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  Section: Biotechnology Methods » Cell Biology and Genetics
 
 
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Black and White Film Development and Printing for Karyotype Analysis

 
     
 
Content
Cell Biology and Genetics
  Cell Cycles
  Meiosis in Flower Buds of Allium Cepa-Acetocarmine Stain
  Meiosis in Grasshopper Testis (Poecilocerus Pictus)
  Mitosis in Onion Root Tip (Allium Cepa)
  Differential Staining of Blood
  Buccal Epithelial Smear and Barr Body
  Vital Staining of DNA and RNA in Paramecium
  Induction of Polyploidy
  Mounting of Genitalia in Drosophila Melanogaster
  Mounting of Genitalia in the Silk Moth Bombyx Mori
  Mounting of the Sex Comb in Drosophila Melanogaster
  Mounting of the Mouth Parts of the Mosquito
  Normal Human Karyotyping
  Karyotyping
  Black and White Film Development and Printing for Karyotype Analysis
  Study of Drumsticks in the Neutrophils of Females
  Study of the Malaria Parasite
  Vital Staining of DNA and RNA in Paramecium
  Sex-Linked Inheritance in Drosophila Melanogaster
  Preparation of Somatic Chromosomes from Rat Bone Marrow
  Chromosomal Aberrations
  Study of Phenocopy
  Study of Mendelian Traits
  Estimation of Number of Erythrocytes [RBC] in Human Blood
  Estimation of Number of Leucocytes (WBC) in Human Blood
  Culturing Techniques and Handling of Flies
  Life Cycle of the Mosquito (Culex Pipiens)
  Life Cycle of the Silkworm (Bombyx Mori)
  Vital Staining of Earthworm Ovary
  Culturing and Observation of Paramecium
  Culturing and Staining of E.coli (Gram’s Staining)
  Breeding Experiments in Drosophila Melanogaster
  Preparation of Salivary Gland Chromosomes
  Observation of Mutants in Drosophila Melanogaster
  ABO Blood Grouping and Rh Factor in Humans
  Determination of Blood Group and Rh Factor
  Demonstration of the Law of Independent Assortment
  Demonstration of Law of Segregation

Purpose
35-mm black and white film (Kodak Technical Pan film) is developed and print enlargements are made from the negatives. The chromosomes from the prints are identified, cut out, and arranged on the karyotype form.


Time Required
  1. 5–20 minutes to develop 1 or 2 rolls of film
  2. 45–60 minutes to print 20 quality prints
Special Supplies
  1. Kodak Technical Pan Film 2415
  2. Kodak Polycontrast III Paper 5" × 7", RC plastic coated
  3. Kodak Ektamatic S30 Stabilizer
  4. Kodak S Activator
  5. Kodak D19 Developer
  6. Photo-Flo 200
Special Equipment
  1. Kodak Ektamatic Print Processor
  2. Beseler Enlarger
  3. Graylab 500 Timer
Procedure
Developing the film
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°C.
  1. In total darkness (no safe light on), remove the film from the cassette and wind into the developing reel. It is important to wind the film correctly to prevent undeveloped areas. Correctly wound film will have no edges protruding. Place the reel in the developing tank and twist the lid of the tank to close. It is now safe to turn on the room lights.
  2. Pour 400 mL of Kodak developer D-19 into the top of the tank to develop the film. Agitate the tank periodically over 4 minutes to ensure the developer is in contact with all parts of the film (prevents uneven film development). After the fourth minute of developing time, pour the developer into a small storage bottle. Developer may be used a total of 3 times.
  3. Briefly rinse the tank with water. Agitate and drain water completely.
  4. Pour 400 mL of fixer into the developing tank and fix for 4 minutes, agitating several times during this time period. Film should not be left in the fixer longer than 4 minutes, because the negative will bleach, or become saturated with fixer. The fixer time should be for 2–4 minutes for high-contrast films, while most continuous tone films should be fixed for 5–10 minutes.
  5. Remove the lid and empty the developing tank. Rinse the film in the tank for 2 minutes with running water at 23°-30°C. Because fixer is heavier than water, be certain to fill and empty the tank several times to prevent the fixer from remaining at the bottom of the tank. The amount of time the film stays wet should be kept to a minimum to prevent film deterioration, e.g., swelling and clumping of the grain may occur, which decreases the sharpness of the image.
  6. Remove the film from the reel and add photo-flo solution to the tank. Empty the tank after 30 seconds. This helps film dry without streaking. Drain film briefly, squeegee, and hang in a dust-free area to dry. Producing 5 × 7 Prints 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. See the steps below.
  7. Load the negative onto the negative carrier. This is done by moving the negative stage lever (16) downward, removing the negative carrier from the stage, opening the carrier, inserting the negative, closing the carrier, and replacing the carrier onto the stage. Pull the stage lever back up to close.
  8. Turn on the enlarger with the graylab timer. The negative stage guide (13) should be positioned on 35-mm and smaller formats. Using the negative stage adjustment knob, lower the stage to the 35-mm mark. Use the negative lock (18) to secure the stage at this position. The elevator motor/control box (3) moves the enlarger head up or down for changes in elevation. Use the elevation switch (4) to move the enlarger head until the image becomes clearly visible. Manual elevation control (5) can be used for precise elevation. By using the motor switch as a scale indicator, a record of the height can be recorded for repeat magnification at a later date. For enlarging a 35-mm negative onto a 5" × 7" print, position the switch to approximately 4 inches on the reference scale. A grain-focusing scope (microsight) should be used to ensure the film is at the best magnification to obtain clear, fine detailed exposures. Place the microsight directly on a piece of white paper for focusing and move the manual elevation control (5) until the grains in the negative are clearly visible on the paper.
  9. The lens on the enlarger is sharpest at an estimated f stop of f8 or f11. Set the aperture and test the exposure using a “test strip,” in which a piece of paper is used to mask portions of the print paper during a series of exposures. For example: 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 or 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 2 exposures.) For each time point, move the masking paper down the paper. Develop the test strip 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 overexposed 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.
  10. Place the print paper on the feed shelf of the Ektamatic film processor with the exposed surface facing downward. The rollers will pull the paper through the activator and the stabilizer sections and out to the other side of the processor. The print can now be viewed under room lights to check the exposure, focusing, etc.
  11. Place the developed print in fixer solution (which is poured in a print tub to a depth that will cover prints) for 5 minutes.
  12. Transfer the print to a washer tub and rinse for 1–2 minutes. Water will travel across the prints with whirlpool action, eliminating the fixer more effectively than with standing water alone.
  13. Hang the prints to dry or dry them flat on a counter top at least 30 minutes.

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 52°C, 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).
  • Kodak Fixer. Slowly pour powdered fixer (680 g) into 3.8 L of water (not above 26.5°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; most continuous tone films should be fixed for 5–10 minutes with agitation.
  • Photo-Flo 200 solution, Kodak. Add 5.5 mL of Photo-Flo to 1.1 liters of water.
  • Scum will appear on the developed film if the Photo-Flo concentration is too high.
 
     
 
 
     




     
 
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