Approved Tests for the Detection of Bacillus anthracis in the Laboratory Response Network (LRN)1
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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.
Page last modified January 28, 2001