**BASIC\
LABORATORY PROTOCOLS\
\
\
FOR THE PRESUMPTIVE\
IDENTIFICATION OF**\
\
\
\
\
***Bacillus anthracis***\
\
\
\
\
**CDC**\
\
**Centers for Disease Control\
and Prevention**\
\
\
\
\
\
\
\
\
\
\
This protocol is designed to provide laboratories\
with techniques to identify microorganisms, in order to\
support clinicians in their diagnosis of potential diseases.

Credits:\
\
**Subject Matter Experts:**\
\
Robbin S. Weyant, Ph.D.\
Chief, Special Bacteriology and Reference Laboratory\
Centers for Disease Control and Prevention\
\
John W. Ezzell, Jr., Ph.D.\
Chief, Special Pathogens Branch\
United States Army Medical Research Institute of\
Infectious Diseases\
\
Tanja Popovic, M.D., Ph.D.\
Chief, Epidemic Investigations Laboratory\
Centers for Disease Control and Prevention\
\
\
**Technical** **Editors:**\
\
Kimberly Quinlan Lindsey, Ph.D.\
Laboratory Education and Training Coordinator\
Bioterrorism Preparedness and Response Program\
Centers for Disease Control and Prevention\
\
Stephen A. Morse, M.S.P.H., Ph.D.\
Deputy Director, Laboratory Services\
Bioterrorism Preparedness and Response Program\
Centers for Disease Control and Prevention

Table of Contents\
\
*Bacillus anthracis* Identification\
\
I. [Introduction]{.underline}\
\
1. Overview\
2. Human infection\
3. Antibiotic therapy\
4. The anthrax vaccine\
\
II. [Laboratory procedures for the identification of *B.
anthracis*]{.underline}\
\
1. General\
2. Collection of clinical specimens\
3. Materials needed for processing of clinical specimens\
4. Isolation from clinical specimens\
5. Incubation and examination of cultures\
6. Differential tests for the presumptive identification of *B.
anthracis*\
6.a. Colony characteristics of *B. anthracis*\
6.b. Gram stain morphology of *B. anthracis*\
6.c. India Ink staining of clinical specimens (i.e. blood, CSF) for
capsule\
6.d. Motility test: wet mount and motility medium\
7. Presumptive identification key for *B. anthracis*\
8. Actions if a presumptive *B. anthracis* colony is identified and
suspected as a bioterrorist threat agent\
9. Listed vendors\
\
III. [Appendix]{.underline}: General overview flowchart

I. Introduction\
\
**1.** **Overview**\
\
Anthrax is a zoonotic disease that is transmissible to humans through
handling or consumption of contaminated animal products. The etiologic
agent of anthrax, *Bacillus anthracis*, is a spore forming gram-positive
bacillus. Although anthrax can be found globally in temperate zones, it
is more often a risk in countries with less standardized and less
effective public health programs. Areas currently listed as high risk
are South and Central America, Southern and Eastern Europe, Asia,
Africa, the Caribbean, and the Middle East. In these regions,
herbivorous wildlife mammals, such as deer, wildebeest, elephants, and
domesticated livestock, such as goats, sheep, cattle, horses, and swine,
are at highest risk for disease. These animals usually become infected
while grazing on contaminated land, eating contaminated feed or drinking
from contaminated water holes. *B. anthracis* spores can remain viable
in soil for many years. Anthrax infrequently occurs in livestock in
North America; however, anthrax outbreaks have been reported among deer
from Louisiana and Texas up through the Midwest and among wood buffalo
in the Northwest Territory in Canada. Animal infections in the United
States are reported most often in Texas, Louisiana, Mississippi,
Oklahoma, and South Dakota. Birds, amphibians, reptiles, and fish are
not directly susceptible to anthrax infection. However, some carnivorous
mammals, such as dogs, lions, and omnivorous mammals such as swine, may
be susceptible to anthrax infection through consumption of meat from
infected animals.\
\
**2. Human infection\
**\
Humans can become infected with *B. anthracis* by handling products or
consuming undercooked meat from infected animals. Infection may also
result from inhalation of *B. anthracis* spores from contaminated animal
products such as wool or the intentional release of spores during a
bioterrorist attack. Human-to-human transmission has not been reported.
