Anthrax Lab Protocol - Office of Public Health Practice

LABORATORY RESPONSE NETWORK (LRN) 

Notice: Protocol subject to change 

Last revised: March 24, 2003 

Level A Laboratory Procedures for Identification of Bacillus anthracis 

I. General: The procedures described below function to rule out or presumptively 

identify B. anthracis from clinical specimens or isolates. These procedures should be 

performed in microbiology laboratories that use Biological Safety Level-2 (BSL-2) 

practices. 

II. Precautions: Refer to Procedure for Laboratory Safety and Decontamination. 

III. Specimen 

A. Acceptable specimens: Collect other specimens if/as clinically indicated (e.g., 

cerebrospinal fluid [CSF], lymph node biopsy). Refer to Appendix for information 

on nasal swabs. 

1. Cutaneous anthrax 

a. Vesicular stage: Aseptically collect vesicular fluid on sterile swabs from 

previously unopened vesicles. Note: The anthrax bacilli are most likely to be 

seen by Gram stain in the vesicular stage. 

b. Eschar stage: Collect eschar material by carefully lifting the eschar’s outer 

edge; insert a sterile swab, then slowly rotate for 2-3 sec beneath the edge 

of the eschar without removing it. 

2. Gastrointestinal anthrax 

a. Blood cultures: Collect appropriate blood volume and number of sets per 

laboratory protocol. In later stages of disease (2-8 days post-exposure) 

blood cultures may yield the organism, especially if obtained before antibiotic 

treatment. 

b. Stool: Transfer ≥5 g of stool directly into a clean, dry, sterile, wide-mouth, 

leak-proof container. 

c. Rectal swab: For patients unable to pass a specimen, obtain a rectal swab by 

carefully inserting a swab 1 inch beyond the anal sphincter. 

3. Inhalational anthrax 

a. Blood cultures: Collect appropriate blood volume and number of sets per 

laboratory protocol. 

b. Sputum: Collect >1 ml of a lower respiratory specimen into a sterile 

container. Inhalational anthrax usually does not result in sputum formation. 

B. Rejection criteria: Use standard laboratory criteria. 

C. Specimen transport and storage: Refer to Shipping Procedure 

1. Swabs: Transport directly to laboratory at room temperature. For transport 

time >1 h, transport at 2-8°C. 

2. Stool: Transport unpreserved stool to laboratory within 1 h. For transport time 

>1h, transport at 2-8°C; Cary-Blair or equivalent transport media is acceptable. 

3. Sputum: Transport in sterile, screw-capped container at room temperature 

when transport time is 1 h, transport at 2-8°C. 

4. Blood culture: Transport directly to laboratory at room temperature. 

ban.la.cp.032403 3/24/03 Page 1 of 18

IV. Materials 

A. Reagents 

1. Gram stain reagents 

2. Catalase reagent (3% hydrogen peroxide) 

3. Motility media (or slide, coverslips, saline for wet mount) 

4. India ink (an optional test) 

5. Sterile saline 

B. Media 

1. 5% sheep blood agar (SBA) or equivalent 

2. Chocolate agar (CA) 

3. MacConkey agar (MAC) 

4. Phenyl ethyl alcohol agar (PEA) 

5. Blood culture bottles 

6. Tubed motility media 

7. Tryptic soy broth (TSB), or equivalent 

8. Thioglycolate broth or equivalent 

C. Equipment/miscellaneous 

1. Blood culture instrument (optional) 

2. Light microscope with 10X, 40X and 100X objectives and 10X eyepiece 

3. Microscope slides and coverslips 

4. Disposable bacteriologic inoculating loops 

5. Incubator, 35-37 o C, ambient preferred (CO 2 enriched is acceptable) 

Disclaimer: Names of vendors or manufacturers are provided as examples of 

suitable product sources; inclusion does not imply endorsement by the Centers for 

Disease Control and Prevention, the Department of Health and Human Services, the 

United States Army, or the Federal Bureau of Investigation. 

V. Quality control: Document all quality control results for the following tests per 

standard laboratory procedure/protocol. 

