ISOLATION AND CHARACTERIZATION OF MICROORGANISM

By :
Name : Sunu Pertiwi
Student ID : B1B015007
Entourage :I
Group :1
Assistant : Dwi Rizki Nurjanah

MICROBIAL SYSTEMATICS LABORATORY REPORT

MINISTRY OF RESEARCH, TECHNOLOGY, AND HIGHER EDUCATION

JENDERAL SOEDIRMAN UNIVERSITY
FACULTY OF BIOLOGY
PURWOKERTO
2017
 I. INTRODUCTION

A. Background

Naturally, microbes in nature are found in mixed populations. To obtain a

pure culture can be isolated starting with serial dilution. Isolation of microorganisms
is the process of taking microorganisms from the environment to be grown in a
medium in the laboratory. The microbial isolation process function is to separating
microbes one with other microbes derived from a mixture of various microbes to be
able to study the properties of culture, morphology and other microbial properties.
This isolation process is important in studying the identification of microbes,
morphological, physiological, and serological tests (Pelczar & Chan, 1986).
Characterization is one of the activities undertaken to observe bacteria and
molds of isolation (isolates). Characterization activities may be based on cytological
properties (cell shape, motility or motility, Gram and endospores), morphological
properties, and physiological properties. The test of morphological properties
includes the properties of the colony, such as size, shape, color and edges, while the
physiological properties test include starch hydrolysis test, fat hydrolysis, protein
hydrolysis and catalase test (Nurjanah, 2007). The phenotypic tests are useful for
identifying a small number of isolates, especially clinical samples, as their software
databases mainly contain clinically important bacteria. However, when using these
methods their limitations must be considered, such as their poor reproducibility, a
result of the plasticity of bacterial growth, the logistic difficulties for large-scale
applications, and their sometimes poor discriminatory power (Moraes, 2013).
Another issue is that bacterial isolates do not express all their genes at the same time,
as gene expression is related to environmental conditions. Considering this, the
identification of such isolates only by biochemical tests can be jeopardized. (Moraes,
2013). A bacterial physiological test is performed to identify bacteria based on their
cell activity (Hadioetomo 1993).
The reason that we used water as a sample because it have relation with
microbiological water quality test. There are many bacteria that can live in water. If
the water is polluted, it can disturb the organism live especially human. Human are
used the water for many ways. If the water are consist of big population of bacteria
(pathogen) it will causes a lot of disease. From this laboratory activity we are able to
know the characteristic of enterobacter and indicate the pollution in water. According
to Afif et al. (2015), Pathogenic bacteria can cause diseases with diarrhea complaints
such as dysentery, typhus, and cholera, through drinking water. Some examples of
pathogenic bacteria are Shigella dysentriae, Salmonella typhi, Salmonella paratyphi.
For non-pathogenic bacteria such as Fecal streptococci, Iron bacteri, and
Actinomycetes. According to Slamet (1996), pollution of waste in a waters has a
relationship with the type and number of microorganisms in these waters. The
densely populated city and rural wastes not only promote the growth of coliform
bacteria but also increase the number of pathogenic bacteria such as Salmonella,
Shigella and Vibrio cholera. Microorganisms commonly found in domestic waste in
large quantities are coliform bacteria, Escherichia coli and Streptococcus faecalis.
Some bacteria that are an indicator of the quality of a waters are coliform, fecal coli
and Salmonella. There are three groups of pollution indicators of coastal waters are
faecal coliform, faecal streptococus and pathogen.
Eschericia coli (E. coli) is a bacterial species that is widely used and accepted
as an indicator organism in the testing of water quality all over the world. Its
presence has proven to be the best indicator for fecal contamination of water supplies
especially in open sources such as rivers. It is also a leading contributor to enteric
and diarrheagenic diseases especially in young children in developing countries. The
standard of water quality is often dictated by the number of microbial agents found in
accordance with acceptable risk levels determined by legislature and WHO
guidelines. However, the absence of indicator organisms in water does not guarantee
safety of drinking water. The presence of bacteria and pathogenic organisms is of
concern when considering the safety and quality of water. Although E. coli might be
detected in the water samples indicating the possible presence of other fecal related
bacterial pathogens (Traore, 2016).
B. Objective

