NATIONAL STANDARD METHOD

EXAMPLE REFERENCE STRAINS FOR

NATIONAL STANDARD METHOD TEST
PROCEDURES

BSOP TP 1

Issued by Standards Unit, Department for Evaluations, Standards and Training

Centre for Infections

EXAMPLE REFERENCE STRAINS FOR NATIONAL STANDARD METHOD TEST PROCEDURES

Issue no: 1 Issue date: 01.12.10 Issued by: Standards Unit, Department for Evaluations, Standards and Training Page: 1 of 10
BSOP TP 1i1
This NSM should be used in conjunction with the series of other NSMs from the Health Protection Agency
www.evaluations-standards.org.uk
Email: standards@hpa.org.uk
STATUS OF NATIONAL STANDARD METHODS
National Standard Methods, which include standard operating procedures (SOPs), algorithms and
guidance notes, promote high quality practices and help to assure the comparability of diagnostic
information obtained in different laboratories. This in turn facilitates standardisation of surveillance
underpinned by research, development and audit and promotes public health and patient confidence
in their healthcare services. The methods are well referenced and represent a good minimum
standard for clinical and public health microbiology. However, in using National Standard Methods,
laboratories should take account of local requirements and may need to undertake additional
investigations. The methods also provide a reference point for method development.

National Standard Methods are developed, reviewed and updated through an open and wide
consultation process where the views of all participants are considered and the resulting documents
reflect the majority agreement of contributors.

Representatives of several professional organisations, including those whose logos appear on the
front cover, are members of the working groups which develop National Standard Methods. Inclusion
of an organisation’s logo on the front cover implies support for the objectives and process of preparing
standard methods. The representatives participate in the development of the National Standard
Methods but their views are not necessarily those of the entire organisation of which they are a
member. The current list of participating organisations can be obtained by emailing
standards@hpa.org.uk.

The performance of standard methods depends on the quality of reagents, equipment, commercial
and in-house test procedures. Laboratories should ensure that these have been validated and shown
to be fit for purpose. Internal and external quality assurance procedures should also be in place.

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
necessary. If you make any changes to this publication, it must be made clear where changes have
been made to the original document. The Health Protection Agency (HPA) should at all times be
acknowledged.

The HPA is an independent organisation dedicated to protecting people’s health. It brings together
the expertise formerly in a number of official organisations. More information about the HPA can be
found at www.hpa.org.uk.

The HPA aims to be a fully Caldicott compliant organisation. It seeks to take every possible
precaution to prevent unauthorised disclosure of patient details and to ensure that patient-related
records are kept under secure conditions1.

More details can be found on the website at www.evaluations-standards.org.uk. Contributions to the

development of the documents can be made by contacting standards@hpa.org.uk.

The reader is informed that all taxonomy in this document was correct at time of issue.

Please note the references are now formatted using Reference Manager software. If you alter or delete text
without Reference Manager installed on your computer, the references will not be updated automatically.

Suggested citation for this document:

Health Protection Agency (2010). Example Reference Strains For National Standard Method Test
Procedures. National Standard Method BSOP TP 1 Issue 1. http://www.hpa-
standardmethods.org.uk/pdf_sops.asp.

EXAMPLE REFERENCE STRAINS FOR NATIONAL STANDARD METHOD TEST PROCEDURES

Issue no: 1 Issue date: 01.12.10 Issued by: Standards Unit, Department for Evaluations, Standards and Training Page: 2 of 10
BSOP TP 1i1
This NSM should be used in conjunction with the series of other NSMs from the Health Protection Agency
www.evaluations-standards.org.uk
Email: standards@hpa.org.uk
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 REAGENTS AND EQUIPMENT ......................................................................................6
3 EXAMPLE REFERENCE STRAINS ................................................................................6
4 PROCEDURE AND RESULTS ........................................................................................9
5 ACKNOWLEDGEMENTS AND CONTACTS ..................................................................9
REFERENCES.......................................................................................................................10

EXAMPLE REFERENCE STRAINS FOR NATIONAL STANDARD METHOD TEST PROCEDURES

Issue no: 1 Issue date: 01.12.10 Issued by: Standards Unit, Department for Evaluations, Standards and Training Page: 3 of 10
BSOP TP 1i1
This NSM should be used in conjunction with the series of other NSMs from the Health Protection Agency
www.evaluations-standards.org.uk
Email: standards@hpa.org.uk
AMENDMENT PROCEDURE

Controlled document BSOP TP 1

reference
Controlled document title Example Reference Strains For National Standard Method Test
Procedures

Each National Standard Method has an individual record of amendments. The current amendments
are listed on this page. The amendment history is available from standards@hpa.org.uk.

