Biothreat Agents Identification Bench Cards for Sentinel Laboratories

Recognize. Rule-Out. Refer.

Biothreat Agent Bench Cards

for the Sentinel Laboratory

For questions, contact your designated LRN Reference Level Laboratory:

(LRN Reference Level Laboratory Name)

(Phone Number)

PHL thanks the Sentinel Laboratory Partnerships and Outreach Subcommittee, the Public

Health Preparedness and Response Committee, the American Society for Microbiology and

the US Centers for Disease Control and Prevention for contributing their time and expertise to

provide substantial guidance on the development of these bench cards.

Special thanks to the Florida Department of Health, Massachusetts State Public Health

Laboratory, Michigan Department of Health and Human Services, Minnesota Department of

Health, Oregon State Public Health Laboratory, San Antonio Metro Health District, Wadsworth

Center at the New York State Department for Health and Wisconsin State Laboratory of

Hygiene for providing subject matter expertise, content review and photos.

This project was 100% ﬁnanced by federal funds. The total amount of funding received for the

Public Health Preparedness and Response program is $1,768,631.

This project was supported by Cooperative Agreement # NU60OE000103 from CDC. Its

contents are solely the responsibility of the author and do not necessarily represent the

ofﬁcial views of the CDC.

INSTITUTION / LRN REF LABORATORY:

Address:

Phone Number:

Website:

EMERGENCY NUMBERS

Laboratory (business hours):

Laboratory (after hours):

Biothreat Coordinator:

Epidemiology Dept. (business hours):

Epidemiology Dept. (after hours):

Duty Ofcer/Other On-Call:

STATE / LOCAL PUBLIC HEALTH LABORATORY DEPARTMENTS

Microbiology:

Virology:

Serology:

Specimen Receiving/Packaging:

State-Specific Information

Safety

Safety Precautions ...................................................................... 5

Preventing Aerosolization ........................................................... 6

Responding to a Biothreat Agent

Laboratory Response Network for Biological Threats............... 7

Responsibilities of the Sentinel Laboratory .............................. 8

Sentinel Laboratory Checklists .................................................. 9

Biological Risk Assessments ....................................................10

Using BSL-3 Practices ...............................................................11

Biothreat Agent Response Algorithm ....................................... 12

Biothreat agent Identiﬁcation

Gram Negative Bacilli/Cocobacilli Rule-Out Algorithm ...........13

Anthrax — Bacillus anthracis

Handling Instructions ................................................................14

Characterization ........................................................................15

Rule-Out Algorithm ....................................................................16

Anthrax — Bacillus cereus biovar anthracis

Characterization ........................................................................ 17

Recommendations .................................................................... 18

Brucellosis — Brucella spp.

Handling Instructions ................................................................19

Characterization ........................................................................20

Rule-Out Algorithm ....................................................................21

Glanders — Burkholderia mallei

Handling Instructions ................................................................22

Characterization ........................................................................23

Rule-Out Algorithm .................................................................... 24

Melioidosis — Burkholderia pseudomallei

Handling Instructions ................................................................22

Characterization ........................................................................25

Rule-Out Algorithm ....................................................................26

Tularemia — Francisella tularensis

Handling Instructions ................................................................ 27

Characterization ........................................................................28

Rule-Out Algorithm ....................................................................29

Plague — Yersinia pestis

Handling Instructions ................................................................30

Characterization ........................................................................ 31

Rule-Out Algorithm ....................................................................32

Appendix

Acronyms ...................................................................................33

Terms and Denitions ............................................................... 34

Identication Tests .................................................................... 37

Resources .................................................................................. 39

TABLE OF CONTENTS

Refer to the ASM Sentinel Laboratory Guidelines and consult with your LRN Reference Laboratory

for other suspect biothreat organisms not routinely seen in the Sentinel Laboratory, such as

Clostridium botulinum, novel inuenza, Smallpox, Staphylococcus aureus enterotoxin B (SEB), Coxiella burnetii, etc.

Identification Systems

Use May Result in Exposure or Misidentification of Biothreat Agents

Using automated or manual identication systems (e.g., MALDI-TOF, Vitek, API 20 NE, Bactec) may result in

exposure to dangerous pathogens, and could result in erroneous identication (e.g., Bacillus anthracis misidentied

as B. cereus; Yersinia pestis misidentied as Y. pseudotuberculosis, etc.).

Filter Extract to Reduce Risk of Contamination or Exposure

If using automated identication systems for bacterial identication and the manufacturer provided an alternate

tube extraction method (most common with MALDI-TOF), it is recommended that the resulting extract be ltered

using a 0.2 μm (or less) lter. This additional step will reduce the risk of laboratory contamination with viable

bacteria and spores.

Handling a Suspected Biothreat Agent

Use a Biological Safety Cabinet & BSL-3 Practices

As soon as a biothreat agent is suspected, perform all further work in a certied Class II BSC using BSL-3 practices

and appropriate BSL-3 PPE.

Contact your LRN Reference Level Lab

If the agent cannot be ruled-out with tests listed within these bench cards, do not attempt further identication

using commercial automated or kit identication systems. Contact your LRN Reference Level Laboratory to refer the

isolate.

Safety Precautions

SAFETY

Aerosolization

Aerosolization can occur during any procedure

which imparts energy into a microbial suspension,

producing aerosols or droplets which may contain

infectious organisms. Aerosols are very small

particles that may remain suspended in the air and

can be inhaled and retained in the lungs. Droplets

are larger particles which can settle onto surfaces

and gloves due to gravity. Droplets may also come

into contact with the mucous membranes of the

person performing the procedure.

Safety Precautions

Laboratory exposures can be decreased by working

in a BSC using BSL-3 practices and appropriate

BSL-3 PPE when a biothreat agent is suspected.

Identied aerosol-generating procedure risks

should be mitigated.

Examples of Aerosol Producing Procedures

• Opening culture plate, snifng plates

(Examining colony morphology/growth)

• Heat xing a slide

• Dispensing pipette tips

• Centrifuge setup/run/unloading

• Vortexing

• Spills or splashes of liquid media

• Subculturing positive blood culture bottles

• Inoculation of media (plate or tube)

• Preparing samples for automated ID systems

• Open ames, sterilizing loops

• Sonicating

• Pipetting

• Catalase test

• Using automated and manual identication

systems (e.g., MALDI-TOF, Vitek, API 20 NE,

Bactec)

Your facility may identify additional aerosol generating procedures based on the laboratory's risk assessments.

Preventing Aerosolization

SAFETY

The LRN-B was founded in 1999 by CDC, FBI and APHL

to coordinate laboratory response to biological, chemical,

radiological threats and other high priority public health

emergencies, including emerging infectious diseases.

