target="_blank" rel="nofollow" href="#ulink_7690b0b6-8c30-5062-89b6-5e4feeaad401">12) has recommended the addition of 1% (by volume) 2,6-di-t-butyl-p-cresol (butylated hydroxytoluene [Sigma Chemical Co. catalog no. B1253]) to the mounting medium. Without the addition of this antioxidant, mounted stained smears eventually become pink; stains protected with this compound remain unchanged in color for many years.
Note Any slide that is protected by a coverglass and is going to be examined with an oil immersion lens must be covered by a no. 1 coverglass. If a no. 2 coverglass is used, the extra thickness may prevent the oil immersion lens from focusing properly.
Giemsa stain is sold as a concentrated stock solution. Each new lot should be tested for optimal staining times before being used on patient specimens. If the blood cells appear to be adequately stained, the timing and stain dilution should be appropriate to demonstrate the presence of malarial and other parasites. Giemsa stain is also available as a powder for those who wish to make up their own stain. The use of prepared liquid stain or stain prepared from the powder depends on personal preference; there is apparently little difference between the two preparations. Directions for preparing the stain follow (4, 5, 9).
Note Quality control for blood film stains can be any negative or positive blood film; it is not necessary for the control slides to contain actual parasites. The actual patient slide being stained serves as its own control; if the WBCs look acceptable, any parasites present will also be acceptable in terms of morphology and staining colors. If the RBCs and WBCs stain correctly, any parasites present would also stain correctly with the same nuclear and cytoplasmic colors as the blood cells (see Fig. 7.4 to 7.8).
Reagents
Stock Giemsa Stain
1. Grind together small portions of stain and glycerin in a mortar, and collect mixtures in a 500- or 1,000-ml flask until all measured material is mixed.
2. Stopper the flask with a cotton plug, cover the plug with heavy paper, and place the flask in a 55 to 60°C water bath for 2 h. Make sure the water in the water bath is above the level of the stain. Shake gently at 30-min intervals.
3. After grinding the powder and glycerin in the mortar, use the 50 ml of methyl alcohol to wash the last bit of stain from the mortar; then pour the alcohol into a small, airtight bottle.
4. Remove the glycerin-stain powder mixture from the water bath, and allow it to cool to room temperature. Add alcohol washing from the mortar, and shake well.
5. Before use, filter through Whatman no. 1 paper into a brown bottle. Although the stain can be used immediately, it is better to let it stand for 2 to 3 weeks with intermittent shaking.
6. Label and store protected from light; the shelf life is 36 months, provided that results are within quality control guidelines.
Note The stock stain is stable for many years; however, it must be protected from moisture. The staining reaction is oxidative; any oxygen in water will initiate the staining reaction and destroy the stock stain. This is why the aqueous working solution of stock stain is good only for 1 day.
10% Stock Solution of Triton X-100
Mix thoroughly, and store at room temperature. This solution will keep indefinitely if kept tightly stoppered.
Stock Buffers
Disodium Phosphate (Dibasic)
Monosodium Phosphate (Monobasic)
Phosphate-Buffered Water (Table 7.6)
Triton-Phosphate Buffer
0.01% Triton-Buffered Water Stock
0.1% Triton-Buffered Water
For thin blood films or a combination of thin and thick blood films, use 0.01% Triton-buffered water; for thick blood films, use 0.1% Triton-buffered water.
Liquid
The commercial liquid stain or the stock solution prepared from powder should be diluted approximately the same amount to prepare the working stain solution. Stock Giemsa liquid stain is diluted 1:10 with buffer for thin blood films; for thick films, a dilution of up to 1:50 may be used. Some people prefer to stain both thick and thin smears for a longer period in a more dilute solution. Phosphate buffer used in dilution of the stock stain should be neutral or slightly alkaline. Phosphate buffer solution may be used to obtain the right pH (Table 7.6). In some laboratories, tap water has a satisfactory pH and may be used for the entire staining procedure and the final rinse. Some workers recommend using pH 6.8 to emphasize Schüffner’s dots.
Procedure for Staining Thin Films
1. Fix blood films in absolute methyl alcohol (acetone free) for 1 min.
2. Allow the slides to air dry.
3. Immerse the slides in a solution of 1 part Giemsa stock (commercial liquid stain or stock prepared from powder) to 10 to 50 parts phosphate buffer (pH 7.0 to 7.2). Stain for 10 to 60 min. Fresh working stain should be prepared from stock solution each day.
Note A good general rule for stain dilution versus staining time is as follows. If the dilution is 1:20, stain for 20 min; if the dilution is 1:30, stain for 30 min, etc. However, a series of stain dilutions and staining times should be tried to determine the best dilution and time for each batch of stock stain.
4. Dip the slides briefly in phosphate-buffered water, or rinse under gently running tap water.
Note Excessive washing will decolorize the film.
5. Drain thoroughly in a vertical position, and allow to air dry. The bottom of the slide should be wiped to remove excess stain before drying.
Procedure for Staining Thick Films
The procedure to be followed for thick films is the same as for thin films, except that the first two steps are omitted. If the slide has a thick film at one end and a thin film at the other, fix only the thin portion and then stain both parts of the film simultaneously. Normally, a stain/buffer dilution of 1:50 (vol/vol) for a staining time of 50 min is recommended for thick films. The longer staining time seems to
