322
Related Techniques
Hypotonic
treatment
Fixation
Spreading
Staining
3. Continue the culture for 4-5 h.
4. Harvest the cells, and suspend the cells in the culture medium by
pipetting or enzyme treatment (see chapter 4 subprotocoll-3).
5. Centrifuge the cell suspension at 150 g for 5 min.
6. Discard the culture medium.
1. Gently disperse the cells in 7 ml hypotonic KCI solution. Keep the cells
in the KCI solution for 15 min at room temperature.
2. Centrifuge the cells at 150 g for 5 min.
3. Gently disperse the cell pellet in 1 ml hypotonic KCI solution.
1. Add 1 ml Carnoy's fixative, and gently mix with a Pasteur pipette.
2. Gently add a further 6 ml Carnoy's fixative, and mix. Keep the cells in
this solution for 15 min.
3. Centrifuge the cells at 150 g for 5 min.
4. Disperse the cells in 7 ml Carnoy's fixative. Keep the cells in this solution for 15 min.
5. Centrifuge the cells at 150 g for 5 min.
l. Disperse the cells in a small amount (0.1-0.3 ml depending on the
number of cells) of Carnoy's fixative.
2. Place a drop of the cell suspension in the center of a cover glass. The
cover glass should be absolutely clean (see Comments), so that drop
of cell suspension spreads instantaneously over the surface of the glass.
3. Air dry the cover glass.
l. Make a 1 in 10 dilution of Giemsa solution with 1/15 M phosphate
buffer (pH 6.8).
2. Stain the cover glass by placing it in a petri dish containing the diluted
Giemsa solution for 30 min.
3. Wash the cover glass in water.
4. Dip the cover glass in methanol for a few seconds to remove excess
dye.
5. Wash the cover glass in water to remove the methanol quickly and
completely.
6. Examine the staining state.
7. If the staining intensity is appropriate, air dry the cover glass. If the
cells are stained too deeply, repeat steps 3-6. If the die has been removed excessively, or the cells are not stained sufficiently, repeat the
staining procedure, starting at step 2.
8. Mount the cover glass on a slide glass using balsam.
Related Techniques
Hypotonic
treatment
Fixation
Spreading
Staining
3. Continue the culture for 4-5 h.
4. Harvest the cells, and suspend the cells in the culture medium by
pipetting or enzyme treatment (see chapter 4 subprotocoll-3).
5. Centrifuge the cell suspension at 150 g for 5 min.
6. Discard the culture medium.
1. Gently disperse the cells in 7 ml hypotonic KCI solution. Keep the cells
in the KCI solution for 15 min at room temperature.
2. Centrifuge the cells at 150 g for 5 min.
3. Gently disperse the cell pellet in 1 ml hypotonic KCI solution.
1. Add 1 ml Carnoy's fixative, and gently mix with a Pasteur pipette.
2. Gently add a further 6 ml Carnoy's fixative, and mix. Keep the cells in
this solution for 15 min.
3. Centrifuge the cells at 150 g for 5 min.
4. Disperse the cells in 7 ml Carnoy's fixative. Keep the cells in this solution for 15 min.
5. Centrifuge the cells at 150 g for 5 min.
l. Disperse the cells in a small amount (0.1-0.3 ml depending on the
number of cells) of Carnoy's fixative.
2. Place a drop of the cell suspension in the center of a cover glass. The
cover glass should be absolutely clean (see Comments), so that drop
of cell suspension spreads instantaneously over the surface of the glass.
3. Air dry the cover glass.
l. Make a 1 in 10 dilution of Giemsa solution with 1/15 M phosphate
buffer (pH 6.8).
2. Stain the cover glass by placing it in a petri dish containing the diluted
Giemsa solution for 30 min.
3. Wash the cover glass in water.
4. Dip the cover glass in methanol for a few seconds to remove excess
dye.
5. Wash the cover glass in water to remove the methanol quickly and
completely.
6. Examine the staining state.
7. If the staining intensity is appropriate, air dry the cover glass. If the
cells are stained too deeply, repeat steps 3-6. If the die has been removed excessively, or the cells are not stained sufficiently, repeat the
staining procedure, starting at step 2.
8. Mount the cover glass on a slide glass using balsam.