Analyzing and Culturing Soil Bacteria
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Transcript of Analyzing and Culturing Soil Bacteria
Isolating, Analyzing, and Identifying a Soil Bacteria Sample collected from Flagstaff, Arizona
Alexander Matic
Bio 305W Section 4
Angelique Krencius
16 October 2015
INTRODUCTION
Soil samples are commonly filled with a plethora of bacteria that are immensely useful in
societal pursuits such as industry, pharmaceuticals, and agriculture. One of the uses of bacteria in
industrial is the utilization of lipase functions in bacteria to initiate esterification, which
ultimately produces plastic or artificial flavoring (Willerding et al. 2011). Among the other uses
of bacteria is the pharmaceutical testing and development of antibiotics used to treat human-
related diseases. Bacterial components, such as dual stranded DNA molecules known as
plasmids, are studied for the purpose of antibiotic development. Plasmids encode various
secondary functions within soil bacterial samples, such as the ability to resist antibiotics.
Thereby, trials involving the application of experimental antibiotics to bacteria with antibiotic-
resistance-encoded plasmids aim to denature the bacterial plasmids via a chemical gradient.
Once the antibiotic resistant capabilities of the bacteria are diminished, the other components of
the antibiotic work to kill the bacteria. (Herrick et al. 2014). Additionally, agriculture can benefit
from the use of bacteria, such as those that break down harmful substances and resist stress.
Scientists in Japan were able to successfully extract dinitrotoluene (DNT) degrading bacterial
genera such as Bacillus, Burkholderia, Variovorax, Raistonia, Methylobacterium, Arthrobacter,
Mycobacterium, Pseudomonas, and Rhodanobacter, which can survive unfavorable conditions
(Koichi, Yoshinori 2014). These bacteria eliminate DNT within regions affected by warfare and
maintain a stress resistance that applies to temperature and pH. The DNT-degrading bacteria, due
to their versatility, are introduced to plant micro-biomes in order to encourage conjugation and
transformation horizontal transfers that spread resistance-encoded plasmids into these
environments (Koichi, Yoshinori 2014). Thereby, plants with modified micro-biomes are able to
grow in stressful environments and thrive in regions with contaminated soils (Thijs et al. 2014).
In the pursuit of bacterial studies, the specific process of collecting and growing bacteria
via environmental isolate or enriched cultures is imperative in providing bacterial samples for
industrial, pharmaceutical, and agricultural purposes. One process of bacteria retrieval that is
used for a handful of strains is the collection, isolation, and growth of a desired strain by
applying a collected sample to a plate of trypticase soy agar (Fitchett 2015, unreferenced
personal communication). Bacteria may be cultivated by the process of enriching cultures, where
conditions are set up to promote the growth of a specified strain of bacteria while preventing
other, unwanted strains from forming. The enrichment culture technique focuses on creating or
maintaining conditions such as pH, temperature, bacterial competition, moisture, and bacterial
food sources. The purpose of these conditions is to force the growth of bacteria that fit into a
very specific niche defined by the set conditions (Chawla et al. 2013). The purpose of the
environmental isolate culture technique is to identify and utilize naturally occurring bacteria
towards practical aforementioned purposes. Furthermore, once a potentially useful bacterium is
found and isolated in a specific region, the enrichment culture technique is used to ensure the
growth of said bacteria (Chawla et al. 2013). Therefore, with the application of these culture
techniques, bacteria can be manipulated to serve the purposes of the modern world.
The purpose of this study was to isolate, characterize, and identify an unknown species of
bacteria collected from soil in Flagstaff, Arizona. Morphological and physiological
characteristics of an unknown species of soil bacteria were compared with characteristics of
known bacterial species using Bergey’s Manual of Systematic Bacteriology (Holt 1984). The
bacteria cultivated in this experiment are predicated to be bacteria adapted to high altitude,
because Flagstaff has alkaline soil, moderate to high slopes, and high precipitation. These
conditions are caused by proximity to the atmosphere, an absence of nitrogen, and topographical
variance, which influence the growth of bacteria such as Deschampsia flexuosa, Dicentra
peregrina, Pinus pumila, and Stellaria nipponica within mountainous regions, like those found
in Japan (Koichi and Yoshinori 2014).
