PAPANICOLAOU 6. Tap water......................................................................................................................rinse STAINING SYSTEM 7.­ Scott’s Tap Water Substitute................................................................................10 dips (Procedure No. HT40) 8. Tap water......................................................................................................................rinse 9. Reagent­Alcohol,­95%.........................................................................................10­dips _______________________________________________ 10. Papanicolaou­Stain­OG­6..............................................................................1.5­minutes INTENDED USE 11. Reagent­Alcohol,­95%.........................................................................................10­dips _______________________________________________ 12. Papanicolaou­Stain­Modified­EA,­OR ­ Papanicolaou­Stain­EA­50,­OR The Sigma-Aldrich Papanicolaou Staining system is intended for staining exfoliative ­ Papanicolaou­Stain­EA­65.............................................................................2.5­minutes cells in cytologic specimens. Papanicolaou staining reagents are for “In Vitro Diagnostic 13. Reagent­Alcohol,­95%,­two­changes..........................................................10­dips­each Use.” 1 14. Reagent­Alcohol,­100%.....................................................................................1­minute Papanicolaou staining techniques, reviewed in a concise report by Street, have 15.­Xylene or xylene substitute, two changes................................................2 minutes each changed little in the past 40 years. The stain is used for examining exfoliative cells of 16. Coverslip and examine mi cro scop i cal ly. ­sputum­as­well­as­vaginal,­cer­vi­cal,­and­other­body­secretions.­In­gen­er­al,­cell­are­fixed­ PROCEDURE 2 (Rapid Papanicolaou Stain) to a slide, treated with a hematoxylin nuclear stain and counterstained with a mix ture of Reagent Preparation: Prepare Papanicolaou Stain OG 6-EA Solution by mixing equal orange G, eosin Y and fast green FCF (a replacement for light green SF yellowish). These 2 volumes of Papanicolaou Stain OG 6 and one of the EA stains. treatments impart char ac ter is tic color to nuclei and cytoplasmic components. _______________________________________________ 1. Wash smears in running water...........................................................................1 minute NOTES: REAGENTS _______________________________________________ a.­This­ step­ removes­ spray-fixative­ and­ the­ timing­ is­ for­ use­ with­ water­ soluble­ fixatives.­Ini­tial­washing­is­important­and­must­be­thorough­to­expose­the­cell­to­ PAPANICOLAOU STAIN OG 6, Catalog No. HT40116/ HT40132/ HT40180/ HT401128 staining. Certified­orange­G,­0.3%­w/v,­phosphotungstic­acid,­0.015%­w/v,­in­denatured­­alcohol. b.­If­slides­have­been­fixed­in­95%­ethanol,­timing­may­be­short­ened­to­a­few­­seconds. PAPANICOLAOU STAIN MODIFIED EA, Cat a log No. HT40232 2. Stain in Hematoxylin Solution Gill No. 2........................................................1-3 minutes Certified­eosin­Y,­0.25%­w/v,­cer­ti­fied­fast­green­FCF,­0.004%­w/v,­phosphotungstic­acid,­ Wash in running tap water. 0.4%­w/v,­in­denatured­alcohol­with­stabilizer. 3. Scott’s Tap Water Substitute.......................................................................20 seconds PAPANICOLAOU STAIN EA 50, Catalog No. HT40316/ HT40332/ HT403128 Wash in running tap water. Certified­ eosin­ Y,­ 0.23%­ w/v,­ cer­ti­fied­ fast­ green­ FCF,­ 0.08%­ w/v,­ cer­ti­fied­ bismarck­ 4. Reagent­Alcohol,­95%..................................................................................20­seconds brown,­0.05%,­phosphotungstic­acid,­0.2%­w/v,­in­denatured­alcohol. 5.­ Stain in Papanicolaou Stain OG 6-EA (prepared above)..............................1-3 minutes PAPANICOLAOU STAIN EA 65, Catalog No. HT40432 6. Rinse­in­two­changes­of­Reagent­Alcohol,­100% Certified­ eosin­ Y,­ 0.23%­ w/v,­ cer­ti­fied­ fast­ green­ FCF,­ 0.01%­ w/v,­ cer­ti­fied­ bismarck­ 7.­ Clear in two changes of xylene or xylene substitute. brown,­0.05%,­phosphotungstic­acid,­0.2%­w/v,­in­denatured­alcohol. 8. Coverslip and examine mi cro scop i cal ly. STORAGE AND STABILITY: _______________________________________________ Store reagents at room temperature (18-26°C). Reagents are stable un til expiration PERFORMANCE CHARACTERISTICS dates shown on la bels. _______________________________________________ PREPARATION: Nuclei are stained blue while cyto plasm displays varying shades of blue, orange, ­ Differentiation­Solution­is­ready­to­use. pink­or­red.