304 Preliminary notes
11. Reimann, E M, Walsh, D A & Krebs, E G, J
biol them 246 (1971) 1986.
12. Lowry, 0 H, Rosebrough, N J, Farr, A L &
Randall, R J, J biol them 193 (1951) 265.
13. Reddi, A H, Ewing, L L & Williams-Ashman,
H G, Biochem j 122 (1971) 333.
14. Kuo, J F, Wyatt, G R & Greengard, P, J biol
them 246 (1971) 7159.
15. Kuo. J F & Greeneard. P. J biol them 245 (1970)
- II ~ I
2493:
16. Gray, J P, Hardman, J G, Bibring, T & Suther-
land, E W, Fed proc 29 (1970) 608.
17. Castaiieda, M & Tyler, A, Biochem biophys res
commun 33 (1968) 782.
18. Gray, J P, Hardman, J G, Hammer, J L, Hoos,
R T & Sutherland, E W, Fed proc 30 (1971) 1267.
19. Hardman, J G, Beavo, J A, Gray, J P, Chrisman,
T D, Patterson, W D & Sutherland, E W, Ann
NY acad sci 185 (1971) 27.
Received August 8, 1972
A simple technique for demonstrating
centromeric heterochromatin
A. T. SUMNER, Medical Research Council, Clinical
and Population Cytogenetics Unit, Western General
Hospital, Edinburgh EH4 ZXU, Scotland
Pardue & Gall [9], in their study of the in situ
hybridization of RNA complementary to
mouse satellite DNA, noticed that the centro-
merit regions of the mouse chromosome
complement, where the complementary RNA
was localized by autoradiography, stained
more darkly with Giemsa than the remainder
of the chromosome. Arrighi & Hsu [I]
adapted this technique, by omitting the
complementary RNA, and obtained darkly
staining centromeric regions on human [I]
and other mammalian [2, 81 chromosomes.
The darkly stained material has become
known as centromeric heterochromatin. This
technique has been widely used not only for
studying the distribution of centromeric
heterochromatin, but also for identifying
chromosomes [5] and studying rearrange-
ments in abnormal cells 141.
Although the original technique may
produce excellent results, it is somewhat
tedious, and the treatment with sodium
Exptl Cell Res 75 (1972)
hydroxide which it involves can be rather
destructive, causing loss of some chromo-
somes and severe distortion of others. We
have previously described the use of barium
hydroxide, which is less destructive than
sodium hydroxide, for the demonstration of
centromeric heterochromatin [lo], but as
originally applied the technique was not re-
liable. The procedure has now been refined
to make it simple, relatively quick, and re-
liable for routine use. By analogy with the
ASG banding technique, from which it only
differs fundamentally in the insertion of an
extra stage, it has been named the BSG
technique (barium hydroxide/saline/Giemsa).
Material and Methods
Human chromosome preparations were made from
cultures of peripheral blood, treated with hypotonic
potassium chloride (0.075 M for 8 min), fixed in 3
changes of methanol/acetic acid (3 : I), and spread by
air-drying in the normal way.
The slides were treated with 0.2 N hydrochloric
acid for 1 h at room temperature, rinsed with de-
ionized water. and nlaced in a freshly Drepared 5 “/b
aqueous sol&on oi barium hydroxihe- octahydrate
at 50°C for 5-15 min. After thorough rinsing in
several changes of deionized water, the slides were
incubated for 1 h at 60°C in 2 x SSC (0.3 M sodium
chloride containing 0.03 M tri-sodium citrate), rinsed
briefly with deionized water and stained for 14 h with
Giemsa (G. T. Gurr’s Giemsa R66. 1 ml to 50 ml of
buffer (pH 6.8) made with Gurr bbffer tablets). Fin-
ally the slides were again rinsed briefly in deionized
water, blotted, allowed to dry thoroughly, soaked in
xvlene and mounted in DPX. The latter part of the
technique, subsequent to the barium hydroxide treat-
ment, is identical to the ASG banding technique [lo].
As with ASG banding, best results are obtained if
slides are left for a few days before performing the
technique.
Results and Discussion
This technique gives results similar to those
previously described [I, 71; fig. 1 shows a
metaphase cell from a human male.
The most critical stage of the technique is
the treatment with barium hydroxide. For
chromosomes from peripheral blood lympho-
cytes the times, concentration and tempera-
ture given were found to be best, but there is
considerable variation in the reaction of

Fig. 1. Metaphase cell (46, XY) from a human male peripheral blood lymphocyte culture.
slides made from different cultures. Too short
a treatment results in a more or less complete
banding pattern being produced, as in the
ASG and related techniques; too long causes
swelling and distortion of chromosomes, as
is found with sodium hydroxide. Treatment
with barium hydroxide at 60°C requires in-
conveniently brief times in the solution, while
at 37°C even after 1 h complete banding
patterns were often produced. However, for
mouse meiotic chromosomes it has been
found that this lower temperature is prefer-
able [3].
The initial treatment with hydrochloric
acid improves the consistency of the tech-
nique. With good preparations, in which
there is very little cytoplasm surrounding the
chromosomes, it is unnecessary. With less
Preliminary notes 305
good preparations it reduces excessive times
in barium hydroxide which might otherwise
be needed, with risk of swelling, and makes
the reaction more uniform from one part of
the slide to another. The acid treatment has
no deleterious action on good preparations,
and thus it is convenient to include it every
time. The purpose of the acid treatment is
to remove surplus protein from the slide,
although there is in fact no evidence that it
acts in this way or that this is its sole action.
This technique is now in routine use in
this Unit, and has been used, as well as on
human lymphocyte and meiotic chromo-
somes, on mitotic chromosomes of various
mammals [6], on mouse/human hybrid cell
lines, and in virus-transformed cell lines.
I thank Mr G. Spowart for his assistance.
Expd Cell Res 75 (1972)
306 Preliminary notes
References
1. Arrighi, F E & Hsu, T C, Cytogenetics 10 (1971)
81.
2. Arrighi, F E, Hsu, T C, Saunders, P & Saunders,
G F, Chromosoma 32 (1970) 224.
3. Chandley, A C & Fletcher, J H. In preparation.
4. Chen, T R & Ruddle, F H, Chromosoma 34
(1971) 51.
5. Chernay, P R, Hsu, L Y F, Streicher, H & Hirsch-
horn, K, Cytogenetics 10 (1971) 219.
6. Evans, H J, Buckland, R A & Sumner, A T, In
preparation,
Exptl Cell Res 75 (1972)
7. Evans, H J, Buckton, K E & Sumner, A T, Chro-
mosoma 35 (1971) 310.
8. Hsu, T C & Arrighi, F E, Chromosoma 34 (1971)
243.
9. Pardue, M L & Gall, J G, Science 168 (1970)
1356.
10. Sumner, A T, Evans, H J & Buckland, R A,
Nature new biol 232 (1971) 31.
Received August 23, 1972