MATERIALS AND METHODS
The original environmental isolate bacteria were isolated from soil within Flagstaff using
a cotton swab, which was applied to a trypticase soy agar plate via a growth streak. After the
initial culture of environmental isolate bacteria was grown, additional trypticase soy agar plates
were saturated with the environmental isolate bacteria via an isolation streak. The isolation
streaks eventually produced a singular type of bacteria; the purity of the culture was confirmed
via wet mount slides, a simple stain, and a Gram stain. The environmental bacteria were
simultaneously observed on trypticase soy agar plates for growth patterns and morphology, and
trypticase soy agar slants were used to observe growth patterns. The environmental isolate
bacteria were streaked onto two screw-top trypticase soy agar slants and left to grow before the
first slant was stored at room temperature for two weeks while the second slant was stored at
fridge temperature for two weeks. The screw-top trypticase soy agar slants were taken out of
storage after the two week period and restreaked onto new trypticase soy agar slants, resulting in
a growth period that determined the favorable storage condition. Bergey’s manual volume 2 was
used to identify the environmental isolate bacteria because the bacteria were positive for the
Gram stain.
Aseptic technique was used with all stains. The simple stain was performed on bacteria
obtained from a trypticase soy agar plate. The purpose of using the simple stain is to observe the
morphology of the environmental isolate bacteria. The bacterial slide was dry mounted, heat
fixed, and stained with methylene blue dye. The slide was observed using bright-field
microscopy with 1000x magnification and oil immersion. The expected positive result of the
simple stain is the observation of blue stained cells.
The capsule stain was performed on bacteria obtained from a trypticase soy agar plate
towards the purpose of confirming whether or not the environmental isolate bacteria has a
capsule surrounding the cell membrane. The positive control organism used for the capsule stain
is Klebsiella pneumonia, which was obtained from a trypticase soy agar plate. The bacterial slide
was dry mounted, where the bacteria was emulsified with congo red dye and spread across the
slide. After the bacteria were emulsified, the sample was air dried and stained with Maneval's
dye. The slide was observed under bright field view with 1000x magnification and oil
immersion. The expected positive result of the capsule stain is the observation of clear halos
around the bacteria, which is stained red along with the background of the sample. The expected
negative result of the capsule stain is the absence of clear halos around the bacteria, which is
stained red along with the background of the sample.
The Gram stain was performed on bacteria obtained from a trypticase soy agar plate. The
purpose of using the Gram stain is to identify the presence or absence of an outer membranous
layer and to identify the thickness of the peptidoglycan layer of the environmental isolate
bacteria. The positive control organism used for the Gram stain is Bacillus megaterium, and the
negative control organism used for the Gram stain is Escherichia coli, both of which were
obtained from a bacterial broth. The bacterial slide was dry mounted and heat fixed, after which
crystal violet dye was used as a stain, Gram's iodine was used as a mordant, 70% ethanol was
used as a decolorizing agent, and safranin dye was used as a counterstain. The slide was
observed under bright field view with 1000x magnification and oil immersion. The expected
positive result of the Gram stain is the observation of purple stained cells, while the expected
negative result of the Gram stain is the observation of pink stained cells.
The acid-fast stain was performed on bacteria obtained from a trypticase soy agar plate.
The purpose of obtaining these bacteria is to observe the morphology of the environmental
isolate bacteria. The positive control organism used for the acid-fast stain is Mycobacterium
smegmatis and the negative control organism used for the acid-fast stain is Bacillus megaterium,
where the Mycobacterium smegmatis was obtained from trypticase soy agar while the Bacillus
megaterium was obtained from a bacterial broth. The bacterial slide was dry mounted and heat
fixed before carbol fuchsin dye was driven into the cells via heat. After the heating, acid alcohol
was used to decolorize the bacteria and methylene blue dye was used to counterstain the sample.
The slide was observed under bright field view with 1000x magnification and oil immersion. The
expected positive result of the acid-fast stain is the observation of pink stained cells, while the
expected negative result of the acid-fast stain is the observation of blue stained cells.
The endospore stain was performed on bacteria obtained from a trypticase soy agar plate.
The purpose of obtaining these bacteria is to observe the morphology of the environmental
isolate bacteria. The control organism used for the endospore stain is Bacillus megaterium. The
bacterial slide was dry mounted and heat fixed before malachite green was driven into the cells
via heat. After the heating, the sample was decolorized with the use of RO water and
counterstained with the use of safranin dye. The slide was observed under bright field view with
1000x magnification and oil immersion. The expected positive result of the endospore stain is
the observation of red or pink cells with green spores, while the expected negative result of the
endospore stain is the absence of visible spores. All methods are from Shand and Fitchett (2015).
