This Power Point Presentation entitled " Cytological Methods" explains steps in preparation of cytological slides to study mitosis in higher plants with the help of root tips procured from onion and garlic bulb and germinating seeds and also to study mitosis a in Charophytes as Chara and Nitella. Also describes meiotic preparations .

1. CYTOLOGICAL METHODS
Prof. Ichha Purak
Department of Botany
Ranchi Women’s College, Ranchi
11/14/2021
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2. METHODS OF SLIDE PREPARATION FOR CYTOLOGICAL STUDIES
A) Plant mitosis (Onion,Garlic,Lens,Vicia,Pea)
B) Mitosis in Charophytes (Chara,Nitella,Tolypella )
C) Plant Meiosis (Onion,Rhoeo discolor )
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3. A. Plant mitosis (Onion,Garlic,Lens,Vicia,Pea)
STEPS :
 Selection of material
 Root tip cutting
 Pretreatment of root tips
 Fixation of root tip
 Storage of root tips
 Staining
 Temporary slide preparation
 Tapping
 Squashing and Sealing
 Making the slide permanent
 Observation
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4. SELECTION OF MATERIAL
Mitosis occurs in all meristematic cells so can be worked out from
root tips, shoot tips, leaf tips etc . Among these root tips are the most actively growing
parts and are devoid of pigments ,so can be used to study mitosis.
For the demonstration of different stages of mitosis, root tips of Allium cepa are the most
ideal material.
Root tips can be obtained easily from bulbs (Onion and Garlic - whole or cloves ) /
germinating seeds (Lens,Vicia,Pea,Gram, Hordeum /Onion,Groundnut etc.)
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5. Method of growing onion/garlic root tips from bulbs
Old roots from bulb of onion or garlic are cut and removed by sharp knife
Bulbs are placed inverted on wide mouth bottle or specimen tube or ice tray
having water or on a plate having wet sand
Left for 1-2 -6-8 days, if temperature is high ,covered by poly bag to keep
moist
White coloured root tips emerge out in bunches in 2-4 days
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7. Methods of growing root tips from seeds of Lens culinaris / Vicia faba /
Gram/Pea /Allium cepa /Trigonella/Groundnut
Fresh and healthy seeds are taken
a) The seeds are washed and soaked in water for one day and then seeds
are spread under the layer of soil or in petri plates with filter papers or
cotton or sand in plate. Or seeds are tied up in wet cloth .
Seeds are kept moist.
b) Generally seeds germinate in 3-4 days.
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Lentil Root tips Green gram root tips