Three forms of anthrax occur in humans: cutaneous, gastro-intestinal,
and inhalational.\
\
2.a. [Cutaneous anthrax]{.underline}\
Cutaneous infections occur when the bacterium or spore enters a cut or
abrasion on the skin, such as when handling contaminated wool, hides,
leather or hair products (especially goat hair) from infected animals.
Skin infection begins as a raised itchy bump or papule that resembles an
insect bite. Within 1-2 days, the bump develops into a fluid-filled
vesicle, which ruptures to form a painless ulcer (called an eschar),
usually 1-3 cm in diameter, with a characteristic black necrotic (dying)
area in the center. Pronounced edema is often associated with the
lesions because of the release of edema toxin by *B. anthracis*. Lymph
glands in the adjacent area may also swell. Approximately 20% of
untreated cases of cutaneous anthrax result in death either because of
the infection becomes systemic or because of respiratory distress caused
by edema in the cervical and upper thoracic regions. Deaths are rare
following appropriate antibiotic therapy, with lesions becoming sterile
within 24 h and resolving within several weeks.\
\
2.b. [Gastrointestinal anthrax]{.underline}\
The gastrointestinal form of anthrax may follow the consumption of
contaminated meat from infected animals and is characterized by an acute
inflammation of the intestinal tract. Initial signs of nausea, loss of
appetite, vomiting, and fever are followed by abdominal pain, vomiting
of blood, and severe diarrhea. The mortality rate is difficult to
determine for gastrointestinal anthrax but is estimated to be 25%-60%.\
\
2.c. [Inhalation anthrax]{.underline}\
This form of anthrax results from inhaling *B. anthracis* spores, and is
most likely following an intentional aerosol release of *B. anthracis*.
After an incubation period of 1-6 days (depending on the number of
inhaled spores), disease onset is gradual and nonspecific. Fever,
malaise, and fatigue may be present initially, sometimes in association
with a nonproductive cough and mild chest discomfort. These initial
symptoms are often followed by a short period of improvement (ranging
from several hours to days), followed by the abrupt development of
severe respiratory distress with dyspnea (labored breathing),
diaphoresis (perspiration), stridor (high-pitched whistling
respiration), and cyanosis (bluish skin color). Shock and death usually
occur within 24-36 h after the onset of respiratory distress, and in
later stages, mortality approaches 100% despite aggressive treatment.
Physical findings are usually nonspecific. The chest X-ray is often
pathognomonic (disease-specific) revealing a widened mediastinum with
pleural effusions, but typically without infiltrates.\
\
*B. anthracis* can be detected by Gram stain of the blood and by blood
culture with routine media, but often not until late in the course of
the illness. Only vegetative encapsulated bacilli are present during
infection. Spores are not found within the blood, partially because
CO~2~ levels in the body inhibit sporulation. Studies of inhalation
anthrax in non-human primates (i.e., rhesus monkeys) showed that bacilli
and toxins appear in the blood within 2-3 days of exposure. The
appearance of toxins coincides with the appearance of bacilli in the
blood, and tests are available to rapidly detect the toxins.\
\
**3.** **Antibiotic therapy\
\
**Most *B. anthracis* strains are sensitive to a broad range of
antibiotics. Penicillin, ciprofloxacin, or doxycycline are usually
recommended for the treatment of anthrax. To be effective, treatment
should be initiated early. If left untreated, the disease is highly
fatal.\
\
**4. Anthrax vaccine\
**\
An anthrax vaccine for humans is licensed for use in the United States.
The vaccine is a cell-free filtrate that contains protective antigen and
alum. The vaccine is reported to be 93% effective in protecting against
cutaneous anthrax. Animal studies have suggested that the vaccine may
also be protective against aerosol challenge. The anthrax vaccine is
distributed by BioPort Corporation, Lansing, Mich.\
\
*B. anthracis* is considered a potential biological warfare threat
agent. The U.S. Department of Defense recommends anthrax vaccination of
all U.S. active duty military personnel. According to the Advisory
Committee for Immunization Practices (ACIP) civilians who should receive
anthrax vaccine include persons who come in contact in the workplace
with imported animal hides, furs, bonemeal, wool, animal hair
(especially goat hair), and bristles and persons engaged in diagnostic
or investigational activities which may put them in contact with anthrax
spores. The vaccine should be administered only to healthy men and women
aged 18-65 years since all studies to date have been conducted
exclusively in that population. Pregnant women should not be vaccinated,
because it is not known whether the anthrax vaccine can cause fetal
harm.\
\
The anthrax vaccination protocol consists of 3 subcutaneous injections
given 2 weeks apart followed by 3 additional subcutaneous injections
given at 6, 12, and 18 months. Annual booster injections of the vaccine
are required to maintain immunity. Mild local reactions consisting of
slight tenderness and redness at the injection site of the skin occur in
approximately 30% of recipients. A moderate local reaction can occur if
the vaccine is given to anyone with a past history of anthrax. Severe
local reactions occur infrequently and consist of extensive swelling of
the forearm in addition to the local reaction. Systemic reactions
characterized by flu-like symptoms occur in fewer than 0.2% of
vaccinees.