The purpose of the catalase test is to evaluate if the bacteria have the capability to defuse
reactive oxygen species via the catalase enzyme, which the bacteria may or may not have. The
catalase test requires that hydrogen peroxide to be used as the primary reagent, where H2O2 is
applied to a glass slide with a sample of environmental bacteria. In the catalase test
Staphylococcus epidermidis is used as the positive control, and Streptococcus lactis is used as
the negative control. The expected positive result of the catalase test is the presence of bubbles,
while the expected negative result of the catalase test is the absence of bubbles. The results for
the catalase test were recorded on October 16th, 2015.
The purpose of the oxidase test is to evaluate if there are oxidase enzymes in the
cytochrome of the environmental isolate bacteria. The catalase test involves applying the
environmental bacteria to p-phenylenediamine squares to potentially initiate a chemically-based
color change. The positive control organism for the oxidase test is Pseudomonas aeruginosa.
The expected positive result for the oxidase test is a color change to dark purple, and the
expected negative result for the oxidase test is the absence of a color change. The oxidase test
results were recorded on October 16th, 2015.
The purpose of the carbohydrate test is to determine if the environmental isolate
bacteria has the capability to ferment various types of sugars. The carbohydrate test involves the
application of glucose, sucrose, mannose, and lactose; the test requires that the environmental
bacteria be emulsified with each carbohydrate broth. The expected positive result for the
carbohydrate test is a color change to yellow while the expected negative result for the
carbohydrate test is an absence of color change with the sample remaining red, and along with
the positive test, the solution can be scored for the production of gas along with acid. The results
for the carbohydrate test were recorded on October 16th, 2015.
The purpose of the oxygen requirement test is to evaluate what type of respiration the
environmental bacteria uses, whether the method used is anaerobic or aerobic. The oxygen
requirement test involves the use of thioglycollate broth with emulsified environmental isolate
bacteria, which is meant to grow at the level at which the bacteria best survives at based on
oxygen availability. The expected positive result for the oxygen requirement test is the growth of
bacteria at the bottom of the test tube, the top of the test tube, or across the entire test tube. The
expected negative control for the oxygen requirement test is an absence of bacterial growth. The
test results were recorded on October 16th, 2015.
The purpose of the nitrate reduction test is to determine if the environmental isolate has
the capability to reduce nitrate to nitrite, ammonia, or nitrogen. The nitrate reduction test uses
nitrate broth, where the environmental isolate bacteria is emulsified within the nitrate broth, then
exposed to Nitrite A/B reagent and zinc powder. The expected positive result of the nitrate
reduction test is the presence of gas in the Durham tube, a color change to red after the
application of Nitrite A/B reagent, and an absence of color change after the application of zinc
powder. The expected negative result of the nitrate reduction test is the absence of gas in the
Durham tube, an absence of color change after the application of Nitrite A/B reagent, and a color
change to red after the application of zinc powder. The results of the nitrate reduction test were
recorded on October 21st, 2015.
The purpose of the motility test is to evaluate if the environmental isolate has flagella
and, thereby, a means to travel. The motility test involves the environmental isolate being
stabbed into a motility medium of triphenyltetrazolium chloride. The expected positive result is
the presence of a red cloud around the stab pathway, while the expected negative result is the
absence of a red cloud around the stab pathway, where the red coloration is contained within the
stab pathway. The test results for the motility test were recorded on October 16th, 2015.
The purpose of the Simmons’ citrate test is to determine whether the environmental
isolate has the capability to transport citrate and use citrate as a carbon source. The Simmons’
citrate test utilizes Simmons’ citrate agar with brom thymol blue as the medium which is stabbed
and streaked with environmental isolate. The expected positive result for the Simmons’ citrate
test is a color change from green to blue, the expected negative result for the Simmons’ citrate
test is the absence of a color change. The test results for the motility test were recorded on
October 16th, 2015.
The purpose of the urea hydrolysis test is to evaluate whether the environmental isolate
bacteria contain urease and whether the bacteria have the capability to break down urea. The urea
hydrolysis test utilizes a urea broth that consists of phenol red, yeast extract, and urea, where the
environmental isolate bacteria is emulsified into the broth and left to grow. The expected positive
result of the urea hydrolysis test is a color change from yellow to pink, while the expected
negative result of the urea hydrolysis test is the absence of a color change. The test results for the
urea hydrolysis test were recorded on October 16th, 2015.
The purpose of the Kligler’s iron agar test is to determine whether the environmental
isolate bacteria is capable of producing hydrogen sulfide. The Kligler’s iron agar test requires
that the environmental isolate bacteria be stabbed into a medium containing ferrous sulfate,
glucose, lactose, and phenol red. The expected positive result for the Kligler’s iron agar test is
the presence of a dark precipitate, while the expected negative result for the Kligler’s iron agar
test is the absence of a dark precipitate. The test results were recorded on October 16th, 2015.
