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Giemsa-staining method

Depertment of Genetics

3. Selection of metaphases

In Giemsa staining, we use a green or yellow filter that provides the best contrast for observing by eye and photography.

When we select the metaphases, we first use low-magnification lenses. There is one basic rule, specifically, not all metaphases that are apparently suitable under low magnification are actually suitable for analysis. (Low magnification implies 10 (eye lens) × 10 (objective lens) = ×100 or 10 × 20 = ×200 magnification; high magnification implies 10 (or 15) × 100 (oil objective or dry lens) = ×1000 (or 1500). Whenever the metaphases selected under low-power magnification are suspicious, moderate magnification, such as ×400 (10 × 40), is recommended for confirmation.

The conditions of metaphases suitable for analysis are as follows:
(1) The 46 chromosomes distribute evenly on the slide in a circular or slightly elliptical shape.
(2) Ideally, chromosomes should not overlap, although some overlap is allowed.
(3) Centromeres should be observed in all chromosomes. When chromosomes overlap, centromeric portions should not overlap.
(4) Chromosomes should be stained evenly. In other words, all the chromosomes should have the same staining density. A chromosome on the edge of the microscopic field with darker or lighter staining should be excluded. This is due to a torsional stress during the air-drying process that may cause differences in chromosome length even between homologous chromosomes. Generally, lighter (darker) staining results in longer (shorter) chromosomes.
(5) The total number of chromosomes should be 46, but chromosomes are difficult to count under low magnification. The possible variation should be 46 ± 1.
(6) Sister chromatids comprising each chromosome should be clearly seen.
(7) Avoid metaphases with heavily condensed chromosomes (too much effect of colcemid) or with visible chromatin spiral. In both cases, centromere positions are not clearly seen.

Of these seven conditions, only (1) may be met under low magnification; the remaining conditions should be met primarily under moderate to high magnification. Let us now consider the examples in Figures 1 to 3. All of these metaphase pictures were taken under ×1000 (10 × 100) magnification.

Figure 1
Figures 1 A to D represent good metaphases for analyses. Chromosomes in Figure 1A are well spread with no overlaps. In contrast, chromosomes in Figures 1B and 1D tend to be shorter, which indicates that the cells are at a late stage of metaphase and that the effect of colcemid was slightly too strong. Chromosomes in Figure 1C also appear good, but, as indicated by the dotted arrow, two chromosomes are attached end-to-end and appear as if they constitute one abnormal chromosome (a dicentric chromosome).

Figure 2
Examples of unsuitable metaphases are shown in Figures 2 and 3. In Figure 2A, there are too many overlapping chromosomes including centromere regions, so it is impossible to identify centromere positions and, hence, karyotyping cannot be achieved. Chromosomes in Figure 2B are not in metaphase but rather in prophase, and individual chromosomes cannot be identified. Figures 2C and 2D represent chromosomes that are too condensed so that centromere positions are not clear. Some chromosomes in Figure 2D are folded, and their lengths cannot be properly measured.

Figure 3
Figure 3A represents two metaphases located side by side. In this picture, the borderline between the two cells can be easily seen because the stage of chromosome condensation is different (dotted line). However, one should keep in mind that two daughter cells may occasionally begin the second mitosis simultaneously when located side by side on the slide. In such cases, conditions of chromosome condensation are very similar in both cells. Hence, the borderline between the two cells cannot be seen clearly. Under such circumstances, the two cells may exchange some chromosome, and one may easily score aberrations that are artifacts.

Figure 3B represents an example of different condensation among chromosomes in one cell. Chromosomes on the left are darkly stained whereas the staining becomes lighter toward the right. Such a metaphase should be avoided as the extended chromosomes may be interpreted as aberrations.

The metaphases selected under low magnification will be used for karyotyping under high magnification. The procedures are as follows.
(1) Score the number of chromosomes. In the beginning, you may use a counter for this purpose, but you will soon be able to count 46 chromosomes in your mind. The total number of chromosomes must be 45, 46, or 47.
(2) Examine the shape and length of each chromosome (karyotyping). Usually, we take microscopic photographs at 2000- to 3000-fold magnification, cut out each chromosome, and arrange the chromosomes in order with their homologues to find an abnormality.
(3) As the procedure is very time consuming, we need to train ourselves to examine chromosomes under the microscope. If you can achieve it, you will no longer need to take photographs of normal cells and cut out each chromosome but only take photographs of metaphases with suspected or definitive abnormalities. However, it is very important to take photographs of any metaphases with suspected aberrations.