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WEEK10 ASSIGNMENT3 COURSE PROJECT
BACILLUS SUBTILIS

Guide Book


1


Week10 Assignment3 Course Project
Bacillus subtilis

Guide Book

Laura Stuckwisch

Biology 2071

Microbiology

May 29, 2012

Instructor: Dr. John Oprandy

South University Online







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BACILLUS SUBTILIS

Guide Book


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Chapter1:
Identifying Morphological Characteristics

The identification of an unknown organism has to start somewhere. The clues as to what microorganism unknown
microorganism #4
were limited to

the morphological characteristics, then the Gram stain color of the unknown microorganism, and
then the biochemic
al test results.
The morphological characteristics include:

Morphological Characteristics of Unknown Microorganism Template


Morphological Characteristics on Nutrient Agar

Unknown
microorgani
sm code no.

Shape

Size

Margin

Pigmentation

Appearance

Texture

Elevation

Optical
Property



4




Irregular

Large

Entire

Tan

Dull

Rough

Raised

Opaque


With these descriptions the best microorganisms morphological characteristics were the
Clostridium
difficile

and
Bacillus subtilis.
Clostridium
difficile

are 4
-
6 mm in diameter, irregular, raised, opaque, and they appear a grey
-
white color after being incubated for 48
hours, (Acumedia, n.d.).
The other hypothesis is that the unknown bacteria are
Bacillus subtilis
.
Bacillus
subtilis

is a Gram
-
positive
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3

bac
terium
, it has a rigid structure outside of the cell, and it has an irregular shape, (Microbe Wiki, 2011). For the hypothesis ther
e had
to be some experiments to take place to help determine what the unknown microorganism was.
Some of the experiments tha
t could be
conducted would be:

1.

Gram
-
staining could be a possible experiment to narrow down the possibilities because this will show whether the bacteria is
gram
-
negative or gram
-
positive. It can show the taxonomy and the cell wall structure of the bacteri
a, (Cowan, M. Talaro, K.
2009, p. 79).

2.

A hanging
-
drop experiment could be used because this allows observation of viable organisms, (Microbugz, n.d.).

3.

Also, the bacteria could be grown on nutrient a
g
ar or blood agar to see how the bacteria grows, and de
termine what their
morphological characteristics are.

Chapter2:
Studying Gram Reaction

The gram nature of the unknown bacteria number four is that the gram stain revealed purple colored rods. Gram staining is
used to distinguish between Gram
-
positive and
Gram
-
negative bacteria. Gram
-
positive bacteria stain
purple

and Gram
-
negative
bacteria stain red, (Microbial Life Educational Resources, 2012). Due to this fact the purple colored rods of the unknown ba
cteria
means that the unknown bacteria is Gram
-
posit
ive.

The hypothesis that seems to fit best with the morphological characteristics from week two and the gram stain from this week
is that
the genus could be either Clostridium
difficile

or
bacillus subtilis
.
Clostridium difficile

is a part of the hypothesis because it fits with
WEEK10 ASSIGNMENT3 COURSE PROJECT
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most of the morphological characteristics.
Clostridium
difficile

are 4
-
6 mm in diameter, irregular, raised, opaque, and they appear a
grey
-
white color after being incubated for 48 hours, (Acumedia, n.d.).

The other hypothesis i
s that the unknown bacteria is
B
acillus
subtilis
.
Bacillus subtilis

is a Gram
-
positive
bacterium
, it has a rigid structure outside of the cell, and it has an irregular shape,
(Microbe Wiki, 2011).


Experiments can be performed to
determine what bacteria are.
Clostridium difficile

is a spore
-
forming bacteria, so if the
bacteria is growing spores that can be used for
Clostridium difficile
. It also lives in the absence of oxygen, so if oxygen is added to the
bacteria and the bacteri
a dies, or if it is grown in a dish with absence of oxygen and it lives then that will help determine if the bacteria
is
Clostridium difficile
, (Kumm, J. 2009).
Bacillus subtilis

is also spore forming so if the bacteria is producing spores in the agar dis
h
this can be and indicator of the bacteria. A test to test for
Bacillus subtilis

is to do a starch hydrolysis test.
Bacillus subtilis

would
need to be grown on starch agar and it would need to be incubated for 24 hours at 37 degrees Celsius. Iodine is
then added to the
plates and the iodine changes color from yellow
-
brown to blue
-
black when it comes into contact with starch. In this test
Bacillus
subtilis
had a positive reaction because it produced a clear zone around the growth, (American Socie
ty for
Microbiology, 2010).
Ba
cillus

produced an enzyme amylase and this hydrolyzed starch in the agar, (American Society for Microbiology, 2010).

