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UK Standards for Microbiology Investigations
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Introduction to the preliminary identification of medically
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important bacteria and fungi from culture
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Issued by the Standards Unit, Microbiology Services, PHE

Bacteriology – Identification | ID 1 | Issue no: dzs+ | Issue date: dd.mm.yy <tab+enter> | Page: 1
of 32
© Crown copyright 2016
Introduction to the preliminary identification of medically important bacteria and fungi from
culture
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 https://www.gov.uk/uk-
standards-for-microbiology-investigations-smi-quality-and-consistency-in-clinical-
laboratories. SMIs are developed, reviewed and revised by various working groups
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which are overseen by a steering committee (see
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https://www.gov.uk/government/groups/standards-for-microbiology-investigations-
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steering-committee).
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The contributions of many individuals in clinical, specialist and reference laboratories
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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.
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For further information please contact us at:
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Standards Unit
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Microbiology Services
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Public Health England 21
61 Colindale Avenue
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London NW9 5EQ

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E-mail: standards@phe.gov.uk
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Website: https://www.gov.uk/uk-standards-for-microbiology-investigations-smi-quality-
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and-consistency-in-clinical-laboratories
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PHE publications gateway number: 2016308

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UK Standards for Microbiology Investigations are produced in association with:

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Logos correct at time of publishing.
Bacteriology – Identification | ID 1 | Issue no: dzs+ | Issue date: dd.mm.yy <tab+enter>|

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UK Standards for Microbiology Investigations | Issued by the Standards Unit, Public Health England
culture
Contents
Acknowledgments ................................................................................................................. 2
Amendment table ................................................................................................................... 4
Scope of document ................................................................................................................ 8
Introduction ............................................................................................................................ 8
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Conventional identification methods of bacteria and fungi ................................................ 9
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Rapid identification methods .............................................................................................. 17
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Technical information/limitations ....................................................................................... 19
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1 Safety considerations .............................................................................................. 21
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2 Target organisms...................................................................................................... 21
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3 Identification ............................................................................................................. 21
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Characteristics of Gram positive cocci ................................................................... 22
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5 Characteristics of Gram positive rods .................................................................... 23

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6 Characteristics of Gram negative bacteria ............................................................. 24

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7 Characteristics of Gram negative bacteria (Continued from previous page) ....... 25

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8 Characteristics of fungi ............................................................................................ 26

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9 Reporting .................................................................................................................. 27
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10 Referrals .................................................................................................................... 27
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11 Notification to PHE, or equivalent in the devolved administrations ..................... 27

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References ........................................................................................................................... 29
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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
standards@phe.gov.uk.
New or revised documents should be controlled within the laboratory in accordance
with the local quality management system.
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Amendment no/date. # <tab+enter>/dd.mm.yy <tab+enter>
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Issue no. discarded. #.# <tab+enter>
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Insert issue no. #.# <tab+enter>
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Section(s) involved Amendment
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Title of the document updated with fungi.
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Addition of fungi to the scope of the document.
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Section on technical limitations updated with
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relevant information. 21
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Whole document. Section on Identification updated with cutting edge

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molecular methods and MALDI-TOF MS.

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Flowcharts updated.
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Additional flowchart on characteristics of fungi

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added.
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References. References updated.

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UK SMI#: scope and purpose

Users of SMIs
Primarily, SMIs are intended as a general resource for practising professionals
operating in the field of laboratory medicine and infection specialties in the UK. SMIs
also 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
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appropriate tests. The documents also provide commissioners of healthcare services
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with the appropriateness and standard of microbiology investigations they should be
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seeking as part of the clinical and public health care package for their population.
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Background to SMIs
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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
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syndrome) stage to the analytical (laboratory testing) and post analytical (result
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interpretation and reporting) stages. Syndromic algorithms are supported by more
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detailed documents containing advice on the investigation of specific diseases and
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infections. Guidance notes cover the clinical background, differential diagnosis, and
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appropriate investigation of particular clinical conditions. Quality guidance notes
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describe laboratory processes which underpin quality, for example assay validation.
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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

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

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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

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found at https://www.gov.uk/uk-standards-for-microbiology-investigations-smi-quality-
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and-consistency-in-clinical-laboratorieshttp://www.hpa-standardmethods.org.uk/.
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Inclusion of a logo in an SMI indicates participation of the society in equal partnership

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and support for the objectives and process of preparing SMIs. Nominees of
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professional societies are members of the Steering Committee and working groups
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which develop SMIs. The views of nominees cannot be rigorously representative of

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the members of their nominating organisations nor the corporate views of their
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organisations. Nominees act as a conduit for two way reporting and dialogue.
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Representative views are sought through the consultation process. SMIs are
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developed, reviewed and updated through a wide consultation process.

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Quality assurance
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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
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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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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 development. The
performance of SMIs depends on competent staff and appropriate quality reagents
and equipment. Laboratories should ensure that all commercial and in-house tests
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have been validated and shown to be fit for purpose. Laboratories should participate
in external quality assessment schemes and undertake relevant internal quality control
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procedures.
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Patient and public involvement
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The SMI working groups are committed to patient and public involvement in the
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development of SMIs. By involving the public, health professionals, scientists and
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voluntary organisations the resulting SMI will be robust and meet the needs of the
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user. An opportunity is given to members of the public to contribute to consultations
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through our open access website.
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Information governance and equality 21
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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

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records are kept under secure conditions. The development of SMIs is subject to PHE
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Equality objectives https://www.gov.uk/government/organisations/public-health-

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england/about/equality-and-diversity.
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The SMI working groups are committed to achieving the equality objectives by
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effective consultation with members of the public, partners, stakeholders and

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specialist interest groups.

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Legal statement
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While every care has been taken in the preparation of SMIs, PHE and the partner
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organisations, shall, to the greatest extent possible under any applicable law, exclude
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liability for all losses, costs, claims, damages or expenses arising out of or connected
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with the use of an SMI or any information contained therein. If alterations are made by
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an end user to an SMI for local use, it must be made clear where in the document the
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alterations have been made and by whom such alterations have been made and also
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acknowledged that PHE and the partner organisations shall bear no liability for such
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alterations. For the further avoidance of doubt, as SMIs have been developed for
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application within the UK, any application outside the UK shall be at the user’s risk.
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The evidence base and microbial taxonomy for the SMI is as complete as possible at
the date 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. (YYYY <tab+enter>). Introduction to the preliminary
identification of medically important bacteria and fungi from culture. UK Standards for
Microbiology Investigations. ID 1 Issue d. https://www.gov.uk/uk-standards-for-
microbiology-investigations-smi-quality-and-consistency-in-clinical-laboratories
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Scope of document
The aim of this document is to provide a guide to the preliminary identification of the
common bacteria and fungi which may be encountered in clinical specimens. It is
intended to lead the user to a more detailed identification method and is designed to
be used for cultures of bacteria and fungi isolated on agar plates and not for direct
identification of bacteria and fungi from clinical samples/smears. It does however
mention rapid methods that could be used in place of the conventional methods
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mentioned in this document.
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For more information on dermatophytes, refer to B 39 – Investigation of dermatological
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specimens for superficial mycoses.
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This SMI should be used in conjunction with other SMIs.
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Introduction
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Identification of bacteria and fungi by diagnostic laboratories is based on phenotypic
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characteristics, usually by direct comparison of an unknown with those of type
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cultures1. Phenotypic characteristics used as a means of identification for fungi rely on
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microscopic morphology for accurate and correct identification2. Greater confidence in
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identification is in direct proportion to the number of similar characteristics. In medical
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microbiology, experience of the types of specimens, the infection and the organisms
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associated with those sites of infection is useful as an aid to preliminary identification.

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When identifying microorganisms it should be remembered that their characteristics

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may be variable. In addition, species within a genus may differ in some characteristics,
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for example Capnocytophaga canimorsus is oxidase positive, whereas

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Capnocytophaga ochracea is oxidase negative. For this reason some genera may
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appear in more than one table or chart. Clinical information should also be taken into
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consideration during the identification process.