VI. Procedure: Refer to Fig. A1a and A1b. 

A. Stains and smears 

1. Gram stain 

a. Procedure: Perform Gram stain procedure/QC per standard laboratory 

protocol. 

b. Interpretation 

(1) B. anthracis is a large gram-positive rod (1-1.5 X 3-5 µm). 

(2) Blood and impression smears: Vegetative cells seen on Gram stain of 

blood and impression smears are in short chains of 2-4 cells that are 

encapsulated, which may be seen on the Gram stain as clear zones 

around the bacilli. Spores are not present in clinical samples unless 

exposed to low CO 2 levels, such as those found in the atmosphere; higher 

CO 2 levels within the body inhibit sporulation. The presence of large 

encapsulated gram-positive rods in the blood is strongly presumptive for 

B. anthracis identification. Refer to Fig. A2. 


(3) Growth on SBA or equivalent medium: B. anthracis forms oval, centralto-subterminal 

spores (1 X 1.5 µm) on SBA that do not cause significant 

swelling of the cell; frequently occur as long chains of bacilli. However, 

cells from growth on SBA regardless of the incubation conditions 

(ambient atmosphere or CO 2 enriched) are not encapsulated. Refer to 

Fig. A3a and Fig. A3b. 

2. India Ink (optional procedure) 

a. Purpose. Used to improve visualization of encapsulated B. anthracis in clinical 

samples such as blood, blood culture bottles, or cerebrospinal fluid (CSF). 

b. Quality control 

(1) Positive control strain: Klebsiella pneumoniae (or laboratory validated 

equivalent) will demonstrate a well-defined clear zone on SBA. 

(2) Negative control strain: E. coli ATCC 25922 (or laboratory validated 

equivalent) will demonstrate no clear zone. 

(3) Method controls: Perform the test with suspensions of fresh cultures of 

the control strains. Control strains should be assayed on each day of 

testing. 

(4) Resolving out-of-control results: Check media, reagents, controls and 

equipment; replace or correct as appropriate. Document corrective 

actions and repeat test. 

c. Procedure 

(1) For the controls, transfer a small amount of growth (1 mm diameter) 

from each control SBA plate (positive control = Klebsiella pneumoniae; 

negative control = Escherichia coli ATCC 25922) into 0.5 ml saline and 

mix. 

(2) For the unknowns, take 100 µl of sample (blood, CSF). Transfer 5-10 µl 

of unknown sample or control to a slide. Place a coverslip on the drop, 

and then add 5-10 µl of India ink to the edge of the coverslip. After the 

ink diffuses across the slide, view the cells using 100X oil immersion 

objective with oil on top of the coverslip. 

d. Interpretation 

(1) Positive result: The capsule will appear as a well-defined clear zone 

around the cells. 

(2) Negative result: No zone will be present. 

e. Reporting/actions 

(1) Clinical specimens with encapsulated (visualized with India ink), grampositive 

rods provide a presumptive identification of B. anthracis. 

(2) Every effort should be made to obtain an isolate for continued testing and 

referral to state public health laboratory. 

f. Limitations 

(1) Interpretation of results requires trained/experienced staff. 

(2) A negative test result should not be used to rule out B. anthracis. 

B. Cultures 

1. Inoculation and plating procedure: Inoculate and streak the following media for 

isolation of the respective specimen types. Note: Standard media should be used 

according to normal laboratory procedures. 

a. Blood cultures: Process following routine laboratory protocol. 

b. Cutaneous swab specimens: Plate directly on media used routinely for 

surface wounds such as SBA, MAC, and broth enrichment, and prepare 

smears for staining. Note: B. anthracis does not grow on MAC. 


c. Stool: Plate directly on appropriate media, such as PEA, SBA, and MAC. 

Note: B. anthracis does not grow on PEA. 

d. Sputum specimens: Plate directly on media used routinely, such as SBA, MAC, 

and CA, and prepare smears for staining. 

2. Incubation 

a. Temperature: 35-37°C 

b. Atmosphere: Ambient preferred 

c. Length of incubation: Hold primary plates for at least 3 days; read daily. 

Examine plates within 18-24 h of incubation. Growth of B. anthracis may 

be observed as early as 8 h after incubation. 