1. Students are able to separate the microbe from the environment into pure culture.
2. Students are able to know the characteristic of microorganism through the
enzymatic test, physiology test, biochemistry test and the observing of the
morphology.
 II. MATERIAL AND METHOD

A. Material

The tools that we used in this laboratory activity are reaction tube, shelf tube,
bunsen, micropipette, filler, petridish, Drugalsky, ose needle, object glass, cover
glass, light microscope, and chop stick sterile.
The material that we used in this laboratory activity are sample of water,
aquades, Nutrient Agar media, Starch Agar media, Protease Agar media, Medium
Mineral + Lipid media, OF basal media, Urease Broth media, SIM A media, Sugar
media (glucose, sucrose, lactose, fructose, galactose and arabinose), Tryptone Broth
media, Simmon’s Citrate media, catalase reagent (H2O2 3%), oxidase reagent
(tetramethyl-p-phenylenediaminedihydrocloride), crystal violet, lugo’s iodine,
alcohol, safranin, paraffin, Nutrient Broth and Peptone Broth media.
B. Method

A. Isolation of sample
1. Water river are take with the opposite of river flow.
2. Make a dilution using 9 ml aquades until 10-7.
3. The second last of dilution are platting in the Natrium Agar media by
spread plate method.
4. Incubated until 2x24 hours at room temperature.
5. Choose 3 kind of isolate bacteria.
6. Make the purification by streak quadrant method.
7. Incubated until 2x24 hours at room temperature.

B. Characterization
Observing the morphology
1. The isolate bacteria that was purified are observe of some character of
macro morphology such as the surface, elevation, the margin, the color,
the size and form of colony.

Reculture of isolates
1. Isolate bacteria that was purified are inoculated in the reaction tube
containing of Nutrient Agar with slant media by streak continue method.
2. Incubated until 3x24 hours at room temperature.
Exoenzyme test
a. Amylolytic test
1. Isolate bacteria (A and B) from isolation process are inoculated
into Starch Agar (SA) media until half of petridish.
2. Incubated until 2x24 hours at room temperature.
3. Drop lugol’s iodine into it.
4. Observed the changing of the reaction.
The interpretation:
(+): there is a clear zone surrounding the isolate.
(-): the clear zone is not formed.

b. Proteolytic test
1. Isolate bacteria (A and B) from isolation process are inoculated
into Protease Agar (PA) media until half of petridish.
2. Incubated until 2x24 hours at room temperature.
3. Observed the result.
The interpretation:
(+): there is a clear zone surrounding the isolate.
(-): the clear zone are not formed.

c. Lipolytic test
1. Isolate bacteria (A and B) from isolation process are inoculated
into Medium Mineral Agar + Lipid media.
2. Incubated until 2x24 hours at room temperature.
3. Observed the result.
The interpretation:
(+): the media changed become yellow.
(-): the media color is not changing.

Endoenzyme test
a. Catalase test
1. Isolate bacteria (A and B) from isolation process are smear into
object glass.
2. Drop the catalase reagent into it.
3. Observed the changing reaction.
 The interpretation:
(+): there is a bubble in the object glass.
(-): the bubbles are not formed.

b. Oxidase test
1. Isolate bacteria (A and B) from isolation process are smear into
merang paper above the object glass.
2. Drop the oxidase reagent into it.
3. Observed the changing reaction.
The interpretation:
(+): there is a changing color on merang paper.
(-): the color merang paper are not changing.

Gram staining
1. Isolate bacteria (A and B) from isolation process are inoculated into
object glass.
2. Drop the aquades into it.
3. Make a fixation until 2-3 time.
4. Drop the Crystal Violet (Gram A), wait until 60 second.
5. Let it until dry.
6. Drop the Lugol’s Iodine (Gram B), wait until 60 second.
7. Let it until dry.
8. Drop the Alcohol (Gram C) until the color is change become clear.
9. Let it until dry.
10. Drop the Safranin (Gram D) wait until 45 second.
11. Let it until dry.
12. Observed under the microscope.
The interpretation:
Gram (+): the color of bacteria cell is purple
Gram (-): the color bacteria cell is red
Physiology test
a. Temperature test
1. Put the isolate bacteria (A and B) as much as 0,1 ml into reaction
tube.
2. Give the label on the reaction tube.
3. Incubated until 2x24 hours at 5oC, room temperature, 37oC and
80oC. Incubated according to the temperature written on the label.
4. Observed the result.
The interpretation:
(+): the media are changing become more turbid
(-): the media are not changing.

