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ISOLATION AND CHARACTERIZATION OF MICROORGANISM

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: Gibran Muhammad Tri R


: B1K014025
:2
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: Willery Yeo

PRACTICAL REPORT OF MICROBIAL SYSTEMATICS

MINISTRY OF RESEARCH, TECHNOLOGY AND HIGHER EDUCATION


JENDERAL SOEDIRMAN UNIVERSITY
FACULTY OF BIOLOGY
PURWOKERTO
2016
I. INTRODUCTION
A. Theoretical Basis
Microbes isolation is dividing one kind of microbe with other microbes or it
mixture so the pure culture can be obtained. Isolation of microbes can be done using
various methods depending on what sample that we want to take. The microbes sample
mostly taken from the soil or water. When taking the sample from soil, usually the
easiest method is using spoon, after the soil taken wrap the spoon with aluminum foil
and sterilize the soil. While when taking the sample from water, it can be from water
spring or tap water. After the sample was taken, isolation can be done by using streak
quadrant method which will let only single colony remain in latest area (Ashok, 2006).
Microbes identification is the method to determine the microbes morphological
characteristic. The identification can be done both when microbes still active or not.
Characterization is essential to understand kind of microbes and what group that
microbes belongs. The characterization also need to understand the physiological
character of microbes, and what factor that determine the species of microbes. Microbes
confirmation is to understand what environment and specific medium that will be used
for them to grow (Prakash, 2012).
Characterization divided into two steps there are classification and identification.
To identified and classify the microbes, first the characteristic of microbes need to be
determined. Classification is grouping the microbes into one taxonomical group. The
identification theory of microbes is comparison between identified microbes and
unidentified microbes. The accuracy level from identification is depend on work
precision from preparation such as medium making, observation precision, reagent
making and coloration (Pelczar, 2007).

B. Objective
The purpose of this practicum are:
1. Student can divide the microbes from it mixture
2. Student can characterize the microorganism based on morphology, physiology,
enzymatic, and biochemical.

B. Discussion

Aseptic technique or sterile technique is used to avoid contamination of sterile


media and equipment during cell culture in isolation. Sterile technique should always be
employed when working with live cell cultures and reagents/media that will be used for
such cultures. This technique involves using flame to kill contaminating organisms, and
a general mode of operation that minimizes exposure of sterile media and equipment to
contaminants. The isolation during aseptic technique will increase the probability to get
pure culture. The isolation usually done with streak quadrant, the streak which done
recur and divide the medium into 4 area. The first area is the area which first contacted
with sample which usually contain most microbes colony. While in last area is the last
area which contain the least colony of microbes and usually can be found a single colony
of microbes (Ashok, 2006).
Soil is the habitat for microbes to grow. In just one gram of soil contain a million
microbes which consist of bacterium, actinomycetes, fungi and protozoa. The diversity
of microbes in soil is affected by the contamination around the soil itself, for example
the garden soil contained lesser microbes than landfills. The garden soil is the common
place for microbe. It also most frequent contacted with human. The garden soil is good
for taking microbe sample because it contain divers microbes on it. When taking the
garden soil for sample usually we should dug in 15-30 cm bellow the surface of soil in
order to get pure soil microbe sample. On a per cell basis, these deeper microbes may,
arguably, have a greater influence on soil formation processes than their counterparts at
the surface due to their proximity to parent material (Eilers, 2012).
The commonly found microbes from garden soil are actinomycetes, while in dry
and frigid soil is commonly found Streptomyces. In order to get pure garden soil colony
of microbes, the serial dilution of soil must be done at least 6 times. The soil microbes
have some characteristic that different from air or water microbes. The soil microbes
usually is anaerobic microbes which dont need any oxygen to grow, there also
facultative anaerobic microbe which they can live with or without oxygen in order to
grow. The other characteristic of soil microbes is they are cosmopolite organism which
mean they can be found nearly everywhere. (Pujiati, 2014).