The purpose of the gelatinase test is to determine whether the environmental isolate can
utilize the polypeptides and amino acids contained within gelatin, which can be broken down
with the presence of the enzyme gelatinase. The gelatinase test requires that the environmental
isolate bacteria be stabbed into a gelatin media that contains protein, peptone, beef extract, and
gelatin. The expected positive result of the gelatinase test is that the gelatin remains in liquid
form after being chilled, and the expected negative result of the gelatinase test is that the gelatin
forms into a solid after being chilled. The test results for the gelatinase test were recorded on
October 21st, 2015.
The purpose of the starch hydrolysis test is to evaluate if the environmental isolate
bacteria is capable of breaking down starch via a-amylases enzymes. The test involves applying
the environmental isolate bacteria, as well as other bacteria, to a plate of starch medium, waiting
for growth, and applying Gram’s iodine. The positive control for the starch hydrolysis test is
Bacillus Megaterium and the negative control for the starch hydrolysis test is Escherichia coli.
The expected positive result of the starch hydrolysis test is the presence of clear zones around the
bacteria, and the expected negative result of the starch hydrolysis is the absence of clear zones
around the bacteria. The test results for the starch hydrolysis test were recorded on October 21st,
2015.
The purpose of the casein hydrolysis test is to evaluate the capability of the
environmental isolate bacteria to break down casein. The casein hydrolysis test involves
applying the environmental isolate bacteria, along with other bacteria, to a plate of casein that
contains protein. The positive control organism for the casein hydrolysis test is Bacillus
megaterium, and the negative control organism for the casein hydrolysis test is Escherichia coli.
The expected positive result of the casein hydrolysis is the presence of clear zones around the
bacteria, and the expected negative result of the casein hydrolysis is the absence of clear zones
around the bacteria. The test results for the casein hydrolysis test were recorded on October 21st,
2015.
The purpose of the lipid hydrolysis is to determine the capability of the environmental
isolate to break down lipids via the use of lipase enzymes. The lipid hydrolysis test involves the
application of environmental isolate bacteria, along with other bacteria, to a plate of spirit blue
agar. The positive control organism for the lipid hydrolysis test is Serratia spp., and the negative
control organism for the lipid hydrolysis test is Staphylococcus epidermidis. The expected
positive result of the lipid hydrolysis test is the presence of a clear zone around the bacteria,
while the expected negative result of the lipid hydrolysis test is the absence of a clear zone
around the bacteria. The test results for the lipid hydrolysis test were recorded on October 16th,
2015.
The purpose of the facultative anaerobe test is to evaluate if the environmental isolate
bacteria is capable of surviving under anaerobic conditions. The facultative anaerobe test
involves placing a streaked trypticase soy agar plate in an anaerobic chamber. The expected
positive results of the facultative anaerobe test is the presence of bacterial life after the
incubation period, the expected negative results of the facultative anaerobe test is the absence of
bacterial life after the incubation period. The test results for the facultative anaerobe test were
recorded on October 21st, 2015.
The purpose of the biofilm test is to determine whether or not the environmental isolate
bacteria contain a biofilm. The biofilm test requires that glucose TSB be emulsified with
environmental isolate bacteria and then mixed with crystal violet solution. The positive control
organism used for the biofilm test is Staphylococcus aureus, and the negative control organism
used for the biofilm test is Staphylococcus epidermidis. The expected positive result for the
biofilm test is the presence of a biofilm layer on the top of the solution, the expected negative
result for the biofilm test is the absence of a biofilm layer. The test results for the biofilm test
were recorded on October 21st, 2015.
RESULTS
The original plate grew colonies of various sizes, some of which were circular and other
of which were filamentous, with the circular colonies being yellow while the filamentous
colonies were white. Purity was confirmed by the absence of oddly shaped molecules and the
presence of clear rods within the wet mounted slide; the simple stain showed blue-stained rods
without the presence of other organisms; the Gram stain showed purple stained rods and did not
include the presence of black colored organisms such as yeast. The environmental isolate
bacteria were found to be white with a filamentous configuration, a filamentous margin, a flat
elevation, and a rhizoid growth pattern. After a two week period, the storage condition test of the
environmental isolate bacteria proved that the bacteria are best suited to (insert results).