National Laboratories

National labs, including the CDC, US Army Medical Research

Institute of Infectious Diseases (USAMRIID), and the Naval

Medical Research Center (NMRC), are responsible for

specialized strain characterization, bioforensics, biothreat agent

activity and handling of highly infectious biological agents.

Reference Laboratories

Reference labs, including state and local public health, military,

veterinary, agriculture, food and water testing laboratories, are

responsible for investigation and conrmatory testing. Facilities

located in Australia, Canada, the United Kingdom, Mexico and

South Korea serve as international reference laboratories.

Sentinel Laboratories

Sentinel labs, comprised of hospital-based and commercial

laboratories, are responsible for the early detection and the

rule-out or referral of potential biothreat agents.

Laboratory Response Network for Biological Threats

RESPONDING TO A BIOTHREAT AGENT

A Sentinel Laboratory:

1. Is familiar with reportable disease guidelines in its jurisdiction, and

has policies and procedures in place to refer clinical and diagnostic

specimens or isolates suspected to contain agents of public health

signicance to the appropriate local or state public health laboratory.

2. Ensures sufcient personnel have met the applicable federal

regulations for packaging and shipping of Category A and B

infectious substances.

3. Has policies and procedures for the collection and referral of

suspect biothreat agents or other emerging threat specimens and/

or isolates to the appropriate LRN Reference Laboratory consistent

with the ASM Sentinel Level Clinical Laboratory Protocols and

Guidelines for Suspected Agents of Bioterrorism and Emerging

Infectious Diseases.

4. If a clinical core laboratory, provides their satellite facilities with

written directions and training as needed for appropriate specimen

collection and handling. Core laboratories should also provide

satellite facilities with procedures for the recognition of the agents

of bioterrorism and assure training at a level commensurate with the

complexity of services offered at that facility.

5. Maintains the capability to perform the testing outlined in the ASM

Sentinel Clinical Laboratory Protocols and must demonstrate annual

competency by participation in prociency testing or exercises, such

as APHL, CDC and the College of American Pathologists Laboratory

Preparedness Exercise (CAP LPX), state-developed prociency/

challenge sets, or other equivalent assessment.

6. Based on its risk assessment, has and utilizes a currently certied

Class II or higher BSC when there is a risk of aerosol production or

when working with a biological threat agent or other emerging threat

organism is suspected.

7. Complies with the practices as outlined in the current edition of the

Biosafety in Microbiological and Biomedical Laboratories guidelines

and those detailed in the Guidelines for Safe Work Practices in

Human and Animal Medical Diagnostic Laboratories.

8. Has a biosafety and biosecurity risk assessment policy and ensures

that such risk assessments are routinely performed as part of their

quality management program.

9. Complies with applicable US Occupational Safety and Health

Administration regulations for bloodborne pathogens and has a

respiratory protection program.

10. Complies with the applicable rules and regulations of the Federal

Select Agent Program.

11. Has policies and procedures for secure storage of any remaining

suspect biothreat or other emerging threat agent material retained

within its facilities until it is transferred or destroyed.

12. Has policies and procedures for nal decontamination/destruction

of any remaining suspect biothreat or other emerging threat agent

material within the required time-frame (e.g., primary specimens or

subcultures retained within its facilities).

Responsibilities of the Sentinel Laboratory

RESPONDING TO A BIOTHREAT AGENT

Laboratory Preparedness

Plans

□

Institutional Emergency or Incident

Response Plan

□

Specic Bioterrorism Response Plan

□

Institutional Risk Assessment Plan

Training

□

Packaging and Shipping of Infectious

Substances

□

Rule-Out of Select/Biothreat Agents

□

Select Agent Regulations

□

Communications and Messaging

Proficiency Testing

□

Prociency test/exercise

(e.g., CAP LPX)

□

Maintain supplies for rule-out testing

Updates

□

Review ASM’s website for updated

Sentinel Level Clinical Laboratory

Protocols

□

APHL trainings

If you have a:

Suspect BT Agent

□

Follow rule-out procedures and

conduct work in a BSC

□

Initiate/maintain communication

with departmental/hospital

leadership and infection control

□

Contact BT personnel at designated

LRN Reference Level Laboratory

□

Ship isolate to designated LRN

Reference Level Laboratory

□

Document courier transfer

(e.g., institutional or commercial

courier tracking number)

□

Secure all potential biothreat

agent(s) and residual samples

□

Document personnel with access

to potential biothreat agent(s)

(biosecurity)

□

Document personnel who have

worked with suspect biothreat agent

and those present in laboratory if

exposure occurred (biosafety)

Confirmed BT Agent

□

Follow directions from designated

LRN Reference Level Laboratory

for the destruction or transfer of all

isolates/specimens

□

Perform risk assessment review

□

Document identication of biothreat

agent(s) with APHIS/CDC Form 4

□

Document disposition of biothreat

agent(s) with APHIS/CDC forms:

• Form 2 to transfer

• Form 4 for destruction

Exposure to a BT Agent:

□

Document any laboratory exposures

with APHIS/CDC Form 3

□

Work with designated LRN

Reference Level Laboratory or

health department for post-exposure

prophylaxis

Sentinel Laboratory Checklists

RESPONDING TO A BIOTHREAT AGENT

Biological Risk Assessment Goals

• Identify hazards associated with handling infectious agents in the

laboratory.

• Identify and implement controls in order to minimize the risk of

exposure to workers and the environment.

• In the clinical lab, focus is primarily on the prevention of laboratory

acquired infections from:

• Spills/splashes to mucous membranes

• Inhalation of aerosols

• Percutaneous inoculation from cuts, needle sticks,

non-intact skin

• Ingestion (e.g., contamination from surfaces, fomites to hands, etc.)

Conducting a Biological Risk Assessment

Risk assessments must be performed regularly based on procedure or

agent, and when there are changes in agents, procedures, equipment

or staff. Risks identied by the assessment should be prioritized and a

mitigation plan should be established based on that prioritization.

Risk assessments require management involvement and support,

knowledge of the hazards and understanding of the work, the environment

and the staff. Ideally, they consist of a multidisciplinary team, depending on

the work.

Consult with your LRN Reference Lab for guidance, and refer to APHL's Risk

Assessment Best Practices for more information.

Biological Risk Assessments

RESPONDING TO A BIOTHREAT AGENT

BSL-3 Practices

• Restrict access to the laboratory.

• Wear additional PPE (solid-front gown, gloves and face/eye protection as a minimum) and respiratory protection

(previously t-tested for use).

• Laboratory personnel must demonstrate prociency prior to handling pathogenic and potentially lethal agents, and

must be supervised by scientists experienced and competent in handling the specic infectious agents present in the

laboratory and associated procedures.

• Do not manipulate organisms or work in open vessels on the bench. All work must take place in a certied Class II or

higher BSC, or other containment equipment. Tape plates shut.