Based on Pelczar (2007) the microbes are divided into some group based on how
they survive in various pH and temperature level:
A. pH
1. Acidophilic microbes, if the microbes can stand in pH between 2- 5 they
include in this group.
2. Netrophilic microbes, if the microbe live in pH between 5.5 8 they include
in this group.
3. Alkalophilic microbes, if the microbes can stand in pH between 8.5 11 they
include in this group.
B. Temperature
1. Psychophilic microbes, if the microbe can live in 0 oC - 20oC temperature they
include in this group.
2. Mesophilic microbes, if the microbes can live in 25 oC - 50oC temperature
they include in this group.
3. Thermophilic microbes, if the microbes can live in 55 oC - 80oC temperature
they include in this group.
4. Hyperthermophilic microbes, ig the microbes can live in 85oC - 100oC
temperature they include in this group.
There also microbe which can live in mesophilic and psychophilc
temperature level which called psychotolerant microbes but they will not grow as
fast as their natural temperature level.
The result of plating in group 2 from 10 -5 and 10-6 dilution we got 9 single
colonies in each dish and one dish contain spreader. In 10 -5 dish contain less colonies
than in 10-6, this result is not correlated with Pelczar (2007) statement which said the
more serial dilution done when in sample the less colonies consist in that dilution. The
result might not related because when serial dilution, the sample confused with early
dilution so the 10-6 has more colonies than in 10-5 dilution (Prakash, 2012).
The isolate which used for characterization is the one with just on it single
colony. First we observe the morphology characteristic of colony. Then take the colony
with ose needle and put in slant medium in order to make stock isolate which later will
be used for characterization. The isolate has various characteristic morphologically
observed and noted. The stock microbes later used for characterization based on
physiology, enzymatic, and biochemical test.

The tests which are conduct to determine the character of microbes in laboratory
are:
1. Morphological observation
The morphological observation is observed directly in colonies of microbes
which observe characteristic such as the surface, size color, shape margin and
elevation of the colonies (). In group 2 the colonies morphology character is
varies in 9 isolate used. For colony surface 5 isolate has shiny surface and 4
isolate has dull surface; for colony size there are 3 pinpoint size colonies and
6 small size colonies; for colony color there are 5 cream color colonies 1
milky white color colony and 3 creamy white color colonies; for colony
shape only 1 colony has irregular shape, the rest has circular shape; for
colony margin only 1 colony has undulate margin and the rest has flat
margin; for the colony elevation all of it has raised elevation.
2. Physiological examination
The physiological examination is consisted of 4 different tests which are:
a. Temperature test, the temperature test is done by put the isolate into NB
medium and incubated with different temperatures, if the isolate become
turbid then the isolate give positive response while if no change in the
medium the isolate give negative response (Pii, 2015). In this practicum
are use 4oC, 37oC, and 80oC. Based on the result only in 37 oC the isolate
give positive response while the rest give negative response.
b. pH test, the pH test is done by put the isolate into NB medium with
different pH on each medium, if the medium become turbid then the
isolate give positive response while if no change in medium the isolate
give negative response (Vilas, 2016). In this practicum we use 3, 5, 7 and
9 pH for the test and the result are every isolate in every pH used are
giving positive response.
c. Salinity test, salinity test is done by put the isolate into NB medium with
different salinity on each medium, if the medium become turbid then the
isolate give positive response, while if no change in medium the isolate
give negative response (Vilas, 2016). In this practicum we use salinity
parameter 0.5%, 0.85%, and 1%. Most isolate give positive response in

0.85% salinity, in 0.5% salinity only 3 isolate give positive response, and
in 1% salinity 6 isolate give positive response.
d. Gram staining, gram staining is done by stain the microbe with gram A,
B, C, and D reagent, if the microbe become violet then it include in gram
(+) group while if become red it include in gram () group (Bradford,
1976). In this practicum by using gram staining method there are 2 gram
positive microbes and the rest is gram negative microbes.
3. Enzymatic examination
The enzymatic examination is consisted of 5 different tests which are:
a. Amylolytic test, the amylolytic test done by streak the isolate into SA
(starch) medium using ose needle, if the isolate give clear zone after
poured with lugol iodine reagent they give positive response while there
are no clear zone they give negative response (Boehringer, 1997). In
group 2 result there only 2 isolate which give positive response, based on
(Ji, 2014) only small amount of soil microorganism can process the
amylum since in soil there are not many amylum contained in soil.
b. Protease test, the protease test done by streak the isolate into PA
(protease) medium using ose needle, if there are clear zone around
colonies the isolate give positive response while there are no clear zone
around colonies the isolate give negative response (Boehringer, 1997). In
group 2 result there only 2 isolate which give positive response, based on
(Ji, 2014) only few of soil microorganism can process the protein.
c. Lipolytic test, the lipolytic test done by streak the isolate into MM + lipid
using ose needle, if there are red spot in medium and the medium color
change into yellow the isolate give positive response while if there are no
or only one change in medium the isolate give negative response
(Boehringer, 1997). In group 2 result all of the isolate give negative
response, based on (Ji, 2014) the soil microorganism mostly could not
process lipid since there only small amount of it contain in soil.
d. Oxidase test, oxidase test done by smear the isolate into merang paper
and dropped by oxidase reagent, if the isolate color change into dark blue
they give positive response while if there are no change in color they give
negative response (Boehringer, 1997). In group 2 there only 2 isolate give
positive response while the rest is negative, based on (Ji, 2014) the soil