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Matrix assisted Laser Desorption/Ionisation – Time of Flight Mass Spectrometry

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(MALDI-TOF MS), is increasingly being applied for organism identification within

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diagnostic microbiology laboratories. Identification using this technology affords the

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opportunity to rapidly and cost-effectively identify bacteria, most yeast species as well
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as some genera of filamentous fungi in comparison with the more traditional

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techniques3. Any identification should be considered alongside phenotypic information

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that is available.
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Taxonomy
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Characteristics
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When classifying microorganisms, all known characteristics are taken into

consideration, but certain differential and distinguishable characteristics are selected
and used for the purpose of identification. Primary identification usually involves a few
simple but fundamental tests such as cellular morphology (usually shown by Gram
(bacteria) or lactophenol cotton blue (fungi)), growth under various atmospheric
conditions, growth on various types of culture media (for example MacConkey agar,
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Sabouraud agar plate culture), catalase and oxidase tests1. Using these few simple
tests it is usually possible to place organisms, provisionally, in one of the main groups
of medical importance.
Principles of identification
There are three basic methods of identification. The first relies heavily on the
experience of the investigator: a judgement is made on the presumptive identity of the
organism based on clinical data, cultural and atmospheric characteristics. A limited
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range of tests are then used to confirm or disprove the hypothesis. This relies heavily
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on a stable pattern of phenotypic characteristics.
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If identification is not made using the first principle, the next approach will be
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subjecting the organism to a battery of tests, such as those found in commercial
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identification systems. The data is collated and compared to standard texts or used to
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create a numerical profile to obtain identification.
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The final method follows a step-by-step approach to identification. Fundamental
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characteristics of the organism are determined by primary identification tests such as
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a Gram stain, oxidase or catalase. Results of these tests indicate secondary or even
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tertiary tests to confirm the identity of the subject. This is a systematic approach and
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does not rely on the expertise of the investigator. The disadvantage of this system
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involves the primary tests, setting up tests unnecessarily, misinterpretation of results
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at this stage can lead the investigator down an incorrect path, which wastes both time
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and resources and may also lead to a misidentification and ultimately incorrect
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diagnosis. It is also a time consuming process; further tests cannot be set up until
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results of the previous investigations are known.

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Conditions under which tests are conducted should be clearly defined as reactions
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may vary between organisms.

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Conventional identification methods for bacteria and

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fungi
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Microscopic appearance
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Bacteria
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Microscopic study and staining reveals the shape (coccus or rod) and the
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characteristic grouping and arrangement of the cells, their size and the presence of
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intracellular inclusions, for example spores. In addition to morphology, the Gram

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stained preparation also divides bacteria in two categories - the Gram positive and the
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Gram negative bacteria1,4.

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For morphological appearance, it is preferable to examine fresh cultures from growth

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on non-selective media. Below is the table 1a which details the terms used for stained
preparations of bacteria using different types of stains.
Fungi
Fungal isolates from clinical samples can broadly be split into either yeast or mould
depending on their predominant growth form. If they exhibit a unicellular growth form
with reproduction by budding to produce individual discrete colonies on culture plates

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then they would be classified as yeast. Moulds demonstrate a filamentous growth form
with long, branching hyphae and a single colony may grow to fill an entire petri-dish.
Moulds reproduce by producing spores often in a very characteristic way and
examination of the mechanism by which spores are produced, the sporing structures
and the spores themselves can aid in identification of the isolate. Microscopic mounts
can be made from mould colonies by either taking a portion of the surface growth from
the colony with a sharp needle and teasing it out in a drop of mounting fluid on a
microscope slide and applying a coverslip, known as a tease or needle mount; or by
using sticky tape to remove surface structures, mounting fungus-side uppermost on a
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microscope slide adding a drop of mounting fluid and coverslip, known as a sticky-
tape mount. There are several types of stains that could be used to highlight
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microscopic characteristics of fungi to aid identification such as lactophenol cotton
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blue, calcofluor white with 10% KOH and India ink. For more information, see TP 39 -
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Staining procedures.
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For morphological appearance of yeasts, it is necessary to grow the cultures on a low
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nutrient agar such as cornmeal agar with tween 80, under micro-aerobic conditions
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which can be achieved by the application of a coverslip over a streak inoculum of the
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culture. This method of inducing production of morphological characteristics is known
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as a Dalmau plate and can be used to look for the production of true hyphae,
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pseudohyphae, arthrospores, chlamydospores and capsules. It is also useful to note
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the colonial characteristics such as smooth, wet, dry or wrinkled colonies and any
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pigment production. Table 1b details the terms used for stained preparations of fungi
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using different types of stains.

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Table 1a: Terms used for stained preparations of bacteria5

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Term Description
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singly, in pairs, in chains, in fours (tetrads), in groups, grape-like

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Arrangement clusters, in cuboidal packets, in bundles, in Chinese letters

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(cuneiform), palisade
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Capsule presence or absence

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spherical, oval or ellipsoidal, equatorial, sub-terminal, terminal, cause

Endospores
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bulging of rod
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Ends round, truncate, pointed

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variation in shape and size, clubs, filamentous, branched, navicular,

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Irregular forms
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citron, fusiform, giant swollen forms

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variation in shape, for example filamentous forms interspersed with

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Pleomorphism
coccobacillary forms
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Spheres (cocci), short rods (coccobacilli), long rods (bacilli),

Shape
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filamentous, curved rods, spirals

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Sides parallel, bulging, concave or irregular
length and breadth. The diameter is measured in micromillimetres

Size
(µm).
Staining even, irregular, unipolar, bipolar, beaded, barred, acid-fast,

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Table 1b: Terms used for stained preparations of yeasts and filamentous fungi6
Term Description
It should be noted that presence of budding is a feature that is useful in

identification of yeasts which may show budding on a narrow or broad base.
Arrangement
The arrangement and mechanism of production of spores is key to the
identification of mould species.
presence or absence
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Capsule Capsules are readily visible in Indian ink preparations or in some histological
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stains.
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presence or absence and with or without branching, whether septate or
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Hyphae
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pauci-septate, true or pseudohyphae in the case of yeast isolates
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Sporangia
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/sporangiospores, presence or absence
Conidia /conidiospores,
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Aleuriospores, Although sporulation is rare in some fungi there are ways to encourage
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sporulation and phenotypic identification will not be possible without spore
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Arthrospores,
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Multiseptate conidia, production.
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Chlamydospores
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Small, large, oval, spherical, cylindrical, slightly curved, ellipsoidal, cresent-
Shape of spores shaped, septate, multi-septate
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Note: some moulds will have more than one type of spore
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There is great variation in size of cells. The diameter is measured in

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Size
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micrometres (µm).
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Cultural appearance1,4
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Bacterial or fungal colonies of a single species, when grown on specific media under
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controlled conditions are described by their colony morphology, characteristic size,

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shape, colour, consistency and sometimes pigmentation. When growth conditions are
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carefully controlled, colonial morphology is important for preliminary identification and

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for differentiating organisms7. However, it should also be noted that growth rate of
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certain organisms are variable, depending on the amount of inoculum (bacterial or

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fungal) present in a clinical specimen.

