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Contents

Chapter Technique Page Nos.

1 Polytene Chromosomes from Chironomous Larva 3 - 4

2 Polytene Chromosomes from Salivary Gland of 5 - 6

Drosophila
3 Culture Method for Drosophila 7 - 8

4 Chromosomes of Musca domestica 9 - 10

5 Meiosis in Grass hopper 11 - 12

6 The Bone Marrow Culture 13 - 15

7 Testes Biopsy 16 - 17

8 Microchromosomes 18 - 19

9 Sex Chromosomes 20 - 21

10 Presence of Inactivated X-Chromosome in 22

Buccal Smear
11 Mammalian Whole Blood Culture (Micro-method) 23 - 25

12 Avian Leucocyte Culture 26 - 29

13 Lymphocyte Culture 30 - 33

14 Feather Pulp (in vivo) 34 - 36

15 Feather Pulp (in vitro) 37

16 Avian Cell Culture (Whole Embryo) 38 - 40

 

 
 ΀5(6($5&+7(&+1,48(6,102/(&8/$5%,2/2*<
*$5* *$5* *(1(7,&6$1'%,27(&+12/2*<΁

17 Transplantation of Ascites Cells in Mouse 41 - 42

18 Cytological Investigation of Mouse Ascites Cells 43 - 44

19 Study of Tumor Cells 45

20 Cytobiometry 46 - 47

21 Demonstration of Constitutive Heterochromatin 48 - 50

22 C-banding in Lymphocytes or Fibroblast Cells 51 - 52

23 Giemsa Banding 53 - 54

24 Reverse Banding 55 - 56

25 Satellite or Highly Repetitive DNA 57

26 Nucleolus Organizer’s Region 58

27 In situ Hybridization 59 - 66

28 Extraction of Genomic DNA from Calotes 67 - 69

29 Agarose Gel Electrophoresis of DNA 70 - 72

30 Polymerase Chain Reaction 73 - 77

31 RT-PCR 78 - 83

32 Production of Pectinase by SSF 84 - 87

33 Preparation of Stains and Reagents 88 - 92

34 References 93 - 96

 

 
 ΀5(6($5&+7(&+1,48(6,102/(&8/$5%,2/2*<
*(1(7,&6$1'%,27(&+12/2*<΁ *$5* *$5*


Polytene Chromosomes from

Chironomous larva

The polyteny in these giant chromosomes occurs due to endomitosis of

metaphase chromosomes which multiply almost 8 times resulting in a
giant (quite long and broad) chromosome.

Stock
1N HCl
Aceto-orcein
Aceto-alcohol, 1:3

Procedure

1. Hold the anterior portion of chironomous larvae with the help of

a needle and stretch the adjacent body with the help of another.

2. Trace out the salivary gland, which appears as two light milky
sacs.

3. Locate the giant cells of the salivary gland which are found in
the outer periphery of the gland.

4. Fix the glands in aceto-alcohol for 5 mins. And treat them with
1N HCl at 57oC for 1½ mins.

5. Stain with aceto-orcein at 57oC for 5 mins.

Observations

A close observation of salivary gland chromosome of Chironomous larva

reveals :

 

 
 ΀5(6($5&+7(&+1,48(6,102/(&8/$5%,2/2*<
*$5* *$5* *(1(7,&6$1'%,27(&+12/2*<΁

• Bands, which are deeply stained with orcein

• Interbands which take a little or no stain

• Puffs which do not take stain and are supposed to be the region
of RNA synhesis.

 

 
 ΀5(6($5&+7(&+1,48(6,102/(&8/$5%,2/2*<
*(1(7,&6$1'%,27(&+12/2*<΁ *$5* *$5*


Polytene Chromosome from Salivary

Gland of Drosophila
The name polytene is derived from poly meaning many and tene - strands
which shows that it is a many stranded structure. Polytene chromosomes,
also known as giant chromosomes, result from duplication of most of the
genetic material and subsequent failure of the duplicates to separate into
distinct chromatids. These somatic chromosomes are found in the
salivary glands of Dipteran flies such as Drosophila and also seen in the
secretory tissues like Malphigian tubules, rectum, gut, footpads of Diptera.

The advantage of studying polytene chromosomes lie in its cytological

YLVLELOLW\ PDSSLQJ LGHQWLI\LQJ VWUXFWXUDO FKDQJHV LQ FKURPRVRPHV DQG
monitoring the synthetic activity of individual genes at the cytological
level.

Stock
Fixative (1: 3 aceto -methanol)
Poel’s Medium
Acetic acid (50%)
Aceto orcein

Procedure

1. Pull out the salivary gland from the third instar larvae on a clean
slide containing a drop of Poel’s salt solution.

2. Fix the material in 1: 3 aceto-methanol for 30 secs.

3. Wipe out the fixative and add a drop of aceto-orcein stain. Keep
it as such for 10-15 mins.
 

 
 ΀5(6($5&+7(&+1,48(6,102/(&8/$5%,2/2*<
*$5* *$5* *(1(7,&6$1'%,27(&+12/2*<΁

4. Wash the glands with 50% acetic acid. Again put a small drop
of 50% acetic acid on the glands, place a coverslip on it and
wrap the slide with a thin sheet of filter paper.

5. Exert a little pressure on the coverslip with thumb to squash and

to absorb the excess fluid. Seal the preparation from all sides of
the coverslip with DPX and observe under a light microscope.

Observation

The Polytene chromosome reveals six arms, five of which (2R, 2L, 3R, 3L
and X) are long and the fourth chromosome is present as a knob. The Y -
chromosome is invisible, being under replication. The two homologues of
each chromosome pair fuse to form just one banded unit. The
centromeres of all chromosomes unite at a common chromocentre. Thus,
the telocentric X chromosome appears to be a single unit. The
metacentric second and third unit seem as left (2L and 2L) and right (2R
and 3R) portions. Darkly stained bands alternated with light stained
interbands are visible on the chromosome arms.

Notes
• The polyteny in these giant chromosomes occurs due to endomitosis of metaphase
chromosomes which multiply almost 8 times resulting in a giant (quite long and
broad) chromosome.

• A close observation of salivary gland chromosome of Drosophila reveals

♣ Bands, which are deeply stained with orcein

♣ Interbands which take a little or no stain

♣ Puffs which do not take stain and are supposed to be the region of RNA synhesis.

Autoradiographic studies reveal that the bands are on the region of active DNA synthesis and
incorporate H3- thymidine while the puffs synthesize RNA and can be labeled with H3- uridine.

 

 
 ΀5(6($5&+7(&+1,48(6,102/(&8/$5%,2/2*<
*(1(7,&6$1'%,27(&+12/2*<΁ *$5* *$5*


Culture Method for Drosophila

Drosophila is a 2 mm long fly, usually abundant during June to August or
November to January in dormitories, pantries or in bath rooms.

Stock
Potato 100 gm
Agar 1 gm
Propionic acid 0.8 gm
Yeast 0.5 gm
Dextrose 1 gm
Water 100 ml
Sterilize the wide mouth bottles and test-tubes. Wash them with sterile
water and dry/ heat sterilize at 160oC.

Procedure

1. Boil, peel and mesh the potatoes to a soft pulp. Beat them with
water, agar and dextrose.

2. Sterilize the contents in a beaker. Cover its mouth with a clean

white paper and tie it with a plastic twin / rubber band.

3. Sterilize it at 15 lb pressure for 10-15 mins in a pressure cooker.

Bringthe temperature down to 60oC and then mix propionic acid.

4. Let the mixture cool down to 40o&WKHQGLVVROYH\HDVWFHOOVLQ

water and pour into the medium.

5. Now transfer the contents into sterilized vials, plug them and
leave them at room temperature.

 

 
 ΀5(6($5&+7(&+1,48(6,102/(&8/$5%,2/2*<
*$5* *$5* *(1(7,&6$1'%,27(&+12/2*<΁

Notes
• Keep the culture vials at 23-25oC only
• Heat sterilize the bottles, if neccessary, to avoid the entry of mites.

