NATIONAL STANDARD METHOD

IDENTIFICATION OF
CLOSTRIDIUM SPECIES

BSOP ID 8

Issued by Standards Unit, Evaluations and Standards Laboratory

Centre for Infections

IDENTIFICATION OF CLOSTRIDIUM SPECIES

Issue no: 3 Issue date: 14.07.08 Issued by: Standards Unit, Evaluations and Standards Laboratory Page no: 1 of 14
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STATUS OF NATIONAL STANDARD METHODS
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Whereas every care has been taken in the preparation of this publication, the Health Protection
Agency or any supporting organisation cannot be responsible for the accuracy of any statement or
representation made or the consequences arising from the use of or alteration to any information
contained in it. These procedures are intended solely as a general resource for practising
professionals in the field, operating in the UK, and specialist advice should be obtained where
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Suggested citation for this document:

Health Protection Agency (2008). Identification of Clostridium species. National Standard Method
BSOP ID 8 Issue 3. http://www.hpa-standardmethods.org.uk/pdf_sops.asp.

IDENTIFICATION OF CLOSTRIDIUM SPECIES

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INDEX
STATUS OF NATIONAL STANDARD METHODS ................................................................................ 2

INDEX...................................................................................................................................................... 3

AMENDMENT PROCEDURE ................................................................................................................. 4

SCOPE OF DOCUMENT ........................................................................................................................ 5

INTRODUCTION ..................................................................................................................................... 5

TECHNICAL INFORMATION/LIMITATIONS ......................................................................................... 5

1 SAFETY CONSIDERATIONS ......................................................................................................... 6

2 TARGET ORGANISMS ................................................................................................................... 6

3 IDENTIFICATION............................................................................................................................. 7
3.1 MICROSCOPIC APPEARANCE ........................................................................................................ 7
3.2 PRIMARY ISOLATION MEDIA .......................................................................................................... 7
3.3 COLONIAL APPEARANCE............................................................................................................... 7
3.4 TEST PROCEDURES ..................................................................................................................... 8
3.5 FURTHER TESTS .......................................................................................................................... 8
3.6 STORAGE AND REFERRAL............................................................................................................. 8
4 IDENTIFICATION OF CLOSTRIDIUM SPECIES - FLOW CHART ............................................... 9

5 REPORTING .................................................................................................................................. 10
5.1 PRESUMPTIVE IDENTIFICATION ................................................................................................... 10
5.2 CONFIRMATION OF IDENTIFICATION ............................................................................................. 10
5.3 MEDICAL MICROBIOLOGIST ......................................................................................................... 10
5.3 CCDC...................................................................................................................................... 10
5.5 CENTRE FOR INFECTIONS ........................................................................................................... 10
5.6 INFECTION CONTROL STAFF ....................................................................................................... 10
6 REFERRALS ................................................................................................................................. 11

7 ACKNOWLEDGEMENTS AND CONTACTS................................................................................ 12

REFERENCES ...................................................................................................................................... 13

IDENTIFICATION OF CLOSTRIDIUM SPECIES

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AMENDMENT PROCEDURE

Controlled document BSOP ID 8

reference
Controlled document title Identification of Clostridium species

Each National Standard Method has an individual record of amendments. The current amendments
are listed on this page. The amendment history is available from [email protected].

On issue of revised or new pages each controlled document should be updated by the copyholder in
the laboratory.

Amendment Issue no. Insert Page Section(s) involved Amendment

Number/ Discarded Issue
Date no.
3/ 2.1 3 1 Front Page NIMAG logo added
14.07.08
All All PDF links amended to
read reference
document title
13 References References reviewed
and updated

IDENTIFICATION OF CLOSTRIDIUM SPECIES

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 IDENTIFICATION OF CLOSTRIDIUM SPECIES
SCOPE OF DOCUMENT
This National Standard Method (NSM) describes the identification of Clostridium species.
There are many species of clostridia, which may be found naturally in animal faeces and the
environment. Only species associated with humans will be discussed in this NSM.

