Name: Angel Gustasiana

NIM: 2440013254
Class: BA46
B. Gram-staining result
Bacillus Cereus Escherichia Coli
Draw a representative field 400x 400x

1000x 1000x

Cell morphology: Shape: bacillus Shape: bacillus

Shape Arrangement: singlebacilli Arrangement: monobacilli
Arrangement Cell color: violet Cell color: pink
Cell color Gram reaction: gram- Gram reaction: gram-
Gram reaction positive negative

C. Motility-test result
Nutrient-agar plate
Bacillus Cereus Escherichia Coli
Draw the distribution of
growth

Motility or Brownian Motile Motile

movement
 D. Respiration-test result
Nutrient broth
Bacillus Cereus Escherichia Coli

Classification according to Obligate aerobes Facultative anaerobes

oxygen requirement

E. Catalase-test result
Bacillus Cereus Escherichia Coli

Catalase reaction: Negative Catalase reaction: Positive

1. Why is it important to use a sterilized loop between streaks when preparing a

streakplate?
Sterilization aims to inhibit microbial growth, kill microbes, and eliminate all microorganisms
present in the tools and materials to be used. Therefore the loop must be sterilized first to
maintain microbiological culture and prevent contamination of the growth medium. In the
scratch procedure, sterile loops obtain uncontaminated microbial cultures (Tortora, Funke,
Case, 2018).

2. Outline the differences between a streak plate and a spread plate.

(Tortora, Funke, Case, 2018).

Differences Streak plate Spread plate

Definition a technique used to isolate technique used to count
and purify bacterial species bacteria in a sample
in a sample.
Purpose to isolate and purify certain to count and measure
bacterial species from bacteria in a sample
bacterial mixtures
 Use of a micropipette not necessary inoculum was taken from the
sample using a micropipette
Inoculating tool sterile inoculation loop or a sterile spreader
cotton swab
Inoculum quantity One loopful of inoculum 100 𝜇𝑙 or 1000 𝜇𝑙
Inoculation method Streaks on the fresh medium Spread the inoculum evenly
in a zig-zag pattern on the surface of the fresh
medium
Sterilizing the inoculation the loop is burned on a The spreader is dipped in
tool bunsen fire so that it becomes 95% alcohol and ignited until
red hot the alcohol runs out. This is
repeated three times

3. Explain the methods to investigate the bacterial cell motility. How to describe the result
of motility test using semi-solid agar?
 The upright took and passed over a Bunsen flame until the flaming
 the tube was bacteria were pounded in the upright NA taken with an aseptic technique
(a test tube containing bacteria was passed over a Bunsen fire) tube with the tube
 Bacteria were taken by inserting an upright exposed to bacterial culture.
 The mouth of the tube is passed back over the Bunsen flame. The
 New and sterile MTM media is taken.
 Open the test tube and bunsen flame
 One Vertically inserted into the media. The
 Pass it over the mouth of the tube is passed back over the flame and
 One passed over the Bunsen flame
 Check daily for up to 7 days.
 Observe the growth zone spreading from the inoculation line.
Motility test results using semi-solid agar can be said to be motile if the diffuse growth extends
out from the inoculation line or spreads throughout the medium to make it slightly opaque.
Meanwhile, it can be non-motile if the growth is limited to the puncture line, with clear
boundaries and leaves the surrounding medium transparent.

4. Name the five major groups in which microorganisms are classified based on their oxygen
requirements. Which factor is responsible for the variation in these groups’ ability to
utilize free oxygen for cellular respiration
 Obligates aerobes are organisms that cannot grow without an abundant supply of
oxygen. This type of microorganism grows on the top of the tube.
 Obligates anaerobes are organisms that can only live in an oxygen-deficient
environment because, for them, oxygen is a poison. Obligate anaerobes are usually
bacteria and live in a variety of places naturally. This type of microorganism grows at
the bottom of the tube.
  Facultative anaerobes are organisms that thrive in the presence of oxygen but also grow
in the absence of oxygen by relying on fermentation or anaerobic respiration. This type
of microorganism grows on the top of the tube and can also be throughout the tube.
 Aerotolerant anaerobes are organisms that do not care about the presence of oxygen.
They do not use oxygen because they usually have a fermentative metabolism, but they
are not harmed by oxygen like obligate anaerobes.
 Microaerophiles are microorganisms that require oxygen to survive but require an
environment containing lower oxygen levels than in the atmosphere. The oxygen level
must be just right for growth, not too much and not too little. The minimum oxygen
level for development, around 1%-10%, is well below the 21% found in the atmosphere.
The factors responsible for the variation in the ability of these groups to utilize free oxygen for
cellular respiration are the enzymes catalase and Superoxide dismutase (SOD). In obligate
aerobes, the presence of the enzyme catalase and SOD allows toxins from oxygen to be
neutralized. In obligate anaerobes, the presence of catalase and SOD enzymes allows toxins
from oxygen to be balanced. In facultative anaerobes, there is a lack of enzymes to neutralize
harmful forms of oxygen. In aerotolerant anaerobes, the presence of one enzyme, soil, allows
the harmful forms of oxygen to be partially neutralized. In Microaerophiles, it produces lethal
amounts of toxic oxygen when exposed to normal atmospheric oxygen (Tortora, Funke, Case,
2018).