Observations

Following is the chronology of development from the day of egg’s release:

Days Stage Diagnosis

<1 Eggs Small eggs mrender the surface of the

medium coarse
1 Embryo

2 Hatching

2-3 Larva White, segmented, worm shaped

creatures with dark mouth.
8-9 Adult Earlier seems fragile & light, but attains
maturity within 5-6 hrs.

Mating

1. Select the virgins from 6-7 hrs age group. To have the virgins only,
empty the bottle containing mature flies. After 7-8 hrs, males will
be segregated, leaving virgins in the population.

2. Select one character (wild) in female and one (mutant) in male or

reciprocate it.

3. Mix 4-6 males with 8-12 females (in the ratio 1:2) in a bottle.
Indicate the date of mating and segregate the progeny.

4. By repeated test crosses/ replicate cross progenies, one can

observe the way of inheritance and deduce monohybrid crosses by
‫ ׶‬2 analysis.

 

 
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*(1(7,&6$1'%,27(&+12/2*<΁ *$5* *$5*


Chromosomes of Musca domestica

Stock
Belar’s Saline
Sodium chloride 6.0 gm
Potassium chloride 0.2 gm
Calcium chloride 0.2 gm
Sodium bicarbonate 0.2 gm
Distilled water 1000 ml
Aceto-alcohol
1N HCl
Gomori’s Haematoxylin

Procedure

1. Take an adult pupa of house fly and remove the testicular or

ovarian tissue by clipping off the abdominal tips.

2. Squeeze the visceral contents in Belar’s saline.

3. Separate the whitish (pupal stage) or brownish pear shaped

mass (testis) from the viscera.

4. Fix the tissue in aceto-alcohol, 1:3 for 5 mins.Hydrolyze in cold

1N HCl for 3 mins and then in hot 1N HCl (60oC) for 5 mins.

5. After hydrolysis, stain the material in Gomori’s haematoxylin for

40-45 mins.

6. Differentiate the tissue in freshly prepared 45% acetic acid for

10 mins.

 

 
 ΀5(6($5&+7(&+1,48(6,102/(&8/$5%,2/2*<
*$5* *$5* *(1(7,&6$1'%,27(&+12/2*<΁

7. Finally, squash it in 45% acetic acid, keep in refrigerator for
about a day, and mount in euparal.

Observations

The diploid count as evident from testicular and ovarian metaphase cells
is 12. Chromosome pair II, VI and X are metacentric while pair III, IV, V
and Y are sub-metacentric. Pair VI carries one or two secondary
constrictions.

During early prophase of Meiosis-I, fine chromatin threads appear and the
heteropycnotic sex chromosome lies on one side of the nucleus. In
metaphase-I, the autosomal bivalents assume the shape of V or J, while
the sex bivalent has a U-shaped appearance.In anaphase-I, the sex
chromosomes show precacious movement.

During prophase-II, the chromosome appear as elongated lightly stained

bodies. At metaphase-II, they appear like V or J. X is difficult to be
recognized as it resembles to chromosome-VI, wheras Y, being the
smallest element of the cell, can be easily distinguished.

Notes :
2% Acetocarmine can also be used in place of Gomorin’s Stain.

 

 
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*(1(7,&6$1'%,27(&+12/2*<΁ *$5* *$5*


Meiosis in Grass hopper

Stock
Aceto-orcein stain (2%)
Acetic acid (45%)
Potassium chloride (0.42%)
Glacial acetic acid
Absolute alcohol
Paraffin wax
Procedure

1. Collect a male grass-hopper from nature and bring it to the

laboratory.

2. Give nasal administration of ether for 30 secs.

3. Make a longitudinal incision on the dorsal side, along the entire

length of the abdomen, in posterio-anterior direction and open
the body cavity in water.

4. Beneath the alimentary canal, within the first three segments of

abdomen, a cream coloured finger like compact mass is visible.

5. Remove this mass (testis) from the body and keep in 0.42%
potassium chloride solution for 15-20 mins. Use 0.9% trisodium
citrate, if potassium chloride is not available.

6. Fix the testicular material in a freshly prepared fixative (acetic

acid- absolute alcohol,1:3). Time of fixation may vary in different
tissues. However, fixing the tissue for 30 mins at room
temperature gives the best chromosome preparations in grass
hopper.
 

 
 ΀5(6($5&+7(&+1,48(6,102/(&8/$5%,2/2*<
*$5* *$5* *(1(7,&6$1'%,27(&+12/2*<΁

7. Then, transfer the material in 70% alcohol and store at < 10oC
(upto a maximum of 120 days). Take out a small testicular
tissue and place it in cavity block with 2% aceto-orcein stain.
Incubate at 58-60oC for 30 mins, mince and preserve in 45%
acetic acid.

8. Place a coverslip and seal the preparation with paraffin wax

evading the entry of air bubbles.

 

 
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*(1(7,&6$1'%,27(&+12/2*<΁ *$5* *$5*


The Bone Marrow Culture

The bone marrow cells are extracted from haemopoietic areas of bone.
The technique is fast, eliminates prolonged incubation and is quite
suitable to field conditions.

The lone disadvantage is that the specimen has to be invariably

sacrificed. Though, bone marrow can be extracted from larger animals
through a sternal puncture (without killing the animal), yet it’s a painful
procedure. Moreover, in young ones, the quantity of cells obtained may
be low while in adult individuals, it is sometimes possible that there may
not be found any mitosis at all since the mitotic activity of the marrow cells
is highly variable in mature animals.

Stock
Colchicine or Veblan 0.5 ml
Ether 5.0 ml
Potassium chloride 0.56 %
Aceto-alcohol, 1:3 100 ml
Sulphuric acid
Giemsa (Merk)
Giemsa powder 0.78 gm
Glycerine 50 ml
Methanol 50 ml
Sorenson’s buffer (4%)
Na2HPO4 0.580 gm
NaH2PO4 0.358 gm
Distilled water 1 litre

 

 
 ΀5(6($5&+7(&+1,48(6,102/(&8/$5%,2/2*<
*$5* *$5* *(1(7,&6$1'%,27(&+12/2*<΁

Procedure

1. Give an intraperitoneal injection of colchicine, @ 0.01 ml/13 gm

of the body weight or Velban @ 0.01 ml/2 gm of the body
weight, 1-2 hours prior to harvesting the cells. The larger the
animal, the more the time it needs to accumulate an adequate
number of metaphase cells.

2. Anaesthetize the specimen by nasal administration of ether.

Make an abdominal incision and remove femur, tibia or
sternum. Cut off the epiphyses and aspirate the bone marrow,
using a hypodermic syringe containing 0.56% KCl solution pre-
warmed at 37oC.

3. Allow to stand the cell suspension at 37oC for another half an

hour and then centrifuge at 500 rpm for 5 min.

4. Decant off the supernatant and fix the pellet in 1:3 aceto-
alcohol. The fixative should be added drop by drop with
constant stirring to obtain a homogenous cell suspension.

5. After 30 min of fixation at room temperature, spin the material at

500 rpm for another 5 min.

6. Repeat the step-5, twice or thrice; then add 5-10 ml of fresh

fixative to give an optimal cell density.

Flame drying and Staining

1. Take commercially available microscopic glass slides

(preferably PIC-1) and submerge overnight in 1:4 sulphuric acid
- water.

 

 
 ΀5(6($5&+7(&+1,48(6,102/(&8/$5%,2/2*<
*(1(7,&6$1'%,27(&+12/2*<΁ *$5* *$5*

2. Clean the slides under running tap water for an hour, dip in 70%
alcohol for 2-4 hr and keep in refrigerator.

3. Take the cell suspension and disperse a large drop on a clean

wet slide. Lift and move the slide gently over the flame of the
spirit lamp or keep the slide on a hot plate until the fixative gets
evaporated.

4. Stain the slides in Giemsa (Merk) solution (diluted 1:50 with

Sorenson’s buffer at pH 6.8 for 15 min.

5. Rinse the slides in tap water, dry in air and mount in DPX.

Notes

• The cells are treated with hypotonic solution which enlarges the size of cells and,
in turn, eases the spreading off the chromosomes.