II\. Laboratory Procedures for the identification of *Bacillus
anthracis*\
\
**1. General\
**\
The procedures described below function to rule out presumptively
identified *B. anthracis* in clinical specimens or isolates. These
procedures should be performed in microbiology laboratories that utilize
Biological Safety Level 2 (BSL 2) practices. Laboratory coats and gloves
shall be worn when processing specimens and performing tests. Safety
glasses or eye shields are recommended. Any activities that bring hands
in contact with mucosal surfaces (for example, eating, drinking,
smoking, or applying make-up) are prohibited. Hands should be washed
before leaving the laboratory. Anthrax vaccination is not required.\
\
Disclaimer. Names of vendors or manufacturers are provided as examples
of suitable product sources and inclusion does not imply endorsement by
the Centers for Disease Control and Prevention, the United States Public
Health Service, Department of Health and Human Services, the United
States Army, or the Federal Bureau of Investigation.\
\
1.a. [Handling of samples]{.underline}\
**\
**For safety considerations, analysis of samples for biological threat
agents is performed within a certified Class II biological safety
cabinet (BSC). Procedures requiring removal of items from a BSC, such as
slides for microscopy, should follow published microbiological practices
and precautions. When using a BSC, assure that the cabinet does not
contain unnecessary items that will interfere with proper airflow and
function. As for any procedure involving infectious materials, standard
personal protective gear should be used, such as latex gloves and
laboratory coats, or disposable over garments. Additional respiratory
protection should also be considered with materials or analytical
procedures determined to be potentially hazardous outside the BSC. Once
a biological agent has been identified, modifications in handling of
samples can then be considered.\
\
1.b. [Decontamination]{.underline}\
\
Commercially available household bleach solutions contain 5.25%
hypochlorite and, when diluted 1:10, are effective in routine
decontamination of surfaces and instruments after working with *B.
anthracis*. Contaminated items such as pipettes, needles, loops, and
microscope slides should be immersed in decontamination solution until
autoclaving. Work surfaces, such as a biological safety cabinet (BSC),
should be wiped down before and after use with decontamination solution.
The method of decontamination of a spill depends upon the nature of the
spill. Spills involving fresh cultures or samples known to have low
concentrations of spores should be flooded with decontamination solution
and soaked for 5 min before cleanup. Spills that involve samples with
high concentrations of spores, involve organic matter, or occur in areas
of lower than room temperature (refrigerators, freezers) should be
exposed to decontamination solution for at least 1 h before cleanup.
Personnel involved in the cleanup of any spill should wear gloves,
safety glasses, and a laboratory coat or gown during the cleanup
process. Respiratory protection should be considered for spills in which
a substantial aerosolization is suspected.\
\
**2. Collection of clinical specimens\
**\
2.a. [Materials]{.underline}\
\
Sporocidal disinfectant\
Sputum cup\
Sterile cotton swabs\
Blood culture collection kit\
Stool collection cup\
\
2.b. [Cutaneous anthrax]{.underline}\
\
2.b.1. Vesicular stage: The organism is best demonstrated in this stage.
