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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
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
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.
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