Bacteria

1. University of Puerto Rico
Cayey Campus
2015 RISE Program
Pérez-Ayala, Michelle C.1, Figueroa-Monsanto, Héctor L.2 , Maricelys3 , and
Cruz, Giovanni4
Department of Biology, University of Puerto Rico at Cayey1, Department of
Chemistry, University of Puerto Rico at Cayey2 , Hugher Hughes Program3, and
RISE Program4
Introduction
Microbiology is the field of science that focuses on the study of the morphology,
application, and relevance of microorganisms, such as bacteria with the use of
microscopes and technical procedures. These microorganisms or specimen,belong to
the Bacteria Domain and Monera Kingdom and are found in moist areas. Among these
places, bacteria can be located on epithelial surfaces, saliva, nail grime, as well as on
top of desks, and even within soils. Actually, these diverse localizations have given
these living organisms specific characteristics that permit them to be either heterotrophic
or non-heterotrophic. Non-heterotrophic or autotrophic microorganisms play an eminent
role in processes, such as nitrogen fixation benefiting plants, but not all bacteria have an
optimistic symbiotic relationship or effect on living organisms.
Heterotrophic organisms are adaptable to many organically formed compositions,
such as humans???, acting positively or negatively. In contrast to the autotrophic
individuals (I don’t think it is appropriate to refer to a bacteria as an individual.),
heterotrophic organisms create negative outcomes on humans. For instance, the
species Treponella palidium causes a skin disease called syphilis. This promotes the
development of sores throughout the body by having contact with bodily fluids through
sexual intercourse. Scientists have focused on not only the nourishment preference of
these microorganisms, but also on their cellular wall composition, which makes them
either gram positive or gram negative.

2. Gram-negative bacteria are more difficult to inhibit with antibiotics compared to gram-
positive ones, due to the double layer of peptidoglycans in their cell wall. Gram-negative
bacteria can be identified under the compound microscope because they retain pink or
red stains from the conservation of safranin during the wet mount and gram staining
methods. In contrast, if the stains are purple, they are classified as gram positive from
crystal violet solution retention followed by the addition and rinse of both iodine and
ethanol. This type of bacteria is more vulnerable to antibiotics because it has only one
layer of peptidoglycan. Surprisingly, uncultured bacteria (gram positive/negative)
methods of recompilation have permitted researchers to find bacterial compounds, such
as teixobactin with the capacity to impede bacteria from emerging (Ling et al. 2015).
Teixobactin is a compound that has shown to be more effective in terms of bacteria
inhibition, compared with other antibiotics, such as ofloaxin (Ling et al. 2015). In addition,
another type of antimicrobial chemical made of lipopeptide is called battacin. It was
isolated from the soil bacterium Paenibacillus tianmuensis and has shown a potent
inhibition effect. For instance, it has the capacity to eradicate bacteria in vitro or in cell
culture, as well as gram-negative bacteria (Qian et al. 2012). Popowska et al. (2012),
have compared the soil bacteria resistance, instead of contrasting antibiotic ability to
inhibit these microrganisms. Specifically, exposing the bacteria to a soil located
underneath animals that contained specific antibiotics with the purpose of testing their
inhibition effects (This sentence lacks clarity.). In other words, uncultured bacterium can
have a wide range of characteristics that can give it the capacity to create resistance to
certain antibiotics.
Both cultured and uncultured bacteria have been the precursors of many antibiotic
chemical compounds that have inhibited disease-causing bacterium. These same
antimicrobials have created resistance or mutations against the same antibiotics. The
resistance effect may occurs 30 years after being placed on the market provoking or
caused serious health issues. For this reason, there is an urgent need to find bacteria-
generated compounds that will serve as long-term effective antimicrobials.
The purpose of this study is to identify if harvested soil collected bacteria will develop
a compound similar in capacity as the teixobactins. Also, in this study, soil cultured
bacteria collected will be grown in order to identify only one type of bacteria genera with