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Colonial shape is determined by the edge and thickness of the colony. The edge may
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be smooth (entire) or irregular and serrated. If the colony is thicker in the centre than
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the edge, it is said to be raised, or it may be relatively uniform - appearing like a disc.
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The texture of the colony is also important. It may vary from dry and friable (easily
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crumbled) to butyrous (buttery) to sticky and the surface may be smooth, wet, dry or
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granular. Some organisms produce a pigmented colony, which can aid in the
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identification process (for example Pseudomonas aeruginosa, Serratia marcescens,

Rhodoturula mucilaginosa, Aspergillus nidulans), although non-pigmented strains
within a species may occur7.
Bacteria
The size of bacterial colonies, assuming favourable growth conditions, is generally
uniform within a species. For example Streptococcus species are small, usually 1mm

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in diameter, whilst Staphylococcus species and species within the family,

Enterobacteriaceae are larger and usually 2 - 3mm in diameter, and those of Bacillus
species are much larger in size and usually 2 - 7mm in diameter.
For example, the growth rate for dimorphic fungi such as Blastomyces dermatitidis,
Histoplasma capsulatum and Coccidioides immitis is slow; about 1 week to 4 weeks
are usually required for viable growth (that is, before colonies become visible)6.
Campylobacter species will yield a good growth when incubated for 48 -72 hours
uninterrupted under microaerophilic conditions. Certain genera of fungi, for example
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Trichophyton violaceum take up to 14 -21 days for visible growth.
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Fungi
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The colonial morphology may vary depending on the type of media used and the
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temperature of incubation, for example Histoplasma capsulatum colonies exhibit
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thermal dimorphism and appear as white to tan mould colonies when grown on
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Sabouraud agar at 30⁰C but when grown on brain heart infusion medium enriched
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with blood at 37⁰C; it grows as a yeast form.
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Table 2a: Terms used in colonial morphology of bacteria6,8,9
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Term Description
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Colour 21
by reflected or transmitted light: fluorescent, iridescent, opalescent,
N
Note: There are many colours ranging from white to yellow, pink, orange, red
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or purple.
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Pigmentation This can be seen on the topside and reverse side of the colony.
B
N
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Consistency (texture) butyrous (buttery), fluffy, mucoid (thick, stringy, and wet), friable,
D
membranous, rugose (wrinkled), dry, moist, brittle, viscous, powdery, velvety,

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glabrose, granular, floccose,

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Edge/margin entire, undulate, lobate, crenated, erose, fimbriate, effuse, filiform, curled,
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wavy,
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Elevation (topography) flat, raised, low convex, convex or dome-shaped, umbonate, with or without
A
bevelled margin, pulvinate, crateriform

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Emulsifiability easy or difficult, forms homogeneous or granular suspension or remains

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membranous when mixed in a drop of water

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Shape/form filiform, spreading, rhizoid, circular, irregular, filamentous, spindle,

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punctiform, radiate,
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Opacity transparent, translucent, opaque

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Size The diameter is usually measured in millimetres. Colony size varies and it is
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also described in terms such as pinpoint, small, medium and large.

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Structure amorphous, granular, filamentous, curled,
Surface smooth, glistening, rough (fine, medium or coarsely granular), concentric

(ringed), papillate, dull or wrinkled, heaped up, contoured, veined,
Degree of growth scanty, moderate or profuse

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For individual bacterial colonial descriptions, see the relevant identification SMI using
link: https://www.gov.uk/government/collections/standards-for-microbiology-
investigations-smi#identification.
Table 2a: Terms used in colonial morphology of yeasts and filamentous
fungi6,8,9
Term Description
Colour Yeast colonies are usually white, cream, yellow, red, pink or brown. Mould
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colonies vary greatly, often in shades of green, red, brown or black and
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the surface colour usually reflects the colour of the spores. For some
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groups such as the dermatophytes looking for reverse pigmentation on
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the underside of colonies can be helpful.
B
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Pigmentation Pigment production may colour the entire colony as with yeast or in some
C
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moulds it may only be the spores that are pigmented. Colonies of some
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moulds may produce diffusing pigments.
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E
Consistency (texture) Fungal colony characteristics are dependent upon whether it is yeast or a
B
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filamentous fungus. They range from cottony or woolly (floccose),
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granular, chalky, velvety, powdery, silky, glabrous (smooth), or waxy.
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P
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Edge/margin entire, undulate, filamentous, lobate, erose (serrated),
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Elevation (topography)
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flat, raised, convex, crateriform, heaped, grooved, folded or wrinkled
N
E
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Size The diameter is usually measured in millimetres. Colony size varies and it
W
is also described in terms such as slow-growing, small, medium and

T
E
large.
B
N
Degree of growth Some fungal colonies are fast growing, covering the entire surface of the
O
agar and taking up all the air-space in a petri-dish whilst other fungi may
D
E
grow in a restricted manner.

LT
U
Note: Yeast colony descriptions can be very similar to bacterial colonies.

S
N
O
Chromogenic agar10
C
S
A
There are several commercially available chromogenic media and these are designed
W
to target organisms with high specificity and sensitivity when present among other
T
N
flora. Chromogenic substrates are incorporated into these media that are broken
E
M
down by enzymes imparting a distinct visible colour to the growing colonies to help in
U
their identification. The use of chromogenic agar has been very useful in the isolation
C
O
and presumptive detection of bacteria and yeasts despite the molecular/cutting-edge

D
IS
technologies such as in the screening of Meticillin resistant Staphylococcus aureus.

H
However, the use of chromogenic media does have its limitations which include its
-T
inability to differentiate between organisms such as Candida dubliniensis and Candida

FT
albicans as both display β-hexosaminidase activity. Their main value for yeast is in
A
R
detecting mixed cultures and not as a definitive identification method.

D
Haemolysis
Some organisms produce haemolysins, which cause lysis of erythrocytes in blood-
containing media7. This haemolysis may be
• β (clear zone around the colony causing a clearing of the medium),

Page: 13 of 32
culture
• α (partial lysis of the red blood cells surrounding a colony causing a greenish
discolouration of the medium),
• α -prime (a small zone of intact red cells with a surrounding zone of
haemolysis) or
• non-haemolytic previously called γ-haemolysis (no haemolysis, no apparent
change).
Note: This is used as a form of identification more in bacteria although yeasts can
16
occasionally cause haemolysis.
20
Resistance properties
R
E
B
Certain organisms exhibit a characteristic inherent resistance to specific antibiotics,
O
T
heavy metals, or toxins6. This characteristic is widely used to establish preliminary
C
O
identification information in bacteria and no longer used for yeasts. For example,
-3
Gram positive organisms grow on Columbia blood agar supplemented with colistin
R
and nalidixic acid inhibiting the growth of Gram negative bacilli.
E
B
M
Testing an isolate’s susceptibility to a particular antibiotic is also useful in
E
T
identification, for example most clinically significant Gram negative bacteria are
P
E
resistant to vancomycin and susceptible to the antibiotics, colistin or polymyxin.
S
21
Growth requirements
N
E
E
Microorganisms can be grouped on the basis of their growth requirements. They are
W
T
as follows:
E
B
Atmosphere1,4
N
O
D
It is usual to divide organisms into five categories according to their atmospheric

E
LT
requirements:
U
•
S
strict aerobes grow only in the presence of oxygen

N
O
•
C
strict anaerobes grow only in the absence of oxygen

S
A
• facultative organisms grow aerobically or anaerobically

W
T
•
N
microaerobic organisms grow best in an atmosphere with reduced oxygen

E
M
concentration (addition of 5-10% CO2 may enhance growth)

U
C
• carboxyphilic (or capnophilic) organisms require additional CO2 for growth

O
D
Temperature1
IS
H
-T
Organisms may also be divided according to their temperature requirements:

FT
• psychrophilic organisms grow at low temperatures 2-5°C (optimum 10-30°C).

A
R
•
D
mesophilic organisms grow at temperatures between 10-45°C (optimum 30-

40°C).
• thermophilic organisms grow very little at 37°C (optimum 50-60°C).
• hyperthermophilic organisms grow at temperatures of 80°C or higher
Most clinically encountered organisms are mesophilic.

