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PLNT3140 Introductory Cytogenetics
Lecture 4, part 2 of 2

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B. Pretreatment of material

The objectives of pretreatment are to

In general, pretreatment causes physical changes in both cytoplasm and nucleus to assist in revealing morphology of chromosomes. There are specific pre-treatments for specific purposes and different pre-treatments for different species.

The tissue is generally pretreated for 12 to 24 hours in a refrigerator or ice-water bath. Depending on the time and the objectives, both the time and temperature can be manipulated to produce the best results. Longer pretreatment will cause the chromosomes to shorten which may be desirable.

Immersion in ice cold water stops most cells at metaphase when chromosomes are most condensed. It also accentuates the densely staining regions or  heterochromatin in the region of the centromere and the telomeres at each end of the chromosome.

There is a tendency for chromosomes to clump together and become sticky so spreading the chromosomes for a clear count is difficult.

Widely used for Graminae species wheat, barley and rye.
  • Immerse tissue in 0.2 to 0.5% colchicine for 1 to 2 hours at room temperature
  • Roots should be washed thoroughly after colchicine treatment to remove the alkaloid which might otherwise remain on the tissue surface and obscure the cell constituents. During washing, the dividing cells tend to enter interphase rapidly. Therefore washing cannot be prolonged. The pretreatment facilitates the penetration of the fixative at the subsequent stages of chromosome preparation.
  • Colchicine is a microtubule inhibitor. Pretreatment with colchicine disrupts spindle fibres,  arresting the cells at metaphase which makes chromosome counts very efficient. 
  • It emphasizes the primary and secondary constrictions and shortens the centromeric region.
  • By far, the most widely-used pretreatment for both plant and animal tissue.
  • The effects of colchicine are reversible and it is possible to induce chromosome doubling in a cell with higher concentrations.

PLEASE NOTE: Chemical pretreatments must be used with caution as their action is to arrest cell division in living tissue. Gloves should be worn when handling the chemicals or making up solutions. Avoid breathing the solutions.

C. Fixation

Fixation is the preservation of all cellular and structural elements in as nearly the natural living condition as possible. The role of the fixative is to fix or stop the cells at the desired stage of cell division without causing distortion, swelling or shrinkage of the chromosomes or with as little chemical and structural change of cell constituents as possible. It is required primarily in order that structures which are obscured or entirely invisible in the living cell may be made clearly visible and secondarily that the soft structures may be hardened sufficiently for further treatment.

Several factors affect fixation including temperature, pH, osmolarity, rate of penetration, rate of chemical and physical changes and length of fixation. Poor fixation makes it impossible to obtain good results from sectioning and staining.

Rules affecting Fixation:

1. The fixative should be carefully chosen for the tissue being studied.
2. Small organisms may be fixed whole eg. algae but larger one should be cut in smaller pieces to allow rapid and uniform penetration of fixative.

The example shows single-celled and multicellular microorganisms from pond water. Since each cell can be surrounded by fixative, and cellular volume is small, fixation can be highly efficient.


3. The volume of the fixative should be approximately 10 to 12 times that of the tissue being fixed.
Remember, the goal is to get the tissue to equilibrate with the treatment. At a 1:1 volume, the final concentration of the treatment in the tissue will be half the original concentration.  By using a higher ratio of treatment to tissue volume, the dilution of the treatment becomes insignificant.

4. Tissues which are waxy, cutinized or suberized may present technical difficulties especially for slower penetrating fixing agents. In plants, the epidermis is usually covered with a hydrophobic cuticle layer that prevents aqueous reagents from entering tissue. Hairy surfaces or air trapped in the material also interferes with the action of fixing agents. Dipping in alcohol before fixation may help or evacuation to remove air when material is in fixing agent.

There are several widely used fixatives.

Physical fixation. A quick freeze in liquid nitrogen is effective in maintaining good cell structure with very little diffusion and no significant change in enzymes. However, if ice crystals form they can rupture cells.

Chemical fixation
The secret of chemical fixatives is the balance between the properties of the reagents. For example the action of acids is to cause swelling while alcohols generally shrink cell structures. A mixture of acid and alcohol properly balances the two properties to maintain the cell structures in as life-like condition as possible. As well, acid denatures proteins, and depurinates DNA, which leads to nicking of the DNA backbone.
Carnoy's solution Glacial acetic acid:ethanol (95%) 1:3
24 hr. room temp
store in cold

Carnoy's solution II Glacial acetic acid:chloroform:ethanol (95%) 1:3:6
24 hr. room temp
store in cold
The chloroform increases the separation of aqueous and organic phases. This modification is used for pollen mother cells.
Propionic acid alcohol solution Propionic acid:ethanol 1:3

good for plants with small chromosomes.

Clearing and Maceration ( Hydrolysis)

Root Tips - Hot hydrolysis with 1N HCl (60 C) for 5 -15 min

Acid hydrolysis breaks peptide bonds in proteins and depurinates DNA, the latter leading to nicks (breakage of one strand) of the DNA backbone. Hydrolysis is a critical step to allow the cells to separate easily. When hydrolysis is complete , only the root tip remains white and the rest is transparent.

D. Staining of chromosomes

If you look at any tissue through a microscope, it will be mostly transparent. You won't see much detail, other than cell walls in plants.
The purpose of staining is to create 
optical contrast so that chromosomes or other cell structures can be seen through the microscope. There are specific stains for specific cell structures.

The stain is specific for DNA in chromosomes and can be used with a microspectrophotometer for measuring DNA content. Today, however, there are better fluorescent stains for DNA such as DAPI.

Treatment:The root tips must be hydrolysed in 1 N HCl (60 C) for 6 to 10 min prior to staining. After hydrolysis, the root tips are rinsed in distilled water and transferred to the Feulgen stain for 1 to 2 h at room temperature. The stained regions show pink and can be cut and placed on a slide. A drop of 1% aceto-carmine is added, then a cover glass.

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PLNT3140 Introductory Cytogenetics
Lecture 4, part 2 of 2

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