Chromosome Science 19: 57-66, 2016 Hirotami T.

IMAI 57

Technical Note

A manual for ant chromosome preparations (an improved air-drying method) and
Giemsa staining

Hirotami T. IMAI

Received: September 29, 2016 / Accepted: Februrary 15, 2017

© 2016 by the Society of Chromosome Research

A. CHROMOSOME PREPARATION II. APPARATUS (Fig. 1)

(1) A transmitted stereomicroscope (x20-x40) for dissecting
I. CHEMICALS
out organs (Fig. 1a).
(1) Colchicine stock solution (1mg/ml, 0.1% solution) (2) A pair of dissecting needles (Fig. 1b).
(a) 50 mg colchicine (3) Four Pasteur pipettes each with a rubber nipple (Fig.
(b) 50 ml distilled water (D.W.) 1c).
* Store in a brown bottle and keep in a refrigerator. (4) Centrifugation tube (Spits tube) (Fig. 1d).
(2) Hypotonic solution (1% Sodium citrate solution) (5) Depression slides (hole slides) (Fig. 1e).
(a) 1 g Trisodium citrate dihydrate (6) Slide glasses (Fig. 1f).
(b) 100 ml D.W. * Store slides in 80% ethanol and dry with washed clean
(3) Colchicine-hypotonic solution (* freshly prepared) gauze as necessary.
10 ml Hypotonic solution with 0.005 % colchicine * If slides are oily, add a drop of fixative I on the slide,
(a) 0.5 ml Colchicine stock solution and clear with paper or washed clean gauze.
(b) 9.5 ml Hypotonic solution (7) Test tube stand (Fig. 1g).
(4) Fixative I (* freshly prepared) (8) Filter paper (blotting paper) (Fig. 1h).
60% 1:1 Acetic-ethanol (9) Rolled filter paper or paper towel (Fig. 1i).
(a) Glacial acetic acid 3 ml (10) Fingerstalls for the thumb and the forefinger (Fig. 1j).
(b) Ethanol (>99.5%) 3 ml * To protect fingers from fixative.
(c) D.W. 4 ml * Cut the tip of fingers of a vinyl glove for right hand.
* Don’t store Fixative I overnight, because it turns rapidly (11) Washed cotton gauze or tissue paper (Fig. 1k).
into ethyl acetate, which is deleterious to mitotic cells. (12) A glass engraving pen for label (Fig. 1l).
* Don’t use old glacial acetic acid and ethanol that are (13) Forceps to open cocoons (Fig. 1m).
near the bottom of a stock bottle or that have been
left for several months. Both chemicals tend to absorb III. SUITABLE ORGANS AND DEVELOPMENTAL STAGES
moisture from the air and chromosomes will be damaged FOR OBSERVING ANT CHROMOSOMES (Fig. 2)
by the contamination of water.
(5) Fixative II (* freshly prepared)
4 ml Absolute 1:1 Acetic-ethanol IV. DISSECTION OF ORGANS (Fig. 3)
(a) Glacial acetic acid 2 ml Dissect out subject organs (brain Fig. 3c, testis or ovary
(b) Ethanol (>99.5%) 2 ml Fig. 3d) in colchicine hypotonic-solution on a depression
(6) Fixative III slide (Fig. 3a) using dissecting needles under a transmitted
2ml Glacial acetic acid (GAA) stereomicroscopy (Fig. 3b).
● Remove as much as possible: fat body, tracheae and

epithelial membranes (especially for brain) (Fig. 3c).

● Use dissecting needles as shown in Fig. 4.

V. A SHORT-TIME TREATMENT OF ORGANS WITH

Hirotami T. Imai (*)
Oiwake1714-3, karuizawa, Nagano-ken, 389-0115, Japan
(Formerly at National Institute of Genetics, Mishima, Shizuoka-ken,
411-8540, Japan)
E-mail: chromohimai@nifty.com
This method is based on Imai 1966, Crozier 1968, Imai et al. 1977 and
1988. Chromosomes prepared with this method can be stained not
only by the conventional Giemsa staining but also FISH (fluorescence
in situ hybridization), e.g. , see Meyne et al. 1995 and Hirai et al. 1996.
COLCHICINE-HYPOTONIC SOLUTION
Transfer the dissected organs to another depression slide
filled with fresh colchicine-hypotonic solution using a
Pasteur pipette (Fig. 5a).
Leave to stand for 20 mines (max. 1 hr) at room
temperature (Fig.5b), by which organs are swollen and the
frequency of mitotic metaphases increase.
● Note that mitotic chromosomes will contract remarkably
by more than 1 hr treatment in the hypotonic solution.
● Use Pasteur pipettes skillfully. Be sure to aspirate the
58 A manual for ant chromosome preparations (an improved air-drying method) and Giemsa staining

Figure 1. Apparatuses used for observing chromosomes of ants. See text for explanation of (a)-(m). (n): Apparatuses at a glance.
 Hirotami T. IMAI 59

Figure 2. Suitable organs and developmental stages for observing ant

chromosomes. The figures were modified from Jpn. J. Genet. and Acta Hy-
menopterologica with permission.

