UK Standards for Microbiology Investigations

Catalase Test

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Issued by the Standards Unit, Microbiology Services, PHE

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© Crown copyright 2014
 Catalase Test

Acknowledgments
UK Standards for Microbiology Investigations (SMIs) are developed under the
auspices of Public Health England (PHE) working in partnership with the National
Health Service (NHS), Public Health Wales and with the professional organisations
whose logos are displayed below and listed on the website
http://www.hpa.org.uk/SMI/Partnerships. SMIs are developed, reviewed and revised
by various working groups which are overseen by a steering committee (see
http://www.hpa.org.uk/SMI/WorkingGroups).
The contributions of many individuals in clinical, specialist and reference laboratories
who have provided information and comments during the development of this

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document are acknowledged. We are grateful to the Medical Editors for editing the
medical content.
For further information please contact us at:

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Standards Unit
Microbiology Services
Public Health England
61 Colindale Avenue
London NW9 5EQ
E-mail: [email protected]
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Website: http://www.hpa.org.uk/SMI
UK Standards for Microbiology Investigations are produced in association with:
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Contents
ACKNOWLEDGMENTS .......................................................................................................... 2

CONTENTS ............................................................................................................................. 3

AMENDMENT TABLE ............................................................................................................. 4

UK STANDARDS FOR MICROBIOLOGY INVESTIGATIONS: SCOPE AND PURPOSE ....... 5

SCOPE OF DOCUMENT ......................................................................................................... 8

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INTRODUCTION ..................................................................................................................... 8

TECHNICAL INFORMATION/LIMITATIONS ........................................................................... 8

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1 SAFETY CONSIDERATIONS ...................................................................................... 9

2 REAGENTS AND EQUIPMENT ................................................................................... 9

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QUALITY CONTROL ORGANISMS ............................................................................ 9

PROCEDURE AND RESULTS..................................................................................... 9

APPENDIX: CATALASE TEST ............................................................................................. 11

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REFERENCES ...................................................................................................................... 12
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Amendment Table
Each SMI method has an individual record of amendments. The current amendments
are listed on this page. The amendment history is available from
[email protected].
New or revised documents should be controlled within the laboratory in accordance
with the local quality management system.

Amendment No/Date. 5/13.03.14

Issue no. discarded. 2.2

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Insert Issue no. 2.3

Section(s) involved Amendment

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Document has been transferred to a new template
to reflect the Health Protection Agency’s transition
to Public Health England.

Whole document.
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Front page has been redesigned.
Status page has been renamed as Scope and
Purpose and updated as appropriate.
Professional body logos have been reviewed and
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updated.
Standard safety and notification references have
been reviewed and updated.
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Scientific content remains unchanged.

Amendment No/Date. 4/21.10.11

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Issue no. discarded. 2.1

Insert Issue no. 2.2
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Section(s) involved Amendment

Whole document. Document presented in a new format.

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References. Some references updated.

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UK Standards for Microbiology Investigations#:

Scope and Purpose
Users of SMIs
• SMIs are primarily intended as a general resource for practising professionals
operating in the field of laboratory medicine and infection specialties in the UK.
• SMIs provide clinicians with information about the available test repertoire and
the standard of laboratory services they should expect for the investigation of
infection in their patients, as well as providing information that aids the
electronic ordering of appropriate tests.

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• SMIs provide commissioners of healthcare services with the appropriateness
and standard of microbiology investigations they should be seeking as part of
the clinical and public health care package for their population.

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Background to SMIs
SMIs comprise a collection of recommended algorithms and procedures covering all

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stages of the investigative process in microbiology from the pre-analytical (clinical
syndrome) stage to the analytical (laboratory testing) and post analytical (result
interpretation and reporting) stages.
Syndromic algorithms are supported by more detailed documents containing advice
on the investigation of specific diseases and infections. Guidance notes cover the
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clinical background, differential diagnosis, and appropriate investigation of particular
clinical conditions. Quality guidance notes describe laboratory processes which
underpin quality, for example assay validation.
Standardisation of the diagnostic process through the application of SMIs helps to
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assure the equivalence of investigation strategies in different laboratories across the

UK and is essential for public health surveillance, research and development activities.

Equal Partnership Working

SMIs are developed in equal partnership with PHE, NHS, Royal College of
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Pathologists and professional societies.

The list of participating societies may be found at
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http://www.hpa.org.uk/SMI/Partnerships. Inclusion of a logo in an SMI indicates

participation of the society in equal partnership and support for the objectives and
process of preparing SMIs. Nominees of professional societies are members of the
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Steering Committee and Working Groups which develop SMIs. The views of nominees
cannot be rigorously representative of the members of their nominating organisations
nor the corporate views of their organisations. Nominees act as a conduit for two way
reporting and dialogue. Representative views are sought through the consultation
process.
SMIs are developed, reviewed and updated through a wide consultation process.

