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Procedure of staining and slide preparation to study mitosis in root tip cells of onion

MATERIALS REQUIRED FOR THIS STUDY

    1. Onion root tip 10. Aceto-carmine solution
    2. Microscope 11. Acetic acid (45 %)
    3. Watch glass 12. Scissors
    4. Cover slip 13. Forceps
    5. Slide 14. Needle
    6. Spirit lamp 15. Fine and coarse cloth piece
    7. Hydrochloric acid (10 %) 16. Soft brush (Camel hair brush)
    8. Iron alum solution (2 %) 17. Sand paper: 1 small piece
    9. Haematoxylin stain (0.5 %)
      Note: In laboratory, students are expected to be equipped with the materials listed in bold and italic letter
    10.    

STAINING

  1. Two to three fixed root tips were taken from fixative and were washed in water for several times in the watch glass.
  2. Water was completely removed and a small quantity of 10% hydrochloric (HCl) acid was given in the watch glass containing the root tips.
  3. The root tips were warmed gently and intermittently over a spirit lam flame for 2-3 minutes, until the roots were sufficiently soft.
  4. The watch glass containing the roots was allowed to cool and then the HCl solution was thrown away and softened roots were washed in water for several times.
  5. The root tips were treated in 2% iron alum solution for 2-3 minutes in the watch glass and then washed with water for several times.
  6. The root tips were stained with 0.5% Haematoxylin stain for 2-3 minutes in the watch glass.
  7. Stained root tips were washed in running water for several times and finally kept in water in the watch glass.

SLIDE PREPARATION

  1. One stained root was taken on a clean slide and the root tip end (about 1 mm) was cut in a small amount of dilute Aceto-carmine solution. The back portion of the root tip was then thrown away.
  2. A small amount of dilute Aceto-carmine or 45% Acetic acid was given on the root tip on the slide.
  3. The slide was then prepared by squash method as follows:
    a) A clean cover slip was placed on the material avoiding air bubble.
    b) By holding the cover slip gently with a finger tip the material was gently tapped with the back end of the needle handle to the cells uniformly.
    c) If air entered under the cover a little amount of the dilute Aceto-carmine was added in the edge of the cover slip, on the other hand if needed excess liquid was blotted out from the edge of the cover slip.
    d) The following activities were repeated as necessary:
    • Gentle tapping
    • Warming
    • Cooling
  4. The slide was then ready for observation under the microscope low power objective (10x) first, then medium power objective (40x). Oil immersion (100x) was never to be used.
  1. Laboratory rules (lab coats, instruments etc.)
  2. Able to prepare slide from root tip cells of onion
  3. Able to spread cells in the slide
  4. Able of handle microscope properly
  5. Able to adjust microscope at the critical illumination
  6. Clean up bench and wash hands

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