b. pH test
1. Put the isolate bacteria (A and B) as much as 0,1 ml into reaction
tube. The reaction tube is containing of media with pH 3, pH7 and
pH 11.
2. Give the label on the reaction tube.
3. Incubated until 2x24 hours at room temperature.
4. Observed the result.
The interpretation:
(+): the media are changing become more turbid.
(-): the media are not changing.

c. Osmotic pressure test

1. Put the isolate bacteria (A and B) as much as 0,1 ml into reaction
tube. The reaction tube is containing of media with the osmotic
0%, 0,5%, 0,85% and 1%.
2. Give the label on the reaction tube.
3. Incubated until 2x24 hours at room temperature.
4. Observed the result.
The interpretation:
(+): the media are changing become more turbid.
(-): the media are not changing.
Biochemistry test
a. Sugar tets
1. Put the isolate bacteria (A and B) as much as 0,1 ml into reaction
tube. The reaction tube is containing of media with some sugar,
which are glucose, sucrose, lactose, fructose, galactose and
arabinose.
2. Give the label on the reaction tube.
3. Incubated until 2x24 hours at room temperature.
4. Observed the result.
The interpretation:
(+): the media are changing become yellow and containing of
bubble.
(-): the media are only changing into yellow or neither.

b. OF test with stab inoculation

1. Put the isolate bacteria (A and B) as much as 0,1 ml into two
reaction tube. Both of the reaction tube are containing of OF basal
media
2. Added 2-3 drop of paraffin into one of the reaction tube.
3. Give the label on the reaction tube.
4. Incubated until 2x24 hours at room temperature.
5. Observed the result.
The interpretation:
(+) oxidative bacteria : the media are changing become yellow
in OF basal media.
(+) fermentative bacteria : the media are changing become yellow
in OF basal media + paraffin.
(+) saccharolytic bacteria : both of the media are not changing in
the color.

c. Urease test
1. Put the isolate bacteria (A and B) as much as 0,1 ml into reaction
tube. The reaction tube is containing of Urease Broth (UB) media.
2. Give the label on the reaction tube.
3. Incubated until 2x24 hours at room temperature.
 4. Observed the result.
The interpretation:
(+): the media are changing become pink.
(-): the color of media is not changed.

d. H2S test
1. Put the chop stick sterile into isolate bacteria (A and B).
2. Make a stab inoculation into reaction tube containing of SIM A
agar.
3. Give the label on the reaction tube.
4. Incubated until 2x24 hours at room temperature.
5. Observed the result.
The interpretation:
(+): there is sediment in the media.
(-): there is not sediment in the media.

e. IMVIC test
Indole test
1. Put the isolate bacteria (A and B) as much as 0,1 ml into reaction
tube containing of Tryptone Borth (TB) media.
2. Incubated until 2x24 hours at room temperature.
3. Added 1-2 drop of covack indole reagent.
4. Observed the changing of the reaction.
The interpretation:
(+): the red ring is formed.
(-): the red ring is not formed.

Methyl Red (MR) test

1. Put the isolate bacteria (A and B) as much as 0,1 ml into reaction
tube containing of Protease Borth (PB) media.
2. Incubated until 2x24 hours at room temperature.
3. Added 1-2 drop of methyl red reagent.
4. Observed the changing of the reaction.
The interpretation:
(+): the color is changed become red.
(-): the color is not changed.
Voges Proskauer (VP) test
1. Put the isolate bacteria (A and B) as much as 0,1 ml into reaction
tube containing of Protease Borth (PB) medium.
2. Incubated until 2x24 hours at room temperature.
3. Added the α-naphtole and KOH 40% with ratio 3:1. And put the
α-naphtole firstly.
4. Observed the changing of the reaction.
The interpretation:
(+): the color is changed become red.
(-): the color is not changed.

Citrate test
1. Put the isolate bacteria (A and B) as much as 0,1 ml into reaction
tube containing Simmon’s Citrate media.
2. Incubated until 2x24 hours at room temperature.
3. Observed the result.
The interpretation:
(+): the color is changed become blue.
(-): the color is not changed.
 III. RESULT AND DICUSSION

Table 3.1 Data character of microbe.