#
Microbiology is used as a generic term to include the two GMC-recognised specialties of Medical Microbiology (which includes
Bacteriology, Mycology and Parasitology) and Medical Virology.

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Quality Assurance
NICE has accredited the process used by the SMI Working Groups to produce SMIs.
The accreditation is applicable to all guidance produced since October 2009. The
process for the development of SMIs is certified to ISO 9001:2008.
SMIs represent a good standard of practice to which all clinical and public health
microbiology laboratories in the UK are expected to work. SMIs are NICE accredited
and represent neither minimum standards of practice nor the highest level of complex
laboratory investigation possible. In using SMIs, laboratories should take account of
local requirements and undertake additional investigations where appropriate. SMIs
help laboratories to meet accreditation requirements by promoting high quality
practices which are auditable. SMIs also provide a reference point for method

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development.
The performance of SMIs depends on competent staff and appropriate quality
reagents and equipment. Laboratories should ensure that all commercial and in-house

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tests have been validated and shown to be fit for purpose. Laboratories should
participate in external quality assessment schemes and undertake relevant internal
quality control procedures.

Patient and Public Involvement EV

The SMI Working Groups are committed to patient and public involvement in the
development of SMIs. By involving the public, health professionals, scientists and
voluntary organisations the resulting SMI will be robust and meet the needs of the
user. An opportunity is given to members of the public to contribute to consultations
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through our open access website.

Information Governance and Equality

PHE is a Caldicott compliant organisation. It seeks to take every possible precaution
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to prevent unauthorised disclosure of patient details and to ensure that patient-related

records are kept under secure conditions.
The development of SMIs are subject to PHE Equality objectives
http://www.hpa.org.uk/webc/HPAwebFile/HPAweb_C/1317133470313. The SMI
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Working Groups are committed to achieving the equality objectives by effective

consultation with members of the public, partners, stakeholders and specialist interest
groups.
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Legal Statement
Whilst every care has been taken in the preparation of SMIs, PHE and any supporting
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organisation, shall, to the greatest extent possible under any applicable law, exclude
liability for all losses, costs, claims, damages or expenses arising out of or connected
with the use of an SMI or any information contained therein. If alterations are made to
an SMI, it must be made clear where and by whom such changes have been made.
The evidence base and microbial taxonomy for the SMI is as complete as possible at
the time of issue. Any omissions and new material will be considered at the next
review. These standards can only be superseded by revisions of the standard,
legislative action, or by NICE accredited guidance.
SMIs are Crown copyright which should be acknowledged where appropriate.

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Suggested Citation for this Document

Public Health England. (2014). Catalase Test. UK Standards for Microbiology
Investigations. TP 8 Issue 2.3. http://www.hpa.org.uk/SMI/pdf.

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Scope of Document
This test is to detect the catalase enzyme present in most cytochrome-containing
aerobic and facultative anaerobic bacteria1. Streptococcus and Enterococcus species
are exceptions.
This SMI should be used in conjunction with other SMIs.

Introduction
The catalase test is used to detect the presence of catalase enzymes by the

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decomposition of hydrogen peroxide to release oxygen and water. Hydrogen peroxide
is formed by some bacteria as an oxidative end product of the aerobic breakdown of
sugars. If allowed to accumulate, it is highly toxic to bacteria and can result in cell
death. Catalase either decomposes hydrogen peroxide or oxidises secondary

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substrates, but it has no effect on other peroxides2.

Technical Information/Limitations
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Media containing whole red blood cells will contain catalase, and could therefore give
a false positive result.
The enzyme is present in viable cultures only. Do not perform on cultures over 24hr
old. Older cultures may give false-negative reactions2.
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A weak catalase or pseudocatalase reaction may be produced by some strains of
Aerococcus species. Some strains of Enterococcus species also produce a
pseudocatalase.
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Hydrogen peroxide is unstable and should be stored in a spark proof fridge. Avoid any
undue exposure to light.
Cultures of anaerobic bacteria should be exposed to air for 30min prior to testing2.
Some inoculating loops or wires can react with the hydrogen peroxide to produce
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false-positive reactions.
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1 Safety Considerations3-13
Refer to current guidance on the safe handling of all organisms and reagents
documented in this SMI.
Catalase testing of bacteria can be hazardous due to the release of bacteria-laden
aerosols by liberated oxygen. All work likely to generate aerosols must be performed
in a microbiological safety cabinet.
Hydrogen peroxide is an irritant.
The above guidance should be supplemented with local COSHH and risk
assessments.

