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Preparation of slide to study of cell division

Dhirendra khare Plant Breeding and Genetics

JNKVV, Jabalpur (India)

Study of cell division

Principle

To Study the cell division (either Mitosis or Meiosis), stained chromosomes are observed with the help of microscope in the killed and fixed tissues.

Fixing and killing of material

To study the cell division the cell has to be killed and fixed at the particular stage before observation

The solution in which material is fixed or killed is known as fixing or killing solution

It is of two types

Farmer’s fluid

Carnoy’s fluid

Fluid for killing and fixing of tissues

Farmer’s fluid Chemical Carnoy’s fluidA B

3 part 95% alcohol 6 part 6 part1 part Glacial acetic acid 3 part 1 part

- Chloroform 1 part 3 part

These chemicals should be prepared fresh immediately before fixing of material

Solution for staining of chromosomes

To observe chromosomes certain stains are used they provide colour to chromosome and create contrast

between cytoplasm and chromosome

In general three types of stains are used❑ Aceto carmine❑ Aceto orcein❑ Feulgen stain

Preparation of aceto-carmine

Chemicals required

Acetic acid and Carmine

First prepare 45% acetic acid solution in distilled water

Boil it

Add 0.5g (1.5 to2.0%)carmine in 100cc boiling 45% acetic acid

Boil it for 1-2 minutes or until there is sudden change to a darker colour

Cool it

Filter it before storage

Preparation of aceto-orcein

Chemicals required

Acetic acid and orcein

First prepare 45% acetic acid solution in distilled water

Boil it

Add 2.0% orcein in boiling 45% acetic acid in a long neck flask

Boil it for 1-2 minutes or until there is sudden change to a darker colour

Cool it

Filter it before use

Mitotic cell division

To study the mitotic cell division following material is required

o Compound microscope o Toothpickso Fresh onion root tips o Distilled watero A pair of forceps o Hydrochloric acid, 1M solutiono Watch glass o Single-edged razor bladeso Aceto-carmine solution

Obtain root tip from onion

Place a healthy onion bulb on a beaker with water at the base.

With in three to four days the root will come out from the base ( nearly 1-2cm long)

Collect these roots for observation

Obtain root tip from onion

Collect an onion root tip from the stock table and place it carefully on a clean glass slide

Obtain root tip from onion

Collect an onion root tip from the stock table and place it carefully on a clean glass slide

Locate the area of the tip where the milky white color changes to a dull white. At this point, cut off the tip and place it into a watch glass.

Obtain root tip from onion

Collect an onion root tip from the stock table and place it carefully on a clean glass slide

Locate the area of the tip where the milky white color changes to a dull white. At this point, cut off the tip and place it into a watch glass.

Discard the remaining part of the root.

With a clean dropping pipette, add 1M HCl to cover the tip completely.

Keep the tip in the solution for 6 to 8 minutes.

With a clean dropping pipette, add 1M HCl to cover the tip completely. Keep the tip in the solution for 6 to 8 minutes.

NowCover the entire tip with distilled water. Allow the tip to soak the water for 2 minutes.

With a clean dropping pipette, add 1M HCl to cover the tip completely. Keep the tip in the solution for 6 to 8 minutes.

NowCover the entire tip with distilled water. Allow the tip to soak the water for 2 minutes.

carefully transfer the tip to a clean glass slide, after 2 minutes.

With a clean dropping pipette, add 1M HCl to cover the tip completely. Keep the tip in the solution for 6 to 8 minutes.

NowCover the entire tip with the distilled water. Allow the tip to soak the water for 2 minutes.

carefully transfer the tip to a clean glass slide, after 2 minutes.

Add one to two drops of aceto-carmine stain to the tip.

With the razor blade, cut the tip into small pieces

With the razor blade, cut the tip into small pieces

Push the material stick on the razor blade back into the solution with the help of toothpick.

With the razor blade, cut the tip into small pieces

Push the material stick on the razor blade back into the solution with the help of toothpick.

Add one more drop of aceto-carmine

With the razor blade, cut the tip into small pieces

Push the material stick on the razor blade back into the solution with the help of toothpick.

Add one more drop of aceto-carmine

Place it over the slide and cover with glass cover slip

With the razor blade, cut the tip into small pieces

Push the material stick on the razor blade back into the solution with the help of toothpick.

Add one more drop of aceto-carmine

Place it over the slide and cover with glass cover slip

Roll it with a piece of blotting paper

With the razor blade, cut the tip into small pieces

Push the material stick on the razor blade back into the solution with the help of toothpick.

Add one more drop of aceto-carmine

Place it over the slide and cover with glass cover slip

Roll it with a piece of blotting paper

Press it down firmly with thumb, inside the blotting paper

With the razor blade, cut the tip into small pieces

Push the material stick on the razor blade back into the solution with the help of toothpick.

Add one more drop of aceto-carmine

Place it over the slide and cover with glass cover slip

Roll it with a piece of blotting paper

The stain coming out from the cover glass will be absorbed by the blotting paper

Press it down firmly with thumb, inside the blotting paper

Now carefully remove the blotter paper that have absorbed excess stain from the edges of the cover glass.

Now carefully remove the blotter paper that have absorbed excess stain from the edges of the cover glass.

