This method of preparing polytene chromosomes; is slightly different from traditional
methods. In the past chromosomes were spread by applying pressure to the slide with
one's thumb or with a vise. Described below is a modified technique of Johng K. Lim's
of the University of Wisconsin-Eau Claire, in which the chromosomes are gently
spread by a wave of stain flowing under the coverslip.
1) For the best results larvae should be grown in well yeasted media at 18°C and care
should be taken not to overcrowd the bottles.
2) On a clean siliconized slide place two drops of 45% acetic acid and a small drop
(2-3 mm) of lacto-aceto orcein stain.
3) In the first drop of 45% acetic acid dissect out the salivary glands from a third
instar larva.
4) Transfer the glands to the second drop of 45% acetic acid and cut off the anterior
ends of the glands. Quite often this will allow the glands to separate from the
membrane and the attached fat body. If this does not occur, carefully dissect away
the fat body.
5) Transfer the "fat free" glands into the staining solution and incubate for 2-3 minutes.
6) Place a clean siliconized coverslip (18 mm square) over the glands in the stain.
7) Place the slide on several sheets of paper towel. With another sheet of paper towel
hold the edge of the coverslip to prevent it from moving.
8) Using a stiff, pointed probe, gently press once over the glands. Then gently tap the
coverslip, starting over the glands, working your way toward the edge in a spiral
pattern.
9) Turn the slide over so that the coverslip is between the paper towel and the slide.
Press gently on one edge of the slide so that the stain flows to the opposite edge.
10) Turn the slide back over and again hold the edge of the coverslip with a paper
towel.
11) Using the probe, gently streak the coverslip with a rapid back and forth motion at
one edge and then gradually move to the opposite edge.
12) Blot off excess stain and examine under the microscope using phase contrast. If
spreading is not sufficient, gently restreak the coverslip.
13) To make the slide permanent, keep the slide overnight at room temperature.
14) Freeze the slide in an ethanol-dry ice bath for 60 minutes)
15) Pop off the coverslip and immediately place the slide in 95% ethanol (room
temperature) for 2-3 minutes.
16) Place the slide in 100% ethanol (room temperature) for 10 minutes.
17) Place a drop of mounting media over the spread and cover with a 22 mm square
coverslip.
Lacto-aceto orcein stain:
Incubate a 2% (w/v) solution of "Gurr's orcein natural" in concentrated lactic acid at
room temperature for several hours, then filter. Heat, but do not boil, a 2% (w/v)
solution of "Gurr's orcein natural" in concentrated glacial acetic acid for several hours,
then filter. Combine the filtered solutions with water in a 1:1:1 ratio.
Gurr's Orcein Natural
Cat. no. 22100
Bio/Medical Specialties
Box 1687
Santa Monica, CA 90406
(213) 829-2373