3. Colony characteristics of B. anthracis 

a. After incubation of SBA plates for 15-24 h at 35-37°C, well isolated colonies 

of B. anthracis 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. There may be often commashaped 

projections from the colony edge, producing the "Medusa-head" 

colony. Refer to Fig. A4. 

b. B. anthracis colonies on SBA usually have a tenacious consistency. When 

teased with a loop, the growth will stand up like beaten egg white; refer to 

Fig. A5. In contrast to colonies of B. cereus and B. thuringiensis, colonies of 

B. anthracis are not β-hemolytic; refer to Fig. A6. However, weak hemolysis 

may be observed under areas of confluent growth in aging cultures and 

should not be confused with β-hemolysis. 

c. When examining primary growth media, it is important to compare the extent 

of growth on SBA plates with that on MAC. B. anthracis grows well on SBA 

but does not grow on MAC or PEA. 

d. B. anthracis grows rapidly; heavily inoculated areas may show growth within 

6-8 h and individual colonies may be detected within 12-15 h. This trait can 

be used to isolate B. anthracis from mixed cultures containing slower-growing 

organisms. 

4. Extent of identification: For the Level A laboratory, identification is limited to 

‘presumptive’ only (see section VII, below, for specific criteria/key 

characteristics). 

C. Motility test: Wet mount or motility medium 

1. Purpose: Used to determine motility of suspected isolates; B. anthracis is 

nonmotile. Two methods are given, the wet mount and the motility medium 

test. 

2. Wet mount procedure 

a. Deliver 2 drops (approximately 0.1 ml) of TSB, or equivalent, into a sterile 

glass tube. Using an inoculating loop, transfer a portion of the suspect colony 

from a 12-20 h culture and suspend the growth in the broth medium. 

b. Alternatively, a loopful of medium from a fresh broth culture can be used. 

c. Transfer 10 µl of the suspension to a microscope slide and overlay with a 

coverslip. 

d. Examine slide under a microscope using the 40X objective (total 

magnification 400X; may also be viewed at 1000X with oil objective). 

e. Discard slide(s) following standard laboratory procedures, such as into 0.5% 

hypochlorite solution. 


3. Motility medium test procedure 

a. Using a sterile inoculating needle, remove a portion of growth from an 

isolated, suspect colony after 18-24 h incubation. 

b. Inoculate the motility medium by carefully stabbing the needle 3-4 cm into 

the medium and then drawing the needle directly back out so that a single 

line of inoculum can be observed. 

c. Incubate the tube at 35-37°C in ambient atmosphere for 18-24 h. 

4. Interpretation of motility results: Lack of motility is unusual among Bacillus 

species and is therefore useful in the preliminary identification of B. anthracis 

isolates. 

a. Wet mount 

(1) Positive result: Motile organisms will be observed moving throughout 

the suspension. Observe that the movement may be sluggish/slower 

than that of the positive controls . 

(2) Negative result: Nonmotile organisms either do not move or move with 

Brownian motion. 

b. Motility test 

(1) Positive result: Motile organisms will form a diffuse growth zone around 

the inoculum stab. 

(2) Negative result: Nonmotile organisms, such as B. anthracis, will form a 

single line of growth that does not deviate from the original inoculum 

stab. 

5. Quality control 

a. Positive control strain: Pseudomonas aeruoginosa ATCC 35032 or laboratoryvalidated 

equivalent will demonstrate motility. 

b. Negative control strain: Acinetobacter spp. ATCC 49139 or laboratoryvalidated 

equivalent will show no motility 

c. Method controls: Perform the test with fresh cultures of the control strains 

using the same method as with unknowns. Control strains should be assayed 

on each day of testing. 

6. Resolving out-of-control results 

a. Check media, reagents, controls and equipment; replace or correct as 

appropriate. Document corrective actions and repeat test. 

b. Check purity and identity of control strains and repeat testing. 

