Theme: Microbiological diagnosis of

anthrax
 Bacillus anthracis

Gram stain

Fluorescent-
Burry-Gins stain labeled
(with Indian ink) antibody test
 Burry-Gins stain
• Emulsify small amount of culture in a drop of Indian ink on a slide.
• Using another slide, spread it out over the entire slide to form a thin
film.
• Let it completely air dry and fix by passing in over flame three times.
• Stain with dilute carbol fuchsin for 1 minute.
• Wash the smear with water.
• Blot dry and observe using oil immersion microscopy.
• The organism will pick up the red dye, background – black while the
capsule will remain colorless.
Detection of capsule in smear-
imprint from tissue

Methylene blue stain

Endospore. Ozheshko stain
 Gansen (Ozheshko) stain
• 1. Place a piece of blotting paper over heat-fix a smear of Bacillus
anthracoides and saturate with carbol fuchsin.
• 2. Holding the slide with forceps, carefully heat the slide in the flame
of a bunsen burner until the stain just begins to steam. Remove the slide
from the heat until steaming stops; then gently reheat. Continue steaming
the smear in this manner for five minutes. As the carbol fuchsin
evaporates, continually add more. Do not let the paper dry out.
• 3. After five minutes of steaming, wash the excess stain and blotting
paper off the slide with water. Don't forget to wash of any dye that got onto
the bottom of the slide.
• 4. Cover the slide with 0,5 % H2SO4 .
• 5. Wash the slide with water.
• 6. Now flood the smear with methylene blue and stain for 1 minute.
• 7. Wash off the excess methylene blue with water.
• 8. Blot the slide dry and observe using oil immersion microscopy.
• With this endospore staining procedure, endospores will stain red while
vegetative bacterial cell will stain blue.
 Nonhemolytic on sheep blood agar
Colonies are nonhemolytic, flat or
slightly convex with irregular edges and
ground-glass appearance.
There are often comma shaped
projections from the colony edge
producing a “Medusa-head” colony.
Flat or slightly raised, gray to white with
a "ground glass" appearance
“Medusa head” colonies of B.anthracis
 Gelatin Hydrolysis test
• Test tube on the top is (+) as indicated by
liquid nature
 Motility test medium

non motile motile

organism organism
 The virulence factors of B. anthracis
• Capsule is anti-phagocytic .
• Exotoxins: A plasmid-encoded, heat-labile,
heterogeneous protein complex made up of 3
components:
– Protective Antigen (PA) binds to surface receptors
on eukaryotic cells and then binds either EF or LF
which enters the cell by endocytosis
– Edema Factor (EF) acts as adenylate cyclase.
– Lethal Factor (LF) activates of macrophages and
production of cytokines which cause necrosis, fever,
shock and death.
 Modes of transmission
• by the cutaneous route
(direct contact with
diseased animals,
hides, wool, brushes,
or bone meal),
• by biting insects,
• by inhalation,
• by ingestion of meat
from diseased animals.
PATHOGENESIS
OF ANTHRAX
Cutaneous
anthrax
Cutaneus anthrax
 Laboratory diagnosis
Specimens: vesicular fluid, fluid from under the eschar, blood,
sputum, or spleen or lymph node aspirates.
• Microscopy:
– stain with methylene blue or Gram stain.
– IF.
• Culture on blood agar plates.
– absence of hemolysis,
– lack of motility,
– gelatin hydrolysis,
– susceptible to lysis by gamma phage
– test with penicillin.
• Biological method: mouse or guinea pig inoculation.
Specimen: tissues and exudates of dead animals
• Ascoly test: ring precipitation test.
 Lysis of Bacillus anthracis by the lytic gamma phage

• The plaque (clear

area) in the region
of confluent growth
is where the gamma
phage was applied.
• The plaque results
from the phage's
ability to lyse the
bacterial cells
Penicilline test
Thermoprecipitation reaction (ring
precipitation test - Ascoli's test)