Page: 14 of 32
culture
Motility11
Many bacteria are observed to be motile and move from one position to another when
suspended in fluid. True motility must not be confused with Brownian movement
(vibration caused by molecular bombardment) or convection currents. Microscopic
examination may indicate whether a motile organism has polar flagellae shown by a
darting zigzag movement or peritrichate flagellae, which cause a less vigorous and
more vibratory movement. Some bacteria may be motile at different temperatures, for
example motile at ambient temperature but not at 37°C, or vice versa (TP 21 - Motility
16
test).
20
Nutrition1
R
E
B
Study of the nutritional requirements of an organism is useful in identification, for
O
T
example the ability to grow on ordinary nutrient media, the effect of adding blood,
C
O
serum or glucose or the necessity for specific growth factors such as X factor (haemin)
-3
and V factor (Nicotinamide Adenine Dinucleotide (NAD)) for the growth of
R
Haemophilus species.
E
B
M
Tests for bacteria4
E
T
P
E
Numerous biochemical tests may be used for the identification of microorganisms
S
(refer to individual identification SOPs). Some such as catalase and oxidase are rapid
21
and easy to perform and may be used for preliminary differentiation purposes. The
N
E
fermentation of glucose may also be used to distinguish between groups of

E
W
organisms. Examples of biochemical tests are:

T
E
•
B
Catalase (TP 8 - Catalase test)

N
O
The catalase test is used to detect the presence of catalase enzymes by the
D
E
decomposition of hydrogen peroxide to release oxygen and water as shown by

LT
the following reaction:

U
S
2 H2O2→2 H2O + O2
N
O
C
Hydrogen peroxide is formed by some bacteria as an oxidative end product of

S
A
the aerobic breakdown of sugars. If allowed to accumulate it is highly toxic to

W
bacteria and can result in cell death. Catalase either decomposes hydrogen
T
N
peroxide or oxidises secondary substrates, but it has no effect on other

E
M
peroxides.
U
C
•
O
Oxidase (TP 26 - Oxidase test)

D
IS
The oxidase test is used to determine if an organism possesses the

H
cytochrome oxidase enzyme. This system is usually present only in aerobic

-T
organisms, which are capable of utilising oxygen as the final hydrogen

FT
acceptor.
A
R
D
• Fermentation of glucose (TP 27 - Oxidation and fermentation of glucose test)

The oxidative-fermentative test is used to determine if bacteria metabolise
carbohydrates oxidatively, by fermentation, or are non-saccharolytic and
therefore have no ability to use the carbohydrate in the media.
Oxidative organisms can only metabolise glucose or other carbohydrates under
aerobic conditions, that is, oxygen is the ultimate hydrogen acceptor. Other
Page: 15 of 32
culture
organisms ferment glucose and the hydrogen acceptor is then another

substance, for example sulphur. This fermentative process is independent of
oxygen and cultures of organisms may be aerobic or anaerobic. The end
product of metabolising a carbohydrate is an acid.
• Different staining methods (TP39 – Staining procedures)
There are many staining procedures commonly used for the identification of
microorganisms. These stains are used to highlight structures in clinical
specimens/isolates, often when viewed with the aid of different microscopes.
16
Stains have different affinities for different organisms and are used to
20
differentiate types of organisms or to view specific parts of organisms. For more
R
E
information on staining procedures, refer to the relevant SMI.
B
O
T
Using these few simple tests it is usually possible to place organisms, provisionally, in
C
O
one of the main groups of medical importance.
-3
The biochemical tests (above) list the common tests used once an organism has been
R
E
isolated on culture plates and colonial appearance and growth requirements have
B
M
been assessed. The lists are not exhaustive and further tests may be needed in
E
T
addition to the common ones described. Refer to
P
E
https://www.gov.uk/government/collections/standards-for-microbiology-investigations-
S
smi#test-procedures for the full list of all UK SMI test procedures. 21
N
E
Serotyping tests
E
W
T
Serotyping is a subtyping method based on the immuno-reactivity of various antigens.

E
B
Selected antisera can be used to classify different bacterial species. This may be
N
based on either carbohydrate or protein antigens from the bacterial cell wall or the
O
D
capsular polysaccharide. There are commercially available serotyping identification

E
LT
kits for the rapid detection of organisms.

U
S
Refer to relevant SMIs for more information.

N
O
C
Tests for fungi

S
A
W
Germ tube test6

T
N
E
This is a presumptive identification test used to differentiate Candida albicans,

M
Candida africana and Candida dubliniensis from other Candida species and other
U
C
yeasts. Strains of all three of these species produce germ tubes when in a liquid
O
D
nutrient environment and incubated at 35ºC for up to 3 hours. Further tests are
IS
required to differentiate between the three species.

H
-T
FT
Rapid urease test12,13

A
This can be used for differentiation between the yeasts, Candida albicans and
R
D
Cryptococcus neoformans. A presumptive identification of C. neoformans is based on

rapid urease production, whilst Candida albicans do not produce urease. Other
species of Cryptococcus, Trichosporon and Rhodotorula can give positive result for
urease test. Occasionally, Candida krusei can give a positive result. For more
information on urease test, refer to TP 36 - Urease test.

Page: 16 of 32
culture
Dermatophyte test medium14

This can be used for isolation and presumptive identification of dermatophytes such as
Microsporum, Trichophyton, and Epidermophyton genera because of a distinct colour
change in the medium. Rapidly-growing species may produce a complete colour
change in the medium within days while the slower-growing species will take longer
periods to change the indicator. Care should be taken during interpretation, as some
non-dermatophytes can also cause a colour change.
Note: The complete classification of dermatophytes is dependent upon microscopic
16
observations of direct and slide culture preparations, along with other biochemical
20
tests.
R
E
Antigen protein tests6
B
O
T
The antigen protein testing can be used in identification of yeasts in culture; however it
C
O
is not useful in the diagnosis of moulds. These assays have now been largely
-3
replaced by the rapid identification techniques seen below.
R
E
B
Rapid identification methods
M
E
T
P
E
Matrix-assisted laser desorption/ionisation - time of flight mass spectrometry
S
(MALDI-TOF MS) 21
N
This technique is a rapid and highly reliable analytical tool for the characterisation of a
E
E
diverse collection of microbes encountered in the clinical laboratory. It has emerged as

W
a new technology for species identification and can be used to analyse the protein
T
E
composition of a microbial cell15. It has been shown to be a powerful technique

B
N
because of its reproducibility, speed and sensitivity of analysis. The advantage of

O
MALDI-TOF MS as compared with other identification methods is that the results of

D
E
the analysis are available within minutes to a few hours rather than several days.
LT
U
This method has been used successfully in the identification of bacteria and yeasts.
S
N
This identification of moulds using this method is being developed. It has equally been
O
C
very useful for direct testing of clinical specimens such as urine, cerebrospinal fluid
S
A
and blood which has resulted in significant improvements to patient care and reduced
W
turnaround time to result3,16,17.

T
N
E
For more information, refer to SMI TP 40: Matrix-assisted laser desorption/ionisation -

M
U
time of flight mass spectrometry (MALDI-TOF MS) test procedure.

C
O
D
Whole genome sequencing (WGS)

IS
H
This is also known as full genome sequencing, complete genome sequencing, or

-T
entire genome sequencing. It is a technique that determines the complete DNA

FT
sequence of an organism's genome at a single time. There are several high-

A
throughput techniques that are available and used to sequence an entire genome
R
D
such as pyrosequencing, nanopore technology, sequencing by synthesis (SBS)

technology, Ion semiconductor sequencing, etc. This sequencing method holds great
promise for rapid, accurate, and comprehensive identification of microbial
transmission pathways in hospital and community settings, with concomitant
reductions in infections, morbidity, and costs.

Page: 17 of 32
culture
WGS has been used successfully to explore the genome of organisms as well as to
identify candidate genes responsible for pathogenesis, and to develop better methods
of strain detection and to advance the understanding of the evolution of organisms.
Examples of such organisms include Shigella species, Escherichia coli O157:H7,
Salmonella enterica serovar Typhi and Yersinia enterocolitica18-21.
Commercial identification Systems (kits/rapid tests)

The use of commercially available identification kits alongside other biochemical
16
reactions may be used to give accurate identification of microorganisms – bacteria
20
and yeasts22. It should be noted that there are no commercial kits for biochemical
R
profiling of filamentous fungi. Laboratories should follow manufacturer’s instructions.
E
B
Ideally, where possible, scores should be available and easily accessible during the
O
T
authorisation process and for audit purposes.
C
O
Note: If a commercial yeast identification kit provides a biochemical profile but also
-3
specifies examination of key morphological features to obtain a numerical profile it is
R
E
not sufficient to identify the organism based only on the biochemical profile.
B
M
E
T
Other identification methods
P
E
Molecular methods have had an enormous impact on bacterial and fungal taxonomy
S
21
and analysis of gene sequences has increased understanding of the phylogenetic
N
relationships of bacteria, fungi and other related organisms. These methods have
E
E
made identification of many species more rapid and precise than is possible with
W
T
phenotypic techniques and have also aided the recognition of numerous new species.
E
B
Some of these methods remain accessible to reference laboratories only and are
N
difficult to implement for routine microbial identification in a clinical laboratory due to

O
D
costs and lack of expertise amongst staff.

