Approved Tests for the Detection of Bacillus anthracis in the Laboratory Response Network (LRN)¹

Test Procedure	Laboratory Level
Test Procedure	A	B	C	D
Gram stain (micromorphology)	x	x	x	x
Capsule (microscopic observation)	x	x	x	x
Routine culture:
1. Colonial morphology	x	x	x	x
2. Hemolysis	x	x	x	x
3. Motility	x	x	x	x
4. Sporulation (microscopic observation)	x	x	x	x
Confirmatory tests:
1. Lysis by gamma-phage		x	x	x
2. Direct fluorescence assay (DFA)(polysaccharide cell wall and capsule)		x	x	x
Antimicrobial susceptibility testing			x	x
Advanced technology (e.g., time-resolved fluorescence [TRF] testing, polymerase chain reaction [PCR] testing)			x	x
Molecular characterization				x

Gram stain
Routine staining procedure for observation of bacterial micromorphology.

Capsule
In Level A laboratories, India ink may be used to visualize encapsulated B. anthracis in clinical specimens by direct examination of peripheral blood, cerebrospinal fluid, or cells grown on media supplemented with sodium bicarbonate. M’Fadyean stain (Level B) and DFA for capsular antigen (Level B) may also be employed. Note that some avirulent strains, such as the veterinary vaccine Sterne strain, do not produce the capsule.

Routine culture
Standard 5% sheep blood agar (SBA) and chocolate agar will support growth. B. anthracis will not grow on MacConkey agar.

Colonial morphology
On SBA, after 15-24 h incubation at 35-37° C, well-isolated colonies 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. Colonies typically have a tenacious consistency; that is, teasing with an inoculating loop causes colony to ‘stand up’ like beaten egg white.

Hemolysis
Colonies of B. anthracis are non-hemolytic. However, weak hemolysis may be observed under areas of confluent growth in aging cultures and should not be confused with beta-hemolysis.

Motility
B. anthracis is non-motile. Working in a biological safety cabinet (BSC) with gloves, prepare routine wet mount and observe microscopically. Alternatively, motility test medium may be used.

Sporulation
Spores will appear in a growing culture after 18-24 h incubation at 35-37° C in a non-CO2 atmosphere. Oval, central to sub-terminal spores that do not appreciably swell may be observed by Gram stain (Level A), wet mount (Level B), or malachite green stain (Level B).

Lysis by gamma-phage
Highly specific for B. anthracis, and when demonstrated concomitantly with the presence of a capsule, provides confirmatory identification.

Direct fluorescence assay (DFA)
Used to detect the galactose/N-acetylglucosamaine cell-wall-associated polysaccharide and capsule produced by vegetative cells of B. anthracis strains. Concomitant demonstration of both antigens provides confirmatory identification.

Antimicrobial susceptibility testing
An array of selected antimicrobics is used to determine their respective minimum inhibitory concentrations using standardized methods against B. anthracis.

Advanced technology
Various advanced detection methodologies include time-resolved fluorescence (TRF), polymerase chain reaction (PCR), etc. These may have future applications within the LRN laboratories.

Molecular characterization
Various tests to determine the molecular characteristics of isolates are done. These include molecular subtyping using multi-locus variable-number tandem repeat analysis (MLVA), sequencing of genes coding for 16S ribosomal RNA.

Serologic tests for potential exposure to B. anthracis are currently being validated and at this time their clinical utility is not known.

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Protocols for Level A tests are publicly available at emergency.cdc.gov/agent/anthrax. Protocols for Level B are available only to laboratories in the LRN. These laboratories include state public health laboratories and many federal laboratories.