­If­observed­results­vary­from­expected­results,­please­contact­Sigma-Aldrich­ Scott’s Tap Water Substitute is prepared by mixing 1 part of Scott’s Tap Water Technical Service for assistance. Substitute­Concentrate­with­9­parts­deionized­water. _______________________________________________ Filter Papanicolaou Staining System reagents before use. PRECAUTIONS: REFERENCES _______________________________________________ Normal precautions exercised in handling laboratory reagents should be followed. Dispose of waste observing all local, state, provincial or national regulations. Refer to 1. Street CM: Papanicolaou Techniques in Exfoliative Cytology. IN Laboratory Technique Safety­Data­Sheet­and­product­labeling­for­any­updated­risk,­hazard­or­safety­information. in­ Biology­ and­ Medicine,­ 3rd­ ed.­ EV­ Cowdry­ Editor,­ Williams­ &­ Wilkins,­ Baltimore,­ _______________________________________________ 1952,­p­253 PROCEDURE 2. Johnson­PL,­Klein­MN:­Ap­pli­ca­tion­of­Papanicolaou­stain­to­par­af­ n­sections.­Stain­ _______________________________________________ Technol­31:223,­1956 SPECIMEN COLLECTION: 3. Liu­W:­A­simplified­cytologic­staining­technic.­Am­J­Clin­Pathol­54:767,­1970 4. A Manual for Cytotechnology, C Keebler, J Reagen, Editors, American Society of All samples must be clearly labeled. Suitable instruments must be used for obtaining Clinical Pathologists Press, Chicago (IL), 1983, p 323 and­preparing­samples.­Follow­manufacturer’s­instructions­for­use.­No­known­test­method­ can­ offer­ complete­ assurance­ that­ blood­ ­samples­ or­ tissue­ will­ not­ transmit­ infection.­ 2021­ Sigma-Aldrich­ Co.­ LLC.­ All­ rights­ reserved.­ SIGMA-ALDRICH­ is­ a­ trademark­ of­ Therefore, all blood derivatives or tissue specimens should be considered potentially infectious. Sigma-Aldrich Co. LLC, registered in the US and other countries. Sigma brand products ­ Smears­are­prepared­and­fixed­immediately­with­a­spray­fixative­or­placed­in­95%­ethanol are sold through Sigma-Aldrich, Inc. Purchaser must determine the suitability of the product(s) for their particular use. Additional terms and conditions may apply. Please see for­a­minimum­of­15­minutes. SPECIAL MATERIALS REQUIRED BUT NOT PROVIDED: product information on the Sigma-Aldrich website at www.sigmaaldrich.com and/or on the­reverse­side­of­the­invoice­or­packing­slip. Differentiation­Solution,­Catalog­Nos.­A3179­or­A3429 Reagent Alcohol, Catalog No. R8382, or Ethanol (absolute) Procedure No. HT40 Hematoxylin Nuclear Stain: Previous Revision: 2020-12 Harris­Hematoxylin­Solution,­Mod­i­fied,­Catalog­No.­HHS16/­HHS32/­HHS80/­HHS128 Revised: 2021-04 Hematoxylin Solution Gill No. 1, Catalog No. GHS116/ GHS132/ GHS1128 Hematoxylin Solution Gill No. 2, Catalog No. GHS216/ GHS232/ GHS280/ GHS2128 Scott’s­Tap­Water­Substitute­Con­cen­trate,­Catalog­No.­S5134 Prepare Scott’s Tap Water Sub sti tute Solution as indicated on bottle label. Xylene or xylene substitute. NOTES:4 Authorized Rep in the EU EN ISO 15223-1 1. Separate staining solutions should be maintained for gynecologic smears and SYMBOLS nongynecologic smears. 2. All­ solutions­ preceding­ the­ hematoxylin­ solutions­ should­ be­ fil­tered­ after­ each­ staining­sequence­and­all­solutions­should­be­filtered­daily. 3. Cellulose­membrane­filters­should­be­dehydrated­(Procedure­1,­step­14­and­15)­in­ isopropyl alcohol to prevent dissolution. 4. Staining times may be varied for individual color preferences. 5.­ Ethanol may be substituted for Reagent Alcohol. 6. Appropriate control slides should be included in each run. MDSS GmbH 7.­ The data obtained from this procedure serves only as an aid to diagnosis and should Schiffgraben 41 be reviewed in conjunction with other clinical diagnostic tests or information. 30175 Hannover, Germany PROCEDURE: PROCEDURE 1 (Standard Method) SIGMA-ALDRICH, INC. 1. Reagent­Alcohol,­95%...................................................................................15­minutes 3050­Spruce­Street,­St.­Louis,­MO­63103­ USA 2. Tap water......................................................................................................................rinse 314-771-5765 3. Hematoxylin solution..................................................................................1-3 minutes NOTE: Time will vary with se lec tion of hematoxylin solution. Technical­Service:­ 800-325-5832­ 4. Tap water......................................................................................................................rinse sigmaaldrich.com/customer-support.html 5.­ Differentiation­So­lu­tion............................................................................20-60­seconds­ To­Order:­ 800-325-3010­ NOTE: Necessary for regressive hematoxylin stains only. sigmaaldrich.com 1-E.