• Evaluate all potential exposures immediately.

• Decontaminate all cultures, stocks and other potentially infectious materials prior to disposal by using an approved

decontamination method, such as autoclaving or chemical disinfection. Decontamination would preferably take place

within the laboratory.

When to Use BSL-3 Practices in a BSL-2 Laboratory

• When working with agents that can be transmitted via inhalation and are normally handled at BSL-3, but a BSL-3

laboratory is not readily available.

• When the laboratory director determines that BSL-3 practices are needed based on a risk assessment.

• When specic high-risk pathogenic organisms are suspected, such as Brucella spp., Coccidioides spp., Blastomyces

dermatitidis, Francisella tularensis, Histoplasma capsulatum, Mycobacterium tuberculosis, MERS, SARS, highly

pathogenic inuenza, Tier 1 Select Agents, etc.

Using BSL-3 Practices

RESPONDING TO A BIOTHREAT AGENT

Perform rule-out testing.

Able to rule-out biothreat agent?

Able to rule-in/conrm

biothreat agent?

Proceed with

routine ID

Notify LRN Reference Laboratory by phone and then ship them

the suspect select agent, using appropriate procedures.

Immediately secure any remaining specimens, isolates and

derivatives until Reference Laboratory's testing is completed.

Notify the Sentinel Laboratory and inquire about

potential biothreat agent exposures there.

Initiate

APHIS/CDC

Form 3

Initiate APHIS/CDC Form 4;

complete sections A & B,

send to Sentinel Laboratory.

If required, immediately

notify CDC.

Complete sections C & D of APHIS/CDC Form 4

and return to LRN Reference Laboratory

(which will send it to CDC Select Agent Program).

If requested, transfer all

specimens, isolates and

derivatives to the Reference

Laboratory, which will

initiate APHIS/CDC Form 2.

Complete Section 2.

Note: Authorization

must be received prior to

biothreat agent transfer

Destroy all related

specimens, isolates,

and derivatives using

approved method.

Note: LRN Reference

Laboratory can

provide guidance

Biothreat Agent Response Algorithm

RESPONDING TO A BIOTHREAT AGENT

Sentinel Laboratory LRN Reference Laboratory

YES

NO EXPOSURES

POTENTIAL EXPOSURES

Notify Sentinel Laboratory

Gram Negative Bacilli/Coccobacilli Rule-Out Algorithm

BIOTHREAT AGENT IDENTIFICATION

Slow growing Gram negative bacilli/coccobacilli

Growth on MAC?

NO OR POOR GROWTH

Catalase positive?

Oxidase positive?

Urea positive?

Urea negative?

Indole negative?

Follow ASM

Brucella

guidelines

Oxidase negative?

Satellite negative?Satellite negative?

Gray, translucent,

non-hemolytic

colonies on BAP?

Grows better on

CHOC than BAP?

No hemolysis

on BAP?

Indole negative?

Follow ASM

Y. pestis

guidelines

Follow ASM

B. pseudomallei

guidelines

Follow ASM

B. mallei

guidelines

Follow ASM

F. tularensis

guidelines

Oxidase positive?

NEGATIVE OR

WEAK POSITIVE

YES

YESYES

YES

YES VARIABLE

YES

Handling Instructions

ANTHRAX — Bacillus anthracis

Safety

Patient specimens can be handled using BSL-2 practices.

As soon as B. anthracis is suspected, perform all further work within a Class II BSC using BSL-3 practices, especially when

performing activities with a high potential for aerosol or droplet production.

Potential Lab Exposures

Ingestion, inhalation, inoculation and direct contact via skin abrasions and mucous membranes.

Specimen Collection

Ideal Time & Temp

Transport

Within Facility

Storage

Cutaneous

Vesicular Stage

Collect uid from intact vesicles on sterile swab(s).

The organism is best demonstrated in this stage.

≤2 h RT ≤24 h RT

Eschar Stage

Without removing eschar, insert swab beneath the

edge of eschar, rotate and collect lesion material.

≤2 h RT ≤24 h RT

Gastrointestinal

Stool

Collect 5-10 g in a clean, sterile, leak proof container. ≤1 h RT ≤24 h 4°C

Blood

Collect per institution’s procedure for routine blood

cultures.

≤2 h RT

Incubate per

lab protocol

Inhalation

Sputum

Collect expectorated specimen into a sterile, leak proof

container.

≤2 h RT ≤24 h RT

Blood

Collect per institution’s procedure for routine blood

cultures.

≤2 h RT

Incubate per

lab protocol

Characterization

ANTHRAX — Bacillus anthracis

Gram Stain

• Large Gram positive rods

(1-1.5 µm x 3-5 µm)

• Direct smears of clinical specimens:

• Short chains (2-4 cells)

• Capsule present

• No spores present

• Smears from culture (BAP or CHOC):

• Long chains

• No capsule present

• Spores in older cultures: oval,

central to subterminal, no swelling

of cell wall

Biochemical/Test Reactions

• Catalase positive

• Non-motile

Colony Morphology

• Grows well on BAP and CHOC

• Aerobic rapid growth as early as 4-8h

• Colonies 2-5 mm on BAP and CHOC

at 24h

• No growth on MAC and EMB

• Flat or slightly convex with irregular

edges that may have comma-like

projections

• Ground-glass appearance

• Gamma hemolytic (non-hemolytic) on

BAP

• Tenacious, sticky colonies, adheres to

agar surface

Common Misidentifications

May not be identied in common

automated ID systems, including MALDI-

TOF, and possible misidentications

include Bacillus megaterium and other

Bacillus species.

Note: Bacillus cereus Group includes B. anthracis, but automated ID systems may not alert

microbiologist beyond this group identication.

Gram stain of blood culture

24h growth on BAP

Irregular-edged colonies

Rule-Out Algorithm

ANTHRAX — Bacillus anthracis

As soon as B. anthracis or B. cereus biovar anthracis is suspected perform all further work in a Class II BSC using BSL-3

practices. If B. anthracis or B. cereus biovar anthracis cannot be ruled out with the tests below, do not attempt further ID

using commercial automated or kit identication systems.

Gram stain morphology

□

Large, Gram positive rods?

Note: Spores may be found in cultures grown in 5% CO

or ambient

atmosphere but not usually observed in clinical samples.

Colony morphology

□

Ground glass appearance?

□

Non-pigmented, gamma hemolytic (no hemolysis) on BAP?

Note: Some strains of B. cereus biovar anthracis may be weakly

hemolytic after 48h

□

No growth on MAC (or EMB)?

Gamma hemolytic (no hemolysis)?

Bacillus anthracis

is ruled out

Bacillus anthracis and

B. cereus biovar anthracis

are ruled out

Catalase positive?