INTRODUCTION
Taxonomy
The genus Clostridium currently contains approximately 100 species. In 1994 the heterogeneity of
this species was confirmed by 16S rRNA gene sequencing. As a result five new genera and eleven
new species were proposed2, none of which appear to be relevant to human infections3.
Characteristics of Clostridium species
Clostridium species are Gram-positive rods (some are Gram-variable), often arranged in pairs or short
chains, with rounded or sometimes pointed or square end. They are often pleomorphic. Clostridium
species vary considerably in their oxygen tolerance. Some species such as Clostridium novyi type A
and Clostridium haemolyticum are among the strictest of obligate anaerobes and may require
extended incubation on pre-reduced or freshly prepared plates and total handling in an anaerobic
chamber. Conversely, Clostridium tertium, Clostridium histolyticum and Clostridium carnis are
aerotolerant and will form colonies on blood agar plates incubated in an atmosphere of air with
5-10% added CO23.

Virtually all of the members of the genus, except Clostridium perfringens, are motile with peritrichous
flagellae and form oval or spherical endospores that may distend the cell. They may be saccharolytic
or proteolytic and are usually catalase-negative. Many species produce potent exotoxins4.
Toxins of Clostridium species
Clinically significant Clostridium species produce a variety of toxins. It is the production of these toxins
which leads to the distinctive clinical features of the diseases they cause, eg tetanus and botulism
result from the production of neurotoxins that are amongst the most lethal substances known to man5.
Clostridial toxins are biologically active proteins that are antigenic in nature and can therefore be
neutralised with specific antisera. Detection of a particular toxin in a patient sample may be diagnostic
and therefore render isolation of the organism unnecessary (eg Clostridium difficile).

Clostridium perfringens is the most commonly isolated Clostridium species. Five types (A-E) may be
distinguished by the combinations of major lethal toxins they produce3.
Principles of Identification
Clues to the identity of certain pathogenic species may be obtained by observing characteristics such
as colonial appearance, Gram stain appearances and the presence or absence of -haemolysis.
Other phenotypic tests may also be applied to obtain a presumptive identification in conjunction with
the use of a good laboratory manual such as the Wadsworth-KTL Anaerobe Laboratory Manual6. It is
important to ensure the culture is pure, as the fine spreading growth of some Clostridium species may
mask contaminating organisms. If confirmation of identity is required, isolates should be referred to
the Anaerobe Reference Laboratory, Cardiff.

If Clostridium botulinum is suspected, samples of patients serum, faeces and implicated foodstuff
should be referred directly to the Food Safety Microbiology Laboratory, Colindale.

TECHNICAL INFORMATION/LIMITATIONS
N/A

IDENTIFICATION OF CLOSTRIDIUM SPECIES

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1 SAFETY CONSIDERATIONS7-18
Hazard Group 2 organisms

Refer to current guidance on the safe handling of all Hazard Group 2 organisms documented
in this NSM.

Laboratory procedures that give rise to infectious aerosols must be conducted in a

microbiological safety cabinet.

The above guidance should be supplemented with local COSHH and risk assessments.

Compliance with postal and transport regulations is essential.

2 TARGET ORGANISMS
Clostridium species reported to have caused human disease4

Commonly isolated
C. perfringens
C. septicum
C. tertium
C. difficile

Rarely isolated
C. novyii type A
C. sordellii

Very rarely isolated

C. tetani
C. histolyticum
C. botulinum

Commonly isolated non-pathogenic clostridia

C. sporogenes
C. ramosum
C. innocuum
C. paraputrificum
C. cadaveris
C. bifermentans
C. fallax
C. clostridioforme

IDENTIFICATION OF CLOSTRIDIUM SPECIES

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3 IDENTIFICATION
3.1 MICROSCOPIC APPEARANCE
(See BSOPTP 39 - Staining Procedures)
Gram stain
Gram-positive rods, which may possess a single endospore. Some species may be Gram-
variable.

Spore stain

Used to determine the shape and position of the spore (phase contrast microscopy is an
alternative option).

C. perfringens (Does not sporulate on ordinary media)

C. botulinum Oval, subterminal
C. difficile Oval, subterminal
C. novyi Oval, subterminal
C. sordellii Oval, subterminal
C. septicum Oval, subterminal
C. tetani Round, terminal
3.2 PRIMARY ISOLATION MEDIA
Agar containing blood incubated anaerobically at 35C - 37C for 40 48 h.
3.3 COLONIAL APPEARANCE
Colonial appearance varies with species and brief descriptions of the most common species
are given here

Organism Characteristics of growth on agar containing blood after

anaerobic incubation at 35C 37C for 40 48 h

C. perfringens Large, smooth, regular convex colonies, but may be rough and flat with
an irregular edge. Usually has a double zone of -haemolysis;
produces lecithinase

C. botulinum/ Large (3 mm), irregularly circular, smooth, greyish, translucent with a

sporogenes fibrillar edge that may spread. Most strains are -haemolytic; produces
lipase

C. difficile Glossy, grey, circular colonies with a rough edge; fluoresce green-
yellow under UV light. They are usually non-haemolytic, with a
characteristic farmyard smell.

