Methods in Parasitology

2. Staining blood films with Giemsa stain

Swiss Tropical Institute, Basel April 2005

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)

2.1 Staining blood films with Giemsa

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.

2.2 Staining blood films with Giemsa

Materials:

Methanol (or Ethanol) Staining dishes Buffer solution Giemsa solution

(Giemsa powder can also be used instead of Giemsa solution)

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 ethanol is 20 minutes!

2.5 Staining blood films with Giemsa

For perfect malaria staining the pH should be correct! (pH 7.2) Commercially available buffer tablets can also be used.

2.6 Staining blood films with Giemsa

Prepare your buffer by using tablets 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

Stock solutions: A: KH2PO4 (Merck Art. 4873) 9.08 g/l = 1/15 molar B: 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)
Parasite pH20C 6,4 6,5 6,6 6,7 Borrelia 6,8 6,9 Normal blood/Leishmania 7,0 7,1 Malaria / Babesia 7,2 7,3 7,4 Trypanosoma Trichomonas 7,45 7,5 ml solution A 73.3 68.2 62.5 56.5 50.4 44.6 38.9 33.4 28.0 23.2 19.2 17.5 15.9 ml solution B 26.7 31.8 37.5 43.5 49.6 55.4 61.1 66.6 72.0 76.8 80.8 82.5 84.1

Buffer by Soerensen

2.8 Staining blood films with Giemsa

Prepare staining solution: Take 94 ml buffer solution pH 7.2 Then add 6 ml Giemsa stock solution

2.9 Staining blood films with Giemsa

Mix 94 ml buffer solution ph 7,2 with 6 ml Giemsa stock solution >> 6% Giemsa solution

2.10 Staining blood films with Giemsa

Fill staining dish with staining solution

Place thin film and thick films into the staining dish. Stain blood slides for 45 minutes
(staining a large number of thin films and thick films use two separate staining dishes)

2.11 Staining blood films with Giemsa

Thin films: Wash the slides with tap water

2.12 Staining blood films with Giemsa

Caution: Thick films need careful rinsing! (since they are not fixed before staining) Mistake: Thick films are not rinsed properly! Blood is lost!

2.13 Staining blood films with Giemsa

Drying For example:

Let air dry Use small incubator 37C Use heating plate (not above
37C)

(do not use excessive heat!)