Abstract
I n this lab, an unknown culture of bacteria was identified through a series of microscopic
and biochemical tests. A streak plate and a backup slant were also made. The bacteria that was
used in this experiment was #42, and it was identified asBacillus cereus.The bacteria was
determined to be a gram positive rod using a Gram stain. Through a catalase test, the bacteria
was found to produce the enzyme catalase. An endospore stain showed that the bacteria could
produce endospores. Finally, it was determined that the bacteria could not ferment mannitol
based on a mannitol fermentation test.
Introduction
acterial identification is needed because bacteria play many important roles in human
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life. For many years, bacteria have been used in biotechnology, medicine, genetic engineering,
and food sciences. Bacteria can be pathogenic, and bacterial identification is needed for
diagnosing diseases and finding their origin. Antimicrobial drugs work differently for different
bacteria species. Therefore, bacterial identification is essential for prescribing effective
treatments for bacterial diseases. Bacteria can also be used to produce antibiotics, amino acids,
and hormones, which can be used in medical treatments (Franco-Duarte et al, 2019). Bacteria are
used in other products as well, including fermented foods such as pickles, sauerkraut, and yogurt.
Because bacteria have different metabolic properties, bacterial identification is essential to
ensure that the right bacteria are being used to make products.
Bacteria can be identified by observing morphological characteristics and performing
biochemical tests. The morphology of a bacteria can be viewed using microscopic tests. Under
the oil immersion lens of a compound light microscope, the morphology and arrangement of the
bacterial cells can be identified. Single-colonies of the bacterial culture may also be isolated
using a streak plate, where the size, color, margins, and elevation of the colonies may be
observed. Morphological characteristics can be used to identify bacteria, because bacteria species
have distinct morphology. Biochemical tests can be used to determine how bacteria produce
energy. Biochemical tests can also determine what types of molecules bacteria can use to
produce energy. Since not all bacteria produce energy in the same way, or are able to utilize the
same molecules, biochemical tests can be used to isolate or identify bacteria.
Aseptic Technique
urpose:
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The aseptic technique was used in all procedures to prevent the contamination of pure cultures
and to prevent the spread of unwanted microbes. All culture media for this lab was inoculated
using an inoculating loop. The inoculating loop was sterilized by being flamed until red-hot
under the bunsen burner. The heat from the flame kills the bacteria by denaturing proteins and
enzymes, and destroying lipids in the lipid membrane. Isopropanol alcohol was used to wipe the
workbench because it can kill bacteria cells by denaturing proteins and dissolving lipid
membranes.
Procedure:
1. Before working, the workbench was wiped with 70% Isopropanol alcohol.
2. The inoculating loop was flamed over the bunsen burner until it was red-hot.
3. While holding the sterile loop, the bacterial culture slant was opened by twisting open the
cap with the index and pinky fingers.
4. While holding the cap, the mouth of the tube was heated through the flame three times.
The cap of the bacterial culture slant was never placed on the workbench.
5. The sterile loop was inserted into the bacterial slant culture and placed on the bacteria
growth.
6. The loop, now holding the bacteria, was taken out of the slant.
7. Before closing the bacterial slant culture, the mouth of the tube was heated three times
again.
8. If the culture media was contained in a tube, the mouth of the tube was flamed three
times after being opened.
9. The culture media was inoculated with the loop holding the bacterial culture.
10.If the culture media was contained in a tube, the mouth of the tube was flamed three
times.
11.After the media was inoculated, the loop was sterilized again by being heated until it was
red-hot.
Backup Slant
urpose of backup slant
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The backup slant was made as an alternative to the working slant in case it became
contaminated. The backup slant can also provide younger bacteria, which might be needed for
certain tests. A younger culture might be needed for a gram stain, for example, because the age
of the cells may affect the strength and thickness of the cell walls.