C. novyi Raised, circular colonies, which become flattened and irregular in old
cultures. Colonies tend to fuse forming a spreading growth with a
double zone of -haemolysis. Type A produces lecithinase and lipase

C. sordellii Grey-white, convex, circular colonies with crenated edges, which may
/bifermentans spread. They may be -haemolytic; produce lecithinase; indole positive

C. septicum Usually produce a thick swarming growth with a narrow zone of

-haemolysis

C. tetani Fine swarming growth (may be difficult to see) which may appear
-haemolytic
IDENTIFICATION OF CLOSTRIDIUM SPECIES
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 Other Colonial appearances vary, but may produce a spreading growth which
Clostridium may or may not be -haemolytic
species

3.4 TEST PROCEDURES

Nagler (see BSOPTP 22 - Nagler test) with C. perfringens antitoxin

C. perfringens lecithinase is inhibited by the antitoxin as is that produced by C. bifermentans

and C. sordellii.

Species other than C. perfringens may produce lecithinase.

Also examine for the production of lipase (pearly layer) on egg yolk agar.

Reverse CAMP test can be used for differentiation of C. perfringens from other Clostridium
species19.

Commercial identification kits

Results should be interpreted with caution in conjunction with other test results.

If clinically indicated refer to the Anaerobe Reference Laboratory for further identification.
3.5 FURTHER TESTS
N/A
3.6 STORAGE AND REFERRAL
If required save the pure isolate in fastidious anaerobe broth or Robinsons cooked meat broth
for referral to the Anaerobe Reference Laboratory.

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4 IDENTIFICATION OF CLOSTRIDIUM SPECIES - FLOW CHART

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5 REPORTING
5.1 PRESUMPTIVE IDENTIFICATION
If appropriate growth characteristics, colonial appearances and Gram stain of the culture are
demonstrated and the isolate is metronidazole susceptible.
5.2 CONFIRMATION OF IDENTIFICATION
Following Nagler plate, or Reverse CAMP test for C.perfringens, commercial identification kit
results and/or Reference Laboratory report.
5.3 MEDICAL MICROBIOLOGIST
Inform the medical microbiologist of all positive cultures from normally sterile sites.

According to local protocols, the medical microbiologist should also be informed of a

presumptive and confirmed Clostridium species. when the request card bears relevant
information eg:
Cases of trauma, penetrating injury, compound fracture or retained foreign body, or
known injecting drug abuse (especially heroin)
Septic abortion
Suspicion of clostridial myonecrosis, (necrotising) myofasciitis, surgical wound infection
(especially in cases with occlusive peripheral vascular disease and/or diabetes mellitus)
Other serious medical conditions eg alcohol or substance abuse, immunodeficiency,
cancer, or persons receiving treatment for cancer (including neutropenia and/or
mucositis)
Food poisoning (especially involving descending paralysis with cranial nerve
involvement) and/or consumption of unusual or imported foods (suspicion of botulism)
Investigation of outbreaks
Pseudomembranous colitis or antibiotic-related diarrhoea
Suspicion of tetanus

Follow local protocols for reporting to clinician

5.3 CCDC
Refer to local Memorandum of Understanding.
5.5 CENTRE FOR INFECTIONS20
Refer to current guidelines on CDSC and COSURV reporting.
5.6 INFECTION CONTROL STAFF
Inform the infection control team of presumptive and confirmed isolates of C. botulinum and C.
difficile.

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6 REFERRALS
6.1 REFERENCE LABORATORY
For identification and for information on the tests offered, turn around times, transport
procedure and the other requirements of the reference laboratory refer to:

Anaerobe Reference Laboratory

NPHS Microbiology Cardiff
University Hospital of Wales
Heath Park
Cardiff CF14 4XW

Telephone +44 (0) 29 2074 2171 or 2378

http://www.hpa.org.uk/cfi/arl/default.htm

For toxin detection and for information on the tests offered, turn around times, transport
procedure and the other requirements of the reference laboratory refer to:

Food Safety Microbiology Laboratory

Centre for Infections
Health Protection Agency
61 Colindale Avenue
London NW9 5HT

http://www.hpa.org.uk/cfi/fsml/default.htm

Contact CfI main switchboard: Tel. +44 (0) 20 8200 4400

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7 ACKNOWLEDGEMENTS AND CONTACTS
This National Standard Method has been developed, reviewed and revised by the National
Standard Methods Working Group for Clinical Bacteriology
(http://www.hpa-standardmethods.org.uk/wg_bacteriology.asp). The contributions of many
individuals in clinical bacteriology laboratories and specialist organisations who have provided
information and comment during the development of this document, and final editing by the
Medical Editor are acknowledged.