• Fixative (aceto-alcohol, 1:3) is a selective medium to maintain the cell plates in

status quo.

 

 
 ΀5(6($5&+7(&+1,48(6,102/(&8/$5%,2/2*<
*$5* *$5* *(1(7,&6$1'%,27(&+12/2*<΁


Testis Biopsy
Stock
Potassium chloride 0.56 %
Acetic acid 60 %
Aceto-alcohol, 1:3
Giemsa (Merk) 0.78 gm
Giemsa powder 50 ml
Glycerine 50 ml
Methanol
Sorenson’s buffer (4%)
Na2HPO4 0.58 gm
NaH2PO4 0.358 gm
Distilled water 1 litre
DPX

Procedure

1. Take an adult animal, dissect out its testes and macerate them
in 0.56% KCl prewarmed at 37oC.

2. Allow the cells to swell for half an hour at the same temperature
(37oC).

3. Transfer the tissue to 60% acetic acid for 6-8 mins.

4. Fix the material in an equal volume of aceto-alcohol, 1:3 and

incubate at 37oC for 10-15 mins.

5. Centrifuge at 500 rpm for 5 mins.

6. Decant off the supernatant and add 10-15 ml of fresh fixative.

 

 
 ΀5(6($5&+7(&+1,48(6,102/(&8/$5%,2/2*<
*(1(7,&6$1'%,27(&+12/2*<΁ *$5* *$5*

7. Repeat the steps 5 and 6 twice and finally add a desired
amount of fresh fixative to have an optimal cell density.

8. Disperse a drop of cell suspension on pre-sterilized glass slide.

Lift the slide on the flame of a spirit lamp.

9. Stain the slide in Giemsa merk solution (diluted 1:50 with

Sorenson’s buffer, pH adjusted to 6.8).

10. Rinse the slides, dry in air and mount in DPX.

Notes
• Study of meiosis should not be attempted after pretreatment, because the
chemical interferes with pairing behaviour and exact pairing of homologous
chromosomes, number of chiasma, etc. are disturbed.

 

 
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*$5* *$5* *(1(7,&6$1'%,27(&+12/2*<΁


Microchromosomes
In addition to the normal chromosomal component, a number of small
spherules or dot shaped structures are consistently visible in mitotic
and/or meiotic cell divisions, whose specific role has not been
corroborated.

Stock
Colchicine or Veblan 0.5 ml
Ether 5.0 ml
Potassium chloride 0.56 %
Aceto-alcohol, 1:3 100 ml
Sulphuric acid
Giemsa (Merk)
Giemsa powder 0.78 gm
Glycerine 50 ml
Methanol 50 ml
Sorenson’s buffer (4%)
Na2HPO4 0.580 gm
NaH2PO4 0.358 gm
Distilled water 1 litre

Any bird specimen, preferably a male bird (in order to study both - mitotic
as well as meiotic division) can serve the purpose.

Procedure

Follow i) the bone marrow technique for mitotic study,

ii) testis biopsy for meiotic study.

 

 
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*(1(7,&6$1'%,27(&+12/2*<΁ *$5* *$5*

Observation

Microchromosomes are visible as dot shaped, often telocentric,

structures. In certain cases, broken satellite or chromatin droplets can
also disguise.

 

 
 ΀5(6($5&+7(&+1,48(6,102/(&8/$5%,2/2*<
*$5* *$5* *(1(7,&6$1'%,27(&+12/2*<΁


Sex Chromosomes
In a mojority of higher animals, there is a pair of chromosomes that
decides the sex of an individual. These chromosomes are referred to as
sex chromosomes.

Morphologically, X is a large, metacentric or submetacentric chromosome

and is responsible for femaleness whereas Y is a small, acrocentric or
telocentric, element that leads to maleness. There are certain variables
too : like ZZ-ZW configuration in birds.

Stock
Colchicine or Veblan 0.5 ml
Ether 5.0 ml
Potassium chloride 0.56 %
Aceto-alcohol, 1:3 100 ml
Sulphuric acid
Giemsa (Merk)
Giemsa powder 0.78 gm
Glycerine 50 ml
Methanol 50 ml
Sorenson’s buffer (4%)
Na2HPO4 0.580 gm
NaH2PO4 0.358 gm
Distilled water 1 litre

Any bird specimen, preferably a male bird (in order to study both - mitotic
as well as meiotic division) can serve the purpose.

 

 
 ΀5(6($5&+7(&+1,48(6,102/(&8/$5%,2/2*<
*(1(7,&6$1'%,27(&+12/2*<΁ *$5* *$5*

Procedure

Follow : i) the bone marrow technique for mitotic study,

ii) testis biopsy to observe meiosis.

Observation

Bone marrow preparations of dog (2n=78), yield typical metacentric X-

chromosomes. On the contrary, bird specimens exhibit Z-W mode of sex
determination. Female is the heterogamatic sex with Z-W constitution.
The Z chromosome is relatively larger.

In man, the biopsied testis reveals a small Y-chromosome terminally

paired with a long X- chromosome.

 

 
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*$5* *$5* *(1(7,&6$1'%,27(&+12/2*<΁


Presence of Inactivated X Chromosome

in Buccal Smear
Stock
Aceto-orcein or aceto-carmine
Glass slides
Cover slip
Procedure

1. Carefully scrap the inside of cheek with scalpel or one edge of a

coverslip.

2. Place a drop of water at the center of a clean slide.

3. Run the edge of coverslip with cheek cells through the drop of
water so that the cells may spread evenly under the coverslip.
Press gently and observe under a phase contrast microscope.

4. Look for definite dark body in these squamous epithelial cells.

5. If only a bright field microscope is available, place a small drop

of aceto-orcein or aceto-carmine stain on the material instead of
water drop.

 

 
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*(1(7,&6$1'%,27(&+12/2*<΁ *$5* *$5*


Mammalian Whole Blood Culture

(Micro-method)
Cells can proliferate, in vitro, when supplemented with essential vitamins
and other components.

Stock
Culture medium 10.0 ml
(use Iscoves, McCoy’s 5A, or RPMI 1640)

Foetal Calf Serum 1.5 ml

PHA-M or Pokeweed mitogen 0.2 ml
(depending upon the species)

Glutamine 0.1 ml
Antibiotic- antimycotic 0.1 ml
Heparin 0.1 ml
(quantity shown above would be required for individual culture).

Procedure
1. Prepare individual culture flask (30 ml) ahead of time and freeze
them.

2. Add 0.5 ml of heparinized whole blood in the culture, mix gently.

3. Incubate the culture for 72 hrs at 37oC.

4. Now add 0.1 ml of colchicine (10 mcg/ml) into the culture, 2 hrs
prior to harvest.

5. Pour the culture into a 15 ml siliconized centrifuge tube. Spin at

1000 rpm for 10 mins.

 

 
 ΀5(6($5&+7(&+1,48(6,102/(&8/$5%,2/2*<
*$5* *$5* *(1(7,&6$1'%,27(&+12/2*<΁

6. Pipette off all but the last 0.5 ml of medium. Resuspend the
pellet gently with a siliconized pipette.

7. Add 10 ml of 0.075 M KCl and incubate at 37oC for 12 mins.

8. Centrifuge at 1000 rpm for 10 mins, decant off the supernatant

and resuspend the cells in the remaining fluid.

9. Add 2-3 drops of cold fixative (methanol : acetic acid, 3:1) and
allow the cells to stand as such for 20 mins at room
temperature.

10. Spin the cell suspension at 1000 rpm for another 10 mins,
pipette off all but 0.5 ml of the fixative and resuspend the pellet.

11. Add 5 ml of fresh fixative and repeat the step 10, twice or thrice,
to clean up the culture thoroughly.

Notes
• The procedure works well with cattles, dogs, cats and human blood.
• F.C.S. may be substituted with Porcein serum for equally good results.

Alternatively, the following method can be employed :

1. Draw 3-4 ml of venous blood into an asceptic syringe,

containing 0.1 ml of heparin (1000 units/ml) solution.