A.1.1 B.1.1 A.1.2 B.1.2 C.1.2 A.1.3 B.1.3 C.1.3
P. Gram + + - - - - - -
Amilolitik + + - - - + - -
Proteolitik + + - - - - - -
Lipolitik - - - - - - - -
Oksidase + + - + + + - -
Katalase + + + + + + + +
Suhu 5oC + + + + + + + +
Suhu Ruang + + + + + + + +
Suhu 37oC + + + + + + + +
Suhu 80oC + + + - + - - -
pH 3 - - + + + + + +
pH 7 + + + + + + + +
pH 11 + + + + + + + +
NaCl 0% + + + + + - + +
NaCl 0,5% + + + + + - + +
NaCl 0,85% + + + + + - + +
NaCl 1% + + + + + + + +
Glukosa - + + - - - - -
Galaktosa - - + - - - - -
Fruktosa + + - - + + + +
Laktosa - - + + + - - -
Sukrosa - - - - - + - -
Arabinosa - - - - + - - -
Indole - + - - - - - -
Methyl Red - - - - - - - -
Voges- - - + + + + + +
Proskauer
Citrate + + - + - - + +
Urease + + + + + + + +
H2S - - - - - - - -
Oksidatif + + + + + - - -
Fermentatif + + + + + - + -
Sakarolitik - - + + + + - +
Picture 3.1 Isolate Bacteria (A and B) Picture 3.2 Isolate Bacteria (A and B)
with dilution 10-6 with dilution 10-7
In microbial purification known term is microbial isolation and pure culture.
Isolation of microbes is to move microbes to their environment by isolating
microbial bacteria needed or in other words microbes that we do not need
immediately removed, so obtained pure culture or pure culture. Pure culture is a
culture whose microbial cells originate from division of a single cell, meaning that
microbes are grown from bacteria that are homogenized in other words bacteria are
isolated to obtain the pure bacteria needed later in practicum activities. The object to
watch out for is the bacteria (Subakti, 2010). In isolating microorganisms, various
methods are used, such as streak plate, spread plate method, dilution method, and
micromanipulator (tea micromanipulator method). Two of the most commonly used
are casting and scratch bowls. Both methods are based on the same principle of
diluting the organism in such a way that individual species of individual species can
be separated from others (Hadioetomo, 1993). The conventional microbiological
methods for bacterial identification are based on morphological and physiological
characteristics such as Gram staining, cell shape, spore formation, enzyme
production and the fermentation of different carbohydrates (Moraes et al., 2013).

Picture 3.3 Gram Staining test

 Based on the observation of group 1 entourage I, both of the bacteria are
belong to gram positive. The pictures are showed that the bacterial cell color is
purple. Gram and spore staining can be performed in the cytological properties of
bacteria. The principle of Gram staining is the ability of cell walls against the basic
dye (Crystalline violet) after washing with alcohol 96%. Gram positive bacteria have
purple color because the cell wall binds the violet crystal stronger, while Gram-
negative cells contain more lipids so the pores easily expand and the violet crystals
dissolve easily during washing with alcohol (Pelczar & Chan, 1986).

Picture 3.4 Isolate A Picture 3.5 Isolate B

Amylolytic test Amyolytic test
Based on the observation of group 1 entourage I, both of the bacteria are
belongs to amylolytic bacteria which is have enzyme amylase. Amylolytics are
bacterial activity in processed starch with the help of enzyme amylase. Enzyme
amylase is an enzyme capable of hydrolyzing starch into simpler compounds such as
maltose and glucose. This enzyme is widely used for industrial purposes. This
enzyme can break down or hydrolyze starch, glycogen and polysaccharide
derivatives by breaking the starch glycosidic bond. The amylase enzyme is divided
into 3 groups: α-amylase, also called endoamylase, β-amylase, also called
eksoamilase and glucoaminase (Morsen & Rehm, 1987).

Picture 3.6 Isolate A Picture 3.7 Isolate B

Proteolytic test Proteolytic test
 Based on the observation of group 1 entourage I, both of the bacteria are
belongs to proteolytic bacteria. Proteolytic bacteria are bacteria capable of degrading
proteins, because they produce extracellular protease enzymes. Proteases are
proteolytic enzymes that catalyze the termination of peptide bonds in proteins. to
determine the ability of microorganisms in secreting proteases that can degrade
proteins, the medium includes skim milk containing the patient. Casein is the main
protein of milk, a micromolecule composed of sub-units of amino acids linked to
peptide bonds. Casein serves as a substrate for protease enzymes. In general,
proteolytic bacteria are bacteria of the genus Bacillus, Pseudomonas, Proteus,
Streptobacillus, Staphylococcus and Streptococcus (Puspitasari, 2011).