microorganism mostly is anaerobic microbes and only few of


microorganism in soil is facultative anaerobic microbes which usually
give positive response in oxidase test.
e. Catalase test, catalase test done by smear the isolate into object glass
using ose needle then dropped by H2O2 reagent, if there are bubble in
isolate they give positive response while if there are no bubble in isolate
they give negative response (Boehringer, 1997). In group 2 there only one
isolate give negative response while the rest is give positive response,
based on (Ji, 2014) most of soil microorganism can undergo catalase test
since they can process the hazardous materials inside soil, which is why
the soil organism usually used for bioremediation.
4. Biochemical examination
The biochemical examination consist 7 different test which are:
a. Indole test, the indole test done by inoculate the isolate to TB medium
using ose needle, after incubation drop the kovack indole into medium, if
the red ring formed in surface of the medium the isolate give response
while if there are no red ring formed in surface the isolate give negative
response (Khan, 2014) In group 2 there only one isolate which give
positive response while the rest give negative response. Based on
(Gaherwal, 2015) the red ring formed because the microorganism react to
kovack indole reagent which most of soil microorganism did not have the
ability to react against kovack indole.
b. Methyl red test, the methyl red test done by inoculate the isolate into PB
medium using ose needle, after incubation the medium dropped with MR
reagent, if the medium change color into red the isolate give positive
response while if there are no change in color the isolate give negative
response (Khan,2014). In group 2 there are no isolate give positive
response in methyl red test. Based on (Gaherwal, 2015) the soil
microorganism actually give positive response in methyl red test if the
incubation conducted is longer since the soil microorganism have slow
response to react in methyl red test.
c. Voges-Proskauer test, the Voges-Proskauer test done by inoculate the
isolate to PB medium using ose needle, after incubation the medium

dropped with -naphtol+KOH 40% reagent, if the medium color change


to red the isolate give positive response while if there are no color change
the isolate give negative response (Khan,2014). In group 2 there are six
isolate give positive response while the rest is negative. Based on
(Gaherwal, 2015) soil microorganism will react to VP test but some of it
has slow response to VP test.
d. Simmons citrate test, the Simmons citrate test is done by streak the
isolate into SC medium using ose needle, after incubation if the medium
change color into blue the isolate give positive response while if there are
no change in medium the isolate give negative response (Khan,2014). In
group 2 there are 4 isolate which give positive response and the rest is
negative. Based on (Gaherwal, 2015) the simmons citrate test is depend
on the microorganism, soil microorganism usually react to this test but
some of them just have slow response to it.
e. Sugar test, the sugar test done by streak the isolate into 6 kind of sugar
medium (Glukosa, Fruktosa, Manosa, Galaktosa, Arabinosa, and
Sukrosa) using ose needle, after incubation if the medium change color
into yellow the isolate give positive response while if the medium not
change the isolate give negative response (Khan,2014). In group 2 there
are no isolate give positive response to any sugar which used for test.
Based on (Cycon, 2012) there only few soil microorganism which can
process sugar, but if taken from soil sample at least there must be one or
two isolate that give positive response. The result not correlated might
because when the isolate inoculated into media it already runs out which
makes the isolate cannot grow.
f. H2S test, the H2S test done by stab the isolate into the bottom of TSIA
medium then streak continue the medium using ose needle, after
incubation if the medium upraised the isolate give positive response
while if the medium not upraised the isolate give negative response
(Khan,2014). In group 2 only 2 isolate give positive response while the
rest is negative. Based on (Cycon, 2012) the soil microorganism is
anaerobic microbe and will produce gas that lift the medium when the

H2S test is conducted. This not related with result might because the
isolate is no longer live in medium when streaked.
g. Urease test, urease test done by inoculate the isolate into UB medium,
after incubation if the medium color change into pink the isolate give
positive response while if there are no change in medium color the isolate
give negative response (Khan,2014). In group 2 there are no isolate give
positive response. Based on (Cycon, 2012) soil microorganism is mostly
react to urease since urease is rich in soil. This not correlated with result
might because when inoculate the isolate into medium, the isolate already
runs out while inoculated from one medium to another which makes the
isolate cannot grow.

IV. CONCLUSION AND SUGGESTION


A. Conclusion
Based on this practicum we can conclude that:
1. Isolation from the garden soil acquired 9 isolate which each isolate has different
characteristic.
2. The morphology, physiology, enzymatic, and biochemical show various result from
each isolate.
B. Suggestion
The observation and treatment for the isolate time is too short so when doing the
examination to each isolate will give undependable result.

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