2. Replace the needle guard immediately in order to avoid

contamination and leakage and mix the contents thoroughly.

3. Keep the syringe upright at ambient temperature for 30 mins - 1

hour to facilitate gravity separation of RBC from 0.5 -1.0 ml of
plasma containig WBC.

4. Bend the needle with a needle guard and squeeze 0.3 - 0.5 ml of
plasma into a culture bottle (or flask) already having 6 ml of
 

 
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*(1(7,&6$1'%,27(&+12/2*<΁ *$5* *$5*

culture medium, 2 ml of foetal calf serum and 0.2 ml of
phytohaemagglutinin.

5. Recap the bottle with a sterile rubber stopper and incubate at

30oC.

6. After 3 days, add a few drops of colchicine (0.04 mg/ml) or

colcemid (0.02 mg/ml) and allow to stand for 1 - 2 hrs.

7. Suspend the cells in the medium and centrifuge at 1000 rpm for
5 mins. Decant off the supernatant and add 2 ml of 0.075 M KCl
with constant mixing.

8. Leave the contents as such for 15 - 30 mins at room

temperature.

9. Finally, add 5 - 10 ml of freshly prepared aceto-alcohol, 1:3.

allow the contents to stand for 5 mins at room temperature.

10. Repeat the step - 9, several times until the suspension becomes
homogenous and free of reddish color.

11. Hold the slide at 45o angle and put a drop of cell suspension
from a hieght of 10 - 20 cms. Pass it through a spirit lamp to burn
the excess methanol.

12. Stain the preparations in Giemsa for 15 mins, rinse in tap water,
dry in air, soak in xylol and mount in DPX.

Notes
• pH may be lowered or raised by adding NaHCO3 or 0.1 M-HCl.
• KCl provides better results than sodium citrate in chromosomal plates prepared
from blood specimens.

 

 
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*$5* *$5* *(1(7,&6$1'%,27(&+12/2*<΁


Avian Leucocyte Culture

Stock
Culture medium
A Mc Coy’s a (Mod) (GIBCO) 10.0 ml
B Iscoves Modified (GIBCO)
Glutamine (GIBCO, DIFCO) 0.1 ml
Antibiotic- antimycotic 0.1 ml
Heparin 0.1 ml
Mitogen
A Pokeweed 0.1 ml
B PHA-M 0.1 ml

Procedure

1. Draw 10 ml blood in a sterile heparinized syringe.

2. Centrifuge it @ 500-600 rpm for 10-20 mins.

3. Transfer 1 - 2 ml of plasma with leucocyte to 10 ml media in a

culture flask.

4. Incubate for 70 - 72 hrs at 41oC. Add a drop of 0.05% colchicine

solution and further incubate for 1- 2 hrs.

5. Centrifuge at 1000 rpm for 10 mins. Decant off all but 0.5 ml of
the supernatant.

6. Resuspend the cells by adding 5 ml of hypotonic solution

(distilled water and chicken serum, 6:1) with constant stirring.

 

 
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*(1(7,&6$1'%,27(&+12/2*<΁ *$5* *$5*

7. Spin at 1000 rpm for 10 mins and decant off all but 0.5 ml of the
suspension.

8. Now fix the cells in 3 ml of freshly prepared aceto-alcohol

(acetic acid and methanol, 1:3) with constant agitation.

Notes
The present technique produces good quality metaphase plates, with well spread chromatids,
for consistent karyotyping. Constant mixing or agitation, at various steps, ensures
homogenous cell suspension and enables the chromosomes to spread evenly during slide
preparation. However, in order to get still longer chromatids, for banding analysis, following
changes can be attempted :
i) Incubate the blood culture with colchicine for 30 mins only.
ii) Use 5 ml of 0.075 M KCl as hypotonic solution, in place of serum water. Serum
inhibits the action of trypsin in banding procedures.

Alternatively, the following technique may be employed for chromosomal

study through leucocyte culture :

Stock
Medium 2.0 ml
(use Iscoves, McCoy’s 5A, or RPMI 1640)

Foetal Calf Serum 2.0 ml

Phytohaemagglutinin 0.2 ml
Heparin 0.1 ml
(quantity shown above would be required for individual culture).
NaHCO3
HCl 0.1 M
KCl 0.075 M
Colchicine/colcemid
Aceto-alcohol, 1:3

 

 
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*$5* *$5* *(1(7,&6$1'%,27(&+12/2*<΁

Giemsa (Merk)
Giemsa powder 0.78 gm
Glycerine 50 ml
Methanol 50 ml
Sterilized syringe with needle guard
Culture flasks/ bottles

Procedure

1. Draw 3-4 ml of venous blood into an asceptic syringe, already

containing 0.1 ml of heparin (1000 units/ml) solution and mix the
contents thoroughly.

2. Replace the needle guard immediately in order to avoid

contamination and leakage.

3. Keep the syringe upright at ambient temperature for 30 mins to

an hour to facilitate gravity separation of RBCs from 0.5 - 1.0 ml
of plasma containing WBC.

4. Bend the needle with a needle guard and squeeze 0.3 - 0.5 ml of
plasma into a culture bottle having 6 ml of culture medium, 2 ml
of foetal calf serum and 0.2 ml of phytohaemagglutinin.

5. Recap the bottle with a sterile rubber stopper and incubate at

37oC. Maintain the pH by adding NaHCO3 or 0.1 M HCl.

6. After 2½ - 3 days, add a few drops of colchicine (0.04 mg/ml) or

colcemid (0.2 mg/ml). Stand as such for 1½ hrs.

7. Suspend the cells in the medium and spin for 5 mins at 1000
rpm.

 

 
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*(1(7,&6$1'%,27(&+12/2*<΁ *$5* *$5*

8. Decant off the supernatant and add 2 ml of 0.075 M KCl with
constant agitation. Leave the contents as such at room
temperature for 15 - 25 mins.

9. Now, add 5 - 10 ml of freshly prepared aceto-alcohol, 1:3. allow

to stand at room temperature for 5 mins.

10. Repeat the step - 9 until the suspension becomes homogenous

and free of red color. Finally add 10 - 15 ml of fresh fixative to
provide an optimal cell density.

11. Hold the slide at 45o angle and put a drop of cell suspension
from a height of 10 -15 cms. Move it over the flame of spirit lamp
to ignite the excess fixative.

12. Stain the preparations in Giemsa for 15 mins.

13. Rinse thorougly in tap water, dry in air, soak in xylol and mount
in DPX.

 

 
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*$5* *$5* *(1(7,&6$1'%,27(&+12/2*<΁


Lymphocyte Culture
Stock

Culture medium

Use Eagle’s MEM/Medium TC 199 or Parker 199/ Mc Coy’s 5.

Dissolve the medium in triple distilled water. For immediate use,
add sodium bicarbonate solution to maintain pH between 7.2 and
7.4 with characteristic red/ pink colouration. Sterilize by passing
through filter into an autoclaved bottle. Further, add :

Penicillin 100-200 units/ml

Streptomycin 50-100 mg/ml
o
Test the sterility by keeping at 37 C for 48 hrs and store in a refrigerator.

Phytohaemagglutinin P#
Heparin sodium#/ Laqueniur
Colchicine/colcemid#
#
Store them at 2oC

Fixative
Glacial acetic acid 1 part
Methanol 3 parts
Absolute alcohol
Rectified spirit
Giemsa stain
Potassium chloride (0.07 M) 0.56 %
Xylol
Hydrochloric acid
Ammonium hydroxide

 

 
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*(1(7,&6$1'%,27(&+12/2*<΁ *$5* *$5*

Glycerol
Sodium bicarbonate
Sodium phosphate monobasic (NaH2PO4)
Sodium phosphate dibasic (Na2HPO4)
Potassium phosphate monobasic (KH2PO4)

Laboratory ware Size

Culture tubes (Bakelite) 15/30 ml
Syringes 1/2/5/10 ml
Needles No. 21/22/23
Pasteur’s pipettes with rubber droppers
Centrifuge tubes 1/10/25/50 ml
Pipettes
Reagent bottles
Staining jars
Glass slides
Coverslips
Bottles 500 ml
Millipore filters, with spare filter pads 0.22/0.20 M

Sterilization

1. Wash the culture tubes, their lids/caps, corks (use white corks
only) syringes, needles, medium bottles, etc. thoroughly.