Ch3:
Suggesting Biochemical Tests


Over the last few weeks
Clostridium difficile

and
Bacillus subtilis

became the most prominent bacteria for the unknown
organism number four based on the morphological characteristics, and gram staining. Now the biochemical test results have co
me
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into play. The biochemical tests results are as follows: hydrogen sulfide p
roduction, (
-
), hemolysis, (gamma), catalase production, (+),
oxidase production, (+), coagulase production, (
-
), and starch hydrolysis, (+).
Bacillus subtilis

is catalase positive and
Clostridium
difficile
is catalase negative.
Clostridium difficile

is
negative for hydrogen sulfide production, which matches the result of the
biochemical test, however,
Clostridium difficile

also tests negative for lecithinase, lipase, and catalase activity in the biochemical test,
(Bergey’s Manual of Systemic Bacteriology
, 2009, p. 773).
Bacillus subtilis

has gamma
-
hemolysis and
Clostridium difficile

has beta
-
hemolysis, (Booth, S. 2000, p. 107). In the oxidase test if the test is positive the bacteria turns purple and the
Bacillus subtilis

turned
purple in the oxidase tes
t, (American Society for Microbiology, 2012).
Clostridium difficile

is a bacteria that is fermentative, catalase,
and oxidase negative, (Quinn, P J. Markey, B K, Leonard, F C, FitzPatrick, E S. Fanning, S. Hartigan, P J. 2011, p. 233).


Based on all of
the results of the biochemical test that was provided for the unknown organism number four and the individual
result found for
Clostridium difficile

and
Bacillus subtilis

it makes the most sense to rule out
Clostridium difficile
. Therefore,
Bacillus
subti
lis
is the identification of the unknown organism number four because it is the only one left and it fits with the morphological
characteristics, gram staining test results, and the biochemical test results.

Ch4:
Problem Analysis Sheet


One of the uncertainties of the Bacillus genera is that they are rod
-
shaped and according to the morphological characteristics
given in week2 the shape of the unknown microorganism number four was irregular. Also, the other possible for the unknown
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microo
rganism was Clostridium and Clostridium and Bacillus have similar morphological characteristics. Clostridium is large in siz
e,
Gram
-
positive, and rod
-
shaped just like the Bacillus genus.


There is a family called Bacillaceae; this family forms endospore
s, it is Gram
-
positive, and rod
-
shaped. This family has two
main divisions which include Clostridium and Baci
llus, (MicrobiologyBytes, n.d.).


To resolve or handle the uncertainties about the unknown organism number four being Clostridium or Bacillus bio
chemical
tests need to be performed. They are very similar in morphological characteristics and some biochemical tests, but they are
not
identical. Morphological characteristics can help narrow down possible genus, but the best way is through biochemical

tests because
they are specific to certain genus.

The results of the biochemical tests can provide the solution to the uncertainties of whether the unknown microorganism is
Clostridium or Bacillus. For example, on the Bergey’s Manual gram stain and mor
phological flow chart it says that both Clostridium
and Bacillus are Gram
-
positive, and that they are both spore forming. It also shows that Clostridium is positive for strict anaerobes
and Bacillus is negative for strict anaerobes. Therefore, the Bergey
’s Manual flowchart and the results of different biochemical tests
can narrow the possibilities down.

Ch5:
Creating a Microbial Profile of the Unknown Microorganism

WEEK10 ASSIGNMENT3 COURSE PROJECT
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The following template is a Microbial Profile to help piece together the unknown organisms test results. The Microbial
Profile:

Microbial Profile Template


Unknown microorganism code number: ___4__________

Tests (which
tests have been
used?)

