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!)