2. Sprinkle a little detergent and boil for 30 mins. Cleanse properly

using a lampbrush.

3. Keep in running water for 3 hrs and then leave in distilled water
overnight.

 

 
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*$5* *$5* *(1(7,&6$1'%,27(&+12/2*<΁

4. Heat dry in an oven at 60-80oC.

5. Wrap the labware in aluminium foil/ paper.

6. Autoclave at 15 lbs for 30 mins.

Notes
• Wash pipettes and small syringes in detergents and rinse thoroughly in tap water
for 3 hrs. Place the glassware in 5% HCl solution and allow to stand overnight.
Rinse thoroughly in pipette washer for an hour or two. Finally rinse in distilled
water and siliconize, if required.

• Once washing is complete, immerse the glassware in 1% solution of silicad (in

water) for 5 secs. Rinse in double distilled water twice or thrice. Dry in an oven at
60-80oC.

Procedure

1. Place 1 ml of heparin in a 5 ml syringe and draw blood via a

venous puncture under strict asceptic conditions.

2. Aspirate the blood from needle and small space below. Wipe
the needle with spirit and cover with a needle cover, or a cotton
swab, soaked in spirit.

3. Allow the syringe to stand vertically with needle pointing upward

for an hour until 30 - 40% of the blood sample gets clear of
RBCs.

4. Transfer the plasma into a culture vial containing 5 ml medium

with bend needle (0.5 - 1.0 ml plasma for 5 - 10 ml culture
medium).

5. Incubate at 37oC for 72 - 96 hrs.

Notes
• The heparinized preparations can be stored upto a maximum of 24 hrs.
• Store in refrigerator until further use.
 

 
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*(1(7,&6$1'%,27(&+12/2*<΁ *$5* *$5*

Cell Harvest

1. Add colchicine : 0.04 mg/ml of culture or colcemid : 0.02 - 0.4

mg/ml of culture 2 hrs before the termination of culture. Maintain
the temperature at 37oC.

2. Spin the culture for 10 mins. and resuspend the pellet in 0.075
M KCl for 20- 25 mins.

3. Centrifuge the culture for 10 mins., decant off the supernatant

and add freshly prepared fixative. Allow to stand for 10 mins
and spin the contents again.

4. Repeat the step 3, twice or thrice and finally add 10 - 15 ml of

fresh fixative to give an optimum cell density.

5. Prepare the slides by air/flame drying method.

 

 
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Feather Pulp (in vivo)

There is an active proliferation of cells at the base of every growing
feather, although some feathers may have more mitotic activity than
others, in the same bird. The same is true between individual birds and
there are evidences that mitotic activity may be greater at some hours of
the day. Wing feathers are usually more active in cell proliferation, but if
they are not available, any mature feather may be pulled to initiate growth
of new ones which would be ready within 7 to 10 days.

Stock
Colchicine or Veblan
Sodium citrate 0.45 %
Acetic acid
Distilled water
Bibulous paper
Giemsa (Merk)
Giemsa powder 0.78 gm
Glycerine 50 ml
Methanol 50 ml
Sorenson’s buffer (4%)
Na2HPO4 0.580 gm
NaH2PO4 0.358 gm
Distilled water 1 litre
DPX

 

 
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*(1(7,&6$1'%,27(&+12/2*<΁ *$5* *$5*

Procedure

1. Inject 0.05% solution of colchicine, intraperitoneally (0.04

POJPRIERG\ZHLJKWIRU\RXQJRQHVPOJPRIERG\
weight for adults).

2. After 1 - 2 hrs, pluck the growing feathers and pinch off 2 - 3 mm

of the pulp from its proximal tip with forceps.

3. Place it into a previously prepared solution of 0.45% sodium

citrate.

4. Keep the pellet of pulp into acid water (acetic acid 5 ml and
distilled water 5 ml). Allow to stand as such for atleast 30 mins
before preparing the squash.

5. Take a piece of pulp from fixative and place it on a clean slide.

Using forceps, smear the pulp in a circular manner until the
area, almost equal to the size of coverslip, becomes covered
with a thin film of cells and the fixative.

6. Place the coverslip over the material and gently tap it with a
blunt object, like rubber-eraser, until the material spreads under
the entire area of the coverslip.

7. Hold the slide between a fold of bibulous/ blotting paper and

press the coverslip gently until the excessive fixative wipes out.

8. Place the thumb over one end of the coverslip and exert a little
pressure to ensure the exit of air-bubbles (if any).

9. Observe the preparation under a phase contrast microscope to

determine the quality of the plates. If good, stain and mount with
a suitable mountanant.
 

 
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*$5* *$5* *(1(7,&6$1'%,27(&+12/2*<΁

Notes

• After the administration of mitotic inhibitor, accumulation of metaphase plates

depends largely upon the body wHLJKWRIWKHVSHFLPHQWKHKHDYLHUWKHELUGWKH
longer the time required. However, an excessive dose of colchicine or excessive
length of treatment results in extremely condensed or even distorted
chromosomes. Au contraire, the shorter span results in fewer metaphase plates
but qualitywise they are certainly better. The recommended dose of colchicine,
enables the bird to survive and be reused within a week or so.

• Avoid usage of specimens with pigmented feathers since the melanophores, in

pigmented feathers, take up stains lavishly. If not possible, examine the bird
thoroughly to find out any unpigmented feather. Else, with pigmented feathers,
use the clear semi-solid pulp from its proximal end.

• Wing feathers from a day old bird provide about 1 mm diameter of pulp and may
be treated with hypotonic solution for 10-15 mins at ambient temperature. Wing
feathers from adult specimen yield a gelatinous mass of 2-3 mm and should be
macerated on the slide before preparing the smear. After smearing, the larger
pieces of pulp may be discarded.

 

 
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Feather Pulp (in vitro)

Stock
Mc Coy’s 5a medium (Mod), 199 F10 or similar medium
Colchicine solution
Hank’s solution
Sodium citrate 0.45%
Acetic acid

Procedure

1. Take 5 ml sample of Mc Coy’s 5a medium (Mod), 199 F10 or

similar complete medium and warm it at 41oC beforehand.

2. Add 0.25 - 0.50 ml of colchicine solution (0.25 ml of 0.05%

solution/25 ml of Hank’s solution = 5 ug/ml). The larger the pulp
size, the higher the concentration.

3. Pluck the feathers, pinch off 2-3 mm of the pulp from its
proximal tip and place it into the culture medium. Incubate at
41oC for 45 mins.

4. Remove the pulp pellets and treat them with 5 ml of 0.45%

sodium citrate for 15 - 20 mins.

5. Fix the material in 50% acetic acid ( 5 - 10 ml) for atleast 1 hr

(maximum upto overnight) before preparing the smear.

 

 
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*$5* *$5* *(1(7,&6$1'%,27(&+12/2*<΁


Avian Cell Culture (Whole Embryo)

Avian fibroblast cells can be cultured from early chickens embryos (7-9
days old), through skin biopsy, or any epithelial or connective tissue. The
present procedure eliminates prolonged incubation periods, enhances cell
growth and minimizes the chances of contamination.

Stock
Calf Serum/ Chicken Serum 7.0 ml
Antibiotic- antimycotic mixture 1.0 ml
L-Glutamine (GIBCO) 1.0 ml
Medium
(Mc Coy’s 5a, Ham’s F10 or any self sufficient medium). Prepare medium
just before use.

Trypsin
(Gibco trypsin 2.5% (1:250) 10X dilution. Dilute with 180 ml of sterile BSS
(Ca & Mg free), either Hank’s or Tyrode’s.

Chick Ringer’s Solution

NaCl 7.2 gm
CaCl2 0.17 gm
KCl 0.37 gm
Distilled water 1000 ml

Procedure

1. Autoclave the glassware to be used. Keep scissors (bent

angle), forceps and other essential tools in 70% ethanol. Flame
dry before use.