Picture 3.8 Isolate A Picture 3.9 Isolate B

Lipolytic test Lipolytic test
Based on the observation of group 1 entourage I, both of the bacteria are
negative in lipolytic test. The result show that media are not changing become
yellow. Lipids (fats) are groups of heterogeneous compounds that are associated both
actual and potential with fatty acids. The properties of fat are generally insoluble in
water (Darmayasa, 2008). Fat-hydrolyzing bacteria are able to convert compounds
into fatty acids and glycerol. Bacteria with the ability of fat hydrolysis will cause a
yellowish red color at the bottom and around the colony (Rahayu et al., 1992).

Picture 3.10 Oxydase test

 The purpose of oxydase test is to know the existence of oxydase enzyme in
bacteria. The exystence of oxydase it can be show by the changing color on paper
oxydase or merang paper (Kismiyati et al., 2009). The result of group 1 entourage I
in this laboratory activity is positive. It is proved by the changing color on merang
paper.

Picture 3.11 Isolate A Picture 3.12 Isolate B

Catalase test Catalase test
Based on the result, the isolate bacteria A and B can make an catalytic
activity. The interpretation positive are characterized by the forming of bubbles.
According to Kismiyati et al (2009), The purpose of catalase test is to know bacteria
in producing catalase enzyme. The workings of the catalase test are H2O2 3%
solution drop on the objects, then suspend bacterial colonies with ose. Enzyme
catalase plays a role in breaking H2O2 (Hydrogen Peroxide) to H2O and O2. Positive
catalase test results are characterized by the presence of oxygen bubbles.

Picture 3.13 Temperature test

Based on the result, the isolate bacteria A and B is resistant of all the
temperature. They can live in low temperature, normal temperature and also high
temperatue. The interpretation of this temperature test is positive it is proved by the
existence of turbid in the media. There are several type of bacteria based on their
ability to survive in temperature:
1. Thermophilic bacteria
Thermophylic bacteria is the microorganism that resistant of high
temperature, with the optimum temperatures reach more than 60oC. one of
the utilization of the thermophilic organism is as a producer of various
enzyme that are thermostable. Enzymes that can be produced from
thermophilic microorganisms include cellulase, amylase, chitinase and lipase
(Rosliana, 2009).
Thermophilic microorganisms are able to synthesize molecules in
high temperature conditions, including enzyme molecules. Biotechnology is
generally attracted to enzymes that produce of this microorganisms to work
under normal conditions, where the enzymes from mesophilic
microorganisms are denatured. This enzyme is targeted for the research and
investigation of thermostable proteins and biocatalyst in modern
biotechnology (Elfita, 2010).
2. Mesophylic bacteria.
Microbes that live at room temperature up to 45oC. Examples:
Methylococcus capsulatus, Azotobacter chroococcum, Clostridium
pasteurianum, Rhizobium leguminosarum, Rhodospirillum rubnum, Bacillus
subtilis, L. Bulgaricus, Clostridium butyricum, Bacillus mascerans and
Clostridium sporongenes (Wati, 2013).
3. Psychohilic bacteria
Microbes that live at low temperatures up to 25oC. Example: Bacteria that
live in the sea (Phototrophs), iron bacteria (Gallionella), Bacillus polymixa,
Pseudomonas, Micrococcus and Clostridium botulinum (Elfita, 2010).
Temperatures affected the growth of microorganisms and the rate of reaction
in the formation of biogas. Biogas production process can occur in two
temperature ranges, ie mesophilic temperature range 25-45oC and
thermophilic temperature range 56-60oC. Higher temperatures will give
higher biogas yields, but at highest temperatures that are too high bacteria
will easily die (Wati, 2013).
 Picture 3.14 pH test
The result of group 1 entourage I, the isolate bacteria A and B can grow in
media with pH 7 and pH 11. But it is more optimum at pH 7. The effect of pH on the
growth and production of lactic acid occurs among 2 mechanisms, namely (1)
Decreasing the pH value due to accumulation of organic acids alters the
physiological state of the cell. Cytoplasmic acidification leads to inhibition of
enzyme activity, consequently the catabolic flux through glycolysis is reduced so that
the rate of synthesis of biochemical energy decreases. Decreasing energy production
along with increased energy use to cope with cytoplasmic acidification causes energy
for biomass synthesis to be limited. Under these conditions, the specific growth rate
decreases progressively, and growth eventually stops. The cellular response to this
phenomenon is to maintain the mRNA of catabolic genes at a significant level,
through gene transcription and improving transcript stability. So translation is
maintained and the intracellular concentration of certain enzymes is increased, as a
partial compensation for inhibitory activity due to a decrease in pH. (2) Lactic acid
stress alters the gene expression profile (Subagiyo et al., 2015).