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.

EXAMPLE REFERENCE STRAINS FOR NATIONAL STANDARD METHOD TEST PROCEDURES

Issue no: 1 Issue date: 01.12.10 Issued by: Standards Unit, Department for Evaluations, Standards and Training Page: 4 of 10
BSOP TP 1i1
This NSM should be used in conjunction with the series of other NSMs from the Health Protection Agency
www.evaluations-standards.org.uk
Email: standards@hpa.org.uk
 EXAMPLE REFERENCE STRAINS FOR
NATIONAL STANDARD METHOD TEST
PROCEDURES
SCOPE OF DOCUMENT
This National Standard Method is designed as a stand alone document giving information on example
reference material that can be used as control strains for the range of test procedures covered in the
National Standard Methods (NSM) Test Procedures. This document contains information on the
reference material and does not include information on how to carry out the test procedure which can
be found in the individual Test Procedures available through the National Standard Methods Website.
In all cases the reference material should be an authenticated reference culture from a recognised
national culture collection. (NB: The organisms are not all necessarily type strains).

Reference materials can be provided by the Health Protection Agency Culture Collections, National
Collection of Type Cultures (NCTC) http://www.hpacultures.org.uk or from equivalent organisations
including the American Type Culture Collection. Where indicated the reference strain has been
validated by NCTC for the test shown. However, the remaining reference strains listed in this
document are commonly used but have not been validated by NCTC.

INTRODUCTION
Use of appropriate reference material alongside the test procedure is crucial to ensure reliability of
results. Appropriate controls are needed to ensure that the test is working within defined limits. If the
reference material fails to give a positive or negative result (as appropriate) for the test it is used in
and it is the appropriate control then the validity of the results are questionable. If this is the case the
reason for failure should be fully investigated and where necessary the test should be run again and a
review of the process performed. The use of controls is recognised as good laboratory practice and a
recognised part of any accreditation process.

TECHNICAL INFORMATION/LIMITATIONS
The reference material on receipt must be rehydrated in accordance with any NCTC (or equivalent)
guidelines. The reference material should be subcultured to appropriate non-selective media and
incubate using the correct atmosphere and temperature. If the culture is to be stored for future use this
should be done in such a way as to ensure optimum recovery. It is suggested that micro Cryovials™,
which contain a cryoperservative, are used. These should be inoculated with young colonial growth (18 -
24 hours old) from the subculture to approximately a 3 - 4 McFarland standard. The vial should be closed
tightly and inverted 4 - 5 times to emulsify the organisms. Do not vortex. The organisms are then bound
to the porous beads. The excess cryopreservative should be aspirated with a sterile pastette leaving the
beads as free of liquid as possible. Re-close the vial finger tight. Label the vial with the corresponding
storage number, NCTC (or equivalent) number, name and date. These beads constitute the reference
bead stock and are stored at -80°C. A second set of beads should be made which constitutes the
working stock culture.

One bead from each working stock should be subcultured to an appropriate non selective medium
monthly, to prepare plate cultures. Under aseptic conditions open the Cryovial™ with a sterile needle or
forceps and remove one bead. The inoculated bead may be directly streaked on the appropriate plate
culture medium. The plates must be clearly labelled with name of organism, date of subculture and
NCTC number (or equivalent). The plate cultures may be subcultured weekly to fresh plates, and every
4th week plates should be made from the Cryovial™ stock as above.

Note: Cryovials™ should be returned to -80°C as quickly as possible as excessive changes in

temperature reduce the viability of the organisms.

