapat di alam dalam keadaan murni. Kebanyakan merupakan

campuran bermacam-macam spesies mikroba. Macam-macam cara mengisolasi
dan menanam mikrobia adalah (Tim Laboratorium Mikrobiologi, 2023):
1. Spread plate method;
2. Pour plate method;
3. Streak platemethod.
a. Spread Plate Method
The spread plate technique is a microbial isolation technique by inoculating
microbial cultures by spreading on the surface of solidified agar media. This
method is done by diluting the culture of microbial cultures. Because the
concentration of microbial cells is generally unknown, dilution needs to be done
in several stages, so that at least one of the dilutions contains separate colonies
(30-300 colonies). Separate microbial colonies allow the colonies to be counted.
b. Pour plate method
This method is basically to inoculate the agar medium that is melting at a
temperature of 45-50 ° C with a suspension of material containing microbes,
and pour it into a sterile Petri dish. After incubation, colonies will be seen
scattered on the surface of the agar which may come from 1 bacterial cell, so
that it can be isolated further.
c. Streak Plate Method
The streaking method is commonly used to isolate microbial colonies on agar
plates to obtain separate colonies, which then form a pure culture. In this
method, a suspension containing microbes is streaked across the surface of an
appropriate agar medium in a petri dish. Following incubation, individual
colonies grow along the streak lines, each potentially originating from a single
microbial cell, allowing for further isolation. Proper streaking ensures the
formation of distinct colonies. Bacteria with flagella often form spreading
colonies, especially on moist plates. To prevent this, it is essential to use agar
plates with a thoroughly dry surface. This approach requires careful handling to
maintain sterility, as well as consistent environmental conditions to promote
optimal microbial growth.

Haikal Ramadhana Akmal (2310942009) II-7

2.4 Microbial Isolation Media

There are 2 media in microbial isolation, namely:

1. Nutrient Agar (NA)
Nutrient agar is a medium for testing water and dairy products. Nutrient Agar (NA)
is also used for the growth of most non-selective microorganisms in the sense of
heterotrophic microorganisms. Based on its shape, this medium is solid because
agar is a compacted material. Nutrient Agar is a simple medium made from beef
extract, peptone and agar (Putri, 2017).
2. Potato Dextrose Agar
Potato dextrose agar is one of the best media used to breed a microorganism, be it
a fungus/fungus, bacteria, or living cells. Potato Dextrose Agar is also a medium
used for the growth of Aspergillus flavus fungus . PDA media is a culture medium
that has asolid consistency. PDA media is a common media used to analyze the
type and number of molds in food products. The problem often encountered with
the use of this media is the frequent failure in morphological observations and
calculations due to the growth of colonies that spread so as to inhibit or cover other
colonies (Putri, 2017).

2.5 Factors Affecting Microbial Isolation

One crucial aspect to consider when culturing microbes is sterilization. Sterilization

Haikal Ramadhana Akmal (2310942009) I I-8

used for solutions that are thermolabile (tend to be damaged or changed)

(Nurmalasari, 2021).

Growth factors are organic compounds that are essential for growth, such as
precursors or components of cellular materials, and cannot be synthesized from
simple carbon sources. Microorganisms, especially autotrophic photolithotrophs,
often grow and reproduce when access to essential energy sources, such as carbon,
nitrogen, phosphorus, and sulfur, is available. These organisms contain the
necessary enzymes and pathways required to synthesize all the cellular components
needed for their survival. Furthermore, growth factors can be defined as
microorganisms lacking one or more essential enzymes. As a result, they are unable
to create all the necessary components on their own and must obtain them from their
precursors or derivatives, which cannot be synthesized from simpler projections.
Growth factors are thus essential for ensuring proper cellular function and
metabolism, particularly when the organism cannot produce these factors
independently.