E
LT
The molecular methods include both detection and typing methods and examples
U
include real-time Polymerase Chain reaction, Pulsed Field Gel Electrophoresis, Multi-
S
N
locus sequence typing, Multiple-Locus Variable-Number Tandem-Repeat Analysis

O
C
also known as VNTR, 16S rDNA sequencing, 18S rRNA sequencing, etc.
S
A
W
Real-time polymerase chain reaction (RT- PCR)

T
N
Real-time polymerase chain reaction (RT-PCR) is the method of choice for sensitive
E
M
detection and precise quantification of minute amounts of targeted DNA sequence.

U
C
When combined with reverse transcription (RT) real-time PCR is the preferred method
O
also for the detection and quantification of RNA23. Benefits of this procedure over
D
IS
conventional methods include its sensitivity, enhanced specificity, and the potential for
H
-T
high throughout as well as accurate quantification. An additional advantage of real-

FT
time PCR is the relative ease and convenience of use compared to some older
A
methods. This method has been used successfully in the identification of bacteria and
R
fungi24.
D
For more information on the key laboratory elements needed when performing
molecular PCR assays can be accessed from Q 4 - Good laboratory practice when
performing molecular amplification assays.

Page: 18 of 32
culture
16S ribosomal RNA (16S rDNA) sequencing

16S rDNA gene sequencing is a genotypic identification method and the hyper
variable regions of 16S rDNA gene sequences provide species-specific signature
sequences useful for bacterial identification It has been used for identifying bacteria or
for phylogenetic studies and has subsequently been found to be capable of re-
classifying bacteria into completely new species, or even genera. For bacterial
identification, 16S rDNA sequencing is particularly important in the case of bacteria
with unusual phenotypic profiles, rare bacteria, slow-growing bacteria, as well as
16
culture-negative infections. It has also been used to describe new bacteria that have
20
never been successfully cultured in laboratories25.
R
E
In addition to all its applicability, sequencing 16S ribosomal DNA lacks widespread
B
O
use due to the technical and cost issues. Other drawbacks are difficulty in
T
C
interpretation of results for most technicians and even for clinical microbiologists as
O
well as automation of the technology so that it could be used routinely in clinical
-3
microbiology laboratories25. Its usefulness is also limited because of the high
R
percentage of sequence similarity between closely related species26.
E
B
M
E
18S ribosomal RNA (18S rRNA), intergenic spacer region (ITS), large ribosomal
T
P
E
subunit (D1-D2) sequencing
S
21
These molecular methods work with different primer pairs in a very similar way to the
16S rDNA sequencing and they have been very useful in the identification of clinically
N
E
relevant fungi (yeasts and filamentous fungi)27. Different approaches are used for
E
W
different groups of fungi with ITS1 and ITS2 proving the most useful for most fungal
T
E
genera.
B
N
O
Technical information/limitations
D
E
LT
U
Colonial morphology
S
N
The colonial morphology characteristics alone may be of little value in identifying

O
C
moulds because of variation among isolates and colonies grown on different culture
S
A
media hence the microscopic morphological features of the organism must be used to
W
supplement the identification of the fungi in question6.

T
N
E
Culture technique
M
U
C
The culturing of bacteria on agar media yield results but there are inherent delays in
O
diagnosis associated with this technique. It is often not possible to isolate filamentous
D
IS
fungi from clinical samples and the slow growth rate of many may delay the diagnosis.
H
-T
Commercial identification Systems

FT
The use of commercially available identification kits alone may be unreliable because
A
R
they may not be able to distinguish between related organisms as well as not being
D
able to identify new species of organism that are not in the accompanying database.
Therefore commercial systems should be used alongside other tests28,29.
Germ tube test
Candida tropicalis may produce pseudo-hyphae which may be misinterpreted as germ
tube production. Experience and expertise are required for correct recognition of germ
tubes30. The production of germ tubes is also dependent on the correct amount of
Page: 19 of 32
culture
inoculum used and a heavy inoculum can inhibit germ tube production. It should be
noted that further tests should be performed to confirm any initial results and to
differentiate Candida strains.
Matrix-assisted laser desorption/ionisation - time of flight mass spectrometry
(MALDI-TOF MS)
Refer to SMI TP40: Matrix-assisted laser desorption/ionisation - time of flight mass
spectrometry (MALDI-TOF MS) test procedure for more information on the technical
limitations.
16
20
Misinterpreted results
R
E
When conflicting results are observed, for example; biochemical profile disagrees with
B
O
serological profile, tests should be repeated from the original plate.
T
C
O
Quality control
-3
Each new lot or shipment of commercial identification systems should be tested and
R
E
verified for positive and negative reactivity using known control strains; ensuring it is fit
B
M
for purpose. Laboratories must follow manufacturer’s instructions when using these
E
T
products.
P
E
S
21
N
E
E
W
T
E
B
N
O
D
E
LT
U
S
N
O
C
S
A
W
T
N
E
M
U
C
O
D
IS
H
-T
FT
A
R
D

Page: 20 of 32
culture
1 Safety considerations31-47
Refer to current guidance on the safe handling of all organisms documented in this
SMI.
As a minimum, it is recommended that the processing of all sample cultures, including
that may result in generation of aerosols should be processed in a microbiological
safety cabinet in CL2 conditions with additional precautions to minimise risk of
aerosols production in accordance with the relevant risk assessment, ACDP and HSE
16
guidelines.
20
R
Processing of diagnostic sample cultures that are assessed to be at higher risk of
E
B
containing hazard group 3 organisms must be undertaken under appropriate
O
T
containment conditions as determined by risk assessment, and as required by
C
O
biological agents: managing the risks in laboratories and healthcare premises. This
-3
will normally be under full CL3 conditions. Such organisms include Mycobacterium
R
species, Brucella species, Bacillus anthracis, Blastomyces dermatitidis, Histoplasma
E
B
capsulatum, Coccidiodes immitis, etc.
M
E
T
The above guidance should be supplemented with local COSHH and risk
P
E
assessments.
S
Compliance with postal and transport regulations is essential. 21
N
E
E
2 Target organisms
W
T
E
B
All medically important bacteria and fungi.

N
O
D
E
3 Identification
LT
U
S
N
Refer to relevant identification SMIs for further information. It should be noted that the
O
most commonly encountered organisms are listed in the following flowcharts below for
C
S
characterisation and identification. This list of organisms is not exhaustive but those
A
W
listed are used as examples for characterisation.

T
N
E
M
U
C
O
D
IS
H
-T
FT
A
R
D

Page: 21 of 32
Introduction to the preliminary identification of medically important bacteria and fungi from culture
16
Characteristics of Gram positive cocci9,48,49
20
4
R
E
B
O
Gram positive cocci
T
C
O
-3
R
E
B
Anaerobic growth only Aerobic or facultative growth
M
E
T
P
E
S
21
Catalase
N
E
Peptostreptococcus
E
W
Gemella morbillorum
T
E
B
Negative Positive
N
O
D
E
LT
U
Streptococcus* Staphylococcus
S
N Enterococcus Micrococcus
O
Abiotrophia Rothia***
C
Aerococcus**
S
Gemella
A
Helcococcus
W
Leuconostoc
T
Pediococcus
N
Rothia***
E
M
U
C
O
* Some species may be anaerobic

D
IS
** May be weak catalase positive

H
-T
*** This organism is pleomorphic (with a variation in the size and shape of cells) catalase variable, catalase test may not be helpful for differentiation
FT
The flowchart is for guidance only.

