Materials:

• Chromosome medium (one bottle per student)--- KaryoMAX Peripheral Blood Karyotyping Medium #41675-018 These bottles usually come in groups of 10 (each bottle conmtaining 5 mL of media).
• Colcemid® solution (10 mL bottle) --- KaryoMAX Colcemid liquid #15212-012
• Giemsa stain (100 mL bottle) --- KaryoMAX Giemsa Stain Stock #10092-013
• Potassium chloride solution, 0.075M (about 10 mL per student) --- KaryoMAX Potassium Chloride Solution, 0.075M #10575-074

  Needed for the class: Centrifuge Glacial acetic acid (ACS grade) Absolute methanol (ACS grade) Refrigerator Incubator (capable of 37C) Frosted glass slides

  Needed for each person Sterile 1 or 5 mL syringe 21-guage needle for the syringe Green-top vacutube 21-guage multidraw needle (for vactube) 15 mL centrifuge tube Pasteur pipette Bulb for the pasteur pipette 70 percent isopropyl alcohol pad

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  Laboratory Procedure
• 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 (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.
• Incubation
  • Mix the medium and blood by gentle inversion and place the bottle in a preheated incubator at 37 C.
  • Incubation for 70 hours
  • Mix gently by inversion twice a day during incubation
• Stopping the cell division at metaphase
  • Pre-warm the Colcemid in the incubator at 37 C. 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 C 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.
• Hypotonic treatment of the red and white blood cells
  • Remove the blood and Colcemid solution from the 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 ¼ to ½ 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 C 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 27 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 C incubator for 17 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 ¼ or ½ 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.
• 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 ½ 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.
• 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 C) 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.

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  This laboratory procedure has been developed by Doug Lundberg. It was published in

The American Biology Teacher , volume 52, No. 2, February 1990. All inquires should be address to Doug Lundberg , lundberg@d20.co.edu

NOTES

A. Calibrating the centrifuge is easier than it appears. You can use a fixed sped centrifuge. Attach a voltage regular (borrowed from the physics teacher) and reduce the voltage, thus slowing down the speed of the centrifuge. You can accurately calibrate the speed using a teacher-invented Rube-Goldberg method. Place a large, sharp pencil in the centrifuge and run it on. To determine the speed, take a piece of clean white paper taped onto a firm background and hold it lightly to the spinning pencil for a one-second count. Count the marks left by the pencil on the paper. Between 9 and 15 marks will represent a rpm of 500 to 900. Adjust the voltage regulator until you obtain 9 to 15 marks.

B. Timing: the laboratory exercise takes two to three hours to finish. Students can come in on Saturday morning to do their part of the experiment. The teacher can arrange a "field trip" for half a day during the school day and have the students stay in his or her room to do the experiment. A substitute teacher can take the regularly scheduled classes to the library for research, study or a written assignment. This procedure may be used for a small number of select student(s) rather than an entire class.

C. Slides are observed under high power (400 X) and then photographed under oil immersion. Students may spend a few days determining the best metaphase spread. Each time a student feels that he or she has found a good spread, the teacher marks their slide close to the spot with a fine-tip black lab marker. When the students are all done, they tell the teacher which dot they want to have photographed. The local hospital's cytology department will often allow the teacher to use the scope and camera (with supervision). It is very helpful when it come to photographing slides. The slides are photographed under oil immersion with black and white film. Make friends with the local hospitals department staff. Explain your goals and ask them for help.

D. Cost: the cost of this experiment is relative to what you may have on hand. A general figure of $30 to $70 per 10 students is a good guess. The cost incurred to an individual by a hospital runs many hundreds of dollars, almost $1,000.

References

Coburn, S.P. & Leyauf, R.B. (1976). Human chromosome analysis biokit. Burlington, NC: Carolina Biological Supply Co.

Kaplan, B.J. (1978) Preparation of the normal karyotype (workbook). Chicago: American Society of Clinical Pathologists.

Macgregor, H.C. & Narley, J.M. (1983). Working with animal chromosome. New York: John Wiley & Sons