8. Cutting the Root tips and Pretreatment
When root tips are about ½ to 1 cm long they are cut with sharp blade or
scissor. Cut root tips are then subjected to pretreatment
For it cut and washed root tips are kept in saturated solution of para di chloro
benzene (Pretreating Agent) for 1/2-1-2 hours.
Pretreatment softens the tissue and clears cytoplasm and helps in better
staining of chromosomes
Paradichloro benzene - It is a white crystalline organic substance sparingly
soluble in water.
Besides PDCB there are several other pre-treating agents.
 8 hydroxyquinoline and Oxyquinoline
 Colchicine
 Bromonapthalene
 Cycloheximide
 Chloral hydrate
 Coumarin
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9. FIXATION OF ROOT TIPS
After Pretreatment, root tips are fixed in any of the fixative agent
FIXATIVE AGENTS :
1) Carnoy’s I Fixative (1:3 acetic acid & alcohol) with few drops of Ferric
Chloride ( Aceto alcohol)
2) Carnoy’s II Fixative (4:3:1 absolute alcohol & chloroform & acetic acid)
3) Ethyl alcohol
4) Formaldehyde
5) Acetic acid
6) Chromic acid
7) Potassium Dichromate
8) Mercuric chloride
Carnoy’s I Fixative is generally used for root tip mitosis .
Root tips after pre-treatment are transferred to fixative for about 24 hours.
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10. STORAGE OF ROOT TIPS
The fixed root tips can be stored for future use in Preservative
70% Ethyl alcohol acts as good preservative
Preparation by using formula – S1V1= S2V2
S- Strength V- Volume
70 cc Absolute alcohol + 30 cc DW = 100 cc 70% Alcohol
77.7 cc 90% Alcohol + 22.3 cc DW = 100 cc 70% Alcohol
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11. STAINING
The chromosomes are colour less so stained to make visible under microscope .
Different Kinds of staining reagent can be used
a) Acetocarmine and b) Aceto orecin ( Prepared solution of 1% 25ml pack
available ) c) Feulgen stain
d) Toluidine Blue, 0.05% solution available in 100ml & 500ml packs
In our laboratory generally Acetocarmine or Aceto- orecin are used to stain root tips,
Meiotic slide preparation ,antheridia of Charophytes, algal materials as
Rhizoclonium,Oedogonium ,Cladophora, Chaetophorales and other material as
Pteridophytes and Gymnosperms .
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12. 1-2 gms of carmine/orecin is dissolved in 100 cc of 45% glacial acetic acid in conical
flask.
The mixture is then placed on tripod stand with wire gauze, heated for about 15 minutes
with the help of burner . While heating the mixture is continuously stirred and then
cooled at room temperature
Filtered and stored in dark coloured bottle.
The preparation is confirmed by blotting paper test.
For staining root tips are taken out of preservative
Placed in a watch glass. Acetocarmine or Acetoorecin solution is added
A few drops of N HCL may be added, which helps in dissolving middle lamella and cells
are separated easily.
The material is gently heated over spirit lamp for about 5- 15 minutes or in oven at 60
℃ or hot plate
For good staining red hot needle can be inserted into acetocarmine Aceto-orecin
solution while heating over spirit lamp
By heating root tips become ready for preparing cytological slides
PREPARATION OF ACETOCARMINE / ACETO- ORECIN ( 1-2 %)
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13. Preparation of temporary cytological slide by Squash Method
Requirements :
Watch glass, spirit lamp, Slides, Cover slips ,Needle,
Blotting paper,Brush,Tapper ,Scissor, Blade, cutex or wax
45% acetic acid in dropping bottle, Acetocarmine solution
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14. Root cap
Mitotic region
cell elongation
region
Root tip
On it a stained root tip is placed ,if root tip is long it is made short by cutting with
razor blade. For good separation,less material should be taken.
In the centre of a clean slide few drops of 45% acetic acid is added .
On it a clean coverslip is placed with the help of needle.
Care should be taken that bubbles are not introduced while adding coverslip.
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15. The slide with material is slightly heated over spirit lamp.
The heated slide is kept in filter paper folds.
The slide is then gently pressed with needle tip or gently tapped with flat
tapper to spread the heated material. (Tapper must have flat surface, may be a
glass rod, brush end etc.)
The spread material appears as spray painting
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16. Observation of cytological slide under microscope
The slide for observation is placed on stage of compound binolular / trinocular
microscope below the objective lens
Different stages of division are located under microscope, first at low power
magnification, attempts are made to see whether cells of root tip are separated and in
metaphase plates chromosomes are separated or not.
If not, few drops of 45% Acetic Acid from the sides of coverslip are added, again gently
heated over spirit lamp and tapped . Again observed under microscope to locate stages
of mitotic division.
During observing under microscope ,care should be taken that slide does not dries up,
for this 45% AA is regularly added from sides of cover slip.
This also helps in removing the excess stain taken by the cytoplasm and enhances
visibility of chromosomes ,develops contrast.
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17. Squashing and Temporary sealing of slide
After proper separation has been done the slide is placed in filter paper folds on
Plain table surface. The slide is pressed or squashed by right hand’s thumb, to bring
the material and chromosomes in one plane.
Again 45% AA from sides of cover glass is added , left for 2 minutes, the slide is
sealed by applying cutex or melted wax on sides of cover glass with help of brush.
This temporary slide can observed for 2-3 days or even a week under microscope for
making diagrams ,taking photographs or Karyotyping purpose. These slides can be
kept in refrigerator.
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18. MAKING THE SLIDE PERMANENT
Wax or cutex sealed slide can not be kept for many days due to precipitation of stain.
It is therefore essential to make the slide permanent by dehydrating solvents and finally
mounting in euperol or DPX.
3 staining troughs are required for turning temporary slide to permanent slide
Steps : 2 Alternative Methods
1. Cutex or wax is removed with the help of blade
2. Slide is dipped in first staining trough containing 45% Acetic acid or 70% Alcohol
3. When coverslip separates from the slide, the coverslip and slide both are transferred
separately by forceps into second trough containing 45% acetic acid and Butanol in
1:1 Ratio or 90% alcohol
4. Then finally in one minute both slide and coverslip are transferred to third staining
trough containing Butanol or Absolute ethyl alcohol
5. Slide and coverslip with the help of forceps are carefully taken out of
the trough,placed on filter paper.
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19. Precautions
As both slide and cover slip contain stained cytological material ,care should be taken to
remember the side on which material is present while transferring them 1st staining trough
to second staining trough.
Slide and coverslip should only be dipped for one minute because if left for longer time
the material may be washed out
Microphotographs of well separated metaphase plates and different stages of mitosis
can be taken by Nikon Digital Camera and Trinocular microscope
with the help of Adaptor or Trinocular microscope with LED or Monitor attachment
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The slide is mounted with euperol or DPX using a new coverslip and coverslip having
cytological material is also mounted using fresh slide with euperol or DPX
So from one temporary slide two permanent slides are formed .