3. the purpose of discovering an antibiotic that is able to inhibit the division capacity of the
bacteria. expand this section.
MATERIALS AND METHODS
Soil Collection from Rhizosphere. Samples were collected from two types of
soils rich in nutrients from the rhizosphere. In order to collect both soil samples,
the rhizosphere digging areas were cleared and a small hole was made on each
surface. Both samples were collected with two individual wrapped plastic spoons
until 25% (1/4 parts) of two zip lock bags were filled, respectively. Pictures were
also taken as evidence of the soil collection. The first collection was from a moist
soil located in an urban area at 26˚C at 146˚ SE Caguas, PR 18˚14’25”N 66˚4’7”
W. The area was surrounded by animals such as ducks, and chickens, among
other. A second sample was collected from a rural area approximately 25 ˚C
during the noon at 8:02a.m. in Patillas, PR 17˚53’31”N 65˚53’18” W.
Weight and Dilution of Soil. One gram of the soil samples was weighted and
diluted, in order to obtain a smaller number of bacteria. Each weighted soil was
added to a 10mL saline H2O and NaCl (0.9%) solution containing test tube and
then mixed on a Vortex, respectively. To diminish the concentration inside each
tube and end up with a fewer number of bacteria, 200µL of each concentration
was transferred to an individual 1.5mL centrifuge tube (100) and 20µl of NaCl
were also added. Five different 1.5mL tubes (10-1, 10-2, 10-3, 10-4, 10-5) were filled
with 180µL of NaCl. To complete the dilution, 20µL were transferred from the 100
to the 10-1 and the same process was repeated until 10-5. The TSA and R2A agar
plates were smeared with 30µL of the 100 and 10-5 tubes to grow the bacteria at
a temperature of 30˚C during twenty four hours incubation.
Purification of Bacteria. The medium plates that showed bacterial growth were
purified to eliminate unnecessary debris and to isolate one type of colony,

4. according to its morphology. In purification #1 to #5, an inoculator loop was used
to swap a group of colonies off the R2A 100 plate onto three areas (North, East,
and South) of a new R2A agar. Using a sterilized loop, the colonies were first
smeared in a zig-zag motion on the North side of the plate. Then, the plate was
closed, the loop was sterilized on the Bunsen burner, the plate cover was
opened, the loop was pressed carefully on a corner of the plate where no
bacteria was present, the bacteria were dragged from the previous section
(North) to the next (East) several times, and at the last movement was zig-zag
motion on the East. The same process was performed for the South. The new
R2A plate was stored inside the incubator at 30˚C and the first R2A 100 plate
was placed in the refrigerator. The same process was performed with the TSA
100 plate, prepared on the previous process using a new TSA plate.
Gram Staining. An inoculator loop was used to swap one colony from a purified
plate and rubbed and mixed with a drop of deionized water onto a microscope
slide. Crystal violet was added followed by water to rinse off the excess purple
dye. The same process was repeated with iodine, ethanol, and saphranin. The
slide was then observed under the microscope to recover an image and
determine the same colony morphology (color, margin, elevation, size, texture,
appearance, pigmentation, and optical property).
Freezing. This process is meant primarily to save a portion of the bacteria for
further use. First, 1.5mL of the broth, equivalent liquid media, and 200µL of
glycerol, which allows the bacteria colony to freeze but not to die at low
temperatures, were deposited in a 2.0mL micro tube. Then the tubes were
labeled for further usage and tests for other parts of investigation.
Electrophoresis. This technique was utilized to characterize the DNA by band
sizes, but in order to have an appreciable amount the DNA must be replicated by
using PCR (polymerized chain reaction). This process seeks to replicate and
amplify the DNA that is to be studied by setting it into different temperatures

