To stain metaphase chromosomes with Giemsa or Leishman’s stain to elicit a
banding pattern throughout the chromosome arms, designated G-Bands. This
G-Banding technique requires a chromosomal pretreatment step of trypsin to
induce chromosome bands.
30 to 60 minutes
- Leishman stain
- Gurr buffer tablets
- Prepare the staining solution the day prior to use. Also, slides should be
aged at least 7–10 days or placed in a 55°C–65°C oven for 45 minutes
before staining, to ensure excellent banding patterns. Aging the slides
helps to eliminate fuzzy banding and increases contrast of the bands.
- Exact timing is important; therefore, a maximum of 5 slides should be
stained at one time. Optimum time in the stain appears to be between
2.5–4 minutes. It is necessary to determine the approximate staining times
for each bottle of stain solution. The exact time will vary by several seconds
depending on the source of cells, age of slides, the cell concentration on
the slide, etc. (refer to the table below)
These are approximate times, and test slides need to be done to determine trypsin
and staining time for each cell line.
- Mix 1 mL 0.25% stock trypsin with 49 mL 0.85% NaCl (working salt solution).
Wait 4 minutes before beginning to stain to allow the trypsin to dissolve.
- Dip oven-dried slides that have cooled to room temperature in the trypsin
solution for 5–30 seconds. The time in trypsin is dependent on slide
preparation conditions, harvesting conditions, material being banded, etc.
Stain a test slide first to determine optimum conditions.
- Rinse slides in 50-mL working salt solution.
- Use a graduated cylinder to mix 15 mL Leishman stain and 45 mL Gurr
buffer just prior to staining, and pour into a coplin staining jar. Stain the
slides for 3 to 4 minutes.
- Rinse slides in distilled water and air dry with compressed air or use a
- Check the slides using a Zeiss Microscope, 100X plan-apochromatic oil
objective, brightfield. See “brightfield photography”.
- Overtrypsinized chromosomes appear fuzzy; somewhat difficult to
recognize exact bands.
- Undertrypsinized chromosomes will have indistinct bands, decreased
contrast; very difficult or impossible to determine bands.
- Adequately trypsinized chromosomes will show telomeres not overly
digested and G-Bands will appear sharp and in contrast.
- If slides are undertreated with trypsin, destain the slides before rebanding.
Quickly dip the slides in 3:1 ethanol:acetic acid 2 or 3 times, or until all
stain is removed. Rinse in distilled water, air dry, and reband.
- Coverslip with 24 × 50 mm #1 coverslip using permount.
- Transfer slides to a microslide box or suitable storage container.
- Trypsin (1:250) USP Grade; porcine parvovirus tested. Prepare a 0.25% (1X)
solution by dissolving 0.25 g trypsin in 100 mL PBS. Aliquot into 1 mL
quantities and store at –20°C for up to 1 year. Thaw just before using.
Lowering thetemperature of the trypsin in the working salt solution will
lower its activity, thus requiring longer exposure times and also increasing
the life of the solution. Test slides should be run to determine optimum
- Gurr Buffer Solution: (1 liter). 1 tablet is dissolved in 1 liter distilled water.
Produces a solution of pH approximately 6.8 at 20°C. Use solution at room
- Working Salt Solution: (1 liter). Dissolve 8.5 g of NaCl in 1 liter distilled
water. Use at room temperature.
- Leishman Stain Solution. Dissolve 1 g Leishman stain into a 500-mL bottle
of methanol. Stir for 4 hours and allow stain to age at least 1 day prior
to use. If stain is not dissolved, filter stain through a 0.45-µm filter.