B. anthracis or B. cereus biovar anthracis not ruled-out. Do not attempt further identication and contact your LRN Reference

Level Laboratory to refer the isolate. Suggested Reporting Language: Possible Bacillus anthracis or B. cereus biovar anthracis

submitted to LRN Reference Level Laboratory for conrmatory testing.

YES TO ALL

NO TO ANY

YES

YES, STOP

Continue with routine

identication

SAFETY

Characterization

ANTHRAX — Bacillus cereus biovar anthracis

Characteristic B. anthracis B. cereus

B. cereus biovar anthracis

Hemolysis

—- + —- —-

Motility

—- + +/—-

Gamma phage susceptibility

+ —- —- —-

Penicillin G

S R S R

Capsule

+ Absent in vitro + +

24 h growth on BAP, 5% CO

of CI (left) and CA (right) strains

Côte d’Ivoire strains, from chimpanzees

Cameroon strains, from gorillas or chimpanzees

Hemolysis

+ ........beta hemolytic on sheep blood agar

– ........non-hemolytic

Motility

+ ........motile

– ........non-motile

+/– ...B. cereus biovar anthracis strains are

usually motile, including those recovered

from gorillas, chimpanzees, and

elephants. B. cereus biovar anthracis goat

strains from Democratic Republic of the

Congo were non-motile.

Gamma phage susceptibility

+ ........susceptible

– ........resistant

Penicillin G

S ........susceptible

R .......resistant

Capsule

+ ........Present

Recommendations

ANTHRAX — B. anthracis & B. cereus biovar anthracis

Sentinel-level laboratories should continue using the existing ASM Sentinel Level Clinical

Laboratory Guideline for B. anthracis to rule out or refer isolates of Bacillus spp. that

produce non-hemolytic colonies with a ground glass appearance and are non-motile. Until

new guidelines are available, the following recommendations should be considered:

1. Suspect Bacillus spp. isolates that are large, catalase positive, Gram positive rods,

and non-hemolytic at 24h incubation in ambient atmosphere or 5% CO

should be

tested for motility. Isolates can appear weakly hemolytic upon extended incubation

(48h) in ambient atmosphere and are more hemolytic in 5% CO

at 48h. Semi-solid

medium is recommended for motility to ensure consistent results.

2. Suspect isolates should be investigated to determine if the isolate is signicant

regardless of motility. If the isolate was recovered from a sterile site or from a wound

culture, follow the local public health guidelines to assess whether the public health

lab or clinical lab should contact the patient’s attending physician to determine

the likely clinical signicance (e.g., does the patient have an anthrax-like clinical

syndrome?). Appropriate travel history should be obtained as well. If the isolate is

deemed signicant, the local LRN reference laboratory should be contacted to obtain

guidance regarding the need to refer the isolate for conrmatory testing.

Handling Instructions

BRUCELLOSIS — Brucella spp.

Safety

Patient specimens can be handled using BSL-2 practices.

As soon as Brucella spp. is suspected, perform all further work within a Class II BSC using BSL-3 practices, especially when

performing activities with a high potential for aerosol or droplet production.

Potential Lab Exposures

Ingestion, inhalation, inoculation and direct contact via skin abrasions and mucous membranes. Brucella spp. have a very

low infectious dose and laboratory workers can acquire brucellosis from direct exposure to samples or cultures.

Specimen Collection

Ideal Time & Temp

Transport

Within Facility

Storage

Acute,

Subacute

or Chronic

Serum

Collect at least 1 mL acute phase specimen without anti-coagulant

as soon as possible after disease onset. Collect a second,

convalescent specimen 14-21 days after acute specimen collection.

~2 h RT -20°C

Blood

Collect per institution’s procedure for routine blood cultures.

Note: Slow-growing in automated blood culture systems, consider extended

incubations up to 2-3 weeks.

≤2 h RT

Incubate per

lab protocol

Bone

Marrow

Collect per institution’s surgical or pathology procedure. ≤15 min RT ≤24 h 4°C

Spleen

or Liver

Collect tissue samples at least the size of a pea. Submit in sterile

container. May add 1-2 drops of saline to keep moist.

≤1 h RT ≤24 h RT

Characterization

BRUCELLOSIS — Brucella spp.

Gram Stain

• Faintly staining, not clustered, tiny

Gram negative coccobacilli

(0.4 µm-0.8 µm)

• May retain crystal violet stain and may

be mistaken for Gram positive cocci

Biochemical/Test Reactions

• Catalase, oxidase and urea positive

Note: Oxidase may be variable and test

should be performed on fresh cultures

(18-24h)

• S. aureus streak negative

(X & V Factor satellite test)

Colony Morphology

• Aerobic, slow growth

• Slow growth seen on BAP and CHOC

(CO

may be required by some strains)

• Poor to variable growth on MAC.

Pinpoint colonies may infrequently

be observed with some strains after

extended blood culture incubation

(7 days)

• Non-mucoid

• Pinpoint colonies at 24h, and easily

visible, discrete, white, non-hemolytic

colonies at 48h (0.5 mm-1 mm)

• Colonies on BAP have no

distinguishing features. They will

appear as white, non-pigmented and

non-hemolytic. Colonies will appear as

raised and convex with an entire edge

and shiny surface

Common Misidentifications

May not be identied in common

automated ID systems, including MALDI

TOF, and possible misidentications may

include: Moraxella spp., Micrococcus spp.,

Corynebacterium spp., “slow growing”

Staphylococcus spp., Oligella ureolytica,

Bordetella bronchiseptica, Haemophilus

spp., Pasteurella spp., Psychrobacter

phenylpyruvicus and Psychrobacter

immobilis.

Gram Stain

48h growth on BAP

72h growth on CHOC

Rule-Out Algorithm

BRUCELLOSIS — Brucella spp.

As soon as Brucella is suspected, perform all further work in a Class II BSC using BSL-3 practices. If Brucella spp. cannot

be ruled out with tests below, do not attempt further ID using commercial automated or kit identication systems.

Gram stain morphology

□

Faint staining, not clustered, tiny (0.4 x 0.8µm), Gram

negative coccobacilli?

Note: May retain crystal violet stain and be mistaken for Gram positive cocci

Growth

□

Subculture positive aerobic blood culture to BAP, CHOC?

□

Aerobic, slow, poorly growing colonies after 24h incubation in

5-10% CO

at 35°C?

Note: Incubate plates for at least two additional days if no growth in 24h.

□

Organism not growing on MAC?

□

Slow growing in automated blood culture systems?

Note: Consider extended incubations up to 2-3 weeks.