5. specifically: 95˚C which denaturalizes the DNA breaking the chains of DNA, next
a temperature of 60 ˚C for the process of annealing were the base pairing is
taking place and finally extending the DNA chains at about 72 ˚C. After the
replication proceeding to run the PCR adding the replicated DNA with an amount
of dye because the DNA does not exhibit color and wouldn’t be visible in the gel.
The DNA, RNA and proteins at the electrophoresis are measured based on a
sample that runs the entire gel better referred to as the ladder. Once the
electrophoresis is finished, it was read and the length of how far the DNA ran
was determined identifying it by its size or charge.
Antibiotic Resistance Test. Using the corresponding media plate with specific
antibiotics randomly testing upon five of any antibiotics in order to detect if our
bacteria has any resistance against any of those. Striking with the selected
antibiotics and placing a test paper with the bacteria broth for easy identification
of any resistance. It was verified after incubating it at 30 ˚C for a day.
Antibiotic Production Test. The bacteria M.Luteous and E. Coli were each
smeared in a media plate. A test paper was soaked with the bacteria broth and
placed in the plates. The broth is a previously fixed mix with 1.5mL of the
equivalent liquid media and left to stir and maintain an aqueous character. This
test will result in the creation of a ring all around that will mean the bacteria does
produce antibiotic, but if not it means it doesn’t present this particular
characteristic.
Oil Degradation. In order to test the susceptibility of the bacteria, a plate that
has only a media of oil was made. The selected bacterium was streaked into the
oiled plate for testing. The main purpose of this test is to verify if the bacteria will
either feed of the oil surface or repel against it.
An inoculator loop was used to swap one colony from a purified plate and rubbed
and mixed with a drop of deionized water onto a microscope slide. Crystal violet was
added followed by water to rinse off the excess purple dye. The same process was

6. repeated with iodine, ethanol, and saphranin. The slide was then observed under the
microscope to recover an image and determine the same colony morphology including
(color, margin, elevation, size, texture, appearance, pigmentation, and optical property).
2.5 Freezing
This process was meant primarily to save a portion of the bacteria for further use.
First, 1.5mL of the broth, an equivalentamount of liquid media, and 200µL of glycerol,
which allows the bacteria colony to freeze but not die at low temperatures, were
deposited in a 2.0mL micro tube. Then the tubes were labeled for further usage and
tests for other parts of investigation.
2.6 Electrophoresis
DNA was replicated and with the Polymerized Chain Recation (PCR).
Electrophoresis was utilized to characterize the DNA by band sizes The PCR setting
involved different temperatures specifically: 95˚C, which denaturalizes the DNA breaking
the chains of DNA, next a temperature of 60 ˚C for the process of annealing were the
base pairing takes place and finally extending the DNA chains at about 72 ˚C. After the
replication, ethidium bromide was added to the DNA copies to work as the fluorescent
tag. The DNA, RNA, and proteins in the electrophoresis were divided based on size. The
DNA with the tag and the DNA ladder that reflects the base pairs were added the
agarose gel. Once the electrophoresis was finished, the gel was observed to compare
the DNA of the bacteria with the 16S gene that codifies for samples that are bacteria.
2.7 Antibiotic Production Test
The bacteria M. luteous and E. coli were tested for antibiotic resistance caused
by mutation by placing each bacterium on agar plates. Four test papers were soaked
with the bacteria broth, previously fixed mix with 1.5mL of the equivalent liquid media
and left to stirred for and maintain an aqueous character. Two papers were placed on
each agar plate with the bacterium.
2.8 Oil Degradation
To test if the bacteria would either feed off the oil surface or repel against it, the
bacterium that was positive on the electrophoresis gel was streaked onto a plate of oil
media for an incubation period of seven days at 30 ˚C.
3. Results
3.1 Soil collection from rhizosphere
The first soil collection area is shown in Figure 1. it includes the flora and lake
that surrounds it. The main animals that commonly pass through this area are chickens,