A
R
D
Bacteriology – Identification | ID 1 | Issue no: dzs+ | Issue date: dd.mm.yy <tab+enter>| Page: 22 of 32
16
5 Characteristics of Gram positive rods9,48,49
20
R
E
B
O
Gram positive rods
T
C
O
-3
R
E
B
M
Anaerobic growth only Aerobic or facultative growth
E
T
P
E
S
21
Short medium length Branching filaments Large rods
N
Coryneform Coccobacilli Coryneform
May be in chains or beaded Straight sides
E
E
May have spores
W
T
E
B
N
O
Nocardia* Arcanobacterium Bacillus (spores)#
D
Clostridium (spores) Corynebacterium
Propionibacterium* Lactobacillus
E
Streptomyces* Listeria Corynebacterium
LT
Actinomyces* Actinomyces* Gardnerella** (non sporing)
Erysipelothrix
U
Bifidobacterium Mycobacterium# Mycobacterium# Kurthia*
S
Eubacterium Gordona Nocardia Oerskovia
N Propionibacterium*
O
Tsukamurella Rhodococcus
C
Actinomadura Rothia*
S
Turicella*
A
Brevibacterium
W
Cellulomonas
T
Dermabacter
N
E
Microbacterium
M
U
*This organism is pleomorphic

C
O
**Gardnerella vaginalis is a Gram variable rod and may usually be differentiated by its microscopic appearance
D
IS
#
These organisms (that is, Mycobacterium tuberculosis and Bacillus anthracis) are hazard group 3 organisms and should be processed in a Containment level
H
3 laboratories. Mycobacterium species should be referred to the Reference Laboratory for full identification
-T
FT

A
R
D
16
6 Characteristics of Gram negative bacteria9,48,49
20
R
E
B
O
Gram negative bacteria
T
C
O
-3
R
E
B
M
E
T
P
Cocci / coccobacilli Rods
E
S
21
N
E
E
W
T
E
Aerobic or facultative Anaerobic growth only Aerobic or facultative Anaerobic growth only
B
N
O
D
E
LT
U
S
N Bacteroides
Acinetobacter
O
Fusobacterium
C
Kingella Veillonella Refer to next flowchart below Mobiluncus

S
A
Moraxella Porphyromonas
W
Neisseria Prevotella
T
N
E
M
U
C
O
D
IS
H
-T
FT

A
R
D
16
7 Characteristics of Gram negative bacteria9,48,49(Continued from previous page)
20
R
E
B
Aerobic or facultative Gram negative rods
O
T
C
O
-3
R
Small and/or
E
Variable length Straight sided Curved
pleomorphic
B
M
E
T
P
Aggregatibacter* Legionella*
E
Oxidase
S
Bordetella* (specific growth Vibrio
Brucella* # requirements)
21
Campylobacter (microaerobic)
Cardiobacterium* Arcobacter (microaerobic)
N
Eikenella* Helicobacter (microaerobic)
E
Francisella*
E
W
Pasteurella*
T
Haemophilus* Negative Positive
E
Bartonella
B
Streptobacillus*
N
Acinetobacter
O
D
E
LT
Pseudomonas
Enterobacteriaceae** # Alcaligenes
U
Stenotrophomonas**
S
Burkholderia
Capnocytophaga***
N Aeromonas
O
Acinetobacter Flavobacterium
C
Consider Gardnerella Capnocytophaga

S
Acidovorax
A
W
Chromobacterium
Comamonas
T
Kingella*
N
E
M
* Some species may be anaerobic

U
C
** May be weak catalase positive

O
D
*** This organism is pleomorphic and catalase variable, and are facultatively anaerobic
IS
#
H
These organisms (that is, Brucella species and species within the family Enterobacteriaceae) are hazard group 3 organisms and should be processed in
-T
Containment level 3 laboratories.

FT

A
R
D
16
8 Characteristics of fungi
20
R
E
B
O
Fungi
Fungi
T
C
O
-3
R
Moulds
Moulds Yeasts
Yeasts
E
(filamentous)
(filamentous) (unicellular)
(unicellular)
B
M
ainute
ainute colonies
colonies
E
tresumptive aalassezia
tresumptive aalassezia species
species
T
P
Germ-tube
Germ-tube positive
positive
E
Candida
Candida albicans
albicans
S
Aseptate
Aseptate Septate
Septate Candida
Candida dubliniensis
dubliniensis
21
hyphae
hyphae hyphae
hyphae Candida
Candida africana
africana
N
E
E
W
Mucoraceous
Mucoraceous
Moulds
Moulds
T
(formerly Monomorphic
Monomorphic Dimorphic
Dimorphic
(formerly
E
Zygomycetes)
B
Zygomycetes)
N
O
D
E
LT
U
S
N
O
C
S
Fusarium
Fusarium species
species
A
Aspergillus
Aspergillus species
species #
Mucor
Mucor species
species Histoplasma species#
Histoplasma species
W
Microsporum
Microsporum species
species #
Rhizopus
Rhizopus species
species Trichophyton
Blastomyces dermatitidis#
Blastomyces dermatitidis Pseudohyphae
Pseudohyphae or
or budding
budding yeast
yeast
Trichophyton species
species # Arthrospores
Arthrospores Pink
Pink or
or red
red colonies
colonies
Basidiobolus
Basidiobolus species Paracoccidiodes brasiliensis#
Paracoccidiodes brasiliensis Candida
Candida species
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species Phialophora species species

Phialophora species # Trichosporon
Trichosporon species
species Rhodotorula
Rhodotorula species
species
Conidiobolus
Conidiobolus species Coccidioides immitis#
Coccidioides immitis
N
species Epidermophtyon Cryptococcus

Cryptococcus species
species
Epidermophtyon floccosum
floccosum Talaromyces Saprochaete
Saprochaete capitata
capitata Sporobolomyces
Sporobolomyces species
species
Lichtheimia
Lichtheimia (previously Talaromyces (previously
(previously Saccharomyces
Saccharomyces species
E
(previously Cladosporium species

Cladosporium species
species #
Absidia)
Absidia) species Penicillium) marneffei#
Penicillium) marneffei
M
species Madurella
Madurella species
species Sporothrix schenckii
Rhizomucor
Rhizomucor species
species Sporothrix schenckii
U
Acremonium
Acremonium species
species
Scedosporium
Scedosporium species
C
species
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* 8
C. glabrata does not form pseudohyphae .
IS
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#
These fungi are hazard group 3 organisms and should be processed in a Containment level 3 laboratory.
-T

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A
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culture
9 Reporting
Refer to individual UK Standard for Microbiology Investigation.
Note:
The results of any identification tests should be entered in the pathology IT system
and should be available to staff validating those results. For automated identification
systems, identification scores (that identify the probability of a correct identification)
16
and organism in the differential list should be entered so that the likelihood of the
20
preferred and alternative identifications can be considered in the context of the clinical
R
E
circumstances and consideration can be given as to when alternative identification
B
O
tests are required. However it should be noted that it is not always feasible to store all
T
C
the alternative identifications from the various identification systems onto the IT
O
system as these systems should have already been validated before put in routine
-3
use.
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E
B
M
10 Referrals
E
T
P
E
S
Refer to individual UK Standard for Microbiology Investigation for further information.
21
N
11 Notification to PHE50,51, or equivalent in the

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E
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devolved administrations52-55
T
E
B
N
The Health Protection (Notification) regulations 2010 require diagnostic laboratories to

O
D
notify Public Health England (PHE) when they identify the causative agents that are
E
LT
listed in Schedule 2 of the Regulations. Notifications must be provided in writing, on

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paper or electronically, within seven days. Urgent cases should be notified orally and
S
N
as soon as possible, recommended within 24 hours. These should be followed up by

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C
written notification within seven days.

