Principle

Conventional staining techniques are used to uniformly stain chromosomes and leave the centromeres constricted, thus enabling the measurement of chromosome length, centromeric position, and arm ratio.

Background

Prior to 1960, when Moorehead and Nowell described the use of Giemsa in their chromosome preparations, conventional cytologic stains such as acetoorcein, acetocarmine, gentian violet, hematoxylin, Leishman's, Wright's, and Feulgen stains were used to stain chromosomes. The Romanovsky dyes (which include Giemsa, Leishman's, and Wright's stain) are now recommended for conventional staining, because the slides can be easily destained and banded by most banding procedures. Orcein-stained chromosomes cannot be destained and banded; therefore, orcein is generally not used in routine chromosome staining. Giemsa stain is now the most popular stain for chromosome analysis (Gustashaw, 1991).

Solutions

• Leishman's stain
  
• pH 6.8 phosphate buffer (Gurr's tablets, Biomedical Specialties cat. # 33199)
  
• Working stain: Leishman's stain diluted 1:4 with buffer
  
• Xylene
  

Procedure

1. Stain slides 3 to 5 minutes in working stain.

2. Rinse well in buffer. If stain is too intense, wash longer in buffer. If it is too weak, restain the slides.

3. Blot dry with bibulous paper. Air dry completely.

4. Rinse in xylene.

5. Mount in neutral mounting medium.

References

Gustashaw, KM. Chromosome Stains. In The ACT Cytogenetics Laboratory Manual, Second Edition, edited by M. J. Barch. The Association of Cytogenetic Technologists, Raven Press, Ltd., New York, 1991.