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Staining Procedures UK Standards for Microbiology Investigations Staining procedures Issued by the Standards Unit, National Infection Service, PHE Bacteriology – Test Procedures | TP 39 | Issue no: 3 | Issue date: 17.05.19 | Page: 1 of 55 © Crown copyright 2019 Staining procedures Acknowledgments UK Standards for Microbiology Investigations (UK 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. UK SMIs are developed, reviewed and revised by various working groups which are overseen by a steering committee (see https://www.gov.uk/government/groups/standards-for-microbiology-investigations- steering-committee). The contributions of many individuals in clinical, specialist and reference laboratories who have provided information and comments during the development of this document are acknowledged. We are grateful to the medical editors for editing the medical content. For further information please contact us at: Standards Unit National Infection Service Public Health England 61 Colindale Avenue London NW9 5EQ E-mail: [email protected] Website: https://www.gov.uk/uk-standards-for-microbiology-investigations-smi-quality- and-consistency-in-clinical-laboratories PHE publications gateway number: 2015075 UK Standards for Microbiology Investigations are produced in association with: Logos correct at time of publishing. Bacteriology – Test Procedures | TP 39 | Issue no: 3 | Issue date: 17.05.19 | Page: 2 of 55 UK Standards for Microbiology Investigations | Issued by the Standards Unit, Public Health England Staining procedures Contents Acknowledgments ................................................................................................................. 2 Contents ................................................................................................................................. 3 Amendment table ................................................................................................................... 5 UK SMI: scope and purpose .................................................................................................. 6 Scope of document ................................................................................................................ 9 Introduction ............................................................................................................................ 9 Technical information/limitations ......................................................................................... 9 Bacteria stains ..................................................................................................................... 11 1 Auramine-phenol stain – 1 (acid fast bacilli) .......................................................... 11 2 Gram stain ................................................................................................................. 13 3 Kinyoun stain (Mycobacterium and Nocardia species) ......................................... 15 4 McFadyean stain ....................................................................................................... 17 5 Modifications of the Kinyoun stain method ............................................................ 18 6 Sandiford’s modification of Gram stain .................................................................. 18 7 Spore stains .............................................................................................................. 19 8 Vincent’s stain (oral bacteria) .................................................................................. 21 9 Ziehl-Neelsen stain (acid fast bacilli) ...................................................................... 22 Fungal stains ........................................................................................................................ 24 1 Grocott-Gomori Methenamine Silver stain (GMS) (fungi) ...................................... 24 2 Lactophenol cotton blue stain ................................................................................. 26 3 Modified Giemsa’s stain (Pneumocystis jirovecii) ................................................. 28 4 India Ink stain ............................................................................................................ 29 5 Potassium hydroxide – calcofluor white preparation (KOH-CFW) (fungi) ............ 31 6 Rapid Field’s stain (Pneumocystis jirovecii) .......................................................... 33 Parasite stains ...................................................................................................................... 34 1 Acridine orange stain (Trichomonas vaginalis)...................................................... 34 2 Auramine-phenol stain – 2 (Cryptosporidium species) ......................................... 35 Bacteriology – Test Procedures | TP 39 | Issue no: 3 | Issue date: 17.05.19 | Page: 3 of 55 UK Standards for Microbiology Investigations | Issued by the Standards Unit, Public Health England Staining procedures 3 Calcofluor stain (Microsporidia) .............................................................................. 36 4 Field’s stain (Plasmodium species)......................................................................... 37 5 Giemsa stain (Dientamoeba fragilis and Blastocystis hominis) ............................ 40 6 Giemsa stain (Plasmodium species) ....................................................................... 41 7 Lugol’s iodine (parasites) ........................................................................................ 43 8 Modified trichrome stain (Microsporidia) ................................................................ 44 9 Modified cold Ziehl-Neelsen’s stain (Cryptosporidum and Isospora species) ..... 45 10 Rapid field’s stain (Dientamoeba fragilis and Blastocystis hominis) ................... 47 Appendix .............................................................................................................................. 48 References ........................................................................................................................... 50 Bacteriology – Test Procedures | TP 39 | Issue no: 3 | Issue date: 17.05.19 | Page: 4 of 55 UK Standards for Microbiology Investigations | Issued by the Standards Unit, Public Health England Staining procedures Amendment table Each UK SMI method has an individual record of amendments. The current amendments are listed on this page. The amendment history is available from [email protected]. New or revised documents should be controlled within the laboratory in accordance with the local quality management system. Amendment number/date 5/17.05.19 Issue number discarded 2.1 Insert issue number 3 Anticipated next review date* 17.05.22 Section(s) involved Amendment Document updated. The lactophenol cotton blue stain methods for clinical specimens and colonies clarified in Whole document. separate titles. More information added in the general technical limitations section. References graded and updated. ∗Reviews can be extended up to five years subject to resources available. Bacteriology – Test Procedures | TP 39 | Issue no: 3 | Issue date: 17.05.19 | Page: 5 of 55 UK Standards for Microbiology Investigations | Issued by the Standards Unit, Public Health England Staining procedures UK SMI#: scope and purpose Users of UK SMIs Primarily, UK SMIs are intended as a general resource for practising professionals operating in the field of laboratory medicine and infection specialties in the UK. 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 appropriate tests. The documents also provide commissioners of healthcare services with the appropriateness and standard of microbiology investigations they should be seeking as part of the clinical and public health care package for their population. Background to UK SMIs UK SMIs comprise a collection of recommended algorithms and procedures covering all stages of the investigative process in microbiology from the pre-analytical (clinical syndrome) stage to the analytical (laboratory testing) and post analytical (result interpretation and reporting) stages. Syndromic algorithms are supported by more detailed documents containing advice on the investigation of specific diseases and infections. Quality guidance notes describe laboratory processes which underpin quality, for example assay validation. Standardisation of the diagnostic process through the application of UK SMIs helps to assure the equivalence of investigation strategies in different laboratories across the UK and is essential for public health surveillance, research and development activities. Equal partnership working UK SMIs are developed in equal partnership with PHE, NHS, Royal College of Pathologists and professional societies. The list of participating societies may be found at https://www.gov.uk/uk-standards-for-microbiology-investigations-smi-quality- and-consistency-in-clinical-laboratories. Inclusion of a logo in an UK SMI indicates participation of the society in equal partnership and support
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