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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.