Note: Refer

to
your
microorganism
identification
flow chart.

Purpose of the
test

Observation
and test
results (+/
-
)

Microbial
characteristics
(what does the
test reveal about
the
microorganism?)

Additional tests
(which
confirmatory tests
will support the
identified
c
haracteristics of
the
microorganism?)

Starch
hydrolysis

Tests to see if
the organism
can produce
exoenzymes,
(Microbugz,
+

This helps to
show the shape of
the
microorganism.

Endospore test to
show if the
microorganism
produces
endospores.

WEEK10 ASSIGNMENT3 COURSE PROJECT
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n.d.).

Catalase

production

Shows that
the organism
is able to
break down
hydrogen
peroxide into
water and
oxygen, (Pier,
G.B. Lyczak,
J.B. Wetzler,
L.M. 2004).

+

This shows any
enzymes in the
microorganism.

Motility test to
show is the
microorganism is
motile or not.

Ce
ll diameter
greater than or
equal to 1um

To rule out
any species
that are less
than this
width.

+

This shows how
large the
microorganism is.


SBA agar

B. subtilis

grows well in
SBA agar

+

B. subtilis

grows
well on this so
this could confirm

WEEK10 ASSIGNMENT3 COURSE PROJECT
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B. subtilis

MacConkey
agar

B. subtilis

does not grow
well on
MacConkey
agar

-

B. subtilis

does
not grow well of
this so this could
rule out other
Bacillus species.


Gamma phage
lysis

No other
Bacillus
species is +
for this test so
this can
confirm or
rule out B.
subtilis

+

This can rule out
or confirm
B.
subtilis.




After all of the weeks of receiving information about the unknown organism number four, such as the morphological
characteristics and biochemical tests, the identification of unknown microorganism number four is
Bacillus
subtilis
. Some of the
characterist
ics of
Bacillus
subtilis

are that it is Gram
-
positive, it is large and rod shaped, and its size is 3 to 5 micrometers by 1.2
micrometers and its colonies form in long chains, (Pier, G.B. Lyczak, J.B. Wetzler, L.M. 2004).

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Bacillus
subtilis

is also endosp
ore forming and it has to have oxygen to maintain normal aerobic metabolism.
Bacillus
subtilis

can also breakdown hydrogen peroxide into water and oxygen which shows that it is catalase
-
positive, (Pier, G.B. Lyczak, J.B.
Wetzler, L.M. 2004).


The starch test shows that the microorganism produces specific exoenzymes, such as a
-
amylase and olig
-
1, 6
-
glucosidease; all
of these enzymes hydrolyze starch which is why the test is called starch hydrolysis, (Microbbugz, n.d.).


The catalase production
test shows that
Bacillus
subtilis

can breakdown hydrogen peroxide into water and oxygen, (Pier, G.B.
Lyczak, J.B. Wetzler, L.M. 2004).


There are different agar plates that can help identify
Bacillus
subtilis
.
Bacillus
subtilis

grows well on SBA agar, b
ut it does not
grow well on MacConkey agar. Therefore, if
Bacillus
subtilis

was tested on both the SBA agar and the MacConkey agar at the same
time and the microorganism seemed to grow better on the SBA agar rather than the MacConkey agar then that would
be confirmation
for
Bacillus
subtilis
, (Centers for Disease Control and Prevention, n.d.).


A gamma phage lysis test can also confirm
Bacillus
subtilis
.
Bacillus

subtilis

had to have a test to be able to quickly confirm
the microorganism because of biot
errorism threats. The gamma phage lysis was first developed in the 1950s by the Centers for
Disease Control and Prevention. The U.S. Army Medical Research Institute of Infectious Disease, (USAMRIID), needed to modify

it
to improve its performance and rel
iability, (U.S. Army Medical Research Institute of Infectious Disease, n.d.). The gamma phage
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lysis is specific to
Bacillus
subtilis
. The gamma phage is a virus and it can enter bacterial cells to cause lysis, (U.S. Army Medical
Research Institute of Infectious Disease, n.d.).


To sum up the unknown microorganism number four:

Morphological characteristics:

1.

Shape: irregular

2.

Size: l
arge

3.