Consider Haemophilus

Consider Francisella

Refer to ASM sentinel procedures

Reincubate

Use internal laboratory procedure

48h growth on BAP

24h growth on CHOC

Brucella spp.

ruled out

YES

YES, STOP

Brucella spp. not ruled-out. Do not attempt further identication and contact your LRN

Reference Level Laboratory to refer the isolate. Suggested Reporting Language: Possible

Brucella spp. submitted to LRN Reference Level Laboratory for conrmatory testing.

Satellite negative at 24-48 hours?

Note: Spot BAP with S. aureus ATCC 25923

Oxidase and catalase positive?

Urea positive?

SAFETY

YES TO ALL

NO TO ANY

Handling Instructions

GLANDERS

—

Burkholderia mallei

MELIOIDOSIS

—

Burkholderia pseudomallei

Safety

Patient specimens can be handled using BSL-2 practices.

As soon as B. mallei or B. pseudomallei are suspected, perform all further work within a Class II BSC using BSL-3

practices, especially when performing activities with a high potential for aerosol or droplet production.

Potential Lab Exposures

Ingestion, inhalation, inoculation, and direct contact via skin abrasions and mucous membranes.

Specimen Collection

Ideal Time & Temp

Transport

Within Facility

Storage

Blood or

Bone Marrow

Collect using standard automated blood culture system per

institution’s procedure for routine blood culture.

≤2 h RT

Delayed entry

depends on

instrument

Sputum/Bronchial

Collect into sterile leak proof container. ≤2 h RT ≤24 h 4°C

Abscess Material

and Wounds

Tissue aspirate, tissue uid preferred to swab alternative. ≤2 h RT ≤24 h 4°C

Urine

Collect at least 1 mL in leak proof container. ≤2 h RT ≤24 h 4°C

Characterization

GLANDERS — Burkholderia mallei

Gram Stain

• Small straight or slightly curved Gram

negative coccobacilli (1.5 µm-3 µm x

0.5-1 µm) with rounded ends

• Cells arranged in pairs, parallel

bundles, or the Chinese letter form

Colony Morphology

• Aerobic

• On BAP:

• Pinpoint to small grey colonies at

24h that may become smooth,

grey, and translucent at 48h with

no distinctive odor

• Non-hemolytic

• On MAC: No growth or pinpoint

colorless colonies after 48h

• No pigment, even on Mueller Hinton

agar

• No growth at 42˚C

Biochemical/Test Reactions

• Catalase positive

• Oxidase variable; most are negative

• Spot indole negative

• Non-motile (Recommend tube test,

not wet mount, due to potential

aerosol production)

• Polymyxin B and colistin no zone,

penicillin resistant, amoxicillin-

clavulanate susceptible

Common Misidentifications

May not be identied in common

automated ID systems, including MALDI-

TOF, and possible misidentications

may include: Burkholderia cepacia,

Chromobacterium violaceum,

Pseudomonas stutzeri, Bacillus spp.,

Pandoraea spp., Ralstonia spp. other

nonfermenting Gram negative bacilli.

Note: B. pseudomallei and B. mallei are arginine positive, unlike other Burkholderia; the

arginine test may be in kit identication systems.

Gram Stain

48h growth on BAP

24h growth on BAP

Rule-Out Algorithm

GLANDERS — Burkholderia mallei

As soon as Burkholderia is suspected, perform all further work in a Class II BSC using BSL-3 practices. If B. mallei cannot

be ruled out with tests below, do not attempt further ID using commercial automated or kit identication systems.

Gram stain morphology

□

Small straight or slightly curved Gram

negative coccobacilli with rounded ends?

□

Cells arranged in pairs, parallel bundles

or the Chinese letter form?

Colony morphology

□

Poor growth at 24h on all media?

□

Better growth of grey, translucent

colonies without pigment or hemolysis

at 48h on BAP?

□

Poor or no growth on MAC in 48h?

□

No distinctive odor (from closed plate)?

Reactions

□

Oxidase-variable?

Consider

Brucella

B. mallei &

B. pseudomallei

ruled out

B. mallei

ruled out

Consider

B. pseudomallei

24h growth on BAP

48h growth on BAP

Burkholderia mallei not ruled-out. Do not attempt further identication and contact your LRN

Reference Level Laboratory to refer the isolate. Suggested Reporting Language: Possible

Burkholderia mallei submitted to LRN Reference Level Laboratory for conrmatory testing.

YES

Spot indole negative, catalase positive,

non-hemolytic on BAP, no pigment?

Polymyxin B or colistin: no zone; amoxicillin-

clavulanate susceptible; penicillin resistant?

No growth at 42°C and no odor?

Non-motile?

SAFETY

NO TO ANYYES TO ALL

YES, STOP

Characterization

MELIOIDOSIS — Burkholderia pseudomallei

Gram Stain

• Straight, or slightly curved Gram negative

rod (2-5 µm x 0.4-0.8 µm)

• Colonies may demonstrate bipolar

morphology in direct specimens and

peripheral staining in older cultures,

which can mimic endospores

Colony Morphology

• Aerobic

• On BAP: small, smooth, creamy colonies

in the rst 1-2 days, that may gradually

change in time to dry, wrinkled colonies

(similar to Pseudomonas stutzeri)

• Poor growth at 24h, good growth at 48h

• Colonies are non-hemolytic and not

pigmented on BAP or Mueller Hinton agar.

• Grows on MAC (may uptake pink dye)

• Distinctive musty, earthy odor is apparent

without snifng or opening plate

• Growth at 42˚C

Biochemical/Test Reactions

• Oxidase positive

• Spot indole negative

• Motile

Note: Tube test, not wet mount, is

recommended due to potential aerosolization

• Polymyxin B and colistin no zone,

penicillin resistant, amoxicillin-clavulanate

susceptible

Common Misidentifications

May not be identied in common automated

ID systems, including MALDI TOF, and

possible misidentications may include:

Burkholderia cepacia*, Chromobacterium

violaceum, Pseudomonas aeruginosa,

Pseudomonas stutzeri, S. maltophilia and

other nonfermenting Gram negative bacilli.

* B. pseudomallei is separated from B. cepacia by a

susceptible amoxicillin-clavulanate test. Although rare

in B. pseudomallei, resistance cannot rule out the

identication.

Note: B. pseudomallei and B. mallei are arginine positive, unlike other Burkholderia; arginine test may be

in kit identication systems. Also, unlike B. mallei, B. pseudomallei grows at 42°C in 48h and is motile.

Gram Stain

24h growth on BAP

48h growth on BAP

48h growth on MAC

Rule-Out Algorithm

MELIOIDOSIS — Burkholderia pseudomallei

As soon as Burkholderia is suspected, perform all further work in a Class II BSC using BSL-3 practices. If B. pseudomallei

cannot be ruled out with tests below, do not attempt further ID using commercial automated or kit identication systems.

24h growth on BAP

48h growth on BAP

Gram stain morphology

□

Gram negative rod, straight or slightly

curved?