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Photographs taken by Nikon digital camera
Antheridial filaments of Chara showing prophase of Mitosis

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Observation under low power of
Microscope
Observation under high power of
Microscope

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Prof (Dr) Ichha Purak.
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DATE – 14/11/2019
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Different stages of Mitosis in Allium cepa
prophase Metaphase Early Anaphase
Late Anaphase
Telophase

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SOME METAPHASE PLATES

27. B. METHODS OF CYTOLOGICAL SLIDE PREPARATION OF CHARA
AND NITELLA AND OTHER CHAROPHYTES
(almost same as onion root tip mitosis)
For cytological studies antheridia/ Globules are selected and stages of mitosis are
observed in the antheridial filaments (mostly show synchronous stages)
The iron alum aceto-carmine squash technique as proposed by Godward (1948) is
employed for cytological preparations.
Using pretreatment with PDCB, Fixation in 1:3 aceto alcohol,preservation in 70%
C2H5OH,staining with 1.5% Acetocarmine solution
Steps are same as Onion Root tip mitosis ,differs only in selection of material,Time of
fixation and Staining and Preparation of slide
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28. FIXATION : After pretreatment material is transferred to fixative . As fixative Carnoy’s I
is used which is a mixture of one part of glacial acetic acid and three parts of absolute
ethyl alcohol with few drops of ferric chloride solution.
The materials can be fixed at the site of collection as well as in the laboratory from
cultures. The material is left in fixative for 24 hours.
Time of Fixation : Material for cytological studies is generally fixed in the morning
hours i.e. 9 A M to 11.30 A M. 12.30 P.M.
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29. For staining antheridia, the apical portions of Chara and Nitella containing Nucule and
globule are heated in 1.5 % acetocarmine in watch glass or wide test tube for about half
an hour.
Some stained reproductive branchlets are transferred to 45% acetic acid
After that 3-4 antheridia are selected under dissecting microscope with the help of sharp
and pointed needle, inserted into antheridia,are picked up and placed in middle of a
clean slide with few drops of 45% Acetic acid and then coverslip is gently added.
After that slide is gently warmed and is then kept in folds of blotting paper.
With the help of tapper the slide is tapped gently so that cells get separated and can be
observed for stages of mitotic division.
During tapping excess stain of cytoplasm is removed by adding 45% acetic acid from
the sides of cover slip repeatedly.
Rest of the processes are same as that of Onion root tip mitosis
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Picking antheridia of Chara with the help of needles under dissecting
microscope

31. Chara corallina
Interphase
Prophase
Anaphase
Telophase
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32. Chara corallina
Metaphase
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33. PLATE SHOWING PROPHASE
,METAPHASE ,ANAPHASE &
TELOPHASE
ANAPHASE
PROPHASE
METAPHASE
Chara fibrosa
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METAPHASE
4 CELLS OF ANTHERIDIAL
FILAMENT SHOWING
METAPHASE
Chara fibrosa ( n=56 )

34. C.METHOD OF CYTOLOGICAL SLIDE PREPARATION TO STUDY
PLANT MEIOSIS (Onion,Rhoeo discolor etc. )
Selection of Material
In higher plants meiosis occurs in pollen mother cells which are present in anthers and
also in megaspore mother cell (only one ) which lies in ovule.
Anthers contain many pollen mother cells (PMC) which can be very easily exposed by
bursting anthers. So anthers are excellent source of meiotic material.
Flower buds of different sizes are collected in morning hours and are fixed in
acetic-alcohol (1:3) for about 24 hours
The buds are preserved in 70% alcohol
Buds of Rhoeo discolor
Buds of Ixora coccinia
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35. For staining buds are dissected to procure anthers
After that 1 or 2 anthers are placed in the centre of a clean slide with few drops of
1.5% acetocarmine and a coverglass is placed on the material.
The slide is gently heated over a spirit lamp flame .
The slide is kept between folds of a blotting paper and a gentle pressure is applied
over the cover glass to induce uniform spreading of pollen mother cells (PMCs) and to
remove excess stain (Tapping not needed)
The cover glass edges are sealed with cutex or wax.
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36. The slides then can be observed under microscope to locate different stages of
meiosis I and II
As prophase 1 sub stages leptotene,zygotene,pachytene,diplotene and diakinesis
(Bivalents ), Anaphase 1 ,Telophase 1 and Metaphase 2 and Anaphase 2 and
Telophase 2 .
Chromosomal abnormalities can also be located as meiotic anaphasic bridge,
translocation heterozygote in Rhoeo discolor anthers, micronuclei formation and
chromatin clumping
Microphotographs can be taken with the help of trinocular microscope having
attachments as adapter and digital camera in of place of eyepiece
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Meiosis I Onion
Meiosis II Onion
Translocation Heterozygote In
Rhoeo discolor
Tetrads (Meiosis II) in
Carica papaya
Hordeum vulgare 2n=14,
7 bivalents
SOME MEIOTIC STAGES

38. REFERENCES
Chromosome Techniques Theory and Practice Third Edition
Arun Kumar Sharma and Archana Sharma .Aditya Books ,New Delhi
College Botany Practical Volume-1 S C Santra, T P Chatterjee and
A P Das. New Central Book Agency 1989
Godward, M B E 1948. The iron-alum acetocarmine method for algae.
Nature 161
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