UK NEQAS CPT Best Methods Handbook Grocott Edition 2. Best Methods Handbook. Grocott. Edition 2 Last Updated September 15th 2006

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1 Best Methods Handbook Grocott Edition 2 Last Updated September 15th 2006 Garry Thompson Page 1 of 7 15/09/2006

2 Run 43 June 2006 Set 2 (See previous table for set 1) Centre No Oxidation 1% Periodic acid 5 5% Chromic acid at 56 degrees 5% Chromic acid 1 8 Wash Distilled Running tap Tap Decolourise % Sodium Metabisulphite 30 sec Wash Well in running tap then several changes of distilled Preheat solution in a microwave, 50 sec at 800 watts (medium/ High) Place slides in solution and heat again on Warm for 1 10% aqueous silver nitrate 5ml + conc ammonia dropwise until precipitate dissolves. Dilute to 50 ml with distilled Heated silver solution and check microscopically after 10 Max time should be ml stock silver + 4mls Borax (?%) + 25 ml distilled Stock = 10 ml 5% silver nitrate ml 3% hexae Tap then distilled soln. prewarmed to 60 in a bath Check microscopically after 20 and then at 5-10 intervals upto about 30 until fungal hyphae are brown against a gold background 25 ml stock Methenae + 25 ml distilled + 2ml 5% Borax Stock Methenae solution- Wash 2 changes of distilled Several changes of distilled Distilled Tone 0.2% Gold Chloride 30 sec. 0.02% Gold Chloride 30 sec. 0.2% Gold Chloride 1 Wash Distilled Distilled Distilled Fix 1% Sodium Thiosulphate 2 5% sodium Thiosulphate 5 5% sodium Thiosulphate 1 Wash Distilled Well in running tap then Tap distilled Counterstain 1% Light Green in 1% Acetic acid 5-1% light green 30 sec 0.2% Light green in 0.2% acetic acid 10 sec Wash Water Tap Water Tap Dehydrate DCM Dry in oven and DCM Garry Thompson Page 2 of 7 15/09/2006

3 Run 43 June 2006 Set 1 (See next table for set 2) Centre No Oxidation 5% Chromic acid 1 5% Chromic acid 30 Fresh 5% Chromic acid 1 Fresh 5% Chromic acid 1 Wash Water Water Distilled Running tap Decolourise Wash 1 Tap then several changes of distilled 5 Hexae silver solution at then exae every 3 until fungi black and background clear Stock hexae- 100 ml 3% hexae + 5ml silver nitrate (? %) 2ml fresh 5% sodium Tetraborate + 25 ml distilled + 25 ml stock hexae silver 2% Sodium Metabisulphite Deionised Distilled Tap for 3 then several changes of distilled Fresh Methenae Hexae silver Nitrate preheated to or 8 at 60degrees in the microwave. Check microscopically until fungi is dark brown 12.5 ml fresh 5% sodium Tetraborate + 50 ml 3%hexae silver + 2.5ml 5% silver nitrate (Analar) Methenae silver soln prewarmed to 60 Check microscopically until fungal hyphae are black Stock - 5ml 10% silver nitrate and 3g hexae to 100 ml distilled Working solution- 25ml silver solution + 5ml 5%sodium Tetraborate and add 25 ml distilled Fresh Incubating soln. 10 prewarmed to 60 Check microscopically after 5 then every 1 to 2 do not obscure internal detail of fungi Hexae silver solution- 2 ml 5% Borax + 25 ml 3% Hexae + 23 ml distilled. Heat to 60 then add 1 ml of 10% Nitrate Wash 3 changes of distilled Rinse in deionised Distilled Several changes of distilled Tone 0.1% Gold Chloride 3 2% Gold Chloride 1 0.1% Gold Chloride 2 0.1% Gold Chloride 4 Wash Water Deionised Tap Fix 5% Sodium Thiosulphate 5 5% sodium Thiosulphate 2 5% sodium Thiosulphate 2 3% sodium Thiosulphate 5 Wash Water Water 1 Distilled Tap Counterstain 0.2% Light Green in 0.2% Acetic acid 1 0.2% Masson light green in 0.2% acetic acid 1 Light green 30 sec. 1% Light green in 1% acetic acid + 9ml distilled Wash Water Water Tap Dehydrate DCM 0.2% Light green in 0.2% acetic acid 1-2 Garry Thompson Page 3 of 7 15/09/2006