VII. Interpretation and reporting 

A. Presumptive identification criteria: Refer to Table A1. 

1. Direct smears from clinical samples, such as blood, CSF, or skin lesion (eschar) 

material: Encapsulated gram-positive rods 

2. From growth on SBA or equivalent media: Large gram-positive rods (may 

stain gram-variable after 72 h of culture). Spores may be found in culture, 

under non-CO 2 atmosphere (but not on direct examination). Spores are 

nonswelling and oval-shaped. 

3. Rapid, aerobic growth, and tenacious colonies on sheep blood agar. 

4. Catalase positive 

5. Nonmotile: In addition to B. anthracis, B. cereus var. mycoides is nonmotile. 

6. Nonhemolytic on SBA, ground-glass appearance of colonies 


B. Rule out: While hemolysis, gram stain morphology, or motility can be used for 

rule out when the result provides clear evidence that the isolate is not B. anthracis 

(e.g., a clearly visible zone of beta hemolysis), a combination of two Level A tests is 

recommended for rule out. 

C. Reporting/action 

1. Consult with state public health laboratory director (or designate) if B. anthracis 

is suspected. 

2. General instruction and information 

a. Preserve original specimens pursuant to a potential criminal investigation and 

possible transfer to an appropriate LRN laboratory. 

b. Environmental/nonclinical samples and samples from announced events 

are not processed by Level A Laboratory; submitter should contact the state 

public health laboratory directly. 

c. The state public health laboratory/state public health department will 

coordinate notification of local FBI agents as appropriate. 

d. Assist local law enforcement efforts in conjunction with guidance received 

from the state public health laboratory. 

e. The state public health laboratory/state public health department may 

request transfer of suspicious specimens prior to presumptive testing. 

f. FBI and state public health laboratory/state public health department will 

coordinate the transfer of isolates/specimens to a higher-level LRN laboratory 

as appropriate; refer to Shipping Procedure. 

3. Immediately notify state public health laboratory director (or designate) and 

state public health department epidemiologist/health officer if B. anthracis 

cannot be ruled out and a bioterrorist event is suspected. 

4. Immediately notify physician/infection control according to internal policies if B. 

anthracis cannot be ruled out. 

5. If B. anthracis is ruled out, proceed with efforts to identify using established 

procedures. 

VIII. References 

Brachman, P.S., and A.M. Friedlander. Anthrax, p. 729-739. In S.A. Plotkin and 

E.A. Mortimer, Jr. (ed), Vaccines. W.B. Saunders, Philadelphia, PA. 

Cieslak, T.J. and E.M. Eitzen, Jr. 1999. Clinical and epidemiologic principles of 

anthrax. Emerg. Infect. Dis. 5:552-555. 

Dutz, W. and E. Kohout. 1971. Anthrax. Pathol. Annu. 6:209-248. 

Gilchrist, M.J.R., W.P. McKinney, J.M. Miller, and A.S. Weissfeld. 2000. 

Cumitech 33, Laboratory Safety, management , and diagnosis of biological agents 

associated with bioterrorism. Coordinating ed., J.W. Snyder. ASM Press, Washington, 

D.C. 

Lew, D. P. 2000. Bacillus anthracis (Anthrax). p. 2215-2220. In G. L. Mandell, J.E. 

Bennett, and R. Dolin (ed), Principles and Practice of Infectious Disease, 5 th ed. 

Churchill Livingston, Philadelphia, PA. 


Logan, N.A. and P.C. Turnbull. 1999. Bacillus and recently derived genera, p. 357- 

369. In P.R. Murray, E.J. Baron, M.A. Pfaller, F.C. Tenover, and R.H. Yolken (ed) 

manual of Clinical Microbiology, 7 th ed. American Society for Microbiology, 

Washington, D.C. 


Figure A1a. Flowchart of B. anthracis Level A procedures. 


an.la.cp.032403 3/24/03 Page 9 of 18

Figure A2. Gram stain of B. anthracis in rhesus monkey blood, magnification 1000X 


Figure A3a. Gram stain of B. anthracis from SBA, magnification 1000X 


Figure A3b. Gram stain of B. anthracis with spores, magnification 1000X 


Figure A4. B. anthracis colony morphology; overnight cultures on SBA. 


