The objectives of pretreatment are to
stop the formation of the spindle
increase the number of metaphase cells by arresting the chromosomes at the metaphase plate
contract the chromosome length for ease of counting and to accentuate the primary constrictions
increase the viscosity of the cytoplasm
facilitate the penetration of the fixative by removing barriers such as the cell wall
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.
pretreatments in ice-cold water.
|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
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.
Chemical pretreatments .
Removing the cell wall is
required so that treatments will be absorbed. This can be done
with enzyme treatments such as protease or cellulase,
enzymes commonly extracted from from snail stomachs. The
alternative is hydrolysis with an acid fixative.
- 8-hydroxy quinoline
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.
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.
|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
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.
|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%)
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
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 solution is a dark red.
Carmine can be replaced by orcein to prepare aceto-orcein.
Image from Barley Genetics Newsletter, USDA
The stain is effective for somatic chromosomes of barley and wheat. Roots are transferred into the stain after pretreatment. The stain acts as a fixative.
2. Feulgen stain.
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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