Procedure:
1. An agar slant was inoculated with bacteria from the working (original) slant.
2. The slant was incubated at 35 C for 48 hours.
escription of original (working) slant
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The bacteria streaks were white with beaded growth. The growth was also slightly raised.
esults
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The backup slant also had white and beaded growth. However, the beaded pattern was only
observable on the margins of the growth. This might be because too much bacteria was
inoculated on the slant. Since there was too much bacterial growth, the beads were closer
together making the growth look filiform/even in the center.
Figure 1: Backup Slant Figure 2: Original Slant
Streak Plate
urpose
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The streak plate is used to observe single colonies of the culture. By obtaining single colonies,
the morphology characteristics of the bacteria could be observed. Morphological characteristics
include size, color, shape, and elevation. Even though bacteria usually have distinguishable
single colonies, more testing is needed for identification. Streak plates can also check for
contamination. If the culture is pure, all of the plated colonies should look similar.
Procedure
1. An agar plate was split into three sections.
2. Using the aseptic technique, the bacteria was inoculated on one section of the plate in a
zig-zag pattern.
3. The loop was sterilized after the first streak.
4. The sterile loop was streaked from the inoculated section of the plate to an uninoculated
section of the plate, also in a zig-zag pattern.
5. The loop was sterilized after the second streak.
6. The sterile loop was streaked from the inoculated section of the second streak to the third
section of the plate, also in a zig-zag pattern.
7. The plate was incubated at 35 C for 48 hours.
esults (description of colonies)
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The colonies were about 4mm in size. They were white and umbonate. The raised sections of the
colonies were shiny but the edges of the colonies were matte. The colonies were circular in form
and had entire margins.
Figure 3: Bottom of streak plate Figure 4: Top view of streak plate
Gram Stain
urpose and Background
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The purpose of this stain is to differentiate between gram positive and gram negative bacteria
based on their cell wall composition. Gram positive bacteria have cell walls with multiple layers
of peptidoglycan, while gram-native bacteria have a thin layer of peptidoglycan surrounded by
an outer layer of lipoproteins, phospholipids, and lipopolysaccharides. In a gram stain, bacteria
cells are first stained with crystal violet dye, which is absorbed by peptidoglycan. Gram's iodine
is applied to react with the crystal violet dye to form a crystal violet-iodine complex (CV-I). The
CV-I complex is insoluble in water. A decolorizing agent (in this case acetone-ethanol) is applied
to wash out the primary stain from the gram-negative bacteria. In gram-negative cells, the
alcohol dissolves the outer lipopolysaccharide layer and the CV-I complex washes out of the thin
layer of peptidoglycan. Even though both gram-positive and gram-negative bacteria can be
decolorized with alcohol, gram-negative bacteria decolorize much faster.
Procedure:
1. A smear was prepared and fixed.
2. The smear was covered with crystal violet for 30 seconds.
3. The slide was rinsed with distilled water. The water was squired above the smear and was
allowed to run down to wash off the dye. The water was not squirted directly on the
smear in order to not disrupt the smear.
4. The smear was covered with Gram’s iodine for 10 seconds.
5. The slide was rinsed with distilled water.
6. The smear was decolorized with acetone-ethanol for 2 seconds.
7. The slide was immediately washed with distilled water.
8. The smear was covered with safranin for 30 seconds.
9. The slide was rinsed with distilled water and blotted with a paper towel.
esults
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The bacteria cells appeared dark purple and were in a rod shape. The cells were also arranged in
a streptobacillus formation (in a chain).
Figure 5: Gram stain with gram positive rods
onclusions
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The bacteria cells are gram positive rods. Because the cells have thick peptidoglycan walls, the
crystal violet iodine complex was not washed out by the decolorizing agent.
ossible Sources of Error
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A few of the rods appeared red or gram negative. This may be because some of the cells are
older and the peptidoglycan layers were degraded, making them more susceptible in the
decolorizing step. This also could be because the alcohol was left on too long.
he results may have been more clear if the backup slant culture was used for the smear instead
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of the working slant. The backup slant culture was younger so the peptidoglycan cells walls
would have probably been thicker.
