June 3, 1990
Rosalie Veile
Purpose:
35 mm black and white film (Kodak Technical Pan film) is developed and print enlargements made from the negatives. The chromosomes from the prints are identified, cut out and arranged on the karyotype form.
Time required:
Special supplies:
Special equipment:
Procedure:
Developing the film:
Note: It is important to develop the film at a constant temperature to prevent excess grain on the film. A pan filled with water is used to hold all solutions and water rinses at 23-25 deg. C.
Producing 5 X 7 prints:
Note: Polycontrast paper by Kodak is a variable contrast paper and works well for negatives of low contrast or underexposed film. Polycontrast filters can also be used to improve the contrast of the prints. In the steps below, refer to figures 1 and 2 to find the enlarger parts indicated by (#).
To expose the print paper, place the shiny side of the paper facing up, and center the paper on the enlarging easel. Set the timer for an exposure time (1-2 seconds). Expose the unmasked portion of the print paper by pressing the red button on the timer (Expose/Hold). The enlarger light will automatically stop when the timer goes off. Move the masking paper to reveal more of the print paper and re-expose. (The portion of the paper that was previously exposed now has two exposures.) For each time point, move the masking paper down the paper. Develop the test strip (follow step 4 below) and decide which exposure time is best to use for the negative.
The film has a much greater capacity for producing detail than the print paper, so it may be necessary to do some dodging (holding back light from the over-exposed dark areas) or burning-in (using more light on a particular area to increase the exposure and darken the print) to bring out more details of the chromosomes. For underexposed areas such as F and G group chromosomes, burning-in will make the arms and satellites more visible. A black piece of paper with a hole cut out can be used as a tool, or even your hand with your fingers spread apart to expose those areas that need darkening.
Solutions:
Developer, Kodak D-19:
Avoid breathing dust when preparing, may be harmful if swallowed.
Slowly add a package (595 g) of developer to 3.8 liters of water at 52deg.C, with stirring until chemicals are dissolved and the solution is completely mixed. Store in a 4 liter brown glass bottle at room temperature for up to 1 year (label bottle with the date prepared).
Slowly pour powdered fixer (680 g) into 3.8 liters of water (not above 26.5deg.C), with stirring until all the powder is dissolved. Fixer can be stored in a well stoppered, brown glass 4 liter bottle for up to 2 months (label bottle with the date prepared). For high contrast films, the fixer time should be for 2-4 minutes; while most continuous tone films should be fixed for 5-10 minutes with agitation.
Add 5.5 ml Photo-Flo to 1.1 litres of water. Note: Scum will appear on the developed film if the photo-flo concentration is too high.
References:
Cytogenetics lab manual
Kodak product enclosures