Soak two dry sterile swabs in vesicular fluid from a previously unopened
vesicle.\
2.b.2. Eschar stage: Rotate two swabs beneath the edge of the eschar
without removing the eschar.\
\
2.c. [Gastrointestinal anthrax\
\
]{.underline}2.c.1. If the patient is able to produce a stool specimen,
stool cultures should be performed. Obtain a stool culture.\
2.c.2. In later stages of disease, blood cultures will yield the
organism, especially if specimens are obtained prior to antibiotic
treatment.\
\
2.d. [Inhalation anthrax\
]{.underline}\
2.d.1. If respiratory symptoms are present and sputum is being produced,
obtain a specimen for culture and smear.\
2.d.2. In later stages of disease (2-8 days post exposure) blood
cultures may yield the organism, especially if specimens are drawn
before antibiotic treatment.\
\
**3. Materials needed for processing of clinical specimens\
**\
5% Sheep blood agar plates \[SBA\] (BD Bioscience or Remel, Inc. or
equivalent)\
MacConkey agar plates (BD Bioscience or Remel, Inc. or equivalent)\
Phenyl ethyl alcohol agar (PEA) plates (for stool specimens)(BD
Bioscience or Remel, Inc. or equivalent)\
Trypticase soy broth (BD Bioscience or Remel, Inc. or equivalent)\
Clean glass microscope slides\
Sterile cotton swabs (commercially available specimen transport swabs
for aerobic culture are preferred)\
Disposable bacteriologic inoculation loops\
Clinical centrifuge with appropriate biocontainment tube holders\
Sporicidal disinfectant (0.5% sodium hypochlorite or 0.5% calcium
hypochlorite)\
\
**4. Isolation from clinical specimens[\
]{.underline}\
**4.a. [Sputum specimens]{.underline}\
\
Inoculate 3 routine media for sputum specimens (i.e. SBA, MacConkey
agar, broth enrichment).**\
\
**4.b. [Blood specimens\
]{.underline}**\
**4.b.1. Routine blood culture methods are sufficient.\
4.b.2. There may be enough organisms in the blood to see them on direct
smears by Gram stain. *B. anthracis* appears as short chains of 2-4
cells which are encapsulated as evidenced by clear zones around the
bacilli. The presence of large encapsulated gram-positive rods in the
blood is strongly presumptive for *B. anthracis* identification.**\
**4.b.3. If blood culture bottle is positive, perform a Gram stain
directly and observe for encapsulated rods. These blood cultures should
also be subcultured to SBA and MacConkey agar plates.\
\
4.c. [Swab specimens]{.underline}**\
\
**4.c.1. Use one swab to inoculate 3 standard media for surface wounds
(e.g., SBA, MacConkey agar, or broth enrichment).\
4.c.2. Prepare a smear for Gram staining with the second swab.**\
\
**4.d. [Stool specimens]{.underline}**\
\
**4.d.1. Routine stool culture methods are sufficient (e.g., SBA,
MacConkey agar, or PEA plates).\
4.d.2. Do not use CVA or hectone agar plates.**\
\
**4.e. [CSF specimens]{.underline}**\
\
**4.e.1. If a clinical centrifuge with appropriate biocontainment tube
holders is available, centrifuge the CSF specimen at 1500 X g for 15
minutes.\
4.e.2. Collect the sediment and prepare a smear for Gram staining.\
4.e.3. Inoculate the remainder of the sediment onto SBA and broth
enrichment media (tryptic soy broth or thioglycollate).**\
\
5. Incubation and examination of cultures\
\
**5.a. Cultures should be incubated at 35-37^o^ C under ambient
conditions.\
\
5.b. Cultures should be examined within 18-24 h of incubation. Growth of
*B. anthracis* may be observed as early as 8 h after inoculation.**\
\
6. Differential tests for the presumptive identification of *B.
anthracis\
***\
**6.a. Colony characteristics of *B. anthracis*\
**\
6.a.1. After incubation of SBA plates for 15-24 h at 35-37^o^ C, well
isolated colonies of *B. anthracis* are 2-5 mm in diameter. The flat or
slightly convex colonies are irregularly round, with edges that are
slightly undulate (irregular, wavy border), and have a ground-glass
appearance. There are often comma-shaped projections from the colony
edge, producing the \"Medusa head\" colony.\
\
6.a.2. Colonies on SBA usually have a tenacious consistency. When teased
with a loop, the growth will stand up like beaten egg white. In contrast
to colonies of *B. cereus* and *B. thuringiensis*, colonies of *B.
anthracis* are not β-hemolytic. However, weak hemolysis may be observed
under areas of confluent growth in aging cultures and should not be
confused with β-hemolysis.\
\
6.a.3. When examining primary growth media, it is important to compare
the extent of growth on SBA plates with that on MacConkey agar plates.
