3. Selection of metaphases
| In Giemsa staining, we use a green or yellow
filter that provides the best contrast for observing by eye and
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:
||The 46 chromosomes distribute evenly on the
slide in a circular or slightly elliptical shape.
||Ideally, chromosomes should not overlap, although some overlap
||Centromeres should be observed in all chromosomes. When
chromosomes overlap, centromeric portions should not overlap.
||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.
||The total number of chromosomes should be 46, but chromosomes
are difficult to count under low magnification. The possible
variation should be 46 ± 1.
||Sister chromatids comprising each chromosome should be clearly
||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.
||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
||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 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
The metaphases selected under low magnification will be used for
karyotyping under high magnification. The procedures are as follows.
||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.
||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.
||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.