7. ducks, and dogs. Both this and the parameters included in Table1. show that there is a
probability of finding bacteria in the gathered soil.
Figure 1. a, Shows the tree next to the soil collection
area where the animals, including humans tread roughly or calmly. b, The whole at the
left of the bag less than a meter away from the tree trunk. c, The whole was about an
inch deep surrounded by green grass and it contained roots from the same tree.
The second soil collection area is shown in Figure 2. it, which includes the flora
and shows absence of no water sources around. Only domestic animals such like dogs,
cats, chickens, iguanas and humans pass through by this area. Figure 2 Both this and
the parameters included in Table1. show that there is a probability of finding bacteria.
Figure 2. a, Shows the soil collection area approximately near to many types of trees. b,
The hole was a fairly deep one with an approximate radius of three inches. c, The hole
within had grass roots, dirt pebbles, and small organisms worm like.
Characteristics First soil collection Second soil collection
1. Temperature (˚C) 26˚C 25˚C
2. Coordinates 18˚14’25”N 66˚4’7” W 17˚53’31”N 65˚53’18” W
3. City, State 146˚ SE at Caguas, PR Patillas, PR
4. Soil Description It had a moist texture with
visible grass and roots mixed
with the soil particles
It was moist during the morning, it
lacked roots, and it had little
organisms.
5. Surroundings The area was surrounded by
animals such as ducks and
chickens, among other
The area included avocado
cannon trees and a dog
Table 1. These parameters reflect the circumstances of the collection area, as well as its
location.
a, b, c,

8. 3.2 Weight and dilution of soil
Approximately, 1.033±0.001 grams of the soil sample was weighted and diluted in six
tubes (100
, 10-1
, 10-2
, 10-3
, 10-4
and 10-5
) to obtain a smaller number of bacteria. Only the
100
tube previously spread in the TSA and R2A agar plates shown in Figure 2.
Demonstrated showed bacteria colonies, after a weekend incubation at about 23˚C.
These bacteria might be either registered in the DNA database or they may ight be a
new genus or species. The 10-5
plate shown in Figure 3. had no bacteria growth and the
plates were discarded. These might have been due to a higher presence of the aqueous
NaCl.
a, b,
Figure 2. a, Shows the TSA 100
colonies with an extensive morphology explained in the
Purification bacteria results. b, R2A 100
bacteria colonies were different from the
TSA’s.
a, b,
Figure 3. a, Shows that the TSA 10-5
solution had no bacteria. b, R2A 10-5
had the same
effect.
3.3 Purification of bacteria
From the first soil collection, The TSA 100
colony morphology is shown in Table
2. and Table 3. shows the morphology for R2A 100
bacteria.
TSA100
morphology Purr.#1 Purr.#2 Purr.#3

9. 1. Shape Circular Circular Circular
2. Margin Curled Curled Entire
3. Elevation Flat Flat Convex
4. Size Moderate Punctiform, small and
moderate
Moderate
5. Texture Smooth Smooth Smooth
6. Appearance Dull Dull Dull
7. Pigmentation Cream Cream-yellow Cream
8. Optical property Opaque Opaque Opaque
Table 2. TSA colony morphology.
R2A 100
morphology
Purr.#1 Purr.#2 Purr.#3 Purr.#4 Purr.#5
1. Shape Circular Circular Circular Circular and
irregular
Circular
2. Margin Entire and
curled
Curled Undulated
and entire
Curled Rhizoid
3. Elevation Flat Flat Raised Flat Flat
4. Size Punctiform
and small
Small or
punctiform
Punctiform,
small,
moderate and
large
Punctiform,
small,
moderate and
large
Punctiform,
small and
moderate
5. Texture Smooth Smooth Smooth Smooth Smooth
6. Appearance Dull Dull Dull Dull Dull
7. Pigmentatio
n
White White White White White
8. Optical
property
Opaque Opaque Opaque Opaque Opaque
Table 3. R2A colony morphology.
TSA 10-5 and
colonies demonstrated different morphology when had a different
purification.Purification #1 for TSA revealed a circular shape, curved margins , flat
elevation, small, smooth texture, dull appearance, cream pigmentation, and an opaque
optical property. The R2A 10-5
bacteria had more defined shape circular, curved with flat
elevation, punctifor, with a smooth texture, dull appearance, white pigmentation, and the
same optical property. For purification #2 the TSA 10-5
plate showed bacteria with
irregular shape, curled margin, flat elevation with sizes that vary from a type of cloth that
covers the whole plate, , smooth texture, dull appearance, a cream-yellow pigmentation,
and an opaque optical property. The R2A colonies showed the same shape, margin, and