4 Run 34 November 22 nd 2004 Centre No Oxidation 5% Chromic acid 1 3% Chromic acid 1 hr Fresh 10% Chromic acid (BDH) 10 Wash Running Running Water 5 then Running tap distilled Decolourise 2% Sodium Metabisulphite 1 30 sec briefly Wash Running 3, Wash 2 In warm distilled x 3 30 sec each Freshly mixed Hexae silver solution at Methenae Hexae silver Nitrate preheated to Check microscopically Methenae silver soln. (BDH) Approx.10 At 60 in a prewarmed slide box in a bath. Check microscopically until optimum staining is achieved Wash Distilled Rinse in distilled Distilled Distilled Freshly prepared 5% Chromium trioxide (VWR) 1 Running Water for 10 Methenae silver (VWR and Alfa) at 60 in a bath. Not preheated. Check microscopically at intervals after the first 25 until required results are reached Tone 0.2% Gold Chloride 2 0.2% Gold Chloride 5 0.1% Gold Chloride 10 sec. 0.2% Gold Chloride 3 Wash Running Water Distilled Water Fix 5% Sodium Thiosulphate 1 5% sodium Thiosulphate 1 5% sodium Thiosulphate 3 3% Sodium Thiosulphate 5 Wash Running Wash Distilled Tap Counterstain 0.2% Light Green in 0.2% Acetic acid 1% light green in 1% acetic acid 10 sec. Dilute Mason light green 5-10 sec. 1ml of 2% Light green in 2% acetic acid + 9ml distilled Wash Running Water Tap Lightly in Light green solution fro a few seconds Dehydrate DCM Rapidly Graded alcohols Garry Thompson Page 4 of 7 15/09/2006

5 Run 29 January 19 th 2004 Centre No Oxidation 5% Chromium Trioxide at in a bath for 20 Freshly prepared 5% Chromium Acid (VWR) 1 Wash Tap Running Water for 10 Decolourise Sodium Bisulphite 30 1% Sodium sec Metabisulphite Wash Running tap 20 Tap 3 the sec then thoroughly in several changes of distilled distilled Pre-warmed Fresh Hexae silver at In the dark for upto 60 Check microscopically after 30 until fungi are a very dark black/ brown 1.25 ml of 5% Nitrate to 25 ml of 3% Methenae. Add 2 ml of 5% Borax to ml of the above solution and add 25 ml of distilled Stored solution in fridge Methenae silver (VWR and Alfa) at 60 in a bath. Not preheated. Check microscopically at intervals after the first 25 until required results are reached 5ml of 5% Nitrate to 100 ml of 3% Methenae. A white precipitate forms but this dissolves on shaking Add 3 ml of 5% Borax to 25 ml of the above solution and add 25 ml of distilled 5% Chromic acid 1 5% Chromic acid 1 Water Tap Tap 1% Sodium Metabisulphite 1 Well in Non-preheated silver soln. At Power 3, in microwave oven for 30 sec. Check for fungi. Repeat in 30 sec bursts. Do not boil 5ml of 5% Nitrate to 100 ml of 3% Methenae. A white precipitate forms but this dissolves on shaking Add 2 ml of 5% Borax to 25 ml of the above solution and add 25 ml of distilled 1% Sodium Metabisulphite 1 Tap for 5 then thoroughly in distilled Pre-warmed Hexae silver at 58. Check microscopically after 15 until fungi are a dark brown colour and background is still clear 5ml of 5% Nitrate (BDH) to 100 ml of 3% Methenae. A white precipitate forms but this dissolves on shaking Add 6 ml of 5% Borax to 50 ml of the above solution Fresh 5% Chromic acid (BDH) 1 1% Sodium Metabisulphite 1 Tap for 5 then thoroughly in distilled Pre-warmed Hexae silver at 58. Check microscopically after until fungi are a dark brown colour and background is still clear 1.25 ml of 5% Nitrate (BDH) to 25 ml of 3% Methenae. A white precipitate forms but this dissolves on shaking Add 2 ml of 5% Borax to 25 ml of the above solution and add 25 ml of distilled Stored at 4 Can be stored at 4 Stored at 4 for some months Wash Distilled Distilled Water Well in distilled Rinse x 3 in distilled Tone 0.2% Gold Chloride 30 sec. Do not overtone 0.2% Gold Chloride 3 0.2% Gold Chloride 3 0.2% Gold Chloride 3 0.1% Gold Chloride 5 Wash Distilled Water Water Distilled Distilled Fix 2% Sodium 5% Sodium 5% sodium 5% Hypo 3 2% Sodium Thiosulphate Thiosulphate 1 Thiosulphate 5 Thiosulphate 5 5 Wash Distilled Tap Well in Tap thoroughly Distilled Counterstain 0.1% Light green (BDH) 45 sec. Do not over counterstain Lightly in 0.2% Light green solution in 0.2% acetic acid fro a few seconds 0.1% Light green (BDH) 30 sec Wash Tap Water rinse Dehydrate Graded alcohols Graded alcohols Absolute Alcohol Alcohol Arzac s or Neutral Red 0.2% light green in 0.2% acetic acid 10 sec Garry Thompson Page 5 of 7 15/09/2006