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A
For the purposes of the Notification Regulations, the recipient of laboratory

W
notifications is the local PHE Health Protection Team. If a case has already been
T
N
notified by a registered medical practitioner, the diagnostic laboratory is still required

E
M
to notify the case if they identify any evidence of an infection caused by a notifiable
U
C
causative agent.
O
D
Notification under the Health Protection (Notification) Regulations 2010 does not
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replace voluntary reporting to PHE. The vast majority of NHS laboratories voluntarily
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report a wide range of laboratory diagnoses of causative agents to PHE and many
FT
PHE Health protection Teams have agreements with local laboratories for urgent
A
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reporting of some infections. This should continue.

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Note: The Health Protection Legislation Guidance (2010) includes reporting of Human
Immunodeficiency Virus (HIV) & Sexually Transmitted Infections (STIs), Healthcare
Associated Infections (HCAIs) and Creutzfeldt–Jakob disease (CJD) under
‘Notification Duties of Registered Medical Practitioners’: it is not noted under
‘Notification Duties of Diagnostic Laboratories’.

Page: 27 of 32
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https://www.gov.uk/government/organisations/public-health-england/about/our-
governance#health-protection-regulations-2010
Other arrangements exist in Scotland52,53, Wales54 and Northern Ireland55.
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20
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B
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P
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21
N
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E
W
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B
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N
O
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A
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References
1. Duerden B, Towner KJ, Magee JT. Isolation, description and identification of bacteria. In: Collier L,
Balows A, Sussman M, editors. Topley and Wilson's Microbiology and Microbial Infections. 9th ed.
Vol 2. London: Arnold; 1998. p. 65-84.
2. Vyzantiadis TA, Johnson EM, Kibbler CC. From the patient to the clinical mycology laboratory: how
can we optimise microscopy and culture methods for mould identification? J Clin Pathol
2012;65:475-83.
16
20
3. Clark AE, Kaleta EJ, Arora A, Wolk DM. Matrix-assisted laser desorption ionization-time of flight
mass spectrometry: a fundamental shift in the routine practice of clinical microbiology. Clin
R
E
Microbiol Rev 2013;26:547-603.
B
O
T
4. Barrow GI, Feltham RKA. Bacterial characters and charcterization. Cowan and Steel's Manual for
C
O
the Identification of Medical Bacteria. 3rd ed. Cambridge: Cambridge University Press; 2003. p. 21-
-3
45, 226.
R
E
5. Rogers HG. Bacterial Morphology. In: Linton AH, Dick HM, editors. Topley and Wilson's Principles
B
M
of Bacteriology, Virology and Immunity. 8th ed. Vol 1. London: Edward Arnold; 1990. p. 17-38.
E
T
P
6. Bailey and Scott's. Diagnostic Microbiology. In: Forbes BA, Sahm DF, Weissfeld AS, editors. 11th
E
S
ed. St Louis: Mosby Inc; 2002. p. 152-3.
21
N
7. Freeman BA, editor. Burrows Textbook of Microbiology. 22nd ed. Philadelphia: WB Saunders
E
E
Company; 1985. p. 21-2

W
T
E
8. Koneman EW, Allen S D, Janda W M, Schreckenberger P C, Winn W J, editors. Color Atlas and
B
Textbook of Diagnostic Microbiology. 5th ed. Philadelphia: Lippincott Williams & Wilkins; 1997. p.
N
O
98-102
D
E
9. Isenberg HD, editor. Clinical Microbiology Procedures Handbook. American Society for
LT
Microbiology; 2004. p. 3.3.2-3.3.2.13

U
S
N
O
10. Perry JD, Freydiere AM. The application of chromogenic media in clinical microbiology. J Appl
C
Microbiol 2007;103:2046-55.
S
A
W
11. Collins CH, Lyne PM, Grange JM, Falkinham JO. Identification methods. In: Collins CH, Lyne PM,
T
Grange JM, Falkinham JO, editors. Collins and Lyne's Microbiological Methods. 8th ed. Arnold;
N
E
2004. p. 89-109.
M
U
C
12. Zimmer BL, Roberts GD. Rapid selective urease test for presumptive identification of Cryptococcus
O
neoformans. J Clin Microbiol 1979;10:380-1.

D
IS
H
13. SEELIGER HP. Use of a urease test for the screening and identification of cryptococci. J Bacteriol
-T
1956;72:127-31.
FT
A
14. Hainer BL. Dermatophyte infections. Am Fam Physician 2003;67:101-8.

R
D
15. Suarez S, Ferroni A, Lotz A, Jolley KA, Guerin P, Leto J, et al. Ribosomal proteins as biomarkers
for bacterial identification by mass spectrometry in the clinical microbiology laboratory. J Microbiol
Methods 2013;94:390-6.
16. NICE. Tests for rapidly identifying bloodstream bacteria and fungi (LightCycler SeptiFast Test
MGRADE, SepsiTest and IRIDICA BAC BSI assay). dg20. 10-2-2016. p. 1-46

Page: 29 of 32
culture
17. Nomura F. Proteome-based bacterial identification using matrix-assisted laser desorption

ionization-time of flight mass spectrometry (MALDI-TOF MS): A revolutionary shift in clinical
diagnostic microbiology. Biochim Biophys Acta 2015;1854:528-37.
18. Peng J, Yang J, Jin Q. The molecular evolutionary history of Shigella spp. and enteroinvasive
Escherichia coli. Infection, Genetics and Evolution 2009;9:147-52.
19. Perna NT, Plunkett G, III, Burland V, Mau B, Glasner JD, Rose DJ, et al. Genome sequence of
enterohaemorrhagic Escherichia coli O157:H7. Nature 2001;409:529-33.
16
20. Thomson NR, Howard S, Wren BW, Holden MT, Crossman L, Challis GL, et al. The complete
20
genome sequence and comparative genome analysis of the high pathogenicity Yersinia
R
enterocolitica strain 8081. PLoS Genet 2006;2:e206.
E
B
O
21. Baker S, Dougan G. The genome of Salmonella enterica serovar Typhi. Clin Infect Dis 2007;45
T
C
Suppl 1:S29-S33.
O
-3
22. Teles F, Seixas J. The future of novel diagnostics in medical mycology. J Med Microbiol
R
2015;64:315-22.
E
B
M
23. Bar T, Kubista M, Tichopad A. Validation of kinetics similarity in qPCR. Nucleic Acids Res
E
T
2012;40:1395-406.
P
E
S
24. Espy MJ, Uhl JR, Sloan LM, Buckwalter SP, Jones MF, Vetter EA, et al. Real-time PCR in clinical
21
microbiology: applications for routine laboratory testing. Clin Microbiol Rev 2006;19:165-256.
N
E
E
25. Woo PC, Lau SK, Teng JL, Tse H, Yuen KY. Then and now: use of 16S rDNA gene sequencing for
W
bacterial identification and discovery of novel bacteria in clinical microbiology laboratories. Clin
T
E
Microbiol Infect 2008;14:908-34.

B
N
O
26. Wang LT, Lee FL, Tai CJ, Kasai H. Comparison of gyrB gene sequences, 16S rRNA gene
D
sequences and DNA-DNA hybridization in the Bacillus subtilis group. Int J Syst Evol Microbiol
E
LT
2007;57:1846-50.
U
S
27. Gharizadeh B, Norberg E, Loffler J, Jalal S, Tollemar J, Einsele H, et al. Identification of medically
N
O
important fungi by the Pyrosequencing technology. Mycoses 2004;47:29-33.

C
S
A
28. Janda JM, Abbott SL. The genus Hafnia: from soup to nuts. Clin Microbiol Rev 2006;19:12-8.
W
T
29. Abbott SL, O'Connor J, obin T, immer BL, anda JM. Biochemical properties of a newly described
N
E
Escherichia species, Escherichia albertii. Journal of Clinical Microbiology 2003;41:4852-4.

M
U
C
30. Hoppe JE, Frey P. Evaluation of six commercial tests and the germ-tube test for presumptive
O
identification of Candida albicans. Eur J Clin Microbiol Infect Dis 1999;18:188-91.