*B. anthracis* grows well on SBA, but does not grow on MacConkey agar.
*B. anthracis* grows rapidly; heavily inoculated areas may show growth
within 6-8 h and individual colonies may be detected within 12-15 h.
This trait can be used to isolate *B. anthracis* from mixed cultures
containing slower-growing organisms.\
\
\
\
\
\
\
![](media/image1.png){width="2.3in" height="1.9in"}\
\
\
\
\
\
**Figure 1**. *B. anthracis* and *B. cereus* colony morphology.
Overnight cultures of *B. cereus*\
(left side of plate) and *B. anthracis* (right side) on SBA.

**6.b. Gram stain morphology of *B. anthracis*\
**\
6.b.1. [Procedure]{.underline}\
\
Perform Gram stain by usual procedures.\
\
6.b.2. [Interpretation of results]{.underline}\
**\
***B. anthracis* is a large gram-positive rod (1-1.5 X 3-5 μm) that
forms oval, central-to-subterminal spores (1 X 1.5 μm) on SBA that do
not cause significant swelling of the cell. Spores are not present in
clinical samples unless exposed to atmospheric levels of CO~2~; CO~2~
levels within the body inhibit sporulation. Vegetative cells seen on
Gram stain of blood and impression smears are in short chains of 2-4
cells that are encapsulated. However, cells from growth on SBA under
ambient conditions, are not encapsulated and occur as long chains of
bacilli. When grown on nutrient agar in the presence of 5% CO~2~ or on
other basal media supplemented with 0.8% sodium bicarbonate, virulent
strains will yield heavily encapsulated bacilli. The capsule can be
visualized microscopically using India Ink.\
\
\
**\
**\
\
![](media/image2.png){width="2.3in" height="1.4in"}\
\
\
\
\
\
\
\
\
\
\
\
\
**Figure 2**. Gram stain of *B. anthracis* from SBA\
Magnification 1,000X.**\
**\
\
\
**\
**

**6.c. India ink staining of clinical samples (blood and CSF) for
capsule\
\
**6.c.1. [Purpose]{.underline}\
\
India ink is useful for improving visualization of encapsulated *B.
anthracis* in clinical samples such as blood, blood culture bottles, or
cerebrospinal fluid (CSF).\
\
6.c.2. [Materials\
]{.underline}\
Microscope slides\
Cover glasses\
India ink (Bactidrop, Remel, Inc., Catalog \# 21-518 or equivalent).\
Microscope with 100X oil immersion objective\
\
6.c.3. [Controls\
]{.underline}\
a) Control strains\
(1) Positive control: *Klebsiella pneumoniae* on SBA or equivalent *\
* (2) Negative control: *E. coli* ATCC 25922 or equivalent\
b) Method controls: Perform the test with suspensions of fresh cultures
of the control strains. Control strains should be assayed on each day of
testing.\
c) Resolving an out-of-control result: check the purity and identity of
the control strains and repeat the test.\
\
6.c.4. [Procedure\
]{.underline}\
a) For the controls, transfer a small amount of growth (1 mm diameter)
from each control SBA plate into 0.5 ml whole EDTA-treated blood or
serum. Mix.\
b) For the unknowns, take 100 μl of sample (blood, CSF)\
c) Transfer 5-10 μl of unknown or control to a slide, place a cover
glass on the drop, and then add 5-10 μl of India ink to the edge of the
cover glass.\
d) After the ink diffuses across, view the cells using 100X oil
immersion objective with oil on top of the cover glass.

![](media/image3.png){width="2.079861111111111in"
height="1.9368055555555554in"}\
\
\
\
\
**Figure 3.** India ink staining of *B. anthracis*\
\
\
\
\
\
\
\
\
6.c.5. [Interpretation of results\
]{.underline}\
The capsule will appear as a well-defined clear zone around the cells
for the positive control. No zone should be present in the negative
control.\
\
ACKNOWLEDGEMENTS:\
CDC would like to thank the following contributors to the India ink
protocol:\
J. Edward Brown, Ph.D., Office of Product Development and Regulatory
Affairs\
COL Kelly Davis, DVM, Chief, Pathology Division\
Teresa G. Abshire, Microbiologist, Special Pathogens Branch\
United States Army Medical Research Institute of Infectious Diseases

**6.d. Motility test: Wet mount or motility medium\
**\
6.d.1. [Purpose]{.underline}\
\
This test determines the motility of suspect isolates. *B. anthracis* is
a nonmotile species. This characteristic is unusual among *Bacillus*
species and is therefore useful in the preliminary identification of *B.