Catalase Test
urpose and Background
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The purpose of this test is to identify whether the bacteria produce the enzyme catalase. Catalase
is produced by most aerobic organisms and can break down hydrogen peroxide into water and
oxygen gas. It is favorable for cells to break down hydrogen peroxide, because the compound is
otherwise lethal to the cell. A positive result of a catalase test is indicated by bubbles from
oxygen gas.
Procedure:
1. The streak plate was used.
2. Five drops of hydrogen peroxide were added on the first streak of the streak plate
esults
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Bubbles were produced when the hydrogen peroxide was added to the plate.
Figure 6: Oxygen bubbles on the streak plate after adding hydrogen peroxide
onclusions
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This bacteria has the enzyme catalase and can utilize aerobic respiration. The bacterial cells used
the catalase enzyme to break down hydrogen peroxide (H2O2) into water (H2O) and oxygen gas
(O2).
Endospore Stain
urpose and Background
P
Bacteria can be identified by the presence and position of their endospores. The position of an
endospore can also indicate the age of the bacteria. Endospores do not metabolize or reproduce.
They form in response to harsh conditions and are resistant to heating and various chemicals.
Even when the bacteria cells die, the endospores can remain dormant for a long period of time.
When the conditions allow, the endospore may return to its vegetative or growing state.
Procedure:
1. Prepare and heat fix a smear.
2. Place a piece of absorbent paper over the smear.
3. Cover the paper with malachite green dye.
4. Steam the slide for 10 minutes. Keep the absorbent paper moist by adding malachite
green throughout the steaming process. I added about 2 drops every minute.
5. Wash the smear with water.
6 . C over the smear with safranin for 30 seconds.
7. Wash the smear with distilled water and blot it dry.
esults
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There were endospores in my bacteria but I was not able to stain them green. Under the
microscope, I could see some clear sections in the bacteria that resembled endospores.
Figure 7: Endospore stain with clear endospores
onclusions
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This bacteria produces endospores.
ossible Sources of Error
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The slide might have not been steamed enough and the malachite green did not penetrate the
endospores. The slide was steamed for about 10 minutes, but the machalite green might have
penetrated the spores better if the slide was steamed for 15 or 20 minutes.
Mannitol Fermentation Test
urpose and Background
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A mannitol fermentation test was conducted to determine whether the bacteria could ferment
mannitol. In this test, a fermentation tube was used to detect acid and gas production. The
fermentation tube contains peptone, phenol red, a Duham tube, and mannitol. A Durham tube is
a small invented tube used to trap [Link] phenol red indicator is red in its neutral state, when
the pH is neutral. Acid is a by-product from fermentation. Acidic conditions will turn the phenol
red indicator yellow. Some bacteria produce both acid and gas from fermentation. When gas is
produced, it will appear as a bubble in the Durham tube. If bacteria cannot ferment the mannitol
in the media, they may still grow oxidatively using peptone. Ammonia is a by-product of
peptone production and is basic. The fermentation tube will remain red or pink if it uses peptone
instead of mannitol.
Procedure:
1. The bacteria was transferred aseptically into the mannitol fermentation media.
2. The fermentation tube was incubated at 35 C for 48 hours.
esults
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The fermentation media was turbid, indicating growth. However, there was no color change in
the media and no gas was observed in the Durham tube.
Figure 8: Inoculated mannitol tube Figure 9: Uninoculated mannitol tube
onclusions
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The bacteria does not ferment mannitol. It grew oxidatively using peptone.
Discussion
he bacteria in this culture was identified asBacilluscereus. Bacillus cereusis a
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toxin-producing facultatively anaerobic bacteria (McDowell, Sands, Friedman, 2023).