Figure 3. Dissection of organs. The figures were modified from Acta

Hymenopterologica with permission.
60 A manual for ant chromosome preparations (an improved air-drying method) and Giemsa staining
organ after allowing an air bubble to enter (Fig. 5a).
Otherwise, the organ sticks to the wall of the pipette and
becomes irrecoverable.
VI. SLIDE MAKING BY AN IMPROVED AIR-DRYING
METHOD
(1) Transfer the material onto a pre-cleaned plain slide
using Pasteur pipette (Fig. 6, Fig. 7).
(2) Tilt the slide 10-20°, to drain off most of the hypotonic
solution (Fig. 8).
(3) Draw off as much as possible of the hypotonic solution
around the organs by using a dissecting needle (Fig. 9).
(4) Tilt the slide 10-20° again; apply several drops of freshly
prepared fixative I (60 % 1:1) on the organ and also over
the surface of the slide, except where it is being held (Fig.
10).
● The fixative should flow over the preparation and drain
off the bottom edge of the slide.
● Repeat this procedure two to three times until the flow of
the hypotonic solution stops.
● The hypotonic solution remaining in the organ is now
completely substituted with Fixative I (Fig. 11).
(5) Remove excessive fixative from the end of the slide
using filter paper or paper towel (Fig. 12).
● Stand the slide vertically (100-120°) on filter paper and
remove fixative.
● This procedure is done more effectively by stroking the
tip of the dissecting needle along the edge of the slide.
(6) Wipe the underside of the slide with tissue paper (Fig.
13), and keep the slide horizontally on the stage of the
stereomicroscope.
(7) Apply 2 or 3 drops of fixative I directory onto the organ
(Fig. 14).
(8) After 15-30 sec, macerate organs as quickly as possible
using dissecting needles (Fig. 15) to enable cells and cell-
mass to spread outwards up to 1-1.5 cm in diameter (Fig.
16).
(9) Most of fixative I spreads out gradually to the edge
of the slide, but partly remains around the cell cluster by
surface tension (Fig. 17).
(10) After 1 or 2 min, fixative I evaporates gradually and
becomes a thin layer in the area of cell spreading (Fig. 18).
The spherical cells floating in the fixative adhere to the slide
Figure 4. Using needles for dissection.
(Fig. 19) and become flat by surface tension (Fig. 20).
● At this stage, the spreading cells are detectable as small
spots under reflected light (Fig. 21).
● A pencil flashlight can be used, but a fluorescent lamp is
inappropriate.
● If it is 20°C and 65-70 % in relative humidity, the speed of
the evaporation of fixative and that of the cells becoming
flat by surface tension will be balanced, and beautiful
spreading of metaphase chromosomes is achieved (Fig.
22).
● If the humidity is lower than 50 %, the fixative evaporates
before the cells become flat, and the chromosomes pile
up (Fig. 23).
● If it is warmer than 25°C and more than 80 % in
humidity, fixative I once spread out will retract again
to the centre of the slide, making the chromosome
preparation difficult to make (Fig. 24).
● This problem can be solved partly by warming the slide
to body temperature, e.g., put it on your arm (Fig. 25).
But I recommend to control for optimal temperature and
humidity conditions.
● As the fixative is attracted to the cells or cell-mass by
surface tension, the fixative first evaporates completely
from the inter cell-mass space, which is detectable as
breakage of the thin membrane of the fixative (Fig. 26).
(11) When the broken fixative membrane covers about half
of the area of spreading cells (Fig. 26), add 2 or 3 drops of
freshly prepared fixative II (Fig. 27). So that fixative I (60%
1:1) moves to the periphery of the slide immediately (Fig.
28), and becomes replaced by fixative II (absolute 1:1).
● The timing to apply fixative II is the most important
factor to obtaining good metaphase spread, especially to
obtain a good C-banded chromosome appearance.
● If fixative II was dropped before the breakage of the
fixative membrane, the chromosomes will become fluffy.
● If fixative II is added too late, cells will have dried
out before spreading, and the chromosomes will be
contracted and piled up as showed in Fig. 23.
(12) Remove fixative I accumulating at the periphery of the
slide with rolled filter paper or paper towel (Fig. 29) and
wait 2-3 mins.
(13) When a patchy appearance of the broken thin
membrane of fixative II (1:1) covers more than half of the
cell spreading area, apply 2 or 3 drops of fixative III (glacial
acetic acid, GAA) to the centre of the spreading cells as
shown in Fig. 30.
● Fixative III (GAA) displaces fixative II (1:1) outward as
shown in Fig. 31.
● Fixative III is effective to remove cytoplasm and clean up
the background of metaphase spreads.
● If we use brains at the late prepupal stage covered by
trachea, interphase nuclei spreading on the slide contain
many granules and the background becomes very dirty.
(14) Remove fixative II (1:1) with rolled filter paper (Fig.
32) again, and allow the slide to dry completely (Fig. 33).
● Put mark date and preparation numbers with a glass
engraving pen (Fig. 34).
● The dried preparations are available for staining for more
than 10 years, if they are stocked in a dry and dark place.
However, the best condition for staining is within one
week.
Chromosomes of Myrmecia croslandi are shown in Figs.
35 and 36.
 Hirotami T. IMAI 61