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2 Reagents and Equipment2,14

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Discrete bacterial colonies on solid medium. The catalase test should not be
performed on colonies taken from media containing whole red blood cells. Colonies
taken from chocolate agar may be tested.

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Hydrogen peroxide solution 3–6 % (supplied in various concentrations by commercial
manufacturers). Hydrogen peroxide is unstable and should be stored in a fridge. Avoid
any undue exposure to light.
Clean capped test tubes (plastic or glass) or Bijoux bottles.
Bacteriological straight nichrome-wire or nichrome-loop or disposable alternative.
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Commercial preparations are available.

3 Quality Control Organisms

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Positive Control
Staphylococcus aureus NCTC 6571
Negative Control
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Streptococcus mitis NCTC 10712

4 Procedure and Results

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4.1 Tube or Bottle Method

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• Place approximately 0.2ml of hydrogen peroxide solution in a test tube or bijoux

bottle
• Carefully pick a colony to be tested with a wire/loop or disposable alternative
• Rub the colony on the inside wall of the bottle, above the surface of the
hydrogen peroxide solution
• Cap the tube or bottle, and tilt it to allow the hydrogen peroxide solution to
cover the colony
• Look for vigorous bubbling occurring within 10s

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4.2 Agar Slant Method

• Add three to four drops of hydrogen peroxide to an overnight growth on an agar
slant, and replace the cap
• Look for vigorous bubbling occurring within 10s

For all methods

Positive Result
Vigorous bubbling indicates the presence of catalase.
Negative Result

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No bubbling.
Note: Control organisms should be tested daily because hydrogen peroxide is
unstable.

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Appendix: Catalase Test

Isolate discrete colony

Tube / bottle method Agar slant method

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0.2ml of hydrogen peroxide

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placed into tube / bijoux bottle

3-4 drops of hydrogen peroxide

solution added to agar slant and
cap replaced
Using a loop a colony is picked
and rubbed on inside wall of tube
above liquid
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The tube / bottle is covered and
tilted till solution covers colony
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Vigorous bubbling within

10s No bubbling
Presence of catalase.
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Positive Negative
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Note:

Positive Control: Staphylococcus aureus NCTC 6571

Negative Control: Streptococcus mitis NCTC 10712

The flowchart is for guidance only.

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References
1. Doelle HW, editor. Bacterial Metabolism. London: Academic Press; 1969. p. 240-6.

2. MacFaddin JF. Optochin Disk Test. Biochemical Tests for Identification of Medical Bacteria. 3rd ed.
Philadelphia: Lippincott Williams and Wilkins; 2000. p. 363-7.

3. Advisory Committee on Dangerous Pathogens. The Approved List of Biological Agents. Her
Majesty's Stationery Office. Norwich. 2004. p. 1-21

4. Advisory Committee on Dangerous Pathogens. Infections at work: Controlling the risks. Her
Majesty's Stationery Office. 2003.

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5. Advisory Committee on Dangerous Pathogens. Biological agents: Managing the risks in
laboratories and healthcare premises. Health and Safety Executive. 2005.

6. Health and Safety Executive. Control of Substances Hazardous to Health Regulations. The Control

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of Substances Hazardous to Health Regulations 2002. 5th ed. HSE Books; 2002.

7. Health and Safety Executive. Five Steps to Risk Assessment: A Step by Step Guide to a Safer and
Healthier Workplace. HSE Books. 2002.

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8. Health and Safety Executive. A Guide to Risk Assessment Requirements: Common Provisions in
Health and Safety Law. HSE Books. 2002.

9. British Standards Institution (BSI). BS EN12469 - Biotechnology - performance criteria for

microbiological safety cabinets. 2000.
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10. British Standards Institution (BSI). Part 2: Recommendations for information to be exchanged
between purchaser, vendor and installer and recommendations for installation. BS 5726 -
Microbiological safety cabinets. 1992.
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11. British Standards Institution (BSI). Part 4: Recommendations for the selection, use and
maintenance. BS 5726 - Microbiological safety cabinets. 1992.

12. Health Services Advisory Committee. Safe Working and the Prevention of Infection in Clinical
Laboratories and Similar Facilities. HSE Books. 2003.
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13. Department for transport. Transport of Infectious Substances, 2011 Revision 5. 2011.

14. Snell JJS, Brown DFJ, Roberts C, editors. Quality Assurance Principles and Practice in the
Microbiology Laboratory. London: Public Health Laboratory Service; 1999. p. 147-8
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