There are three main classes of growth factors: (1) amino acids, (2) purines and
pyrimidines, and (3) vitamins. Amino acids are essential for protein synthesis,
purines and pyrimidines are required for nucleic acid synthesis, and vitamins are
small molecules that often form some or all of the enzyme cofactors. Examples of
microorganisms that require these factors include certain bacteria. Some
microorganisms need several vitamins; for instance, Enterococcus faecalis (lactic
acid bacteria) requires eight different vitamins to grow. These growth factors play
a critical role in the proper functioning of enzymes.

Positive interactions, often referred to as cooperative relationships, involve the

formation of colonies between similar microorganisms (due to quorum sensing
processes). Various types of microorganisms, whether of the same or different
species, can also produce protective factors with specific chemical structures. These
are known as extracellular polymeric substances (EPS), or extracellular matrix
(ECM). This process is called biofilm formation. When microorganisms form
biofilms, their pathogenicity is enhanced, and resistance to antimicrobial agents,
including disinfectants or antibiotics
Haikal Ramadhana Akmal (2310942009) I I-8
MINISTRY OF HIGHER EDUCATION, SCIENCE, AND TECHNOLOGY
DEPARTEMENT OF ENVIRONMENTAL ENGINEERING
ENVIRONMENTAL MICROBIOLOGY LABORATORY
Kampus Unand Limau Manis Padang 25163
Telp. (0751) 7862901, Fax (0751 72566)

Cultivation media for microorganisms is a substance composed of a mixture of

nutrients that microorganisms use for growth and reproduction. A good culture
medium, especially one that is easy to prepare, inexpensive, simple to make, and
easy to apply, is essential. Culture media are available in both solid and liquid
forms. A medium is a substance that can be used as a growth environment for
microorganisms such as bacteria (Putri Amelya, 2023).

2.6 Applications of Microbial Isolation in Environmental Engineering

The application of microbial isolation in the field of Environmental Engineering is

Bacteria play many roles in human life and in environmental engineering.

Haikal Ramadhana Akmal (2310942009) I I-10

then grow it in an artificial medium so that a pure culture is obtained. Microbial

isolation is important to know and determine the type, study the culture,
morphology, physiology, and characteristics of the microorganisms sampled. The
application of microbial isolation in the field of environmental engineering,
microbial isolation can be used to determine the presence of pollution involving
microorganisms in it, so that it can be known and solutions can be taken how to
minimize the source of pollution produced by these microorganisms to the
environment. Microorganisms have many benefits for the environment, for example
using microbes as environmentally friendly insecticides, microorganisms are used
in the process of environmental bioremediation (Sabbathini, 2017).

Haikal Ramadhana Akmal (2310942009) I I-11

CHAPTER III

EXPERIMENT PROCEDURE

3.1 Tools

The tools used in microbial isolation practicum are:

1. Petri dish.
2. 1 mL measuring pipette.
3. 10 mL measuring cup.
4. Test tubes and test tube racks.
5. Spirtus.
6. Colony counter.
7. Analytical balance.
8. 100 mL Erlenmeyer.
9. Spatula.
10. Suction ball.
11. Paralon pipe.
12. Shaker

3.2 Material

Materials used in microbial isolation practicum are:

1. NA media.
2. PDA media.
3. 96% alcohol.
4. 0.1% Buffered Peptone Water (BPW) solution.
5. Aquadest.
6. Sample.

3.3 How it works

All equipment is washed and dried :

1. All equipment is washed and dried.

2. Petri dishes (petridish) are wrapped in paper and sterilized in an ovenor autoclave.
INISTRY OF HIGHER EDUCATION, SCIENCE, AND TECHNOLOGY
DEPARTEMENT OF ENVIRONMENTAL ENGINEERING
ENVIRONMENTAL MICROBIOLOGY LABORATORY
Kampus Unand Limau Manis Padang 25163
Telp. (0751) 7862901, Fax (0751 72566)