The stains provided results based on the morphology and color of the environmental
isolate bacterial cells. The simple stain was rod-shaped, as indicated by the observation of rod-
shaped blue-stained cells (Table 1). The Gram stain was positive, as indicated by the observation
of purple cells (Table 1). The capsule stain was negative, as indicated by the observation of a
dark blue background, red cells, and an absence of a clear zone around each cell (Table 1). The
endospore stain was positive, as indicated by pink cells with green spores within and around the
cells (Table 1). The acid-fast stain was negative, as indicated by blue cells (Table 1).
Bubbles were observed for the catalase test, so the test was positive (Table 2). The
reaction area turned dark purple for the oxidase test, so the test was positive (Table 2). The
medium was yellow and gas was absent for the glucose test, so the test was positive for acid
(Table 2). The medium was yellow and gas was absent for the sucrose test, so the test was
positive for acid (Table 2). The medium was red and gas was absent for the lactose test, so the
test was negative for reaction but positive for growth (Table 2). The medium was yellow and gas
was absent for the mannose test, so the test was positive for acid (Table 2). The test tube retained
turbidity across the entire test tube and on a band around the top for the oxygen requirements
test, so the test was positive for facultative anaerobes (Table 2). (insert nitrate results here). A red
cloud formed around the stab pathway for the motility test, so the test was positive (Table 2). A
green medium was observed for the Simmons’ citrate test, so the test was negative (Table 2). The
medium was pink for the urea hydrolysis test, so the test was positive (Table 2). No dark
precipitate was formed within the stab pathway for the Kligler’s iron agar test, so the test was
negative (Table 2). (insert gelatinase results here). (insert starch results here). The test for casein
was inconclusive because the positive control and the casein plates in lab were all defective,
therefore no experiment could be performed. Clear zones were observed around the bacteria for
the lipid hydrolysis test, so the test was positive (Table 2). (insert fac anaerobe results). (insert
biofilm results).
Table 1. Gram, simple, capsule, acid-fast, and endospore stain results for environmental
isolate bacteria cells taken from Flagstaff soil.
Characteristics Results
Cell Morphology Rod
Gram Stain Positive
Capsule Stain Negative
Endospore Stain Positive
Acid-Fast Stain Negative
Table 2. Biochemical test results for environmental isolate bacteria taken from Flagstaff
soil.
Characteristics Result
Catalase Positive
Oxidase Positive
Carbohydrate Fermentation Positive (glucose, mannose, sucrose)
Growth Without Reaction (lactose)
Oxygen Requirements Facultative Anaerobe
Nitrate Reduction
Motility Positive
Simmons’ Citrate Negative
Urea Hydrolysis Positive
Kligler’s Iron Agar Negative
Gelatinase
Starch Hydrolysis
Casein Hydrolysis Inconclusive
Lipid Hydrolysis Positive
Facultative Anaerobe Positive
Biofilm
DISCUSSION
The purpose of the environmental isolate project is to identify a bacterium based on
biochemical tests performed on isolated bacterial samples obtained from soil in Flagstaff,
Arizona. The purpose was accomplished as environmental bacteria were successfully isolated
from Flagstaff soil and identified through a series of stains and biochemical tests. The ability to
utilize catalase allows the organism to defuse oxidative species that can cause harm to the cells.
The presence of oxidase allows the organism to perform the electron transport chain in a very
efficient manner. The ability to ferment monosaccharides allows the environmental isolate
bacteria to establish electron carriers that revitalize energy production (González-Cabaleiro et al.
2015). Because the environmental isolate is a facultative anaerobe, these bacteria can use both
fermentation and aerobic respiration when oxygen is present and absent. (Insert explanation of
nitrate). The presence of motility allows the organism to escape predators and to perform both
aerotaxis and chemotaxis. The absence of citrase means that the environmental isolate cell does
not have the potential to create bioluminescence, which is not universal among all citrase
positive cells. Citrase is typically used to form new colonies that establish symbiotic
relationships with animals; therefore the environmental isolate does not have symbiotic contact
with animals (Septer et al. 2015). In this case, the environmental isolate bacteria is unable to
accommodate host organisms with bioluminescence for camouflage, sexual attraction, and
predation, therefore this bacteria cannot receive safety or nutrition from a host organism (Pooley
2011). The environmental isolate is urea positive, meaning that the bacteria have a steady supply
of nitrogen for use in amino acids, which are constructed into proteins (Oliveira et al. 2009). The
inability to produce H2S for the Kligler’s iron agar test indicates that the organism is unable to
utilize cysteine found within sulfur compounds, which can be used as an antioxidant
(Maheswari, 2014). (insert explanation of gelatinase). (insert explanation of starch). The ability
to breakdown lipids indicates that the organism is able to separate fatty acids from glycerol,
which allows the organism to utilize another energy source. (insert explanation of biofilm).
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