2. Work in a sterile hood, equipped with a UV lamp. Leave the

lamp glowing all the time except when working under the hood.
 

 
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*(1(7,&6$1'%,27(&+12/2*<΁ *$5* *$5*

3. Before starting, turn off the UV lamp and place a towel, soaked
in Betadine solution, on the floor of the hood.

4. Cleanse your hands and arms.

5. Take a fertile egg (preincubated for 24 hrs), wipe the outer shell
with 70% ethanol and let it dry.

6. Break egg shell carefully and discard albumin. Place yolk into a
specimen dish, partially filled with sterile Ringer’s solution.

7. Using sterile scissors and forceps, dissect out the blastula

(germ spot). Try to remove as little yolk as possible.

8. Swish the blastula in Ringer’s solution to make it free from

excess yolk and to remove the adhering vitelline membrane.

9. Transfer the embryo with the help of a pipette into a sterile

bottle containing 0.5 - 1 ml of Hank’s or Tyrode’s BSS ( without
Ca and Mg ).

10. Repeat the procedure with a few more eggs (depending on the
age of the embryos) to have 2- 3 embryos per bottle.

11. Gently separate the embryos in BSS to produce a homogenous

cell suspension.

12. Prepare two flasks (25 cm3) each with 3 ml of culture media.
Add 1 ml of embryo cell suspension to each flask. Incubate at
41oC.

Sub-culture or Cell Harvest

For Sub-culture at 48 hrs when the cells are 75-100% confluent,

they may be subdivided into new flasks.

 

 
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*$5* *$5* *(1(7,&6$1'%,27(&+12/2*<΁

For Cell Harvest at 24 - 48 hrs, when cells begin to adhere to the
bottom of the flask, colchicine @ 0.01 ml ( 0.05% solution) may be
added to each flask from one to several hours.

• Remove the cells from the flask by using 1 ml of 0.025% trypsin

in BSS. Place flask in a water bath at 37oC and watch for cells
to loosen.

• Now transfer the cell trypsin suspension into a centrifuge tube

containing 0.025 ml of chicken serum. Centrifuge at 1000 rpm
for 10 mins.

• For Sub-culture, discard the supernatant and resuspend the

residual pellet in culture media. Transfer the contents into a new
flask with appropriate dilution and incubate.

• For Chromosomal study, remove the supernatant, resuspend

the pellet and add 2 - 3 ml of 0.45% sodium citrate for 10 mins.
Spin at 1000 rpm for 10 mins.

• To obtain air dried preparations, fix the pellet in aceto-

methanol,1:3 for 30 mins, changing the fixative twice or thrice.

 

 
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*(1(7,&6$1'%,27(&+12/2*<΁ *$5* *$5*


Transplantation of Ascites Cells

in Mouse
Stock
Rectified spirit
Normal Saline 0.85% of NaCl in Distilled Water

Procedure

1. Hold the neck of the mouse firmly with thumb, 1st and 2nd fingers
of the left hand and tail of the mouse with little finger.

2. Wipe the surface of the abdomen with a swab of cotton soked in

rectified spirit.

3. Insert carefully, the needle of a sterilized syringe into the

peritoneal cavity. Take care to avoid injury of the internal
organs, which can lead to haemorrhage.

4. Draw about 0.5 ml of ascites fluid and place the fluid in a sterile
tube.

5. Add equal quantity of normal saline, mix the contents

thoroughly.

6. Now, hold a fresh mouse and wipe its abdominal surface as

before.

7. Take 0.2 ml of diluted fluid, containing the ascites fluid (about

2.6 X 105 cells), in a sterilized needle and inject the same into
the peritoneal cavity of the mouse. Wipe out the pricked
(injured) part of the abdomen.

8. Take the weight of the mouse and leave it back into the cage.
 

 
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*$5* *$5* *(1(7,&6$1'%,27(&+12/2*<΁

9. After a week or so, weigh the mouse again and calculate the
growth of ascite cells in terms of ‘increase in weight’.

 

 
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*(1(7,&6$1'%,27(&+12/2*<΁ *$5* *$5*


Cytological Investigation of
Mouse Ascites Cells
Stock
Ether
Colchicine 0.05% in Normal Saline
Hypotonic solution Normal Saline 1 part
Distilled Water 3 parts

Procedure

1. Take a mouse 5- 6 days after transplantation.

2. Hold the neck of the mouse firmly with thumb, 1st and 2nd fingers
of the left hand and tail of the mouse with little finger.

3. Wipe the surface of the abdomen with a swab of cotton soked in

rectified spirit.

4. Inject 0.2 ml of 0.05% colchicine solution. After 2 hrs, administer

ether and take out the ascites fluid.

5. Mix the ascite fluid and hypotonic saline in the ratio 1:4 and
incubate at 37oC for 45 mins with constant stirring to avoid
sedimentation of cells.

6. Spin the cells at 800 - 900 rpm for about 10 mins. Decant off the
supernatant and add aceto-alcohol,1:3 (pre-chilled at 4oC)
dropwise with gentle squeezing with the help of a Pasteur’s
pipette. Allow to stand at 4oC for an hour.

 

 
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*$5* *$5* *(1(7,&6$1'%,27(&+12/2*<΁

7. Spin the cells at 2000 rpm for 5 mins, discard the fluid, add
fresh fixative dropwise again and leave the contents at 4oC for
30 mins.

8. Repeat the step -7 thrice.

9. Take a grease free slide (dipped in 80% alcohol), place a drop

of suspension containing ascite cells on the middle of the slide
and blow violently in one direction, allowing preparation to dry.

10. Stain with 5% Giemsa solution in phosphate buffer at pH 7 for

about 15 mins. Rinse the slides in distilled water and let them
dry overnight.

11. Mount the preparation, if desired.

 

 
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*(1(7,&6$1'%,27(&+12/2*<΁ *$5* *$5*


Study of Tumour Cells

Stock
Hypotonic Glucose
Glucose 0.60% 1 part
NaCl 0.70% 1 part
Sodium Citrate 0.44% 1 part
Acetic-orcein
Orcein 2%
Acetic acid 65 %

Procedure

1. Crush the neoplastic tissue (tumour) into small pieces.

2. Transfer the pieces into a centrifuge tube and keep it as such

for 3- 5 mins.

3. Spin the cells, decant-off the supernatant and incubate the

contents at 37oC for 20- 30 mins.

4. Spin the suspension again, collect the cells and fix in 50%
acetic acid pre-chilled at 4oC.

5. After 2- 3 days, stain the cells in acetic-orcein.

6. Allow to stand for 5 - 10 mins at room temperature and squash

the cells by exerting a little pressure on the cover-slip with a
rubber eraser.

 

 
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*$5* *$5* *(1(7,&6$1'%,27(&+12/2*<΁


Cytobiometry
Morphometric analysis of chromosomes can be carried out from enlarged
photomicrographs of at least five to ten well spread metaphase plates of
each sex.

The Percentage Relative length (%RL), the Arm Ratio (r) or the
Centromeric Index (CI) can be taken as parameters to develop karyotypes
and idiograms. These can be calculated as follows :

Percentage Relative Length of macrochromosome

Length R
% L = Total Haploid Macrochromosomal Length X 100

Length of long arm of the chromosome

Arm Ratio r = Length of short arm of the chromosome

Length of the short arm

C
Centromeric Index I = Total length of that chromosome X 100

Now, classify all the chromosomes as per criterion suggested by De Boer

(1976) : chromosomes more than 7.5% TCL (total chromosomal length)
DV ODUJH   - 7.5 as medium and chromosomes below 7.5% as small.
Measure all micro chromosomes together in the form of total genome,
that they constitute.

For construction of idiograms, classify all macrochromosomes according

to the position of their centromere (after Levan et. al, 1964).