It is good practice to record all subcultures on a record sheet. If any contamination is evident on the
working cultures before the normal replacement time, fresh ones should be prepared from the reference
bead stock.
EXAMPLE REFERENCE STRAINS FOR NATIONAL STANDARD METHOD TEST PROCEDURES
Issue no: 1 Issue date: 01.12.10 Issued by: Standards Unit, Department for Evaluations, Standards and Training Page: 5 of 10
BSOP TP 1i1
This NSM should be used in conjunction with the series of other NSMs from the Health Protection Agency
www.evaluations-standards.org.uk
Email: standards@hpa.org.uk
 1 SAFETY CONSIDERATIONS2-8
Refer to current guidance on the safe handling of all organisms documented in this NSM.

All work likely to generate aerosols must be performed in a microbiological safety cabinet.

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

2 REAGENTS AND EQUIPMENT

Incubator both oxygen and carbon dioxide.
Anaerobic jars

3 EXAMPLE REFERENCE STRAINS

3.1 TABLE OF EXAMPLE REFERENCE STRAINS

National Standard Method Example Reference Strain

BSOP TP 2 – Aesculin Hydrolysis Test Positive control Enterococcus faecalis
NCTC 12697*

Negative control Streptococcus agalactiae

NCTC 8181*

BSOP TP 3 – Agglutination Test

N/A
BSOP TP 5 – Bile Solubility Test Positive control Streptococcus pneumoniae
NCTC 12977

Negative control Streptococcus mitis

NCTC 10712

BSOP TP 8 – Catalase Test Positive control Staphylococcus aureus

NCTC 6571*

Negative control Streptococcus mitis

NCTC 10712*

BSOP TP 10 Coagulase Test Positive control Staphylococcus aureus

NCTC 6571*

Negative control Staphylococcus haemolyticus

NCTC 4276*

BSOP TP 12 Deoxyribonuclease Test Positive control Staphylococcus aureus

NCTC 6571

Negative control Staphylococcus haemolyticus

NCTC 4276
BSOP TP 19 Indole Test Positive control Escherichia coli
NCTC 10418*

Negative control Proteus mirabilis

NCTC 10975*

EXAMPLE REFERENCE STRAINS FOR NATIONAL STANDARD METHOD TEST PROCEDURES

Issue no: 1 Issue date: 01.12.10 Issued by: Standards Unit, Department for Evaluations, Standards and Training Page: 6 of 10
BSOP TP 1i1
This NSM should be used in conjunction with the series of other NSMs from the Health Protection Agency
www.evaluations-standards.org.uk
Email: standards@hpa.org.uk
BSOP TP 21 Motility Test Positive control Proteus mirabilis
NCTC 10975*

Negative control Acinetobacter lwoffii

NCTC 5866*
BSOP TP 22 Nagler Test Positive control Clostridium perfringens
NCTC 8359

Negative control Clostridium difficile

NCTC 11204

BSOP TP 24 ONPG (ß-Galactosidase) Test Positive control Escherichia coli

NCTC 10418
or
Neisseria lactamica
NCTC 10617

Negative control Proteus mirabilis

NCTC 10975
or
Neisseria gonorrhoeae
NCTC 8375

BSOP TP 25 Optochin Test Positive control Streptococcus pneumoniae

NCTC 12977

Negative control Streptococcus mitis

NCTC 10712

BSOP TP 26 Oxidase Test Positive control Pseudomonas aeruginosa

NCTC 10662*

Negative control Escherichia coli

NCTC 10418*

BSOP TP 27 Oxidation and Fermentation of Gram Negative rods

Glucose
Positive control Oxidation Pseudomonas aeruginosa
NCTC 10662*

Fermentation Escherichia coli

NCTC 10418*

Negative control No action Acinetobacterlwoffii

NCTC 5866*
Gram Positive Cocci

Positive control Oxidation Micrococcus luteus

NCTC 4351*

Fermentation Staphylococcus aureus

NCTC 8532*

Negative control No action OF basal medium

without carbohydrate

EXAMPLE REFERENCE STRAINS FOR NATIONAL STANDARD METHOD TEST PROCEDURES

Issue no: 1 Issue date: 01.12.10 Issued by: Standards Unit, Department for Evaluations, Standards and Training Page: 7 of 10
BSOP TP 1i1
This NSM should be used in conjunction with the series of other NSMs from the Health Protection Agency
www.evaluations-standards.org.uk
Email: standards@hpa.org.uk
BSOPTP 29 – Porphyrin Synthesis (ALA) Positive control Haemophilus parainfluenzae (non X
Test dependent)
NCTC 7857*

