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 37^o 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 37^oC.

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 37^oC 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 37^oC 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 37^oC 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 (37^oC) 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.