Group Centromeric position Arm Centromeric Nomenclature Sign

Ratio Index
I Median point 1.0 47.5 - 50.0 Metacentric M

 

 
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*(1(7,&6$1'%,27(&+12/2*<΁ *$5* *$5*

II Median region 1.0 - 1.7 37.5 - 47.5 Metacentric m

III Sub median region 1.7 - 3.0 25.0 - 37.5 Submetacentric Sm

IV Sub terminal region 3.0 - 7.0 12.5 - 25.0 Subtelocentric St

V Terminal region • 7.0 02.5 - 12.5 Acrocentric t

VI Terminal point 00.0 - 02.5 Telocentric T

Now, arrange the macrochromosomes, according to their decreasing

relative length, into morphologically similar pairs. Microchromosomes can
also be placed separately in descending order of size.

In case of heterogamatic sex (ZW), the total (haploid) chromosomal

length can be obtained by substracting the W and adding another Z before
dividing the TCL by 2.

Notes
• Photomicrographs can be taken at an initial mangnification of 1500 X using an oil
immersion objective. Use a sophisticated Optiphot Zeiss Camera with
photophone flourescence facility. If not available, a 35 mm reflex camera, without
lens, can be adopted to take the photomicrograph using Kodak technical print film
Tri-X-pan. A tungsten lamp (12V- 55W) can be used as the source of illumination.

• When arm lengths are unequal, each chromosome is oriented with its shorter arm
(p) upward and its longer arm (q) downward.

• In animals, with a high diploid count, errors may creep in while measuring very
small chromosomes owing to certain facts :
i) WKHH[DFWPLFURFKURPRVRPDOHQWLW\LVXVXDOO\KDUGWRGHWHUPLQH
ii) they can escape from the metaphase plates when the cell suspension is dropped
RQWKHVOLGH
iii) they can lie under the macrochromosomes or indistinguishably close to another
PLFURFKURPRVRPHDQG
iv) small spots of dye or stained dust can be misinterpreted as microchromosomes.

 

 
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*$5* *$5* *(1(7,&6$1'%,27(&+12/2*<΁


Demonstration of
Constitutive Heterochromatin
The position of centromere in a chromosome can be easily detected
through banding analysis. Being heterochromatic in nature, the
centromeric region takes dark stain with Geimsa after denaturation with
phosphate buffer. These dark regions on the chromosomes are known as
C-bands.

Stock
0.2 N Hydrochloric acid
HCl (12N) 16.66 ml
Water 983.34 ml
Barium Hydroxide 5%
2 X SSC (Saline Sodium Citrate)
NaCl 8.77 gm
Sodium Citrate 4.41 gm
Distilled water 500 ml
Sodium Hydroxide
Giemsa (merk)
Giemsa powder 0.78 gm
Glycerine 50 ml
Methanol 50 ml
Sorenson’s buffer (4%)
Na2HPO4 0.580 gm
NaH2PO4 0.358 gm
Distilled water 1 litre

 

 
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*(1(7,&6$1'%,27(&+12/2*<΁ *$5* *$5*

Alcohol - 70%, 100%
Procedure

1. Take four to six days aged slides, bearing well spread

metaphase plates.

2. Expose the slides to 0.2 N HCl for 1 hr at room temperature and

rinse them thoroughly in nano pure water.

3. Keep the slides in a couplin jar containing freshly prepared 5%

aqueous solution of Ba (OH)2 for 1.5 - 2 mins at 41oC.

4. The Barium hydroxide is a supersaturated solution, and may not

dissolve completely. Therefore, rinse the slides under running
tap water and place them in 0.05N HCl for 15 - 30 secs, shaking
the slides manually to remove the adhering scum. Again, rinse
twice in nanopure water.

5. Now reassociate the preparations in 2 X SSC (pH adjusted to

7.0 by adding 5- 6 drops of 1N HCl/ NaOH) for 1 hr at 60oC.

6. After rinsing twice, in nanopur water, stain the slides either in :

• 4% Giemsa stain for 20-PLQVRU

• Carbol fuschin stain

as follows-

i) Rehydrate the slides in 100% → → 70% alcohol → → nanopure

water.

ii) Transfer the slides to the jar containing stain for 20-30 mins.

iii) Dehydrate in nanopure water → → 70% alcohol → → 100%

alcohol. Allow 30-40 secs for each step.

 

 
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*$5* *$5* *(1(7,&6$1'%,27(&+12/2*<΁

iv) If the staining is adequate, the slides can be made permanent.

Notes
Another technique, after Yunis & Yaskinch (1971), also gives comparable results:
i) Denature the air-dried metaphase preparations by keeping them in 0.06 M –
Phosphate buffer (pH 6.8) at 100oC for 10 mins.
ii) Stop the reaction by placing the slides in the same buffer at 0oC for a few
seconds.
iii) Incubate the slides at 65oC for 30 mins in the same buffer.
iv) Lastly, stain with Giemsa for 20 mins.
C-banding stains only certain bands, usually at the kinetochore, which are composed of
constitutive heterochromatin and alternates with G-bands.

 

 
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C- Banding in Lymphocytes or
Fibroblasts Cells
Stock
Acetic acid 50 %
Alcohol 70/90/95/100 %
Gelatin 0.1 %
Chrome alum 0.01 %
HCl
NaOH
Pancreatic RNase 100 mg
2 X SSC 1 ml
(Commercially available RNase should be heated in a boiling waterbath for
5-10 mins.)

Procedure

1. Fix the lymphocytes or fibroblast cells (duly harvested after

colcemied and hypotonic solution treatments) in 50% acetic
acid.

2. Take alcohol cleaned slides and coat them with a solution

containing 0.1% gelatin and 0.01% chrome alum. Allow them to
dry.

3. Smear the cells, already fixed in 50% acetic acid, on to subbed

(coated) slide.

4. Rinse the preparations in 90% alcohol, twice and air dry. Keep
them in 0.2 N HCl at room temperature for 30 mins.

 

 
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*$5* *$5* *(1(7,&6$1'%,27(&+12/2*<΁

5. Again rinse the slides, several times, in distilled water. Leave
them to dry in air.

6. Treat the preparations with pancreatic RNase, at 37oC in a

moist chamber, for 60 mins.

7. Now, rinse the slides again & again in 2 X SSC, 70% ethanol,
95% ethanol and finally dry in air.

8. Place the slides in 0.07 N NaOH for 2 mins and rinse them in
three to four changes of 70% and 95% ethanol.

9. Incubate the slides in 2 X SSC at 65oC overnight. Then rinse,

once again, in 70% and 95% ethanol.

10. Stain the preparations in Giemsa for 15- 30 mins.

11. Finally, rinse the slides in distilled water, air dry and mount.

 

 
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*(1(7,&6$1'%,27(&+12/2*<΁ *$5* *$5*


Giemsa Banding
Banding technique provide a reliable tool for distinguishing chromosomes
in metaphase stage. These techniques include in situ hybridization,
demonstration of C-bands, G-bands, Q-bands, R-bands and so on.
Following is the technique employed for demonstration of Giemsa bands.

Stock
Trypsin solution (0.25%)
Trypsin powder (Difco) 0.3625 gm 1 part
Salt solution (GKN) 250 parts
Glucose 0.0054 M
KCl 0.0054 M
NaCl 0.14 M
Na2CO3 0.042 M
Saline solution 0.9 %
Giemsa stain
Sorenson’s buffer (4%)
Na2HPO4 0.580 gm
NaH2PO4 0.358 gm
Distilled water 1 litre
Sodium bicarbonate
DPX

Procedure

1. Take 6 - 8 days old metaphase plates and flood them in 0.25%

trypsin solution (dissolve 0.3625 gm of trypsin powder {Difco

 

 
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*$5* *$5* *(1(7,&6$1'%,27(&+12/2*<΁

1:250} in salt solution GKN, pH - 7.8 or dissolve the same
amount of trypsin in 100 ml of 0.9% saline).

2. Time required may vary with each sample, so test one slide and
then adjust the time accordingly for the remaining ones.

3. Rinse the preparation briefly in 0.9% saline (no pH adjustment

is required) and wash thoroughly in double distilled water.

