An Inexpensive and Simple Method for Preparing Chromosome Spreads ∗ †

Abstract

The extent of recent literature in cytogenetics conceals the fact that relatively few laboratories perform chromosome analyses as a routine procedure, and even fewer analyze populations of a size desirable for extraction of statistically valid information. A major deterrent is probably cost of time and money; and a minor one, possibly, the feeling that tissue culture techniques require highly specialized equipment and skill. In fact, the basic technique is quite straightforward and fully amenable, without loss of reliability, to simplification and economy. The method described here permits efficient study of large numbers of individual specimens with a minimum of effort, materials and equipment.

Reagents. 1. Phytohaemagglutinin (PHA).

a. Wash red kidney beans (Phaseolus vulgaris) in water.

b. Grind in a pestle and extract overnight at 4°C in normal saline using 10 ml per gram of bean.

c. Centrifuge to clear the supernate, sterilize by filtration and store frozen.

2. Medium.

a. 80% Minimum Essential Medium Eagle (in its spinner formulation and with added glutamine) and 20% pooled human serum (outdated bank blood does very well).

b. Add 50 units per ml of preservative free heparin and 0.2 to 0.5 ml of PHA per 100 ml. The amount of PHA varies with the strain of Phaseolus used and the concentration of the extract and should be ascertained for each batch.

c. Dispense the medium with needle and syringe in 2 ml lots into nontoxic rubber stoppered, evacuated test tubes, and replace the vacuum with room or alveolar air using a needle and plastic tube containing cotton (those found in transfusion sets are quite satisfactory). Store frozen.