Preparation of cell culture
Using a 21-guage multi-draw needle
and a green top vacutube (both supplied by
the local
hospital, if you are lucky), a qualified
technician withdraws blood from the students.
Prepare
a sterile 5 mL syringe
with a 21-guage needle.
Wipe the top of the green top tube [Bottle with needle] (containing blood)
with an
isoproponal
alcohol pad.
Insert the needle on the syringe into the green top and withdraw a
few milliliters of blood.
Open the bottle of chromosome medium and place
five to ten drops of blood into the
medium. Sterile technique must
be used because it is possible to cause major
contamination during this procedure.
2. Incubation
Mix the medium and blood by gentle inversion and place the bottle in a
preheated incubator
at 37o
C.
Incubation for 70 hours
Mix gently by inversion twice a day during
incubation
3. Stopping the cell division at metaphase
Pre-warm the Colcemid in the incubator at 37oC.
CAUTION: Colcemid can be dangerous, so handle with care. Colcemid is a mitotic spindle inhibitor. If splashed on skin, rinse immediately and seek medical help.
Add 0.05 mL (50 microliters) of prewarmed
37oC Colcemid
to the culture. Mix gently and
put the culture back into the incubator.
Incubate for 30 to 60 minutes.
NOTE: Up
to this point the teacher has done everything. This is where students begin
their
part.
4. Hypotonic treatment of the red and white blood cells
Remove the blood and Colcemid solution from the [Centrifuge] incubator
and mix gently.
Put the entire contents of the bottle into a conical centrifuge tube. If
conical tubes are not
available, regular tubes can be used.
Centrifuge for six minutes at 500
- 900 rpm (see notes at the end of lab regarding centrifuge
speed).
After six minutes, turn off the centrifuge and wait for a complete stop.
Carefully remove the
tube.
Remove the supernate (clearish fluid on top)
with a pasteur pipette. Be very careful not to
disturb the button of cells on the
bottom. Make sure that the bulb of the pipette
is
depressed before it is inserted into the
test tube. Leave some fluid (anywhere from * to *
mL) on the top of the button of cells. When
withdrawing fluid, keep the pipette tip
against the side of the test tube
to avoid any shaky movements.
Add one mL of warmed
37oC hypotonic solution to
the tube. Mix by flicking the tube with
your finger. Now add another nine
mL of hypotonic solution. The hypotonic
solution should
not be in contact with the cells for more
than a total of 24 minutes. Excess exposure
may
cause rupture of the white blood cells.
Throughly mix all the hypotonic fluid with the cells. This is done by drawing
all the mixture
at the bottom of the tube into a pasteur
pipette and forcing it out again. Do this two or three
times to thoroughly mix.
Place the mixed solution into the 37oC
incubator for nine minutes
The fixative solution must be made fresh. While the hypotonic solution
is working, make
up the fixative solution as follows:
add three parts chilled absolute
methanol (or as close
as you can get) to
one part galacial acetic acid.
Both chemicals should be as pure as
possible.
After nine minutes, centrifuge for
six minutes at 500 to 900 rpm.
Remove the supernate. leaving * or * mL of fluid on top of the button of
cells. At this time
you probably have a small whitish or reddish
film at the bottom and slightly up the side of
the tube. The film contains large quantities
of red blood cell debris and the enlarged white
blood cells. Your entire experiment,
up to this point, has been to isolate that film at
the bottom of the tube.
5. Fixing the cells
Add 5 mL of fixative
solution to the centrifuge tube.
With a pasteur pipette, mix the fixative and button of cells by drawing
the mixture into the
pasteur pipette and forcing it out
again. Do this three of four times. Place
this solution of
cells and fixative into a refrigerator
for 30 minutes. Make sure the
test tube is covered
with aluminum foil because of
the smell. The 30 minutes
in a refrigerator is a minimum;
actually, it is possible to
keep cells in the refrigerator overnight.
During this time,
practice dropping water on slides
(instructions to follow).
After refrigeration, centrifuge the tube for six
minutes at 500 to 900 rpm.
Remove the supernate and add another 6 mL
of cold fixative and mix as you just did in
the
instructions above.
Centrifuge the tube for six minutes at 500
- 900 rpm.
Repeat the above two steps.
Remove the supernate leaving about * mL of fluid at the bottom of the tube.
It is this
remaining material that you will drop
on your slides in the next section. If you cannot see
any material at the bottom of the
test tube, do not despair; proceed as though there is
visible material present. It
is often very difficult to see.
6. Making the chromosome slides
The slide must be exceptionally clean. Use new, factory
pre-cleaned, frosted slide.
The chromosome separation seems
to work best if the slides are chilled in the freezer first.
Lay five or six slides next to each other
on paper toweling with no separation between
them.
Withdraw the entire contents of the centrifuge tube into a pasteur pipette.
Be careful not
to draw the fluid
any farther than necessary into the pipette.
The cells have a
tendency to attach to
the sides of the pipette.
From a height of about 18 inches,
drop two or three drops of fluid onto each side.
Allow the slides to dry thoroughly. In fact, the best way to 'cure'
the slides are to place
them in the incubator
(37oC) overnight.
Stain the slide by immersion in fresh giemsa stain
for 7 - 10 minutes.
Remove the slides from the stain and rinse in distilled
water until ALL the excess stain is
removed.