anthracis* isolates. Two methods are given: the wet mount and the tubed
motility test.\
\
6.d.2. [Materials\
]{.underline}\
a) For wet mount procedure**\
**Precleaned microscope slides\
Cover glasses\
Sterile distilled water\
Disposable bacteriologic inoculating loop\
Light microscope with 40X objective and 10X eyepiece\
Sterile glass tube\
\
b) For tubed motility test\
Tubed motility media (Remel, Inc., Catalog \# 06-1408 or equivalent), 5
ml per tube\
Sterile disposable 1 μl inoculating loop or needle\
\
6.d.3. [Controls]{.underline}\
a) Control strains\
(1) Positive control: *Pseudomonas aeuroginosa* ATCC 35032 or
equivalent\
(2) Negative control: *Acinetobacter* spp*.* ATCC 49139 or equivalent\
b) Method controls: Perform the test with fresh cultures of the control
strains using the same method as with unknowns. Control strains should
be assayed on each day of testing.\
c) Resolving an out-of-control result: Check the purity and identity of
the control strains and repeat the test.\
\
6.d.4. [Procedures\
]{.underline}Wet mount and motility[\
\
]{.underline}a) [Wet mount procedure]{.underline}\
(1) Deliver 2 drops (approximately 0.1 ml) of sterile distilled water
into the sterile glass tube.\
(2) Using the inoculating loop, sample a suspicious colony from a 12-20
h culture and suspend the growth in the water. (Alternatively, a loopful
of medium from a fresh broth culture can be used).\
(3) Transfer 1 drop of the suspension to the microscope slide and
overlay with the cover glass.\
(4) Examine the slide under the microscope using the 40X objective
(total magnification = 400X).\
(5) Discard slides in 0.5% hypochlorite solution.\
\
b) [Motility test medium procedure]{.underline}\
(1) Using the sterile inoculating needle, remove some growth from an
isolated, suspicious colony of an 18-24 h culture.\
(2) Inoculate the motility tube by carefully stabbing the needle 3-4 cm
into the medium and then drawing the needle directly back out so that a
single line of inoculum can be observed.\
(3) Incubate the tube aerobically at 35-37^o^ C for 18-24 h.\
\
c) [Interpretation of results]{.underline}\
(1) For wet mount: Motile organisms can be observed moving randomly
throughout the suspension. Nonmotile organisms either fail to move or
move with Brownian motion.\
(2) For motility test medium: Nonmotile organisms, such as *B.
anthracis*, will form a single line of growth that does not deviate from
the original inoculum stab. Motile organisms will form a diffuse growth
zone around the inoculum stab.\
\
**7.** **Presumptive identification key for *B. anthracis\
*\
**7.a. From clinical samples, such as blood, CSF, or lesion material:
encapsulated gram-positive rods.\
7.b. Gram-positive, broad rod, spore-positive: *Bacillus* species.\
7.c. Spores are nonswelling and oval shaped; ground glass appearance of
colonies: *Bacillus* morphology group 1 (includes *B. anthracis*, *B.
cereus*, *B. thuringiensis*, and *B. cereus* var. mycoides)\
7.d. Nonmotile: *B. anthracis* and *B. cereus* var. mycoides\
7.e. Nonhemolytic: presumptive *B. anthracis*\
\
**8.** **Actions if a presumptive *B. anthracis* colony is identified
and suspected as a bioterrorist threat agent\
\
**8.a. Preserve original specimens pursuant to a potential criminal
investigation.\
8.b. Contact local FBI, state public health laboratory, and state public
health department.\
8.c. Local FBI agents will forward isolates to a State health department
laboratory as is necessary. Consultation with a state health department
laboratory is strongly encouraged as soon as *B. anthracis* is suspected
as a bioterrorist threat agent.\
\
**9. Listed vendors\
\
**American Type Cell Culture \[ATCC\], 800-638-6597\
Remel, Inc., 800-255-6730\
Becton-Dickinson Bioscience \[BD\], 800-675-0908

III\. Appendix*\
\
*\
***Bacillus anthracis* *ID Overview* *Flowchart\
***