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

ml solution A

ml solution B

6,4

73.3

26.7

6,5

68.2

31.8

6,6

62.5

37.5

6,7

56.5

43.5

6,8

50.4

49.6

6,9

44.6

55.4

7,0

38.9

61.1

7,1

33.4

66.6

7,2

28.0

72.0

7,3

23.2

76.8

7,4

19.2

80.8

Trypanosoma

7,45

17.5

82.5

Trichomonas

7,5

15.9

84.1

Buffer by Soerensen
Borrelia

Normal blood/Leishmania

Malaria / Babesia

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!)