b. Giemsa (Triton X-100) Technique for Staining Blood Films. Giemsa stain,
modified by the addition of polyethylene glycol monoisooctyl phenol ether (Triton X-
100), is one of the best methods for demonstrating malaria, trypanosomes, and filarial
parasites in blood films. Giemsa stains the organism deeply and such preparations
resist fading for long periods. The addition of Triton X-100 reduces transfer of parasites
from slide to slide during mass staining and enhances the parasite staining properties of
the Giemsa stain.
(1) Reagents. Use the Azure B type Giemsa stain certified by the
Commission for the Standardization of Biological Stain.
(a) Stock Giemsa Stain. Preparation of this stain requires the following
Giemsa Powder CP................................ 0.6 gm
Glycerine (Neutral) CP (C3H5(OH)3) ....... 50.0 ml
Methanol, Absolute (Acetone-Free) ....... 50.0 ml
Use only chemically clean, dry glassware to prepare this stain.
STEP 1: Weight out 0.6 grams of Giemsa stain (dry powder) and place a small amount
of stain in a dry mortar.
STEP 2: Measure out 50 milliliters of glycerin in a cylinder and add a small amount of
glycerin to the mortar. Thoroughly grind the stain and glycerin together. Pour
off into a flask.
STEP 3: Repeat the addition of stain and glycerin with grinding until all the stain has
been mixed with glycerin. Rinse the mortar and pestle with the remaining
glycerin and pour this into the flask.
STEP 4: Measure out 50 milliliters of absolute methyl alcohol. Pour some of this into
the mortar and rinse the mortar and pestle with a portion of this. Pour the
washings into a separate bottle and stopper tightly.
STEP 5: Place the glycerin-dye mixture in a 55to 60 C waterbath for six to eight hours
and shake periodically.
STEP 6: Remove the container of glycerin-dye mixture from the waterbath. Cool and
add the washing from the mortar and pestle and the remainder of the alcohol
to the glycerin-dye mixture. Shake the resulting solution well and stopper the