Negative control Haemophilus influenzae (X

dependent)
NCTC 11931*

BSOPTP 30 Potassium Hydroxide Test Positive control Escherichia coli

NCTC 10418

Negative control Staphylococcus aureus

NCTC 6571

BSOPTP 32 Changing the Phase of

Salmonella Test N/A

BSOP TP 34 Thermonuclease Activity Test Positive control Staphylococcus aureus

NCTC 6571

Negative control Staphylococcus haemolyticus

NCTC 4276

BSOP TP 36 Urease Test Positive control Proteus mirabilis

NCTC 10975*

Negative control Escherichia coli

NCTC 10418*

BSOP TP 38 X and V Factor Test X and V factor Haemophilus influenzae

NCTC 11931*

V factor only Haemophilus parainfluenzae

NCTC 10665*

*The reference strain is validated by NCTC for the test shown.

We have no validation data for the other strains.

EXAMPLE REFERENCE STRAINS FOR NATIONAL STANDARD METHOD TEST PROCEDURES

Issue no: 1 Issue date: 01.12.10 Issued by: Standards Unit, Department for Evaluations, Standards and Training Page: 8 of 10
BSOP TP 1i1
This NSM should be used in conjunction with the series of other NSMs from the Health Protection Agency
www.evaluations-standards.org.uk
Email: standards@hpa.org.uk
4 PROCEDURE AND RESULTS
See relevant Test Procedure National Standard Methods Website.

5 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, Department for Evaluations,
Standards and Training, Centre for Infections, Health Protection Agency, London.

For further information please contact us at:

Standards Unit
Department for Evaluations, Standards and Training
Centre for Infections
Health Protection Agency
Colindale
London
NW9 5EQ

E-mail: standards@hpa.org.uk

EXAMPLE REFERENCE STRAINS FOR NATIONAL STANDARD METHOD TEST PROCEDURES

Issue no: 1 Issue date: 01.12.10 Issued by: Standards Unit, Department for Evaluations, Standards and Training Page: 9 of 10
BSOP TP 1i1
This NSM should be used in conjunction with the series of other NSMs from the Health Protection Agency
www.evaluations-standards.org.uk
Email: standards@hpa.org.uk
REFERENCES
1. Department of Health NHS Executive: The Caldicott Committee. Report on the review of patient-
identifiable information. London. December 1997.

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

3. Public Health Laboratory Service Standing Advisory Committee on Laboratory Safety. Safety
Precautions: Notes for Guidance. Public Health Laboratory Services (PHLS). London. 1993.

4. HSE L5 Control of Substances Hazardous to Health Regulations. Approved Code of Practice and
Guidance. 5th ed. HSE Books; 2002.

5. Health and Safety Executive. 5 Steps to Risk Assessment: A Step by Step Guide to a Safer and
Healthier Workplace. HSE Books. 2002.

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

7. HSE Health Services Advisory Committee. Safety in Health Service Laboratories. Safe Working
and the Prevention of Infection in Clinical Laboratories and Similar Facilities. 2. HSE Books. 2003.

8. Health and Safety executive, editor. Biological agents: Managing the risks in laboratories and
healthcare premises 5A.D. HSE Books. Suffolk. 2008.

EXAMPLE REFERENCE STRAINS FOR NATIONAL STANDARD METHOD TEST PROCEDURES

Issue no: 1 Issue date: 01.12.10 Issued by: Standards Unit, Department for Evaluations, Standards and Training Page: 10 of 10
BSOP TP 1i1
This NSM should be used in conjunction with the series of other NSMs from the Health Protection Agency
www.evaluations-standards.org.uk
Email: standards@hpa.org.uk