6 Run 19 June 3 rd 2002 UK NEQAS CPT Best Methods Handbook Grocott Edition 2 Centre No Oxidation 5% Chromic acid 1 hr 5% Chromium VI Oxide 1 5% Chromium trioxide 1 5% Chromic acid (BDH) 45 Wash Water 2 Running 30 sec. Water for 10 Tap Decolourise 2% Sodium Metabisulphite 30 sec 1 Wash Wash 2 Running 3 then several changes of DIST/ DI/ RO Hexae silver preheated Pre-warmed silver soln. At to in the dark Check microscopically after 10 Check at 3 intervals. Cell walls should be black and internal structures brown Wash Rinse in distilled Several changes of DIST/ DI/ RO Tone 0.1% tetra-chloroauric acid 4 1 Water for 5 then thoroughly in distilled Pre-warmed silver at 60. Use two Coplin jars and transfer to fresh silver when silver deposits on first Coplin jar. Check microscopically until fungal hyphae are black/ brown Thoroughly in distilled Gold Chloride until grey/ gold in colour Wash Rinse in DIST/ DI/ RO De-ionised Tap 2 Thoroughly in distilled solution (BDH Analar) at 56 and check every 5 Remove when straw coloured. Rinse in distilled 0.2% Gold Chloride 2 Fix 5% Hypo 3 3-5% sodium Thiosulphate 2% Hypo 2 2% Thiosulphate 5 Wash Wash 2 Running 3 Tap for a few utes Tap Counterstain 0.04% light green in 0.04% 0.04% acidified Light green 0.2% acidified Light green Lightly in 1% light green (BDH acetic acid 5 20 sec. CI40295) 1 Wash Rinse Tap Distilled Dehydrate DCM Rapidly Garry Thompson Page 6 of 7 15/09/2006

7 Run 11 January 29 th 2001 UK NEQAS CPT Best Methods Handbook Grocott Edition 2 Centre No. GT Oxidation 5% Chromic acid 45 Wash Tap Well in Tap the 3 times in distilled Decolourise Wash 1% sodium Metabisulphite 2 Distilled thoroughly Hexae, 56, check every 5 and remove when straw coloured, BDH Analar 5% Chromic acid 1 hr 5% Chromic acid 1 hr 5% Chromic acid in microwave on LOW for 5 Hexae, 60 preheated in the dark BDH analar reagents made up fresh Wash Distilled Well in de-ionised Fix? Running tap 1% sodium Metabisulphite 1 Running tap 5 then 3 changes of de-ionised Pre-warmed Ammoniacal silver nitrate at 60 for 40, BDH Analar reagents 3x de-ionised 2% sodium thiosulphate 2 Dip in 0.2% gold chloride Running tap then distilled Methenae in microwave on LOW for 6 20 sec. Check and repeat at 5 sec intervals upto 7 max Quickly in distilled, no delay in silver Tone 0.2% Gold Chloride 2 0.2% gold chloride 2 0.2% Gold chloride 1 Wash Tap Water 3x de-ionised Distilled Water 5% Chromic acid 1 hr Water 1% sodium bisulphite 1 Water 5-10 then several changes of distilled Methenae at 56 for Check micro every 3 until fungi are black 3x distilled 0.1% gold chloride 3 Fix 2% sodium 5% sodium 2% sodium 5% sodium 5% hypo 5 thiosulphate 1 thiosulphate 3 thiosulphate 2 thiosulphate 5 Wash Tap Running tap Distilled Water Counterstain 1% aqueous light 0.2% light green % light green 1% light green in 1% 0.1% light green 10 green sec acetic acid sec Wash Distilled Briefly in tap Tap Water Dehydrate Blot 95% alcohol Garry Thompson Page 7 of 7 15/09/2006