Note: May demonstrate bipolar morphology

at 24h and peripheral staining, like

endospores, as cultures age.

Colony morphology

□

Poor growth at 24h, but good growth of

smooth, creamy colonies at 48h on BAP?

Note: May develop wrinkled colonies in

time

□

Non-hemolytic?

□

Strong musty/earthy odor (apparent without

opening plate), growth on MAC in 48h?

□

Non-pigmented on Mueller Hinton agar

and BAP?

Reactions

□

Oxidase positive, spot indole negative?

Growth on MAC?

Oxidase positive and

spot indole negative?

Polymyxin B or colistin; no zone or

growth on B. cepacia selective agars

No hemolysis on BAP; not pigmented

Burkholderia pseudomallei not ruled-out. Do not attempt further identication and contact your

LRN Reference Level Laboratory to refer the isolate. Suggested Reporting Language: Possible

Burkholderia pseudomallei submitted to LRN Reference Level Laboratory for conrmatory testing.

Not Burkholderia

Rule out other agents such as

B. mallei, Brucella and Francisella

Not Burkholderia

Consider Chromobacterium violaceum

or indole-negative Vibrio spp.

YES

SAFETY

YES TO ALL NO TO ANY

YES, STOP

Handling Instructions

TULAREMIA — Francisella tularensis

Safety

Patient specimens can be handled using BSL-2 practices.

As soon as F. tularensis is suspected, perform all further work within a Class II BSC using BSL-3 practices, especially when

performing activities with a high potential for aerosol or droplet production.

Potential Lab Exposures

Ingestion, inhalation, inoculation, and direct contact via skin abrasions and mucous membranes. Francisella tularensis has

a very low infectious dose and laboratory workers can acquire Tularemia from direct exposure to samples or cultures.

Specimen Collection

Ideal Time & Temp

Transport

Within Facility

Storage

Sputum or

Throat

Collect routine throat culture using a swab or expectorated sputum collected into a

sterile, leak proof container.

≤2 h RT ≤24 h 4°C

Bronchial or

Tracheal Wash

Collect per institution’s procedure in an area dedicated to collecting respiratory specimens

under isolation or containment circumstances (i.e., isolation chamber or “bubble”).

≤2 h RT ≤24 h 4°C

Blood

Collect per institution’s procedure for routine blood cultures. ≤2 h RT

Incubate per

lab protocol

Biopsy, Tissue,

Scrapings,

Aspirate

or Swab

Submit in sterile container. For small tissue samples add several drops of sterile

normal saline to keep tissue moist. For swabs, collect by obtaining rm sample of

advancing margin of the lesion; place swab in transport package to keep moist with

the transport medium inside packet.

≤2 h RT ≤24 h 4°C

Serum

Collect at least 1 mL without anticoagulant. Collect acute specimen as soon as

possible after onset and a convalescent specimen >14 days after acute.

≤2 h RT 4°C

Characterization

TULAREMIA — Francisella tularensis

Gram Stain

• Tiny, Gram negative coccobacilli

(0.2-0.5 µm x 0.7-1.0 µm)

• Poor counterstaining with safranin

(basic fuchsin counterstain may

increase resolution)

• Pleomorphic

• Mostly single cells

Colony Morphology

• Aerobic, fastidious

• No growth on MAC or EMB

• Scant or no growth on BAP; may

grow on primary culture, not well on

subculture

• Slow growing on CHOC, TM or BCYE:

1-2 mm after 48h

• Colonies are opaque, grey-white,

butyrous with smooth and shiny

surface

Biochemical/Test Reactions

• Oxidase negative

• Catalase negative or weakly positive

• Satellite negative

• Beta-lactamase positive

Common Misidentifications

May not be identied in common

automated ID systems, including MALDI

TOF, and possible misidentications

may include: Aggregatibacter

actinomycetemcomitans, Haemophilus

inuenzae, Oligella spp. and

Psychrobacter spp.

Gram Stain

Gram stain of a blood culture

48h growth on CHOC

Rule-Out Algorithm

TULAREMIA — Francisella tularensis

As soon as Francisella is suspected, perform all further work in a Class II BSC using BSL-3 practices. If F. tularensis cannot

be ruled out with tests below, do not attempt further ID using commercial automated or kit identication systems.

Gram stain morphology

□

Pleomorphic?

□

0.2–0.5 µm by 0.7–1.0 µm faintly

staining, Gram negative coccobacillus?

□

Mostly single cells?

Colony morphology

□

Aerobic and fastidious?

□

No growth on MAC/EMB

□

Scant to no growth on BAP after 48h?

Note: may grow on primary BAP culture, but

not on subculture.

□

Slow growth on CHOC, TM or BCYE?

□

1-2 mm gray to grayish-white colonies

on CHOC after 48h

□

Colonies opaque, grey-white, butyrous

with smooth and shiny surface?

24h: Growth on CHOC but not BAP?

48h: Growth better on CHOC than BAP?

Are colonies satellite negative?

Oxidase negative and either

catalase weakly positive or negative?

β-lactamase positive?

No growth on MAC?

Francisella tularensis not ruled-out. Do not attempt further identication and contact

your LRN Reference Level Laboratory to refer the isolate. Suggested Reporting Language:

Possible F. tularensis submitted to LRN Reference Level Laboratory for conrmatory testing.

Consider

Haemophilus

Francisella tularensis ruled out.

Continue with routine identication

48h growth on BAP

48h growth on CHOC

YES

SAFETY

NO TO ANYYES TO ALL

YES, STOP

Handling Instructions

PLAGUE — Yersinia pestis

Safety

Patient specimens can be handled using BSL-2 practices.

As soon as Y. pestis is suspected, perform all further work within a Class II BSC using BSL-3 practices, especially when

performing activities with a high potential for aerosol or droplet production.

Potential Lab Exposures

Ingestion, inhalation, inoculation, and direct contact via skin abrasions and mucous membranes.