Figure 5. A short-time treatment of organs with colchicine-hypotonic solution. See text for details. The figures were
modified from Acta Hymenopterologica with permission.
62 A manual for ant chromosome preparations (an improved air-drying method) and Giemsa staining
 Hirotami T. IMAI 63

Figure 6-34. See text for details. The figures were modified from Acta Hymenopterologica with permission.
64 A manual for ant chromosome preparations (an improved air-drying method) and Giemsa staining

Figure 35. Mitotic metaphase chromosomes of Myrmecia croslandi.

Figure 36. Polymorphic karyotypes of Myrmecia croslandi.

 Hirotami T. IMAI 65

B. GIEMSA STAINING in the ant Myrmecia (pilosula) n = 1. Chromosoma 98: 456-460.

Imai HT, Hirai H, Satta Y, Shiroishi T, Yamada M and Taylor RW (1991)
I. CHEMICALS (Fig. 37) Phase specific Ag-staining of nucleolar organizer regions (NORs)
(1) Giemsa’s stock solution (Merck; 100 ml) and kinetochores in the Australian ant Myrmecia croslandi. Jpn J
(2) 3 % Giemsa’s working solution Genet 67: 437-447.
Imai HT, Taylor RW, Crosland MWJ and Crozier RH (1988) Modes of
Phos. Buf. 50 ml :
spontaneous chromosomal mutation and karyotype evolution in
Giemsa S. Sol. 1.5 ml
ants with reference to the minimum interaction hypothesis. Jpn J
(3) Sorensen’s Phosphate buffer Genet 63: 159-185.
M/15 (pH 6.8) Imai HT, Taylor RW and Crozier RH (1994) Experimental bases for
(a) Na2HPO4 …… 4.75 g the minimum interaction theory. I. Chromosome evolution in ants
(b) KH2PO4 ….… 4.50 g of the Myrmecia pilosula species complex (Hymenoptera: Formici-
(c) D. W. ……… 1,000 ml dae: Myrmeciinae). Jpn J Genet 69: 137-182.
Meyne J, Hirai H and Imai HT (1995) FISH analysis of the telomere
II. APPARATUS FOR CHROMOSOME STAINING sequences of bulldog ants (Myrmecia: Formicidae). Chromosoma
(Fig. 38) 104: 14-18.

(1) 2 l Beaker
(2) Staining bottle
(3) Spatula
(4) Forceps
(5) Micropipette (1 ml)
(6) Pasteur pipette
(7) Slide holder
(8) Filter paper or paper towel

III. PROCEDURE OF GIEMSA STAINING

(1) After dr ying slides for at least 1 day at room
temperature, stain preparations using freshly prepared
Giemsa working solution (3 % in Phos. Buff.) for 10 mins
at room temperature (Figs. 39 and 40).
● If slides are several months or years old, 30 min to 1 hr

is required for staining. Chromosome spread prepared

between 1 day ~ 1 week old is ideal.
● After churning (bubbling) Giemsa’s working solution

with a Pasteur pipette, remove bubbles with a spatula,

and then remove dirty membrane matter of Giemsa
sediment floating on the surface with filter paper or
paper towel.
● Dip slides quickly into the staining bottle before the

surface is covered by dirty membrane again.

(2) After 10 min staining, each slide is rinsed by a single
passage through a 2 l beaker irrigated with running tap-
water (strong flow with air bubbles), and drained standing
against a vertical surface or a slide holder if available (Fig.
41).
● Before observing the chromosomes, clean (remove

Giemsa sediment) the back of the slide with tissue paper.

References
Crozier RH (1968) An acetic acid dissociation, air-drying technique
for insect chromosomes, with acetolactic orcein staining. Stain
Technology 43: 171-173.
Hirai H, Yamamoto M-T, Ogura K, Satta Y, Yamada M, Taylor RW and
Imai HT (1994) Multiplication of 28S rDNA and NOR activity in
chromosome evolution among ants of the Myrmecia pilosula spe-
cies complex. Chromosoma 103: 171-178.
Hirai H, Yamamoto M-T, Taylor RW and Imai HT (1996) Genomic
dispersion of 28S rDNA during karyotypic evolution in the ant ge-
nus Myrmecia (Formicidae). Chromosoma 105: 190-196.
Imai HT (1966) The chromosome observation techniques of ants and
the chromosomes of Formicinae and Myrmicinae. Acta Hymenop-
terologica 2(3): 119-131.
Imai HT, Crozier RH and Taylor RW (1977) Karyotype evolution in
Australian ants. Chromosoma 59: 341-393.
Imai HT and Taylor RW (1989) Chromosomal polymorphisms involv-
ing telomere fusion, centromeric inactivation and centromere shift
66 A manual for ant chromosome preparations (an improved air-drying method) and Giemsa staining

Figure 37-41 See text for details.