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 fluid

A B
3 part 95% alcohol 6 part 6 part
1 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 Toothpicks
o Fresh onion root tips
o Distilled water
o A pair of forceps
o Hydrochloric acid, 1M solution
o Watch glass
o Single-edged razor blades
o 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.

Now
Cover 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.

Now
Cover 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.

Now
Cover 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

Press it down firmly with thumb, inside the blotting paper

The stain coming out from the cover glass will be absorbed by 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.
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).

Constantly make fine adjustments of focusing to view the

chromosomes clearly.
Identify the stage of cell under division
 Mitosis

It has following major stages

Interphase

Telophase Prophase

Anaphase Metaphase
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 disappeared
Spindle 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 Fixative
o 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.
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.

Rotate the nose piece to fix the high power objective.

Find the same cell(s).

Constantly make fine adjustments of focusing to view the

chromosomes clearly.
Anther
Pollen sacs
[2n]

Diploid cell
[2n]
Diploid cell Pollen grain
(2n) (n+n)

Microspore (n)
Identify the stage of cell under division
 Meiosis

It has following major stages

Meiosis I Meiosis II

Interphase Interphase

Telophase Prophase Telophase Prophase

Anaphase Metaphase Anaphase Metaphase

 INTERPHASE

The chromosomes are

indistinguishable from one another
 PROPHASE

Chromosomes are prepared for

division by shortening and thickening
of chromatids
Nucleolus is visible
 PROPHASE I

It has following sub-stages

Leptotene

Zygotene

Pachytene

Diplotene

Diakinensis
 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 I
Chromosomes 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 I
The chromosomes begin to move towards pole centromere do not split
and the chromatid associated with each centromere move as a unit
The actual reduction of chromosome number occur at this stage
Anaphase I
 Telophase
The 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 II
The centromere splits lengthwise in the
chromosome and the chromatids begin to move
towards pole
 Telophase II
The 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
The 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)
 Allergenic Pollen (SEM x1,000)

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

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.