10. elevation. The sizes were small or linear due to the streaking. They , smooth texture, dull
appearance, white pigmentation, and an opaque optical property. The R2A (puur3)
bacteria colonies had a cloch like appearence, an undulated and entire margin, flat
elevation, punctiform at the edges, small, moderate and large sizes, smooth texture, dull
appearance, white pigmentation, and opaque optical property. The R2A (puur.4) showed
irregular shape, curled margin, flat elevation, all sizes, smooth texture, dull appearance,
white pigmentation and opaque optical property. R2A (purr.5) presented linear shape, an
entire margin, flat elevation, punctiform size, smooth texture, dull appearance, cream
pigmentation and opaque optical property.
3.4 Gram staining
From the first soil collection, both the TSA and R2A colonies showed purple
pigmentations. The colonies were Streptobacillus gram negative, according to Figure 4.

11. a, b,
c,
d,
Figure 4. a,b, Shows that the R2A 100
colonies are gram negative and Streptobacillus.
c,d, sShows the Streptobacillus gram negative bacteria for TSA 100
.
From the second soil sample according to Figure 5. when doing Gram staining the
TSA Streptococcus colored in purple therefore tested to be gram positive. Also the R2A
was proven to be Streptobacillus pink therefore tested to be gram negative.

12. Figure 5.
3.5 Freezing
From the first soil sample two tubes Figure 6. each with 1.5 mL of broth, 0.2 mL of
glycerol and the R2A colonies.
a, b,
c, d,
Figure 6. a, Freezing of TSA bacteria. b, TSA back up. c, R2A frozen bacteria. d, R2A
back up.
3.6 Electrophoresis

13. The R2A copies of the PCR were positive for the 16S gene, according to the
electrophoresis gel in Figure 7. This shows that the DNA copies belonged to bacteria.
Figure 7. a, The R2A DNA in line 4, without counting the DNA ladder, was positive to the
16S gene.
3.7 Antibiotic production test
M. Luteous was inhibited only by TSA 100
, according to Figure 6 a,b,. E. coli on Figure
8. c,d, was not inhibited neither by TSA nor by R2A. This shows that TSA bacteria might
have an antibiotic property on M. luteous.
a, b,

14. c, d,
Figure 8. a, b, Show that TSA bacteria had an antibiotic resistance on M. luteus, but R2A
did not. c, d, E. coli was had no inhibition.
3.8 Oil degradation
The oil degradation test for the bacteria was negative, according to Figure 9. This
shows that the bacteria will not be useful to degrade oil in the development of oil-derived
products.
Figure 9. The plate was from another oil test. It shows the same negative result that
displayed both soil collection bacteria.
4. Cited Literature
Ling LL, Schneider T, Peoples AJ, Spoering AL, Engels I, Conlon BP, Mueller A,
Sch¨aberle TF, Hughes DE, Epstein S et al. 2015. A new antibiotic kills pathogens
without detectable resistance. Nature 517, 455–459. doi: 10.1038/nature14098.
Popowska M, Rzeczycka M, Miernik A, Krawczyk-Balska A, Walsh F, Duffy B. 2012.
Influence of soil use on prevalence of tetracycline, streptomycin, and erythromycin
resistance and associated resistance genes. Antimicrobial Agents and Chemotherapy
53 (3): 1434-43. doi: 10.1128/AAC.05766-11.

15. Qian CD, Wu XC, Teng Y, Zhao WP, Li O, Fang SG, Huang ZH, Gao HC. 2012. Battacin
(octapeptin b5), a new cyclic lipopeptide antibiotic from Paenibacillus tianmuensis
active against multidrug-resistant gram-negative bacteria. Antimicrobial Agents and
Chemotherapy 56(3): 1458-1465. doi: 10.1128/AAC.05580-11.