Georgia Department of Human
Resources Division of Public Health
Two Peachtree Street NW
Suite 15-470 Atlanta, Georgia 30303-3186 Tel: (404) 657-2700 Fax: (404) 657-2715
Laboratory Procedures for the identification of Bacillus
anthracis
The procedures described below function to rule out
presumptively identified Bacillus 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.
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.
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 minutes 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 hour 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.
Materials
required:
·
Sporocidal
disinfectant
·
Sputum
cup
·
Sterile
cotton swabs
·
Blood
culture collection kit
·
Stool
collection cup
Cutaneous
anthrax
·
Vesicular
stage: The organism is best demonstrated in this stage. Soak two dry sterile swabs in vesicular
fluid from a previously unopened vesicle.
·
Eschar
stage: Rotate two swabs beneath the
edge of the eschar without removing the eschar.
·
If
the patient is able to produce a stool specimen, stool cultures should be
performed.
·
In
later stages of disease, blood cultures will yield the organism, especially if
specimens are obtained prior to antibiotic treatment.
·
If
respiratory symptoms are present and sputum is being produced, obtain a
specimen for culture and smear.
·
In
later stages of disease (2-8 days post exposure) blood cultures may yield the
organism, especially if specimens are drawn before antibiotic treatment.
·
Class
II Biological Safety Cabinet
·
5%
Sheep blood agar plates [SBA] (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)
·
Motility
media
·
Microscope
·
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)
·
37
°C incubator
·
Inoculate
3 routine media for sputum specimens (i.e. SBA or broth enrichment).
·
Routine
blood culture methods are sufficient.
·
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 in which clear
zones around the bacilli may be evident. The presence of large encapsulated
gram-positive rods in the blood is strongly presumptive for B. anthracis
identification.
·
If
blood culture bottle is positive, perform a Gram stain directly and observe for
rods. These blood cultures should also be subcultured to SBA plates.
·
Use
one swab to inoculate 3 standard media for surface wounds (e.g., SBA or broth
enrichment).
·
Prepare
a smear for Gram staining with the second swab.
·
Routine
stool culture methods are sufficient (e.g., SBA or PEA plates).
·
Do
not use CVA or hectone agar plates.
· If a clinical centrifuge with appropriate biocontainment tube holders is available, centrifuge the CSF specimen at 1500 X g for 15 minutes.
·
Collect
the sediment and prepare a smear for Gram staining.
·
Inoculate
the remainder of the sediment onto SBA and broth enrichment
Nasal
swab specimens
(asymptomatic persons with known inhalation exposure to B. anthracis
or to credible anthrax threats)
·
Streak
a nasal swab from the patient on SBA and incubate at 37șC overnight. The
next day, check for Gamma hemolysis around the colonies and perform Gram stain.
·
Non-hemolytic
colonies that show gram positive rods upon microscopic examination and are
non-motile on the motility media (or by wet mount), are suspected to be Bacillus
species. Although B. anthracis is a
spore-forming bacterium, the bacilli may or may not contain spores.
·
In
addition, the presence of capsules can be checked microscopically after
staining with India ink.
·
Cultures
should be incubated at 35-37° C under ambient conditions.
·
Cultures
should be examined within 18-24 hours of incubation.
·
Growth
of B. anthracis may be observed as early as 8 hours after inoculation.
·
After incubation of SBA plates for
15-24 hours at 35-37° 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.
·
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 beta-hemolytic.
However, weak hemolysis may be observed under areas of confluent growth
in aging cultures and should not be confused with beta-hemolysis.
B.
anthracis
grows rapidly; heavily inoculated areas may show growth within 6-8 h and
individual colonies may be detected within 12-15 hours. This trait can be used to isolate B.
anthracis from mixed cultures containing slower-growing organisms.
Interpretation of results:
B.
anthracis
is a large gram-positive rod (1-1.5 X 3-5 ”m) that forms oval, central-to
subterminal spores (1 X 1.5 ”m) on SBA that do not cause significant swelling
of the cell. Spores may not always be
observed from a 24-hour SBA plate. Absence of spores alone should not be used
to rule out B. anthracis. Spores are not present in clinical samples
unless exposed to atmospheric levels of CO2; CO2 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% CO2 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.
Confirmation of B. anthracis at the state lab. GPHL has the reagents
necessary for direct fluorescent antibody (DFA) test for the capsules and the
cell wall polysaccharides. If both stainings are positive, B. anthracis is confirmed.
In addition, phage lysis can be performed at GPHL as another confirmatory test.
The DFA test is a rapid test and only takes 4 hours to complete.
Patient testing should be handled by the local hospital labs and clinics, as a part of their routine procedures described above. In case a local hospital laboratory is unable to perform the presumptive tests, the specimens should be sent to a contract lab and/or to GPHL for testing.
If
bioterrorism is suspected, local hospitals and clinics should contact
local law enforcement officers/FBI for referring the specimens to GPHL. In
addition, any suspected B. anthracis
culture should be submitted to GPHL for confirmatory testing.
Reference:
http://www.bt.cdc.gov/Agent/Anthrax/Anthracis20010417.pdf
Dr. Elizabeth Franko 404-327-6803 Answering service
Dr. Mahin Park 404-327-7905 770-578-4104
Marsha Ray 404-327-7990/1
Bill Cheek 404-327-7990/7