No. 9] Proc. Japan Acad., 47 (1971) 729
161. Differential erential Staining o f Human Chromosomes by Treatment with Urea*'
By Yukimasa SHIRAISHI* * ) and Tosihide H. YOSIDA* * * (Comm. by Yoshimaro TANAKA, M. J. A., Nov. 12, 1971)
Arrighi and Hsu (1971), and Somner et al. (1971) have already described the use of a solution of HC1, RNase, NaOH, and SSC at 65°C for differential staining of human chromosomes. Their staining methods have fairly facilitated the identification of individual chromo- somes. However, more detailed morphological criteria of the identi- fication of the each chromosome are still remained, and their tech- niques are also still complicated.
A very simple technique for differential staining of human chro- mosomes was found by us. The principle of the technique is that the air dried slides are pretreated with urea solution and stained with Giemsa. The present paper deals with a preliminary note on this technique.
Material and methods. The material was obtained from human male leucocytes cultured according to routine phytohemagglutinin method. The technical procedure for the differential staining of human chromosomes is as follows :
1. Air-dried slides are prepared from leucocyte cultures ac- cording to routine technique by Moorhead et al. (1960). Natural air drying is desirable for making of slides.
2. Newly prepared slides are treated with 8 M urea solution at 37°C for 15 to 20 minutes. New slides are much better than the old ones, but the old slides and slides already stained can be used by increasing the temperature of the urea solution (about 40-50°C) or extension of treatment time for about 1 to 7 hours.
3. Rinse slides thoroughly with distilled water to avoid the precipitation of urea.
4. Slides are lightly stained with Giemsa, since over staining induces loss of details. For this purpose, the stock solution of Giemsa was diluted about 60 to 80 times with distilled water. The average
*' Contribution No . 857 from the National Institute of Genetics, Japan.
Supported by a grant-in-aid from the Ministry of Education of Japan No. 92332.
`' Department of Anatomy
, School of Medicine, Kanazawa University, Kanazawa 920, Japan.
**' Department of Cytogenetics
, National Institute of Genetics, Misima 411, Japan.
730 Y. SHIRAJSHI and T. H. Y0sIDA [Vol. 47,
Fig. 1. Metaphase chromosomes of a human male treatment technique (46, XY).
leucocyte by urea
Fig. 2. Idiogram of the cell shown in Fig. 1. Note on the banding patterns in each chromosome pair.
No. 9] Staining of Human Chromosomes by Treatment with Urea 731
staining time is 15 minutes at room temperature.
5. Rinse the slides with distilled water and dry in air.
Finding and remarks. Metaphase chromosomes represented by the present technique are shown in Fig. 1. Karyotype analysis made
on the basis of the differential staining pattern is in Fig. 2. Each pair of homologous chromosomes displays a characteristic banding pattern. The appearance and location of darkly stained bands are consistent, and readily identified in metaphase chromosomes. Chromo- somes Nos. 1, 2 and 3 belonging to the group A exhibit the differential specific banding patterns in each pair. In the group B, pair No. 4 is clearly distinguished from pair No. 5. Pair Nos. 6 to 12 and X in the group C are fairly possible to identify from each other. Another important observation in the present study is that a particular ad- vantage is also gained in the identification of the group D, E, F and G, and the Y chromosome. Pair Nos. 13, 14 and 15 in the group D which are characterized by similar acrocentrics are distinguishable from each other in their specific banding patterns.
The present methods may also apply to the study of other mam- malian chromosomes such as of mouse, rat and Chinese hamster. The staining technique by pretreatment with 8 M urea solution seems to be the advantage by appearance of the higher contrast in the banding pattern when compared with some other methods. Especially the present technique is useful for identification of small chromosomes in mammals.
Arrighi, F. E., and T. C. Hsu: Cytogenetics, 10, 81-86 (1971).
Moorhead, P. S., P. C. Nowell, W. J. Mellman, D. M. Battips, and D. A. Hungerford:
Exp. Cell Res., 20, 613-616 (1960).
Somner, A. T., H. J. Evans, and R. A. Buckland: Nature, New Biology, Vol. 232, July 7, 31-32 (1971).