Chromosome Preparation

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Chromosome Preparation
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Introduction

• every cytologist has his favourite technique for
  chromosome preparations.
• different cell types (animal or plant somatic
  or germline cells) may need different techniques.
• none is ideal for all purposes, though successful
  in practised hands, require considerably
  experience before they give consistently good
  results.
• basically, the chromosome preparation involves a
  few major steps Pre-treatment, Fixation/Storage,
  Squashing and Staining.

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Pre-treatment

• mainly for karyotype study.
• preventing the formation of microtubules,
• dividing cells are arrested at mitotic
  metaphase, thus increases the mitotic index.
• chromosomes are more contracted and shorter,
  making chromosome counts easy.

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Pre-treatment

• for root tips and meristematic tissues, the
  treatment is 4-6 hours in aerated pre-treatment
  solution (0.05 colchicine) at temperature below
  18C.
• for animal cells (white blood cells), the
  treatment involves incubation of dividing cells
  in 0.05 colchicine solution at 1-2 hours at
  37C.
• colchicine inhibits the activity of the spindle
  during cell division, resulting in the
  accumulation of cells with chromosomes scattered
  through the cytoplasm instead of aggregated on
  the spindle equator.

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Fixation

• fixation is an attempt to kill the material
  rapidly in such a way that the internal
  structures are preserved in a life-like form.
• it involves denaturation of proteins.
• treatment incubate tissues/cells in fixative
  (absolute ethanol, chloroform, acetic acid,
  formalin)

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Squashing

• for plant material and perhaps to a lesser
  extent for animal material.
• to spread cells out so that the observation on
  chromosome can be done easily.
• treatment press down on coverslip, without
  lateral movement, through blotting paper.

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Staining

• until the late 1960s and early 1970s, chromosome
  spreads were stained with Feulgens reagent (a
  purple dye that reacts with the sugar molecules
  in DNA) or with aceto-carmine (deep red dye) and
  aceto-orcein.
• intercalating agent (with insertional property
  insert between the base pair of DNA) is being
  used more routinely now.
• intercalating agent Quinacrine (fluorescent),
  Giemsa (non-fluorescent), etc.

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Preparation of cells for cytological analysis
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Mitotic chromosomes (metaphase) of incense cedar
(Calocedrus decurrens) stained by aceto-carmine