giemsa stain
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Transcript of giemsa stain
2. Staining blood films with Giemsa stain
Swiss Tropical Institute, Basel
April 2005
Methods in Parasitology
2.0 Staining blood films with Giemsa
Contents• 2.1 Materials• 2.4 Fixation of thin films• 2.5 Preparing Giemsa stain• 2.10 Staining blood films
Production Team:Yvette Endriss, Elisabeth Escher & Birgit Rohr Prof. Hanspeter Rohr (NeoCortex Foundation)Prof. Niklaus Weiss (STI)
For perfect malaria staining, thin and thick films should be made on separate slides. The pH of the buffer solution should be 7.2 .
Thin films will be fixed with Methanol.
Thick films should not be fixed (to allow haemolysis)!
Therefore avoid exposure of the thick films to methanol vapour when thin and thick films are on the same slide.
2.1 Staining blood films with Giemsa
2.2 Staining blood films with Giemsa
Materials:
• Methanol (or Ethanol)• Staining dishes• Buffer solution• Giemsa solution
(Giemsa powder can alsobe used instead of Giemsasolution)
2.3 Staining blood films with Giemsa
Fixation for thin films only!
2.4 Staining blood films with Giemsa
Fixation time for thin films with methanol:
15 – 30 seconds
Important note:Fixation time for ethanolis 20 minutes!
2.5 Staining blood films with Giemsa
For perfect malaria staining the pH should be correct!(pH 7.2)
Commercially availablebuffer tablets can also be used.
2.6 Staining blood films with Giemsa
Prepare your buffer by usingtablets of Soerensen (see next slide) Combine two stock solutions in a certain proportion
KH2PO4 and
Na2HPO4 · 2 H2O
Control the pH before use!
2.7 Staining blood films with Giemsa
Buffer by Soerensen
84.115.97,5Trichomonas
82.517.57,45Trypanosoma
80.819.27,4
76.823.27,3
72.028.07,2Malaria / Babesia
66.633.47,1
61.138.97,0Normal blood/Leishmania
55.444.66,9
49.650.46,8Borrelia
43.556.56,7
37.562.56,6
31.868.26,5
26.773.36,4
ml solution Bml solution ApH20°CParasite
Stock solutions:A: KH2PO4 (Merck Art. 4873) 9.08 g/l = 1/15 molarB: Na2HPO4 2 H2O (Merck Art. 6580) 11,87 g/l = 1/15 molar
(or Na2HPO4 anhydricum (Merck Art. 6586) 9.47 g/l = 1/15 molar)
2.8 Staining blood films with Giemsa
Prepare staining solution:
Take 94 ml buffer solutionpH 7.2
Then add6 ml Giemsa stocksolution
2.9 Staining blood films with Giemsa
Mix 94 ml buffer solutionph 7,2 with6 ml Giemsa stock solution>>
6% Giemsa solution
2.10 Staining blood films with Giemsa
Fill staining dish withstaining solution
Place thin film and thickfilms into the stainingdish.
Stain blood slides for45 minutes
(staining a large number of thinfilms and thick films use twoseparate staining dishes)
2.11 Staining blood films with Giemsa
Thin films:Wash the slides withtap water
2.12 Staining blood films with Giemsa
Caution:Thick films need careful rinsing!(since they are not fixedbefore staining)
Mistake:Thick films are notrinsed properly! Blood is lost!
2.13 Staining blood films with Giemsa
Drying
For example:• Let air dry• Use small incubator 37°C• Use heating plate (not above
37°C)
(do not use excessiveheat!)