Picture 3.15 Osmotic pressure test

Salinity is the concentration of all ions (Cl-, SO42-, CO32-, Na+, Mg2+, K+)
dissolved in water and expressed in grams per liter or parts per thousand or promill
(Boyd, 1982). The salinity test showed that isolate A and B with all the treatment are
have positive interpretation. But the medium with 0.85% of salinity was very
optimum, According to Slamet (1996), optimum bacteria grow at 0.85% salinity.
Salinity test function is used the viability of microorganism in salinity condition.

Picture 3.16 Sugar test

In this laboratory activity the result of group 1 entourage I are, positive
reaction is only isolate B degrade glucose and isolate A and B degrade fructose. The
other reaction is negative. The color of medium are not change from red to yellow. It
is accordance with Hidayat (2012), positive results are indicated by the change in the
color of the sugar medium from the red. Kismiyati (2009), The purpose of sugar test
is to determine the ability of bacteria that can degrade sugar and produce organic
acids which from each of type of sugar, such as glucose, sucrose, maltose arabinose,
manitol and inositol.

Picture 3.17 Indole test

Indole test is the one process of IMVIC test. The first, put the isolate bacteria
into Tryptone Broth media as much as 0,1 ml. And then, incubated until 2x24 hours
at room temperature. After that add the Covack Indole reagent into it. This technique
is different with Hadioetomo (1993), his technique is start from taken a separate
colony using ose needle, then inoculated into SIM media and incubated for 24 hours
at 37ºC. After incubation is added 10-12 drops of Kovac reagent. In this laboratory
activity, only isolate B have positive interpretation. It can be show by the formation
of red ring during the added of Covack Indole reagent it accordance with
Hadioetomo (1993), the positive test is characterized by the formation of a red
coating on the top of the culture.

Picture 3.18 Methyl Red test

The second process of IMVIC is Methyl Resd test. The first, put the isolate
bacteria into Protease Broth media as much as 0,1 ml. And then, incubated until 2x24
hours at room temperature. After that add the Methyl Red reagent into it. This
technique is a little bit different with Hadioetomo (1993), the media that his used is
MRVP and the incubation until 24 hours at 37oC. In the MR test, added 3-4 drops of
methyl red indicator. Group 1 entourage I have result of both isolate bacteria (A and
B) are negative. The media are not absolutely change become red. If the result is
positive, the media marked by the color change become red, which is meaning the
media have acidic condition (Hadioetomo, 1993).

Picture 3.19 Voges-Proskauer test

Generally, if the result of MR test is positive, the result of VP test is negative,
But in this laboratory activity the result of VP test of group 1 entourage I is also
negative. The media of isolate A and B is just change become orange-brownish. The
positive interpretation, the color of media will change into red after added of α-
napthol dan KOH. The result of fermentation of Voges-Proskauer test is asetil metil
karbinol (Volk & Wheeler, 1993).

Picture 3.20 Citrate test

Citrate test is using Simmons Citrate media. At the first we inoculated isolate
bacteria (A and B) using ose needle into media and then incubated until 2x24 hours
at room temperature. The technique of incubation also different with Hadioetomo
(1993), his incubated the media at 37ºC for 24 hours. The result of group 1 entourage
I is positive. Isolate A and B have positive interpretation. The media color are change
from green become blue. It is accordance to Sudarsono (2008), the positive test is
marked by the change of medium color become blue.