Margin: entire

4.

Pigmentation: tan

5.

Appearance: dull

6.

Texture: rough

7.

Elevation: raised

8.

Optical Property: opaque

Biochemical tests:

1.

Gram
-
positive

2.

Hydrogen sulfide production:
-

3.

Hemolysis: gamma

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4.

Catalase Production: +

5.

Oxidase Production: +

6.

Coagulase Product
ion:
-

7.

Starch Hydrolysis: +

With the research and test results that have been found on this unknown microorganism and the morphological characteristics t
hat
have been given the identification of the unknown micro
organism number four is
Bacillus subtilis
.

Ch6:
Determining Useful and Harmful Characteristics of the Microorganism


Bacillus subtilis

is a microorganism that is found in water, soil, air, and decomposing plant residue. This microorganism
produces an endospore so that it can survive in extreme heat and desiccation in the environment, (Biotechnology Program under

the
Toxic Substances Con
trol Act, 2011).
Bacillus subtilis

does not have the traits to caused disease in humans.
Bacillus subtilis

is used
to produce enzymes and specialty chemicals, (Biotechnology Program under the Toxic Substances Control Act, 2011).


Usually the microorganism of
Bacillus subtilis

is found in the water, soil, air, and decomposing plant residue, but it can also be
found in the human body. When it is found in the human body it is usually found on the skin, or in the intestinal tract, (Ki
rk, E. n.d.).
Even though it can be found in the human body it does not usually colonize on the human body, (Kirk, E. n.d.).


It is rare for a clinical disease to be caused by
Bacillus subtilis
. It is said that
Bacillus subtilis

is not a human pathogen

because
its virulence characteristics are low. The only reason why an infection would occur from
Bacillus subtilis

would be if the
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microorganism that was challenging the person was high in number, or the person’s immune status was very low, (Biotechnolog
y
Program under the Toxic Substances Control Act, 2011).


Bacillus subtilis

contributes to nutrient cycling. It contributes to nutrient cycling when it is biologically active due to the
enzymes that it produces, (Biotechnology Program under the Toxic Su
bstances Control Act, 2011).


Ch7:
Determining Measures to Control the Microorganism


The microorganism
Bacillus subtilis
is not a pathogen against humans, and it can be used as a control for other organisms.
Bacillus subtilis
is an organism that occurs naturally in the soil, water, air, and decomposing plant material. It can be used in different
situation as a biological control, (Cornell University, n.d.).

There have been four different strains that were tested for their an
timicrobial potential. The four strains that were tested were
UMAF6614, UMAF6619, UMAF6639, and UMAF8561. These strains were tested for effectiveness against powdery mildew, and
antifungal activity, (PubMed, 2009).


Two strains, UMAF6614 and UMAF6639 we
re found to have high antimicrobial activity in vitro, and they worked best against
Xanthomonas compestris pv. Cucurbitae and Pectobacterium cartovorum subsp. carotovorum, (PubMed. 2009).


The type of heat that kills
Bacillus subtilis
is dry heat. When
Bacillus subtilis

endospores are exposed to moist heat for 15
minutes at 121 degrees Celsius and 134 degrees Celsius it reduces the interleukin
-
6 inducing capacity to 57% and 63%. When the
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Bacillus subtilis
endospores are exposed to dry heat at 220 degree
s Celsius for 45 minutes it reduces the interleukin
-
6 to a capacity of
2%, (Elsevier, 2005).
Bacillus subtilis
is best destroyed by dry heat because dry heat causes DNA damage,(PubMed, 2009).


Bacillus subtilis
is not harmful to humans and it can grow q
uantities of enzymatic substances that can be used in the
food industry, and it is also used in detergent manufacturer facilities, (Hogan, 1998).


Bacillus subtilis
can produce enzymes and special chemicals. It can produce amylase, protease, inosine, ri
bosides, and amino
acids.
Bacillus subtilis
is one of those bacteria that do not cause us harm and are actually beneficial. They can be used in the food
industry and detergent manufacturers, (Biotechnology Program Under the Toxic Substances Control Act (
TSCA), 1997).

