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Model D Bacteriological Diagnosis of Anthrax
 Objective
Isolation and confirmation of Bacillus anthracis from clinical material.
Processing of specimens
Handle all specimens as per guidelines provided in
sections/SOP for biosafety measures (see 5.2, 5.5)
Vesicular fluid
 Prepare two smears.
Stain one by Gram stain and other by
polychrome methylene blue stain.
Follow the method and interpretation given in
SOP C.
Inoculate vesicle fluid onto sheep blood agar
(SBA) and nutrient broth (NB) using swab or Pasteur pipette.
Incubate plates at 37 °C for 18–24 hours.
Read the plates for colony characters:
Examine NB for floccular
deposit and surface pellicle and use this suspension for checking motility of
the isolate either by hanging drop method or by any other reliable method.
Hanging drop preparation 15
B. anthracis is a non-motile
organism. This characteristic is unusual among other Bacillus species.
Place a cover slip on a small piece of
paper on the table inside the BSC.
Place a small drop of mineral oil in
each corner of the cover slip.
Place a flat drop of the broth culture
in the centre of the cover slip, taking care not to have contact with the
oil.
Invert a cavity slide over the cover
slip and press gently to allow the oil to spread and form a complete seal.
Turn the slide, so that the cover slip
is on top.
Inspect the drop to be sure it is
"hanging" freely.
Examine under the microscope, focussing first on the edge of the drop with low power
and then switching to high power magnification.
Ensure proper light adjustment by using
light reflected by the concave mirror, partially closing the iris diaphragm
and lowering the condenser, so as to have dim but adequate light to clearly
view the hanging drop.
Observe the motility:
Discard the slide and cover slip into 10
% hypochlorite solution.
A preliminary report on culture may be sent with
presumptive diagnosis.
Examination of culture smears
Pick up a very small portion of the suspected
colony with a sterile loop.
Mix on a slide with a loopful
of sterile normal saline.
Spread out gently to form a thin smear.
Allow the smear to air dry and heat fix.
Stain with Gram stain and malachite green.
Observe spores.
Malachite green stain for spores 16
Dry the films and fix with minimal flaming.
Place the slide over a beaker of boiling
water, resting it on the rim with the bacterial film uppermost.
When, within several seconds, large droplets
have condensed on the underside of the slide, flood it with 5% aqueous
solution of malachite green. Leave to act for one minute and let the water
continue to boil.
Wash in cold water.
Treat with 0.5% safranine
or 0.05% basic fuchsin for 30 seconds.
Wash and dry.
This method colours the spores
green and the vegetative bacilli red.
Modified Ziehl-Neelsen
stain for spores 16
Air dry and heat fix the smear.
Cover the smear with carbol
fuchsin.
Heat for 3-5 minutes.
Do not allow the stain to boil.
Wash with water.
Decolourize with
alcohol.
Wash with water.
Counterstain with methylene blue.
Wash with water.
Dry in air.
Observe under oil immersion.
Spores will be stained red and vegetative forms blue.
Special tests
Induction of capsule formation 17
In blood
Transfer a pinhead quantity of growth from a
suspect colony to 2.5 ml defibrinated sheep or
horse blood in a sterile test tube or small bottle.
Incubate 5–18 hours at 37 oC.
Transfer a drop with a 1 m l
loop immersed to the bottom of the unshaken bottle or tube to a microscope
slide and make a thin smear.
Stain and examine as described in SOP C.
On bicarbonate agar plates
Plate the suspect colony onto
bicarbonate/serum agar.
Incubate overnight at 37 °C under a 10–20% CO2
atmosphere .
Make smears, stain and examine as described in
SOP C. Although the capsule stains well when produced by this method, it does
not appear so well circumscribed as when produced in vivo or in blood as
described above.
Susceptibility to penicillin G
Inoculate the organisms to be tested in 1.5 ml
nutrient broth.
Incubate for two hours at 37 ° C.
Adjust the turbidity to Mc’Farland’s
0.5 standard.
The organism is inoculated on the entire
surface of a Mueller Hinton agar/blood agar plate with a sterile swab.
Let the inoculum dry
for a period not exceeding 15 minutes.
Place a penicillin G 10 units
disc over it.
Press the disc gently to ensure even contact
with the medium.
Use B. cereus as penicillin-resistant control
and Staphylococcus aureus ATCC as
penicillin-sensitive strain.
Incubate the plate for 18 hours at 37 ° C.
Measure the zone of inhibition around the
disc.
Susceptibility to penicillin is indicated by a zone of
inhibition of growth (³ 29mm) around the disc.
Diagnostic gamma bacteriophage
test
Inoculate a blood agar plate evenly over its
entire surface with a loopful of the test culture.
Place a loopful or a
small drop (10–15 m l) of phage suspension in the center of the plate.
Allow to dry (do not spread).
Incubate at 37 ° C for 6 hours to overnight.
Observe for phage-induced bacterial lysis indicated by area of no growth.
Include a culture of B. anthracis
as a positive control (the Sterne strain will do).
CSF/ascitic fluid
Do macroscopic examination.
Do not centrifuge the fluids, as they are
almost always haemorrhagic.
Proceed with smear and culture as described in
SOP D.3.1
Suggested procedure for isolation and identifictaion of B. anthracis and confirmation of
diagnosis
Suggested procedure for isolation and identification
of B. anthracis
and confirmation of isolate from environmental material
Blood sample
Incubate the inoculated blood culture bottle
at 37 ° C.
Examine the bottle after 18–24 hours
incubation.
Observe the growth as indicated by turbidity.
With a sterile syringe and needle aspirate the
broth (do not open the bottle) and make smears.
Stain smears as described in SOP C.
If the smear is suggestive of B. anthracis,
subculture on blood agar medium and proceed with identification as
described in SOP D.3.1.
If there is no growth in the broth, mix the
broth gently and reincubate the bottle for three
days.
When growth occurs proceed with subculture and
identification as in D.3.1.
Safety
measures
Perform all procedures in appropriate
containment facility.
Seal the plates and tubes before incubation.
Discard the plates/tubes after appropriate
disinfection/autoclave into autcclave bags.
Autoclave and incinerate as in SOP A.3.1.
Dispose of as infectious waste as in SOP
A.3.1.
Quality
control
Media are supplied by the manufacturers for use only
after vigorous quality checking of their products. Still, all batches of
media prepared, whether from dehydrated materials or from ingredients should
be subjected to quality control for sterility and performance before use.
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