3. The workplace is cleaned by spraying 96% alcohol, and allowing a few minutes
to dry.

MICROBES IN SOIL

4. Soil weighed as much as 5 grams.

5. The soil was put into a 100 mL glass erlenmeyer with an additional 45 mL of 0.1%
BPW solution. The sample was homogenized using a shaker; at 160 rpm for 10 minutes
(10-1).
6. 1 mL of sample was taken from the erlenmeyer and put into the first dilution
tube (10-2) aseptically (from suspension preparation) with 9 mL of 0.1% BPW
solution.
7. 1 mL was taken from tube 10-2 with a measuring pipette then transferred to tube
10-3 aseptically then homogenized. The transfer continued until the last dilution
tube in the same way until tube 10-6.
8. 1 mL of sample was taken from the 10-6 dilution, then put into 3 Petri
dishes (1 mL of sample each).
9. NA media was poured into 2 petri dishes that already contained samples
and PDA media, poured into 1 other petri dish.
10. Then put NA and PDA media into a petri dish, then homogenized by rotating
the petri dish to form a figure 8.
11. Petri dishes were covered and allowed to solidify.
12. then incubated for 24 hours and then observed.
13. the shape of the colonies is noted and examined for fine structure.
14. record the results:
a. The number of growing colonies was counted using a colony counter.
b. Colonies that grow are viewed and recorded.
1) Shape (whether point-shaped, round, regular, thread-shaped, thin, thick,
convex, flat)
2) The fringe

3) Surface (whether flat, shiny, rough, irregular dull)

4) Color

Haikal Ramadhana Akmal (2310942009) I-2

INISTRY OF HIGHER EDUCATION, SCIENCE, AND TECHNOLOGY
DEPARTEMENT OF ENVIRONMENTAL ENGINEERING
ENVIRONMENTAL MICROBIOLOGY LABORATORY
Kampus Unand Limau Manis Padang 25163
Telp. (0751) 7862901, Fax (0751 72566)

MICROBES IN THE AIR

15. NA media or PDA media is poured into petri dishes (work near the fire).
16. Petri dishes were covered and allowed to solidify.
17. Petri dishes were left open for 30 minutes.
18. Then wrapped in paper and incubated for 24 hours and observed.

Haikal Ramadhana Akmal (2310942009) I-3

BIBLIOGRAPHY

Deny. 2020. Identifikasi Morfologi Mikroba Pada Ruangan Water Closet Jurusan
Biologi. Makassar: Universitas Negeri Makassar.

Harti, Agnes S. 2014. Mikrobiologi Kesehatan. Yogyakarta: CV Andi.

Istiamah, dkk. 2018. Teknologi Bioproses. Malang: Universitas Brawijaya: Press.

Mayasari, Ulfayani. 2022. Mikrobiologi. CV Media Sains
Indonesia: Bandung.
Marnila. 2016. Isolasi Dan Karakteristik Mikroba Isolat Bakteri Asam Laktat (Bal)
Asal Saluran Pencernaan Doc Broile. UIN Alauddin: Makasar.

Nurmalasari. 2021. Dasar-Dasar Mikrobiologi dan Penerapannya. CV Widiana

Media Utama: Bandung.

Padoli. 2016. Mikrobiologi dan Parasitologi Keperawatan. Jakarta Selatan:

Kementrian Kesehatan Republik Indonesia.

Putri. 2017. Mikrobiologi. BPPSDM Kementrian Kesehatan.

Putri Amelya. 2023. Identifikaasi Mikroba Udara Isolat Pink di Laboraturium

Mikrobiologi. Padang: Universitas Negeri Padang.

Sabbathini. 2017. Isolasi dan Identifikasi Bakteri Genus Sphingomonas dari Daun
Padi (Oryza Sativa) di Area Persawahan Cibinong. Semarang: Universitas
Diponegoro.

Solikah, Ed Lusi. 2019. Teknik-teknik Isolasi atau Penanaman Mikroba. Jurnal

Farmasi: UNIDA.

Tim Laboratorium Mikrobiologi Lingkungan. 2023. Modul Praktikum

Mikrobiologi Lingkungan. Padang : Universiita Andalas.

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