Ch8:
Listing Lab Safety Procedures for Handling Microorganism


Bacillus subtilis

is a microorganism that is found in water, soil, air, and decomposing plant residue. This
microorganism produces an endospore so that it can survive in extreme heat and desiccation in the environment, (Biotechnology

Program under the Toxic Substances Con
trol Act, 2011).
Bacillus subtilis

does not have the traits to caused disease in humans.
Bacillus subtilis

is used to produce enzymes and specialty chemicals, (Biotechnology Program under the Toxic Substances Control
Act, 2011).


The type of heat that k
ills
Bacillus subtilis
is dry heat. When
Bacillus subtilis

endospores are exposed to moist heat for 15
minutes at 121 degrees Celsius and 134 degrees Celsius it reduces the interleukin
-
6 inducing capacity to 57% and 63%. When the
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Bacillus subtilis
endosp
ores are exposed to dry heat at 220 degrees Celsius for 45 minutes it reduces the interleukin
-
6 to a capacity of
2%, (Elsevier, 2005).
Bacillus subtilis
is best destroyed by dry heat because dry heat causes DNA damage, (PubMed, 2009).


Bacillus subtilis
is considered a biosafety level 1. Biosafety level 1 means that the microorganisms are not known to cause
disease in healthy adults, (Nuaire, n.d.). The laboratory safety guidelines for biosafety level 1 are minimal. Standard mic
robiol
ogical
safety practices are required, and the laboratory facilities that handle biosafety level 1 microorganisms are required to hav
e a bench
top sink. For
Bacillus subtilis
there is no requirement for safety equipment, (Nuaire, n.d.).


Preserving
Bacil
lus subtilis
is best with the R
-
K fixation. The R
-
K fixation is a freeze
-
etching technique that can produce
structural changes that occur during the procedure of chemical fixation. This is called the Ryter
-
Kellenberger, (R
-
K), fixation, (The
Journal of C
ell Biology, 2012).


The training for handling
Bacillus subtilis
is the training that is required for handling biosafety level 1 microorganisms.
Training for handling biosafety level 1 is standard microbiological practices, (SMPS), safety equipment trai
ning, and training on
laboratory facilities. Also, laboratory personnel must be supervised by a scientist that has training in microbiology, (UTK/
UTIA/GSM
Biosafety, 2008).


All microorganisms in biosafety level 1 including
Bacillus subtilis
are deconta
minated before disposal. They are
decontaminated by a decontaminating method, such as autoclaving. Then, it is placed in a durable, leak
-
proof container with a
biohazard sign on the outside, (MOBSA, 2009).

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Ch9:
Identifying Additional Characteristics of
the Microorganism


The microorganism
Bacillus subtilis
does produce resting cells and these resting cells are called endospores, (Todar, 2012).
The microorganism does have a capsule of an S
-
layer, also known as proteinaceous surface layer. This S
-
layer is a crystalline layer of
proteins on the surface or gly
coprotein subunits, (Todar, 2012). The microorganism
Bacillus subtilis
has a capsule that contains poly
-
D, or L
-
glutamic acid, (Todar, 2012).


Bacillus subtilis
has motility and it able to have swarming motility by using their flagella, (KenyonCollege,
2011).
Bacillus
subtilis
does grow in the presence of oxygen, (Todar, 2012). This organism is an obligate aerobe which
means

that it has to have
molecular oxygen to supply it for it
to

be able to grow, (PearsonEducation, 2006).
Bacillus subtilis
is rod
shaped. This rod shape is
maintained by two polymers, known as peptidoglycan a
nd teichoic acids, (PubMed, 2006
).






