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How to Perform an Allium Cepa Test

Onions (Allium cepa) are used to assess environmental hazards by allowing developing roots to come into contact with substances to be tested. After sufficient exposure, the roots are taken off, stained and macerated so that cell chromosomes can be seen under a light microscope. Toxicity is evidenced if chromosome abnormalities or cell division abnormalities are detected. Root tips are used because the cells there are actively growing and dividing, giving a good chance to see chromosomes during different stages of cell division.

Things You'll Need

  • Onions
  • Knife
  • Water
  • Beakers or other containers to hold liquids and onions
  • Test liquids
  • Razor blade or scalpel
  • Vials
  • Aceto-alcohol fixative
  • Microscope slides
  • Cover slips
  • Hydrochloric acid
  • Stainless steel macerating rod
  • Carbol fuchsin stain
  • Aceto-orcein stain
  • Acetic acid
  • Canada balsam
  • Light microscope
  • Camera attachment for microscope
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Instructions

  1. Setting Up the Test

    • 1

      Remove the outer dry skin of 12 onions. Scrape the bottom of the onion around the bottom plates without damaging the area from which the roots will emerge.

    • 2

      Select beakers that have a mouth wide enough to hold the bottom of the onions immersed in the test liquids. Fill the beakers with distilled water and place the onions in the beakers in contact with the water. Shield the sides of the beakers with aluminum foil to simulate underground darkness. Change the water daily.

    • 3

      Examine the onion bases after two days. Roots should have formed. Evaluate the root growth and discard the two onions with the weakest root growth.

    • 4

      Set up a second series of five beakers each containing a sample of the material to be tested, such as wastewater or industrial runoff. Put five of the onions into these beakers. Retain the other five in distilled water as test controls. Allow the roots to grow for another 24 hours.

    Preparing the Roots

    • 5

      With a scalpel or razor blade, harvest five to six root tips from each onion, recording which were controls and which were test subjects. Keep roots from individual onions separate in small labeled vials.

    • 6

      Fix root tips immediately in aceto-alcohol in a 1:3 ratio. Place the root tips for an onion on a microscope slide and macerate them in 1N hydrochloric acid, which is the same as 1 molar hydrochloric acid, at 60 degrees Centigrade for three minutes. Stain macerated tips with carbol fuchsin stain, a mixture of phenol and basic fuchsin that binds to elements in cell walls so they become more visible under light microscopy.

    • 7

      Add 2 percent aceto-orcein stain in 4 percent acetic acid. This colors the chromosomes. Stir with the macerating rod. Apply a cover slip, pressing it down to squash the root tips. Either seal the edges with quick-drying fingernail polish or permanently mount the preparations in Canada balsam, resin from the balsam fir tree mixed with either essential oils or xylene. Number the slides so the results will be recorded but not apparent whether each is a test subject or a control.

    Recording Results

    • 8

      Place the microscope slide on the stage of a light microscope. Examine the field under lower magnification, switching to higher magnifications to examine chromosomes.

    • 9

      Record the chromosomal aberrations on a data sheet, recording information by the coded number for each onion. Include incompletely separated or broken chromosomes, micronuclei (extra nuclei formed near the end of cell division that can contain chromosomal debris), chromosome rings and sticky and disturbed chromosomes in different stages of cell division. Photograph each slide.

    • 10

      Analyze the data using statistical analysis. Interpret the results to evaluate whether or not the test solution is genotoxic.

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