5. Consider the culture type in which growth was distributed throughout the entire medium
and explain why the growth was more abundant toward the surface of the medium in
some cultures, whereas other cultures showed an equal distribution of growth throughout
the tubes.
There are types of cultures that grow more towards the surface, and other types of cultures
spread throughout the medium. This is due to the oxygen demand of the bacterial culture. The
growth of the culture with more towards the surface of the media means that it means that the
bacterial culture needs oxygen to survive. Cultures showing growth on the surface can be
defined as obligate aerobic bacteria in which the bacteria cannot tolerate the presence of low
concentrations of oxygen. In addition, microaerophiles that require oxygen for multiplication
but in lower concentrations than those found in room air, and facultative anaerobes that can
also reproduce. Classified into the type of culture that is spread throughout the medium because
of its ability to grow in the presence or absence of oxygen. Meanwhile, other types of cultures
that are spread throughout the medium do not require oxygen to survive. Cultures showing
growth throughout the medium are aerotolerant anaerobes that grow more in the absence of
oxygen and maybe facultative anaerobes. Another type of culture that produces more on the
bottom of the medium is the obligate anaerobic culture. The bacteria cannot tolerate the
presence of oxygen, even in low oxygen concentrations.

6. Illustrate the chemical reaction involved in the degradation of hydrogen peroxide in the
presence of catalase.
The catalyst makes up the chemical reactions involved in the degradation of hydrogen peroxide
faster because it provides an alternative pathway with lower activation energy to take up the
 response. When a catalyst is added, alternative ways can form water and oxygen gas. The rate
of the catalyzed reaction will increase because this alternative pathway has lower activation
energy. The following illustrates the chemical reaction involved in the degradation of hydrogen
peroxide in the presence of catalase (Vlasits, Furtmüller, Jakopitsch, Zamocky, and Obinger,
2010).

(1) H2O2(aq) + I-(aq) → OI-(aq) + H2O(l)

(2) H2O2(aq) + OI-(aq) → H2O(l) + O2(g) + I-(aq)

+
Hasil: 2H2O2(aq) →2H2O(l) + O2(g)
Iodide ion reacts with hydrogen peroxide to produce water and ion hipoiodit, OI-. Then, the
hypoiodite ion will react with another hydrogen peroxide molecule to have water, oxygen gas,
and iodide ion. That is why the catalyst is never consumed in the reaction because it is reformed
at the end of the multi-step reaction.

7. Which bacterial strains can be biochemically differentiated using the catalase test
Bacterial strains that could be distinguished biochemically using the catalase test were Bacillus,
Escherichia, Streptococcus, and Staphylococcus. Escherichia and Staphylococcus strains
showed positive results, which was indicated by the number of bubbles produced. In
Streptococcus, Bacillus showed negative results, which was indicated by no or very few
bubbles produced (Sepriana, Jekti, and Zulkifli, 2017).

References:
Tortora, G. J., Funke, B. R., & Case, C. L. (2018). Microbiology: an Introduction. New York:
Pearson.
Sepriana, C., Jekti, D. S. D., & Zulkifli, L. (2017). Bakteri Endofit Kulit Batang Tanaman
Cengkeh (Syzygium Aromaticum L.) Dan Kemampuannya Sebagai Antibakteri. Jurnal
Penelitian Pendidikan IPA, 3(2), 52-59.
Vlasits, J., Furtmüller, P. G., Jakopitsch, C., Zamocky, M., & Obinger, C. (2010). Probing
hydrogen peroxide oxidation kinetics of wild-type Synechocystis catalase-peroxidase (KatG)
and selected variants. Biochimica et Biophysica Acta (BBA)-Proteins and Proteomics, 1804(4),
799-805.