Specimen Selection

Ideal Time & Temp

Transport

Within Facility

Storage

Pneumonic

Sputum or

Throat

Collect routine throat culture using a swab or expectorated

sputum collected into a sterile, leak proof container

≤2 h RT ≤24 h 4°C

Bronchial

or Tracheal

Wash

Collect per institution’s procedure in an area dedicated to

collecting respiratory specimens under isolation or containment

circumstances (i.e., isolation chamber or “bubble”)

≤2 h RT ≤24 h 4°C

Septicemic Blood

Collect per institution’s procedure for routine blood cultures ≤2 h RT

Incubate per

lab protocol

Bubonic

Tissue or

Aspirate

Submit in sterile container, may add 1-2 drops of saline to keep

moist

≤2 h RT ≤24 h 4°C

Characterization

PLAGUE — Yersinia pestis

Gram Stain

• Plump Gram negative rods

(0.5 x 1-2 µm) seen mostly as single

cells or pairs, and may demonstrate

short chains in liquid media

• May exhibit bipolar, “safety-pin”

appearance that is not seen on

Gram stain, may be exhibited by

Giemsa stain or Wright's stain

Colony Morphology

• Facultative anaerobe

• Slow growing at 35˚C, better growth

at 25-28˚C

• Grey-white, translucent pinpoint

colonies at 24h, usually too small to

be seen

• On BAP:

• After 48h: colonies approximately

1-2 mm in diameter, gray-white to

slightly yellow and opaque

• Older cultures (~96h): “Fried

egg” or “hammered copper”

appearance (under magnication)

• Little to no hemolysis

• Lactose non-fermenter at 48h on

MAC or EMB

Biochemical/Test Reactions

• Catalase positive

• Oxidase, urease (at 35˚C) and indole

negative

Common Misidentifications

May not be identied in common automated

ID systems, including MALDI TOF, and

possible misidentications may include:

Shigella spp., H

S(-) Salmonella spp.,

Acinetobacter or Pseudomonas spp. and

Yersinia pseudotuberculosis.

Gram Stain

48h growth on BAP

24h growth on BAP at 25°C (left) and 35°C (right)

Rule-Out Algorithm

PLAGUE — Yersinia pestis

48h growth on MAC

Fried egg appearance at 96h

(magniﬁed)

As soon as Yersinia is suspected, perform all further work in a Class II BSC using BSL-3 practices. If Y. pestis cannot be

ruled out with tests below, do not attempt further ID using commercial automated or kit identication systems.

Gram stain morphology

□

Gram-negative plump rods,

0.5 x 1-2 µm?

Note: Seen mostly as single cells or pairs,

and may demonstrate short chains in liquid

media.

Colony morphology

□

Facultative anaerobe?

□

Slow growing at 35°C with better growth

at 25-28°C?

□

Either pinpoint colonies or no growth on

BAP after 24h

□

Colonies are 1-2 mm, gray-white to

slightly yellow and opaque on BAP after

48h?

□

Non-lactose fermenter on MAC/EMB?

□

“Fried egg” or “hammered copper” on

BAP in older cultures (~96h), when

magnied?

□

Little to no hemolysis on BAP?

Oxidase negative, catalase positive

and indole negative?

Yersinia pestis not ruled-out. Do not attempt further identication and contact your LRN

Reference Level Laboratory to refer the isolate. Suggested Reporting Language:

Possible Y. pestis submitted to LRN Reference Level Laboratory for conrmatory testing.

Yersinia pestis ruled out.

Continue routine identication

YES

Urease negative at 35°C?

SAFETY

YES TO ALL

NO TO ANY

YES, STOP

APHL ..............Association of Public Health

Laboratories

ASM ...............American Society for Microbiology

BAP .................Blood agar plate

BCYE ...............Buffered Charcoal Yeast Extract

BSC .................Biological safety cabinet

BSL ..................Biosafety Level (1 - 4)

BT .....................Biothreat

CDC .................Centers for Disease Control and

Prevention

CHOC ..............Chocolate agar

EMB .................Eosin Methylene Blue agar

LRN .................Laboratory Response Network

MAC ...............MacConkey agar

MALDI TOF .... Matrix Assisted Laser Desorption/

Ionization Time of Flight Mass

Spectrometer

NF ....................Non-fermentor

PPE ..................Personal Protective Equipment

RT ....................Room Temperature

TM ....................Thayer Martin agar

TTC...................2,3,5-Triphenyltetrazolium

chloride

Acronyms

APPENDIX

Administrative controls

Changes in work procedures such as written

safety policies, work practices, rules, supervision,

schedules and training with the goal of reducing

the duration, frequency and severity of exposures

to hazardous materials or situations.

Aerobic

Requiring oxygen.

Aerosolization

The generation of liquid droplets or particles, ve

microns or less in diameter, that can be inhaled

and retained in the lungs.

Anaerobic

Requiring the absence of oxygen.

Antimicrobial

An agent that kills microorganisms or suppresses

their growth and multiplication.

Antiseptic

A substance that inhibits the growth and

development of microorganisms without

necessarily killing them. Antiseptics are usually

applied to body surfaces.

Barriers

Any method used to separate workers, the outside

community and the environment from hazardous

material; includes primary and secondary barriers.

Barriers, Primary

Specialized laboratory equipment with

engineering controls designed to protect against

exposure to hazardous laboratory materials,

including, but not limited to, biologic safety

cabinets, chemical fume hoods, enclosed

containers, bench shields, animal cages, and

engineered sharps injury-protection devices

(e.g., safety needles, safety scalpels, and sharps

containers).

Barriers, Secondary

Facility design and construction features to

include, but not be limited to, directional air

ow, entrance airlocks, controlled-access

zones, HEPA-ltered exhaust air, facility controls,

decontamination equipment, eyewash stations,

protective showers, and sinks for hand washing.

Biohazardous materials

Infectious agents or hazardous biologic materials

that present a risk or potential risk to the health

of humans, animals, or the environment. The risk

can be direct through infection or indirect through

damage to the environment. Biohazardous

materials include certain types of recombinant

DNA, organisms and viruses infectious to humans,

animals, or plants (e.g., parasites, viruses,

bacteria, fungi, prions, and rickettsia), and

biologically active agents (e.g., toxins, allergens,

and venoms) that can cause disease in other

living organisms or cause signicant impact to the

environment or community.

BSL-1

Biosafety Level 1 is suitable for work involving

well-characterized agents not known to

consistently cause disease in immunocompetent

adult humans, and present minimal potential

hazard to laboratory personnel and the

environment.

BSL-2

Biosafety Level 2 builds upon BSL-1. BSL-

2 is suitable for work involving agents that

pose moderate hazards to personnel and the

environment. (Most Sentinel Laboratory facilities

fall under the denition of BSL-2).

BSL-3

Biosafety Level 3 is applicable to clinical,

diagnostic, teaching, research, or production

facilities where work is performed with indigenous

or exotic agents that may cause serious or

potentially lethal disease through the inhalation

route of exposure.

Terms and Definitions

APPENDIX

BSL-4

Biosafety Level 4 is required for work with

dangerous and exotic agents that pose a high

individual risk of aerosol-transmitted laboratory

infections and life-threatening disease that is

frequently fatal, for which there are no vaccines or

treatments, or a related agent with unknown risk

of transmission.

Containment

Methods used to shield or protect personnel, the

immediate work environment, and the community

from exposure to hazardous, radiologic, chemical,

or biologic materials.