D
IS
H
31. European Parliament. UK Standards for Microbiology Investigations (SMIs) use the term "CE
-T
marked leak proof container" to describe containers bearing the CE marking used for the collection
FT
and transport of clinical specimens. The requirements for specimen containers are given in the EU
A
in vitro Diagnostic Medical Devices Directive (98/79/EC Annex 1 B 2.1) which states: "The design
R
must allow easy handling and, where necessary, reduce as far as possible contamination of, and
D
leakage from, the device during use and, in the case of specimen receptacles, the risk of
contamination of the specimen. The manufacturing processes must be appropriate for these
purposes".
32. Official Journal of the European Communities. Directive 98/79/EC of the European Parliament and
of the Council of 27 October 1998 on in vitro diagnostic medical devices. 7-12-1998. p. 1-37.

Page: 30 of 32
culture
33. Health and Safety Executive. Safe use of pneumatic air tube transport systems for pathology
specimens. 9/99.
34. Department for transport. Transport of Infectious Substances, 2011 Revision 5. 2011.
35. World Health Organization. Guidance on regulations for the Transport of Infectious Substances
2013-2014. 2012.
36. Home Office. Anti-terrorism, Crime and Security Act. 2001 (as amended).
16
37. Advisory Committee on Dangerous Pathogens. The Approved List of Biological Agents. Health and
20
Safety Executive. 2013. p. 1-32
R
E
38. Advisory Committee on Dangerous Pathogens. Infections at work: Controlling the risks. Her
B
O
Majesty's Stationery Office. 2003.
T
C
O
39. Advisory Committee on Dangerous Pathogens. Biological agents: Managing the risks in
-3
laboratories and healthcare premises. Health and Safety Executive. 2005.
R
E
B
40. Advisory Committee on Dangerous Pathogens. Biological Agents: Managing the Risks in
M
Laboratories and Healthcare Premises. Appendix 1.2 Transport of Infectious Substances -
E
T
Revision. Health and Safety Executive. 2008.
P
E
S
41. Centers for Disease Control and Prevention. Guidelines for Safe Work Practices in Human and
21
Animal Medical Diagnostic Laboratories. MMWR Surveill Summ 2012;61:1-102.
N
E
E
42. Health and Safety Executive. Control of Substances Hazardous to Health Regulations. The Control
W
of Substances Hazardous to Health Regulations 2002. 5th ed. HSE Books; 2002.
T
E
B
43. Health and Safety Executive. Five Steps to Risk Assessment: A Step by Step Guide to a Safer and
N
O
Healthier Workplace. HSE Books. 2002.

D
E
LT
44. Health and Safety Executive. A Guide to Risk Assessment Requirements: Common Provisions in
U
Health and Safety Law. HSE Books. 2002.

S
N
O
45. Health Services Advisory Committee. Safe Working and the Prevention of Infection in Clinical
C
Laboratories and Similar Facilities. HSE Books. 2003.

S
A
W
46. British Standards Institution (BSI). BS EN12469 - Biotechnology - performance criteria for
T
microbiological safety cabinets. 2000.

N
E
M
47. British Standards Institution (BSI). BS 5726:2005 - Microbiological safety cabinets. Information to
U
C
be supplied by the purchaser and to the vendor and to the installer, and siting and use of cabinets.
O
D
Recommendations and guidance. 24-3-2005. p. 1-14

IS
H
48. Baer H, Davis CE. Classification and identification of bacteria. In: Braude AI, editor. Medical
-T
Microbiology and Infectious Diseases. Philadelphia: WB Saunders Company; 1981. p. 9-20.

FT
A
49. Bruckner DA, Colonna P, Bearson BL. Nomenclature for aerobic and facultative bacteria. Clin Infect
R
D
Dis 1999;29:713-23.
50. Public Health England. Laboratory Reporting to Public Health England: A Guide for Diagnostic
Laboratories. 2013. p. 1-37.
51. Department of Health. Health Protection Legislation (England) Guidance. 2010. p. 1-112.
52. Scottish Government. Public Health (Scotland) Act. 2008 (as amended).

Page: 31 of 32
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53. Scottish Government. Public Health etc. (Scotland) Act 2008. Implementation of Part 2: Notifiable
Diseases, Organisms and Health Risk States. 2009.
54. The Welsh Assembly Government. Health Protection Legislation (Wales) Guidance. 2010.
55. Home Office. Public Health Act (Northern Ireland) 1967 Chapter 36. 1967 (as amended).
16
20
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B
O
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O
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S
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T
N
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M
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D
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UK Standards for Microbiology Investigations

Issued by the Standards Unit, Microbiology Services, PHE

London NW9 5EQ

PHE publications gateway number: 2016308

UK Standards for Microbiology Investigations are produced in association with:

Logos correct at time of publishing.

Bacteriology – Identification | ID 1 | Issue no: dzs+ | Issue date: dd.mm.yy <tab+enter>|

Amendment table ................................................................................................................... 4

Scope of document ................................................................................................................ 8

5 Characteristics of Gram positive rods .................................................................... 23

6 Characteristics of Gram negative bacteria ............................................................. 24

7 Characteristics of Gram negative bacteria (Continued from previous page) ....... 25

8 Characteristics of fungi ............................................................................................ 26

11 Notification to PHE, or equivalent in the devolved administrations ..................... 27

Bacteriology – Identification | ID 1 | Issue no: dzs+ | Issue date: dd.mm.yy <tab+enter>|

Whole document. Section on Identification updated with cutting edge

molecular methods and MALDI-TOF MS.

Additional flowchart on characteristics of fungi

References. References updated.

Bacteriology – Identification | ID 1 | Issue no: dzs+ | Issue date: dd.mm.yy <tab+enter>|

UK SMI#: scope and purpose

Standardisation of the diagnostic process through the application of SMIs helps to

assure the equivalence of investigation strategies in different laboratories across the

Equal partnership working

Pathologists and professional societies. The list of participating societies may be

Inclusion of a logo in an SMI indicates participation of the society in equal partnership

which develop SMIs. The views of nominees cannot be rigorously representative of

developed, reviewed and updated through a wide consultation process.

Bacteriology – Identification | ID 1 | Issue no: dzs+ | Issue date: dd.mm.yy <tab+enter>|

PHE is a Caldicott compliant organisation. It seeks to take every possible precaution

to prevent unauthorised disclosure of patient details and to ensure that patient-related

Equality objectives https://www.gov.uk/government/organisations/public-health-

effective consultation with members of the public, partners, stakeholders and

specialist interest groups.

Bacteriology – Identification | ID 1 | Issue no: dzs+ | Issue date: dd.mm.yy <tab+enter>|

Suggested citation for this document

Bacteriology – Identification | ID 1 | Issue no: dzs+ | Issue date: dd.mm.yy <tab+enter>|

associated with those sites of infection is useful as an aid to preliminary identification.

When identifying microorganisms it should be remembered that their characteristics

for example Capnocytophaga canimorsus is oxidase positive, whereas

consideration during the identification process.

Matrix assisted Laser Desorption/Ionisation – Time of Flight Mass Spectrometry

(MALDI-TOF MS), is increasingly being applied for organism identification within

diagnostic microbiology laboratories. Identification using this technology affords the

as some genera of filamentous fungi in comparison with the more traditional

techniques3. Any identification should be considered alongside phenotypic information

When classifying microorganisms, all known characteristics are taken into

results of the previous investigations are known.

may vary between organisms.

Conventional identification methods for bacteria and

intracellular inclusions, for example spores. In addition to morphology, the Gram

Gram negative bacteria1,4.

For morphological appearance, it is preferable to examine fresh cultures from growth

Bacteriology – Identification | ID 1 | Issue no: dzs+ | Issue date: dd.mm.yy <tab+enter>|

using different types of stains.

Table 1a: Terms used for stained preparations of bacteria5

singly, in pairs, in chains, in fours (tetrads), in groups, grape-like

Arrangement clusters, in cuboidal packets, in bundles, in Chinese letters

Capsule presence or absence

spherical, oval or ellipsoidal, equatorial, sub-terminal, terminal, cause

Ends round, truncate, pointed

variation in shape and size, clubs, filamentous, branched, navicular,

citron, fusiform, giant swollen forms