Organism Chracteristics:
The etiologic agent of anthrax, Bacillus anthracis, is an aerobic, spore and capsule forming, nonmotile, Gram-positive bacillus. B. anthracis appears as short chains of large (1-1.5 X 3-5 microns) cells that form oval, central to sub-terminal spores (1 X 1.5 microns) on sheep's blood agar plates. Spores do not cause significant swlling of the cell. 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 clinical specimens are in short chains of 2-4 cells that are encapsulated. However, cells from growth on SBA in the absence of CO2 tension are not encapsulated and occur as long chains of bacilli.

India ink preparations are useful for improving visualization of encapsulated B anthracis in clinical samples such as blood, blood culture bottles or CSF. The presence of large encapsulated Gram-positive rods in a specimen is strongly presumptive for B anthracis indentification.

B anthracis is a nonmotile species. This characteristic is unusual among Bacillus species and is therefore useful in the preliminary idenfication of B anthracis  isolates.

Suggested Specimens:

a. Cutaneous form:

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. 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.

1. 5% Sheep's blood agar plates
2. MacConkey agar plates
3. Phenyl ehtyl alcohol agar plates (for stool specimens).
4. Trypticase soy broth (or other general purpose enrichment broth).
5. Blood culture media (if needed for blood culture specimens).
6. Motility test media

Procedure:
1. Inoculation of media:

1. Sputum specimens: Inoculate SBA, MacConkey agar and a trypticase soy broth.
2. Blood specimen: Routine blood culture methods are sufficient. There may be enough organisms in the blood to seem them on direct smears by Gram stain.
3. Swab specimens: Use one swab to inoculate SBA, MacConkey agar, and a Trypticase Soy broth. Prepare a smear for Gram staining with the second swab.
4. Stool specimens: Inoculate SBA, MAC and PEA plate.

1. Plates and broth media: Culture should be incubates in the absence of CO2 tension at 35-37 degrees C. Cultures should be examined within 18-24h of incubation. Growth of B anthracis may be observed as early as 8h after inoculation.
2. Blood culture media: Follow routine blood culture protocols.

Plated media: After incubation of SBA for 15-24h at 35-37 degrees C, isoated colonies of B anthracis are 2-5mm in diameter. The flat or slightly convex colonies are irregularly round, with edges that are slightly undulate (irregularm wavy boarder), 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 wills tand up like beaten egg white. In contrast to colonies of B. cereus and B. thuringiensis, colonies of B anthracis are not beta hemolytics. However, weak hemolysis may be observed under areas of confluent growth in aging cultures and should not be confused with beta hemolysis.

When examining primary growth media, it is importnatn to compare the extent of growth on SBA plates with that on the MacConkey agar plate. B anthracis grows well on SBA, but does not grow on MacConkey agar. B anthracis grows rapidly. Heavily inoculated areas may show growth within 6-8h and individual colonies may be detected within 12-15h. This trait can be used to isolate B anthracis from mixed cultures containing slower growing organisms.

General purpose broth:
Subculture and examine for characteristic organisms id they are not found on plated media

Blood culture media:
If a blood culture bottle is positive, perform a Gram stain directly and observe for encapsulated rods. Positive blood cutures should be subcultured to SBA and MacConkey agar plates.

4. Motility test: Interpretation of results.
Using standard wet mount or tube media motility tests employed in your lab, interpret results as follows:

• Wet mount: Motile organisms can be observed moving randomly throughout the suspension. Nonmotile organisms either do not move or move with Brownian motion.
• Tubed Media Motility test: 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.

1. Gram stain of clinical samples show sncapsulated, large, spore forming, Gram-positive rods.
2. Spores and nonswelling and oval shaped.
3. Ground glass appearance of colonies
4. Nonmotile
5. Nonhemolytic