PLNT3140 Introductory Cytogenetics - Lecture 4, part 2 of 2

PLNT3140 Introductory Cytogenetics
Lecture 4, part 2 of 2

B. Pretreatment of material

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.

1. Physical pretreatments such as ice-cold water. Pretreatment with ice cold water is widely used for the chromosomes of the Graminae species wheat, barley and rye. Immersion in ice cold water stops most cells at metaphase when chromosome contraction is highest. It also accentuates the 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.

The chromosomes are generally pretreated for 12 to 24 hours in a refrigerator. 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.

2. Chemical pretreatments .

All chemical pretreatments have similar effects.

Colchicine - The most commonly used chemical pretreatment is the alkaloid colchicine, used at a low concentration (0.2 to 0.5% for 1 to 2 hours at room temperature).

Colchicine is a microtubule inhibitor. Pretreatment with colchicine disrupts spindle fibres, arresting the cells at metaphase which makes chromosome counts very efficient. 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 suitable in the analysis of most higher organisms. It emphasizes the primary and secondary constrictions and shortens the centromeric region. The effects of colchicine are reversible and it is possible to induce chromosome doubling in a cell with higher concentrations.

Other chemical pretreatments are:

8-hydroxy quinoline - An antioxidant which is very effective for plants with large chromosomes because it accentuates primary and secondary constrictions. This technique combines the advantages of a pretreatment ( both chemical and cold temperature) with those of fixation in hydrochloric acid and of heat treatment.

The excised root tips are pretreated in a 0.002 M solution of 8-hydroxy quinoline (0.058 g dissolved in 200 cc warm distilled water) at 10 °C, followed by running tap water for 3-4 hours. This results in shortening of chromosomes ( as with colchicine) and facilitates spreading by inactivation of the spindle fibres. The unaffected centromere region becomes extremely clarified as the two arms of the chromosomes undergo heavy contraction at the opposite ends. The secondary constrictions are also clarified including the Nucleolar Organiser Region.

Mono-bromonapthalene has the same effect as colchicine. It arrests cell growth, results in inhibition of spindle formation and also in shortening and straightening of chromosomes. It is barely water soluble so a saturated aqueous solution has to be used for pretreatment from 2 to 4 h at room temperature. Chromosomes after pretreatment do not respond to most of the fixing agents because the chromosomes undergo chemical changes and the cytoplasm is altered so that it does not allow the penetration of certain fixatives. Farmers fixative (95% alcohol and glacial acetic acid in a 3:1 ratio) is a suitable fixative. The root tips after pretreatment with mono-bromonapthalene and using the proper fixative are easier to handle and give good results with most staining procedures

Paradichlorobenzene is very effective for plants with small chromosomes. It is also used as a saturated aqueous solution for 2 to 21/2 hours at room temperature.

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.

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.

C. Fixation

Killing and fixation are two distinct processes but are usually carried out by means of a single mixture of various reagents.

Killing, the sudden and permanent termination of the life processes of all the cells of the organism or tissue concerned, precedes fixation since the reagents that do the killing usually penetrate the tissues faster than those that cause hydration or 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.

Cell structures are defined as

a) clearly recognised entities in the living cells eg. nuclei, chromosomes, plastid

b) objects possessing a bounding membrane eg. vacuoles- not clear in living cells

c) entities that are not visible in living cells, appear under widely diverse conditions and after fixation eg. the spindle fibres.

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 to give desired results.

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.

3. The volume of the fixative should be approximately 10 to 12 times that of the tissue being fixed.

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 treatment. 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.

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.

1. Carnoy's solution.

1 part glacial acetic acid to 3 parts ethanol (95-100%).

This fixative is prepared fresh for roots and anthers and the material is kept in the fixative for 24 h at room temperature then stored in the cold.

2. Carnoy's solution II.

1 part glacial acetic acid, 3 parts chloroform and 6 parts ethanol. This modification is used for PMC. The chloroform increases the definition where oils and fats may interfere.

3. Propionic acid alcohol solution

1 part Propionic acid, 3 parts ethanol (95-100%) and 1g 100 ml fixative for meiotic materials only.

This fixative is good for plants with small chromosomes.

Clearing and Maceration ( Hydrolysis)

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

Hydrolysis is a very 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

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.

1. Aceto-carmine stain - 1 g of carmine powder is added to 100 ml of 45% boiling glacial acetic acid , then cooled rapidly and filtered into a dark bottle. The stain solution is a dark red.

Carmine can be replaced by orcein to prepare aceto-orcein.

Acetocarmine-stained barley chromosomes.
Image from Barley Genetics Newsletter, USDA
http://wheat.pw.usda.gov/ggpages/bgn/12/12p47.html

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.

1 g basic fuchsin is dissolved in 200 ml boiling distilled water and the mixture is shaken, cooled and filtered. 30 ml 1 N HCl is added, then 3 g potassium metabisulphite and 0.5g activated charcoal. The bottle must be closed tightly, wrapped in foil and stored in the dark. The feulgen stain fades in the light. The stain is specific for DNA in chromosomes only and can be used with a microspectrophotometer for measuring DNA content.

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.

E. Slide Preparation

The material is spread on the slide and stained using a drop of stain. A cover slip is added and the slide is heated gently.

The material is then spread by pressing or tapping the cover slip with thumb or needle etc. It is important not to allow lateral movement of cover slip to occur as this causes the cells to be distorted. A temporary preparation may be sealed with nail polish or paraffin wax.

To make the slide permanent, put it on a block of dry ice which causes it to freeze from bottom up. The cells stick to the slide and the cover slip can be removed with razor blade. The water content of cells must be replaced with mounting medium which are resins such as Canada Balsam.

F. Data Recording and Micrometry

The use of the Camera Lucida will be demonstrated in the laboratory. A combination of mirrors and prisms is used to bring images and pencil into same plane for drawing. It is important to make sure everything is parallel or perpendicular to avoid distortion. The image produced by the Camera Lucida is better than a photo because you can focus through the specimen and tell which chromosome is on top and provides a better representation of what is there. A photograph has restricted depth of focus and requires a much higher quality of preparation.

Finders. The mechanical stage reading uses the Vernier scale- see laboratory notes. The scale works well on one microscope but the readings cannot be transferred from one microscope to another.

The objective slide marker is mounted in the nosepiece, and is spring loaded.

A field finder is an alternative way of marking the specimen. These have 2500 squares in 25 mm square area of slide and use the co-ordinates A-5 etc. Micrometer are used for measurements of arm lengths or arm ratio. They are fitted into the eyepiece and must be calibrated for each objective with the stage micrometer.

Digital Imaging

Altthough our student lab doesn't have digital imaging equipment, most research labs you are likely to work in do. Good examples of what you can do with digital imaging can be found on at web sites for companies that specialize in microscopes. One example is from Zeiss (http://www.zeiss.com) under "Documentation."

http://www.zeiss.com/4125681F004CA025/Contents-Frame/286BA4D22B14DEE985256B4A007C3686

In particular, have a look at "Interactive Measurement".

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