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OPTOCHIN TESTIssue no: 2.1 Issue date: 09.12.10 Issued by: Standards Unit, Department for Evaluations, Standards and Training Page no: 1 of 9

BSOP TP 25i2.1This NSM should be used in conjunction with the series of other NSMs from the Health Protection Agency

www.evaluations-standards.org.ukEmail: [email protected]

NATIONAL STANDARD METHOD

OPTOCHIN TEST

BSOP TP 25

Issued by Standards Unit, Department for Evaluations, Standards and TrainingCentre for Infections


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STATUS OF NATIONAL STANDARD METHODSNational Standard Methods, which include standard operating procedures (SOPs), algorithms andguidance notes, promote high quality practices and help to assure the comparability of diagnosticinformation obtained in different laboratories. This in turn facilitates standardisation of surveillanceunderpinned 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 standardfor clinical and public health microbiology. However, in using National Standard Methods, laboratoriesshould take account of local requirements and may need to undertake additional investigations. Themethods also provide a reference point for method development.

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

Representatives of several professional organisations, including those whose logos appear on the frontcover, are members of the working groups which develop National Standard Methods. Inclusion of anorganisations logo on the front cover implies support for the objectives and process of preparingstandard 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 amember. The current list of participating organisations can be obtained by [email protected].

The performance of standard methods depends on the quality of reagents, equipment, commercial andin-house test procedures. Laboratories should ensure that these have been validated and shown to befit 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 Agencyor any supporting organisation cannot be responsible for the accuracy of any statement orrepresentation made or the consequences arising from the use of or alteration to any informationcontained in it. These procedures are intended solely as a general resource for practising professionalsin 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 theoriginal document. The Health Protection Agency (HPA) should at all times be acknowledged.

The HPA is an independent organisation dedicated to protecting peoples health. It brings together theexpertise formerly in a number of official organisations. More information about the HPA can be foundat www.hpa.org.uk.

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

1.

More details can be found on the website at www.evaluations-standards.org.uk. Contributions to thedevelopment of the documents can be made by contacting [email protected].

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

Suggested citation for this document:Health Protection Agency (2010). Optochin Test. National Standard Method BSOP TP 25 Issue 2.1.http://www.hpa-standardmethods.org.uk/pdf_sops.asp.

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


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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 REAGENTS AND EQUIPMENT........................................................................................6

3 QUALITY CONTROL ORGANISMS .................................................................................6

4 PROCEDURE AND RESULTS .........................................................................................6

4.1 PURE COLONY ..............................................................................................................64.2 SPECIMEN ....................................................................................................................6

5 ACKNOWLEDGEMENTS AND CONTACTS....................................................................7

APPENDIX: OPTOCHIN TEST................................................................................................8

REFERENCES.........................................................................................................................9


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AMENDMENT PROCEDURE

Controlled documentreference

BSOP TP 25

Controlled document title Optochin Test

Each National Standard Method has an individual record of amendments. The current amendmentsare 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 inthe laboratory.

AmendmentNumber/Date

Issue no.Discarded

InsertIssueno.

Page Section(s) involved Amendment

2/

01/12/2010

1.1 2 5 Optochin Test Reworded

Report of occasional Optochinresistant strain added toTechnical Information andLimitations

7 Acknowledgementsand Contacts

Contact details added

8 Appendix Flowchart added

9 References Revised

3/

9/12/2010

2 2.1 6 Quality control

organisms

Negaitive control amended

from Streptococcus viridanstoStreptococcus mitis

8 Appendix Flowchart amended to correct14 mm radius to 14 mmdiameter zone size


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OPTOCHIN TEST

SCOPE OF DOCUMENTSusceptibility to optochin (ethlyhydrocupreine hydrochloride) is a simple and reliable method ofdifferentiating Streptococcus pneumoniaefrom other alpha-haemolytic streptococci

2.

INTRODUCTIONThe optochin test detects an organisms susceptibility to the chemical optochin. The chemical teststhe fragility of the bacterial cell membrane and causes S. pneumoniae to lyse due to changes insurface tension.

The optochin test is widely used in the form of filter paper discs, impregnated with ethylhydrocupreine

hydrochloride, which are applied directly to inoculated plates before incubation3,4.

TECHNICAL INFORMATION/LIMITATIONSSome viridans streptococci may produce a small zone of inhibition, ie


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1 SAFETY CONSIDERATIONS7-13

Refer to current guidance on the safe handling of all organisms and reagents documented inthis National Standard Method.

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.

Compliance with postal and transport regulations is essential.

2 REAGENTS AND EQUIPMENT14

Filter paper discs impregnated with 5g of ethylhydrocupreine hydrochloride.

Bacteriological straight wire/loop (preferably nichrome) or disposable alternative.

3 QUALITY CONTROL ORGANISMS

15

Positive control Streptococcus pneumoniaeNCTC 12977Negative control Streptococcus mitisNCTC 10712

NB: Strains are not validated by the HPA National Collection of Type Cultures to give thisresult

4 PROCEDURE AND RESULTS

4.1 PURE COLONY

Streak a blood plate with the organism to be tested

Place an optochin disc in the centre of the inoculum

Incubate at 35-37C for 18-24 hours in 5% CO2

Examine for zones of inhibition

4.2 SPECIMEN

Streak the specimen on a blood agar plate

Place an optochin disc on the edge of the primary inoculum

Incubate at 35-37C for 18-24 hours in 5% CO2

Examine for zones of inhibition.

Positive result Zone of inhibition of 14mm diameter indicating test organism isS. pneumoniae.

Negative result No zone of inhibition indicating test organism is not S. pneumoniae.A zone < 14mm diameter please see Technical Information sectionfor further test requirements.


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5 ACKNOWLEDGEMENTS AND CONTACTSThis National Standard Method has been developed, reviewed and revised by the NationalStandard Methods Working Group for Clinical Bacteriology(http://www.hpa-standardmethods.org.uk/wg_bacteriology.asp). The contributions of manyindividuals in clinical bacteriology laboratories and specialist organisations who have providedinformation 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 UnitDepartment for Evaluations, Standards and TrainingCentre for InfectionsHealth Protection AgencyColindaleLondonNW9 5EQ

E-mail: [email protected]


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APPENDIX: OPTOCHIN TEST

Pure colony Specimen

Streak a blood agar plate with

organism to be tested. Placeoptochin disc in centre of

inoculum

Incubate at 35-37 C for

18-24 hrs in 5% CO2

Positive

Zone of inhibition of

= 14mm radiusfromthe edge of disc

Negative

No zone of inhibition

or < 14mm etc


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