Professional Documents
Culture Documents
By :
Name : Sunu Pertiwi
Student ID : B1B015007
Entourage :I
Group :1
Assistant : Dwi Rizki Nurjanah
A. Background
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. 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.
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
A. Conclusion
The suggestion of this laboratory activity is nothing. For the assistant keep do
the best.
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