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Reference

Acymedia, (n.d.).
Clostridium difficile agar (7385),
Retrieved from
http://www.neogen.com/Acumedia/pdf/ProdInfo/7385_PI.pdf

American Society for Microbiology (2010).
Microbe library,
Retrieved from
http://www.microbelibrary.org/library/laboratory
-
test/3172
-
the
-
starch
-
hydrolysis
-
test

American Society for Microbiology, (2012).
Oxidase test,
Retrieved fro
m
http://www.microbelibrary.org/index.php/library/laboratory
-
test/3287
-
oxidase
-
test

Bergey, (2009).
Bergey’s Manual of Systemic Bacteriology,
3
rd

edition, Pub
lished by Springer Science + Business Media

Biotechnology Program under the Toxic Substances Control Act, (2011).
Bacillus subtilis final risk assessment,
Retrieved from
http://www.epa.gov
/oppt/biotech/pubs/fra/fra009.htm

Booth, S. (2000).
Microbiology pearls of wisdom,
Lincoln, NE, Published by Boston Medical Publishing Corporation

Cowan, M. Talaro, K., (2009).
Microbiology a systems approach,
2
nd

edition,

New York, NY, Published by
McGraw Hill, Retrieved
from myeclassonline.com

Cornell University, (n.d.).
Bacillus subtilis,
Retrieved from
http://web.pppmb.cals.cornell.edu/resourceguide/mfs/01b
acillus_subtilis.php

Definiction.Net, (2011).
Staphylococcus,
Retrieved from
http://www.definitions.net/definition/Staphylococcus

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Dictionary.com, (2012).
Micrococcus,
Retrieved from
http://dictionary.reference.com/browse/micrococcus

Elsevier, (2005).
Dry and moist heat sterilization cannot inactivate pyrogenicity of gram positive microorganisms,
Retrieved from
http://www.sciencedirect.com/science/article/pii/S0928098705002253

Farlex, (2012).
Blood agar,
Retrieved from
h
ttp://medical
-
dictionary.thefreedictionary.com/blood+agar

Hogan, S. (1998).
Bacillus subtilis decoded,
Retrieved from
http://ec.europa.eu/research/rtdinf17/17e10.html

KenyonCollege, (2011).
B
acillus subtilis,
Retrieved from
http://microbewiki.kenyon.edu/index.php/Bacillus_subtilis

Kirk, E. (n.d.).
Bacillus subtilis,
Retrieved from
http://web.mst.edu/~microbio/BIO221_2009/B_subtilis.html

Kumm, J. (2009).
General characteristics of clostridium difficile,
Retrieved from
http://bioweb.uwlax.edu/bio203/s2009/kumm_jakl/growth&adapt.htm

Microbial Life Educational Resources, (2012).
Gram staining,
Retrieved from
http://serc.carleton.edu/microbelife/research_methods/microscopy/gramstain.html

MicrobiologyBytes, (n.d.).
Bacillus,
Retrieved from
http://www.microbiologybytes.com/video/Ba
cillus.html

Microbugz, (n.d.).
Starch hydrolysis,
Retrieved from
http://www.austincc.edu/microbugz/starch_hydrolysis.php

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Microbugz, (n.d.).
Wet mount and hanging drop,
Retrieved from
http://www.austincc.edu/microbugz/wet_mount_and_hanging_drop.php

MicrobeWiki, (2011).
Bacillus subtilis,
Retrieved from
http://microbewiki.kenyon.edu/index.php/Bacillus_subtilis

MOBSA, (2009).
The laboratory biosafety levels,
Retrieved from
http://www.mobsa.org/index.php?option=com_content&view=article&id=16&Itemid=25

Nuaire, (n.d.).
Selecting a biological safety cabinet,
Retrieved from
http://www.nuaire.com/bio
logical
-
safety
-
cabinets/selection
-
guide/

PearsonEducation, (2006).
Oxygen requirements,
Retrieved from
http://academic.pgcc.edu/~kroberts/Lecture/Chapter%206/requireme
nts.html

Pier, G.B., Lyczak, J.B. Wetzler, L.M. (2004).
Bacillus anthracis,
Retrieved from
http://www.ppdictionary.com/bacteria/gpbac/anthracis.htm

PubMed, (2009).
The iturin
-
like lipopeptides are essential components in the biological control arsenal of bacillus subtilis against bacterial
diseases of cucurbits,
Retrieved from
http://www.ncbi.nlm.nih.gov/pub
med/22066902

PubMed, (2012).
Shape determination in Bacillus subtilis,
Retrieved from
http://www.ncbi.nlm.nih.gov/pubmed/17981078

PubMed, (2009).
Spores of bacillus subtilis: their resistance to a
nd killing by radiation heat and chemicals,
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