The National Standard Methods are issued by Standards Unit, Evaluations and Standards
Laboratory, Centre for Infections, Health Protection Agency, London.

For further information please contact us at:

Standards Unit
Evaluations and Standards Laboratory
Centre for Infections
Health Protection Agency
Colindale
London
NW9 5EQ

E-mail: [email protected]

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REFERENCES
1. Department of Health NHS Executive: The Caldicott Committee. Report on the review of patient-
identifiable information. London. December 1997.

2. Collins MD, Lawson PA, Willems A, Cordoba JJ, Fernandez-Garayzabal J, Garcia P, et al. The
phylogeny of the genus Clostridium: proposal of five new genera and eleven new species
combinations. Int J Syst Bacteriol 1994;44:812-26.

3. Koneman EW, Allen S D, Janda W M, Schreckenberger P C, Winn WC J, editors. Color Atlas and
Textbok of Diagnostic Microbiology. 5th ed ed. Philadelphia: Lippincott Williams & Wilkins; 1997.
p. 709-84

4. Holt JG, Krieg N R, Sneath P H A, Staley J T, Williams S T, editors. Bergey's Manual of

Determinative Bacteriology. 9th ed ed. Baltimore: Williams and Wilkins; 1994. p. 560

5. Hatheway CL. Toxigenic clostridia. Clin Microbiol Rev 1990;3:66-98.

6. Jousimies-Somer H, Summanen P, Citron D, et al. Anaerobic Bacteriology Manuel. Anaerobic

Bacteriology Manuel. Sixth ed. Star Publishing Company; 2002. p. 54.

7. Advisory Committee on Dangerous Pathogens 2004 Approved List of Biological Agents.

http://www.hse.gov.uk/pubns/misc208.pdf. p. 1-17.

8. Health and Safety Executive, editor. Biological Agents: Managing the risks in laboratories and
healthcare premises. 5 A.D.

9. Public Health Laboratory Service Standing Advisory Committee on Laboratory Safety. Safety
Precautions: Notes for Guidance. 4th ed. London: Public Health Laboratory Service (PHLS); 1993.

10. Control of Substances Hazardous to Health Regulations 2002. General COSHH. Approved Code
of Practice and Guidance, L5. Suffolk: HSE Books; 2002.

11. Health and Safety Executive. 5 steps to risk assessment: a step by step guide to a safer and
healthier workplace, IND (G) 163 (REVL). Suffolk: HSE Books; 2002.

12. Health and Safety Executive. A guide to risk assessment requirements: common provisions in
health and safety law, IND (G) 218 (L). Suffolk: HSE Books; 2002.

13. Health Services Advisory Committee. Safety in Health Service laboratories. Safe working and the
prevention of infection in clinical laboratories and similar facilities. 2nd ed. Suffolk: HSE Books;
2003.

14. NHS Estates. Health Building Note 15. Accommodation for pathology services. 1st ed. London:
Her Majesty's Stationary Office (HMSO); 1991. (Out of print - 2nd edition in press).

15. BS EN 12469: 2000. Biotechnology - performance criteria for microbiological safety cabinets.
London: British Standards Institution (BSI); 2000.

16. BS 5726: 1992. Microbiological safety cabinets. Part 2. Recommendations for information to be
exchanged between purchaser, vendor and installer and recommendations for installation.
London: British Standards Institution (BSI); 1992.

17. BS 5726: 1992. Microbiological safety cabinets. Part 4. Recommendations for selection, use and
maintenance. London: British Standards Institution (BSI); 1992.

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18. Advisory Committee on Dangerous Pathogens. The management, design and operation of
microbiological containment laboratories. Suffolk: HSE Books; 2001.

19. Buchanan AG. Clinical laboratory evaluation of a reverse CAMP test for presumptive identification
of Clostridium perfringens. J Clin Microbiol 1982;16:761-2.

20. Health Protection Agency. Laboratory Reporting to the Health Protection Agency. Guide for
diagnostic laboratories. 2008.

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