Facultatively anaerobic organisms can utilize both aerobic and anaerobic metabolism depending
on the presence of oxygen (Johnson & Case, 2019). Morphologically, Bacillus cereus strains are
usually thin bacilli of about 3-4 micrometers in size with square ends (Ramarao et al, 2020).
Bacillus cereusis commonly found in soil, on vegetables,and in many processed and
unprocessed foods (McDowell, Sands, Friedman, 2023).
Bacillus cereuswas first discovered in 1887, whenit was isolated on a gelatin plate from
the air in a cowshed by Frankland and Frankland (Frankland & Frankland, 1887).Bacillus cereus
was classified as a disease causing organism in the 1950s, with the first described outbreaks of
diarrheal disease in hospitals in Norway from 1947-1949 (Stenfors Arnesen, Fagerlund, Einar
Granum, 2008).
The pathogenicity ofBacillus cereusis associatedwith the production of exoenzymes
(McDowell, Sands, Friedman, 2023). Exoenzymes are enzymes that are secreted by a cell and
perform functions outside of the cell (Johnson & Case, 2019) . When ingested, these toxins can
cause two types of gastrointestinal diseases: the diarrhoeal and emetic(vomiting) syndromes. The
diarrhoeal syndrome occurs when a large number of vegetativeBacillus cereuscells are ingested
and produce enterotoxin in the small intestine (McDowell, Sands, Friedman, 2023). The emetic
syndrome is caused by the emetic toxin cereulide. Cereulide is produced directly in food, rather
than in the small intestine. The toxin is resistant to heat and will not be eliminated by most
cooking methods. Even if vegetative cells are not present, emetic syndrome can still occur when
cereulide is ingested directly (Stenfors Arnesen, Fagerlund, Einar Granum, 2008).
Bacillus cereusinfections are usually self-limitedand do not require any targeted
treatment. The durations of both the diarrhoeal and emetic syndromes are usually less than 24
hours (Stenfors Arnesen, Fagerlund, Einar Granum, 2008). Most patients receive symptomatic
care with oral hydration. However, intravenous fluid hydrations may be needed in severe cases
(McDowell, Sands, Friedman, 2023).
igure 10: Scanning electron microscopy image ofB.cereuscells with square ends (Ramarao et
F
al, 2020)
Resources
Franco-Duarte, R., Černáková, L., Kadam, S., Kaushik, K. S., Salehi, B., Bevilacqua, A., Corbo,
M. R., Antolak, H., Dybka-Stępień, K., Leszczewicz, M., Relison Tintino, S.,
Alexandrino de Souza, V. C., Sharifi-Rad, J., Coutinho, H. D. M., Martins, N., &
Rodrigues, C. F. (2019). Advances in Chemical and Biological Methods to Identify
Microorganisms-From Past to Present.Microorganisms,7(5), 130.
[Link]
Frankland, G.C., Frankland, P.F., 1887. XI. Studies on some new micro-organisms obtained from
air. Philosophical Transactions of the Royal Society of London. (B.) 178, 257–287..
[Link]
Johnson T. R., Case C. L., 2019, Introduction of Laboratory Experiments in Microbiology,
Pearson Education, Inc., Twelfth Edition
McDowell RH, Sands EM, Friedman H.Bacillus Cereus.[Updated 2023 Jan 23]. In: StatPearls
[Internet]. Treasure Island (FL): StatPearls Publishing; 2023 Jan-. Available from:
[Link]
Ramarao, N., Tran, S. L., Marin, M., & Vidic, J. (2020). Advanced Methods for Detection of
Bacillus cereus and Its Pathogenic Factors.Sensors(Basel, Switzerland),20(9), 2667.
[Link]
Stenfors Arnesen L., Fagerlund A., Einar Granum P., 2008. From soil to gut:Bacillus cereusand
its food poisoning toxins,FEMS Microbiology Reviews,Volume 32, Issue 4,Pages
579–606,[Link]
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