Decontamination

The removing of chemical, biologic, or radiologic

contamination from, or the neutralizing of it on, a

person, object, or area. Any process for removing

and/or killing microorganisms. The same term is

also used for removing or neutralizing hazardous

chemicals and radioactive materials.

Disinfectant

A chemical or mixture of chemicals used to kill

microorganisms, but not necessarily spores.

Disinfectants are usually applied to inanimate

surfaces or objects.

Disinfection

A physical or chemical process of reducing or

eliminating microorganisms from a surface or

space, but not necessarily spores.

Droplet nuclei

The residue of dried droplets of infectious agents

that is easily inhaled and exhaled and can remain

suspended in air for relatively long periods or be

blown over great distances.

Droplet spread

The direct transmission of an infectious agent

by means of the aerosols produced in sneezing,

coughing, or talking that travel only a short

distance before falling to the ground.

Engineering controls

Refers to methods to remove a hazard or place

a protective barrier between the worker and the

workplace hazard, which usually involves building

design elements and specialized equipment.

Exposure

Having come into contact with a cause of, or

possessing a characteristic that is a determinant

of, a particular health problem.

Fomite

An inanimate object that can be the vehicle for

transmission of an infectious agent (e.g., bedding,

towels or surgical instruments).

Incident

An unexpected event that causes or has the

potential to cause loss, injury, illness, unsafe

conditions, or disruptions to normal procedures.

Incubation period

The time interval from exposure to an infectious

agent to the onset of symptoms of an infectious

disease.

Infection

Invasion of the body tissues of a host by an

infectious agent, whether or not it causes disease.

Medical surveillance

Monitoring of a person who might have been

exposed to an infectious, chemical, radiologic, or

other potentially causal agent, for the purpose of

detecting early symptoms.

Mitigate

To correct identied deciencies and to make

a hazard less severe. This includes corrective

actions taken as a result of an inspection or audit,

or after an incident.

Mode of transmission

The manner in which an agent is transmitted from

its reservoir to a susceptible host.

Terms and Definitions

APPENDIX

Personal protective equipment (PPE)

Items worn by laboratory workers to prevent direct

exposure to hazardous materials, including gloves,

gowns, aprons, coats, containment suits, shoe

covers, eye and face shields, respirators, and masks.

Risk

The probability that an event will occur (e.g., that

a person will be affected by, or die from, an

illness, injury, or other health condition within a

specied time or age span).

Risk assessment

A process to evaluate the probability and

consequences of exposure to a given hazard, with

the intent to reduce the risk by establishing the

appropriate hazard controls to be used.

Risk factor

An aspect of personal behavior or lifestyle,

an environmental exposure, or a hereditary

characteristic that is associated with an increase

in the occurrence of a particular disease, injury,

or other health condition.

Routes of exposure

Paths by which humans or other living organisms

come into contact with a hazardous substance.

Three routes of exposure are breathing

(inhalation), eating or drinking (ingestion), and

contact with skin (dermal absorption).

Sharps

Items capable of cutting or piercing human skin.

Examples include hypodermic needles, syringes

(with or without attached needles), Pasteur

pipettes, scalpel blades, suture needles, blood

vials, needles with attached tubing, and culture

dishes (regardless of presence of infectious

agents). Also included are other types of broken

or unbroken glassware that have been in contact

with infectious agents (e.g., used microscope

slides and cover slips).

Sterilization

The use of physical or chemical process to

completely destroy or eliminate all classes of

microorganisms and spores.

Symptom

Any indication of disease noticed or felt by a

patient.

Transmission (of infection)

Any mode or mechanism by which an infectious

agent is spread to a susceptible host. Airborne

transmission is the transfer of an agent

suspended in the air (considered a type of

indirect transmission). Direct transmission

is the immediate transfer of an agent from a

reservoir to a host by direct contact or droplet

spread. Indirect transmission is the transfer of an

agent from a reservoir to a host either by being

suspended in air particles (airborne), carried by

an inanimate objects (vehicleborne), or carried by

an animate intermediary (vectorborne).

TTC

2,3,5-Triphenyltetrazolium chloride, indicator dye

within motility test medium.

Universal precautions

Guidelines recommended by CDC for reducing

the risk for transmission of bloodborne and

other pathogens in hospitals, laboratories, and

other institutions in which workers are potentially

exposed to human blood and body uids. The

precautions are designed to reduce the risk

for transmission of microorganisms from both

recognized and unrecognized sources of infection

in hospitals, laboratories, and other institutions to

the workers in these facilities.

Virulence

The ability of an infectious agent to cause severe

disease, measured as the proportion of persons

with the disease who become severely ill or die.

Zoonosis

An infectious disease that is transmissible from

animals to humans.

Terms and Definitions

APPENDIX

Urea

Look for pink color change

Negative Positive

Identification Tests

APPENDIX

Spot Indole

Look for color change, varies by

reagent; Cinnamaldehyde preferred

Cinnamaldehyde:

positive is blue

Benzaldehyde:

positive is pink

Arginine Dihydrolase (Decarboxylase)

Look for pink/purple color change

Uninoculated

Base

Positive Base Negative Positive

Controls

NF Base Positive

Catalase

3% Hydrogen peroxide: look for bubbles

Negative Weak Positive Positive

Safety Note: Recommended to perform this test in a

BSC, covered petri dish or tube to contain aerosols

Oxidase

Tetramethyl reagent: look for purple color change

Negative Positive

X/V Factor Satellite Test

Use Staphylococcus aureus-streaked media

or X and V growth factor-impregnated discs

Negative

Growth is not isolated to area immediately

adjacent to S. aureus streak or X and V factors

Positive (Satellite)

Growth occurs only along S. aureus streak/

X and V factors

Motility

Negative (Non-motile)

Growth only in line of inoculum; no fuzziness or

spreading; media is clear

Intermediate

Start to see growth outside line of inoculum

(appears fuzzy), media still clear

Positive (Motile)

Distinct growth outside line of inoculum into the

media, which is not clear

Safety Note: Avoid wet mount motility tests, which are hazardous

due to the potential for creating an aerosol. Perform a tube motility

test instead, and always in a BSC.

Identification Tests

APPENDIX

Negative: Brucella growing across entire plate

Positive: Haemophilus growing only around

the Staphylococcus aureus streak

Negative Intermediate Positive

With 2,3,5-Triphenyltetrazolium chloride (TTC)

TTC: Colorless medium dye, turns red when reduced by

bacteria. Inhibits some bacteria; look for growth away

from line of inoculum.

Negative Positive

No Additives

APHL

Public Health Preparedness & Response Program

aphl.org/programs/preparedness/Pages/default.aspx

Lab Biosafety & Biosecurity Resources

aphl.org/programs/preparedness/Pages/Strengthening-Lab-Biosafety-

Biosecurity.aspx

State Public Health Laboratories Emergency Contact