Figure A5. Tenacious colonies of B. anthracis on SBA 


Figure A6. B. anthracis and B. cereus colony morphology; overnight cultures of B. cereus 

(left side of plate) and B. anthracis (right side) on SBA. 


Table A1. Presumptive identification of B. anthracis 

Lab 

Level 

Type of sample 

Presumptive identification 

Characteristic 

Method 

A Clinical sample 1. Gram-positive rods 

Gram stain 

AND 

2. Capsule 

India ink stain 

A Isolate 1 Spore-former 

Gram stain 

AND 

2. Colony morphology 

Observation on SBA 

AND 

3. Nonhemolytic 

Observation on SBA 

AND 

4. Nonmotile 

Motility medium or 

Wet mount 


IX. Appendix: Nasal specimens for Bacillus anthracis screening 

A. General: Nasal specimens (nares culture) should ONLY be used to support a 

confirmed exposure to B. anthracis or during an ongoing epidemiologic 

investigation. Gram stain of nasal specimens for B. anthracis spores is not 

recommended. Refer to limitations section below. 

B. Materials: Swab (Dacron, rayon or other synthetic swabs are preferred over cotton) 

and transport medium for culture. 

C. Procedure 

1. Selection 

a. The specimen of choice is a swab specimen taken at least 1 cm inside the 

nares. 

b. Lesions in the nose require samples from the advancing margin of the lesions. 

2. Method 

a. Carefully insert the moistened swab (saline, sterile water) at least 1 cm into 

the 

nares. 

b. Firmly sample the inside of the nares by rotating the swab and leaving it in 

place for 10 to 15 sec. 

c. Withdraw the swab, insert it into its transport container, and submit the 

sampling unit to the laboratory for culture. 

3. Labeling 

a. Label the swab container with patient information. 

b. Indicate, if possible, the degree or likelihood of exposure. 

4. Transport 

a. Transport the specimen to the laboratory as soon as possible. 

b. Do not refrigerate specimens for culture. 

5. Culture: Heat Shock 

a. Remove the swab from transport container and place it into 1.5 ml of sterile 

saline or a nutrient broth such as trypticase soy broth, brain heart infusion 

broth, or equivalent. Vigorously twist the swab, and recap the tube. 

b. Leave the swab in the tube. Place the broth suspension into a 65 o C water bath 

for 30 min. 

c. Plate 100-200 µl of broth on 5% sheep blood agar plate and incubate at 

35-37 o C for 18-24 h. Many B. anthracis will have visible growth in 12-18 h; 

observe for characteristics of B. anthracis. 

D. Interpretation: Observe colony morphology for typical Bacillus colonies, look for 

lack of hemolysis, perform Gram stain, and evaluate for B. anthracis characteristics 

as described in the Level A laboratory protocol. 

E. Reporting: If B. anthracis cannot be ruled out, submit the isolate to the state public 

health laboratory/department for confirmation. Refer to Level A reporting. 

F. Limitations: Nasal cultures taken to evaluate for the presence of anthrax spore 

have not been evaluated for sensitivity or specificity. Nasopharyngeal and throat 

specimens are not recommended for anthrax screens and should not be submitted. 

Nasal cultures are NOT recommended for screening those who are asymptomatic and 

without known exposure. 


G. Procedure Notes 

1. Anterior nares cultures, without an indication of the presence of a lesion, are 

routinely examined only for presence of Staphylococcus aureus and β-hemolytic 

streptococci. Because of the unknown sensitivity of this method for detecting B. 

anthracis spores, interpret negative results with caution. 

2. Anterior nares cultures cannot be used to predict a subsequent infection with B. 

anthracis, and should not be submitted in lieu of blood and other appropriate 

specimens from symptomatic patients. 

3. Anaerobic cultures are not done on nasal specimens. B. anthracis produces 

spores in culture only when grown in air. 

4. Nasal swabs may also be plated directly onto sheep blood agar prior to or without 

heat shocking, however normal nasal flora may overgrow very low numbers of 

Bacillus colonies. 

5. Pediatric needs: Use the same procedure substituting a small fine-wire or 

nasopharyngeal swab to sample the anterior nares.

Anthrax Lab Protocol - Office of Public Health Practice

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