The slide is placed on the stage of the microscope to observe the stages of Mitosis cell division.

Locate the cells of the root tip under microscope in low power

Locate the cells of the root tip under microscope in low power

Slowly focus the objective looking for small darkly stained structures inside the cells which appear large and rounded.

Rotate the nose piece to fix the high power objective. Find the same cell(s).

Locate the cells of the root tip under microscope in low power

Slowly focus the objective looking for small darkly stained structures inside the cells which appear large and rounded.

Rotate the nose piece to fix the high power objective. Find the same cell(s).

Locate the cells of the root tip under microscope in low power

Slowly focus the objective looking for small darkly stained structures inside the cells which appear large and rounded.

Constantly make fine adjustments of focusing to view the chromosomes clearly.

Identify the stage of cell under division

Mitosis

Interphase

Prophase

MetaphaseAnaphase

Telophase

It has following major stages

INTERPHASE

The chromosomes are indistinguishable from one another

Nucleolus is visible

PROPHASE Chromosomes are prepared for division by shortening and thickening of chromatids

Nucleolus is visible

Metaphase Chromosomes are arranged in random manner on the equitorial plate of the cell

Nuclear membrane and Nucleolus are disappearedSpindle fibres are visible

Anaphase The centromere splits lengthwise in the chromosome and the chromatids begin to move towards pole

Telophase The chromosomes have completed their movement towards the pole and begin to disperse inside the

nuclear membrane

Interphase

Meiosis cell division

To study the meiosis cell division following material is required

o Compound microscope o Watch glass o Fixativeo Aceto-carmine solution

Obtain anther from bud

Select a sufficiently developed old bud at morning or evening time in case of pulses Collect a range of bud sizes including very small ones. Check the stages in each before collecting more material.

Placed it in the Farmer’s or Carnay’s fixative

Obtain anther from bud

select inflorescence that may appear 5-7 days later. It may be located inside the tightly rolled sheaths by feelings of fingers. Collect the inflorescence by opening the sheath with the help of razor blade.In an inflorescence of cereal the oldest anthers are in spikelets slightly above the middle .

Placed it in the Farmer’s or Carnay’s fixative

Obtain anther

The anthers from the collected material is placed on a slide and smeared with the help of needle in acetocarmine stain.

Obtain anther

The anthers from the collected material is placed on a slide and smeared with the help of needle in acetocarmine stain.It is covered with a cover slip.

Obtain anther

The anthers from the collected material is placed on a slide and smeared with the help of needle in acetocarmine stain.It is covered with a cover slip.

Heat it gently.

Rotate the nose piece to fix the high power objective. Find the same cell(s).

Locate the cells under microscope in low power

Slowly focus the objective looking for small darkly stained structures inside the cells which appear large and rounded.

Constantly make fine adjustments of focusing to view the chromosomes clearly.

Diploid cell [2n]

Pollen sacs [2n]

Anther

Microspore (n)

Pollen grain (n+n)

Diploid cell (2n)

Identify the stage of cell under division

Meiosis

It has following major stages

Meiosis I Meiosis II

Interphase

Prophase

MetaphaseAnaphase

Telophase

Interphase

Prophase

MetaphaseAnaphase

Telophase

INTERPHASE

The chromosomes are indistinguishable from one another

PROPHASE

Chromosomes are prepared for division by shortening and thickening

of chromatidsNucleolus is visible

Diakinensis

Diplotene

Pachytene

PROPHASE I

It has following sub-stages

Zygotene

Leptotene

PROPHASE I

Diakinesis

The chromosome continue to contract and the nucleolus begin to disappear

Early Prophase I

Mid prophase

Late prophase

Diakinesis

A summary of the stages of synapsis and desynapsis

Metaphase IChromosomes are arranged in random manner on the

equitorial plate of the cell Nuclear membrane and Nucleolus are disappeared

Spindle fibres are visible

Metaphase I

Anaphase IThe chromosomes begin to move towards pole centromere do not split

and the chromatid associated with each centromere move as a unitThe actual reduction of chromosome number occur at this stage

Anaphase I

TelophaseThe chromosomes have completed their movement towards the pole and begin to disperse inside the

nuclear membrane each nucleus at this stage is now one n rather than 2n

Telophase

Prophase II

Meiosis 2

Metaphase II

Chromosomes are arranged in random manner on the equitorial plate of the cell

Metaphase II

Meiosis 2

Anaphase IIThe centromere splits lengthwise in the

chromosome and the chromatids begin to move towards pole

Telophase IIThe chromosomes have completed their movement towards the pole and begin to disperse inside the nuclear membrane

Four reproductive cells are arranged in a formation

Telophase IIThe chromosomes have completed their movement towards the pole and begin to disperse inside the nuclear membrane

Four reproductive cells are arranged in a formation

Telophase II

Interphase II

Tetrads in the "callose special wall".

Tetrads completing the callose stage.

Released microspores.

Bicellular pollen

Pollen grain (n+n)

poplar, alder, timothy grass, ragweed, sagebrush, scotchbroom

Allergenic Pollen (SEM x1,000)

Pollen germination on the stigma.When pollen lands on the stigma, it germinates and forms a pollen tube that continues to elongate until it penetrates the style, enters the ovary and then enters the micropyle and deposits sperm cells in a specific location within the embryo sac.