Picture 3.21 Urease test

Group 1 entourage I, the isolate bacteria A and B have positive result. The
color of media is change become pink. It is accordance to Lim (2006), interpretation
of results: negative (-): no change of media color becomes pink / pink, meaning
germs do not break urea to form ammonia. Positive (+): does not change the color of
the media to pink, meaning germs break down urea to form ammonia. The purpose
of this test is to determine whether germs have urease enzymes that can decompose
urea to form ammonia. The urea medium contains phenol red indicator. (Lim, 2006).
 Picture 3.22 H2S test
H2S test function is to differentiate the type of bacteria based on the ability to
break down dextrose molecule, lactose, sucrose and releasing sulfida. In addition,
TSIA test is to know the bacteria producing H2S. There are two media that used in
this test: slant media and butt media (Kismiyati, 2009). The isolate bacteria (A and
B) of group 1 entourage I is positive. The positive interpretation is the media form
sendimentation.

Picture 3.23 OF Basal Picture 3.24 OF Basal + Parafin

Based on the observation, the result of group 1 entourage I the bacteria
belong to oxidative-fermentative bacteria. Because one tube with the OF basal media
are changing become yellow and also the other tube that contain of OF basal media +
paraffin. The change become yellow is caused of the decreasing the pH in media.
The condition of the media is acid. It is according to Hugh & Leifson (1953),
Oxidative and fermentative organisms will produce an acid reaction. The acid
reaction produced by the oxidative organisms is apparent first at the surface and
extends gradually downward into the medium. The different between oxidative and
fermentative bacteria is the oxidative bacteria only can produce acid if the condition
of media consist of oxygen while fermentative can produce acid in the media with
and without oxygen. In fermentation process, the glucose molecule first is
phosphorylated and then split into two triose molecules which undergo further
changes. This process is independent of oxygen. In oxidation process, the glucose
molecule is not split into two triose molecules, but the aldehyde group is oxidized to
a carboxyl group forming gluconic acid. Further oxidation may take place to form
various products such as 2-ketogluconic acid.
Some bacteria apparently both ferment and oxidize carbohydrates. The
medium will not demonstrate oxidation of a carbohydrate which is actively
fermented by the organism. If the fermentation is weak or slow, oxidative
metabolism may be demonstrated. The group of bacteria commonly called paxacolon
bacteria apparently may oxidize lactose, ferment lactose, or carry out both reactions
(Hugh & Leifson, 1953).
Fermentation process involved complex microbial activities and biochemical
changes. The microorganisms responsible for the fermentation are yeasts, lactic acid
bacteria (LAB) and acetic acid bacteria (AAB), moreover species of Bacillus sp,
other bacteria and filamentous fungi could also grow with consequent influence on
quality of the process. First main event regards microbial activities on the
mucilaginous pulp that results in the production of alcohol and acids, and an
increasing of temperature. Secondly, fermentation is essential for the death of the
embryo of the seed, which takes place through the production of heat and acetic acid.
This is also important to eliminate bitterness and astringency and, lastly, for the
formation of aroma precursors. High sugar concentration, high temperatures, pH
changes, ethanol production, metabolism of organic acids and as a result their
concentrations, turn fermentation into a stressful environment for microorganism
growth (Visintin et al., 2015).
 IV. CONCLUSION AND SUGGESTION

A. Conclusion

The conclusion of this laboratory activity are:

1. To separate the microorganisms from the environment to be grown in a medium
in the laboratory is by isolation process. The isolation of microorganism include
of several process such as serial dilution and purification.
2. The characteristics of isolate A:
 Isolate A is belongs to gram positive, coli-fecal bacteria and oxidative-
fermentative bacteria.
 Have several enzymes such as amylase, protease, oxidase, catalase and
urease but doesn’t have lipase.
 Can live at low, normal and high temperature, at pH 7 and 11, and in several
type of osmotic pressure (0%, 0,5%, 0,85% and 1%).
 They only can processed and used fructose as a source of nutrient.
The characteristic of isolate B:
 Isolate B is belong to gram positive, coli-fecal bacteria and oxidative-
fermentative bacteria.
 Have several enzyme such as amylase, protease, oxidase, catalase and urease
but doesn’t have lipase.
 Can live at low, normal and high temperature, at pH 7 and 11 and in several
type of osmotic pressure (0%, 0,5%, 0,85% and 1%).
 Can processed and used glucose and fructose as a source of nutrient.
B. Suggestion

The suggestion of this laboratory activity is nothing. For the assistant keep do
the best.
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