4. Stain in freshly prepared 4% buffered Giemsa (merk) for 10 - 15

mins at pH 6.8 and examine the slide under a light microscope.
If the stain is too weak, rinse with saline, restain as long as
neccessary and rinse thoroughly in tap water. If too intense,
wash the slide in 4% PO4 buffer solution (buffer solution =
Na2HPO4 0.58 gm + NaH2PO4 0.358 gm in 1 litre of distilled
ZDWHUDWURRPWHPSHUDWXUHS+DGMXVWHGWRZLWK - 6 drops
of 10% NaHCO3).

5. Air dry the preparation and examine under bright field optics to
determine the quality of banding. If satisfactory, mount the
preparation in DPX.

 

 
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Reverse Banding
C-T Banding allows the recognition of all chromosomes by staining the
regions rich in constitutive heterochromatin as well as the bands
belonging to reverse system (R-Banding).

Stock
Ba(OH)2
2 X SSC
Stains all
Basic fuchsin
NaOH
Formamide
Water

Procedure

1. Prepare the chromosome spreads from cultured peripheral

lymphocytes.

2. Treat the slides with saturated solution of Ba(OH)2 at pH 13.2

for 5- 10 mins.

3. Incubate them in 2 X SSC at 60oC for 10 - 30 mins and stain in

0.005% solution of Stains all (4, 5, 4, 5’ - dibenzo - 3, 3’ - diethyl - 1
- 1 - methyl - WKLDFDUERF\DQLQH EURPLGH6HUYDLQD1 formalde-
hyde mixture).

Notes
• Divalent cations present in the alkaline pretratment contribute to the band
formation with the C.T. technique.

 

 
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*$5* *$5* *(1(7,&6$1'%,27(&+12/2*<΁

• If Stains all is not available, rinse the slides (after incubation in 2 X SSC) in
deionized water and transfer them to 0.1% solution of basic fuchsin in a 1 : 1: 1
mixture of 0.1 NaOH : formamide : water (pH adjusted to 10.2 with 1 N HCl)

• The characteristics of R- banding pattern produced with basic fuchsin are

comparable with Stains all.

 

 
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*(1(7,&6$1'%,27(&+12/2*<΁ *$5* *$5*


Satellite or Highly Repetitive DNA

Stock
Na2HPO4 (0.06 M) 6.30 gm
Water 500 ml
Citric acid (0.06 M) 1.06 gm
Water 125 ml
Giemsa stain
Phosphate buffer

Procedure

1. Prepare the metaphase plates from bone marrow cells,

following the flame drying technique.

2. Keep the slides in 0.06 M Phosphate buffer (Na2HPO4 + Citric

acid, pH 6.8) at 80- 100oC for 10 mins.

3. Cool down immediately to 0oC.

4. Reincubate in phosphate buffer at 65oC severally, at an interval

of 20- 30 mins.

5. Stain with Giemsa solution.

 

 
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*$5* *$5* *(1(7,&6$1'%,27(&+12/2*<΁


Nucleolus Organizer’s Region

Stock
Trichloro-acetic acid (5%)
HCl (0.1N)
Phosphate buffer
Giemsa stain

Procedure

1. Incubate the air dried preparations in 5% trichloro-acetic acid

(TCA) at 85 - 90oC for 30 mins.

2. Rinse the slides in tap water and reincubate in 0.1 N HCl at

60oC for 30- 45 mins.

3. Rinse the slides thoroughly in tap water and stain in buffered

Giemsa (diluted 1:10, pH 7.0).

Observation

In good metaphase cells, NOR appears as distinct purpulish-red spots, at

the satellite regions, in all acrocentric chromosomes.

 

 
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In situ Hybridization
In situ hybridization studies on chromosomes provide an approach to
genetic mapping of the sequence of interest. When one of the
hybridization partner remains in situ, using a given labeled polynucleotide
(DNA or RNA) probe, location of the homologous sequences in cells can
be determined. The pattern of functional organization or its expression
can also be studied conveniently by this technique at cellular or at organ
level.

Stock
20X SSC
Sodium chloride 3 M
Sodium citrate 0.3 M
Gelatin solution 0.1%
Gelatin 100 mg
Distilled water 100 ml
(warm at 70oC for 1 hr.)

Sodium acetate 3 M
(pH 5.2 with the help of glacial acetic acid)

RNase 10 mg /10 ml
Alcohol 70/90/100 %
Tris
(pH 7.5, pH 8.0, pH 9.5) 1 M
Sodium chloride 5 M
EDTA 0.05 M
TE
Tris
 

 
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*$5* *$5* *(1(7,&6$1'%,27(&+12/2*<΁

10 mM (pH 8.0)
EDTA 1 mM
Digoxigenin-dUTP labeled DNA probe
Salmon sperm DNA 10 mg/ml
Hybridization mix

Buffers
I - 7ULVP0S+1D&OP0
II - Blocking reagent in Buffer - I 0.5% w/v
III - Tris 100 mM, pH 9.5 1D&OP00J&O2 50 mM
IV - 7ULVP0S+('7$P0

Developer (to be prepared fresh)

Nitroblue Tetrazolium 4.5 μl
(NBT 75 mg/ml in Dimethyl formamide)

5-Bromo-4-Chloro-3-Indolyl Phosphate 3.5 μl

(50 mg/ml in Dimethyl formamide)

Buffer - III to make 1 ml

Stains
Safranin 100 mg
Distilled water 100 mg
(Dissolve Safranin powder in water at room temperature. The prepared stain can be stored
and used later on.)

Entellan mountant

Laboratory ware
Three incubators, preset at 37o, 42o and 60oC.
Sterilized glass slides and cover glasses

 

 
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*(1(7,&6$1'%,27(&+12/2*<΁ *$5* *$5*

Slide racks
Slide trays
Couplin jars
Magnetic stirrer
Micropipettes
Pipette tips
Plastic box
Forceps

Procedure

Step - I :

Labeling of probe DNA (with digoxigenin dUTP) by random priming

method

* 1-3 μg of DNA can be labeled per standard reaction.

1. Take desired amount of linear DNA in an eppendorf tube and

denature by heating in boiling water for 10 mins.

2. Quickly chill the test-tube on ice and add the following in

sequential order :
Hexanucleotide mix 2 μl
dUTP labeled mix 2 μl
Distilled water to make 19 μl
Klenow enzyme (3-5 units) 1 μl

3. Incubate the contents at 37oC for 1 hr. Stop reaction by adding

0.8 ml of 0.5 M EDTA (Final concentration 20m M)

4. Precipitate labeled DNA by the addition of :

Salmon sperm DNA (10 mg/ml) 2.0 μl
 

 
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*$5* *$5* *(1(7,&6$1'%,27(&+12/2*<΁

Lithium chloride (4 M) 2.5 μl
Prechilled (- 20oC) ethanol 75 μl

5. Allow to stand at - 70oC for 2 hrs.

6. Centrifuge the tubes at 12,000 rpm for 30 mins at 4oC.

7. Decant off the supernatant and wash the pellet with 70%
ethanol.

8. Dry at room temperature.

9. Dissolve in required amount of TE.

The labeled probe can be stored at - 20oC for more than two years.

Step-II :

Test for efficacy of DIG labelling

1. Take a small piece of nylon membrane, wet it with 2X SSC and

blot (under vacuum) 10 pg, 1 pg and 0.1 pg of the prepared
probe. Allow to dry at room temperature (~ 30 mins).

2. Cross-link the probe DNA with the membrane either by 3 - 4

mins exposure to UV on a transluminator or by heating the filter
at 70oC for 2 hrs.

3. Wash the filter briefly in Buffer- I.

4. Incubate in Buffer - II at room temperature for 30 mins to block

the membrane surface for specific binding of antibody to be
used afterwards. Briefly rinse in Buffer - I.

5. Incubate in Anti-DIG Antibody-enzyme conjugate (1 μl in 4 ml of

buffer - I) for 30 mins at room temperature.

6. Wash twice in Buffer - I at an interval of 15 mins.

 

 
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*(1(7,&6$1'%,27(&+12/2*<΁ *$5* *$5*

7. Briefly rinse in Buffer - III

8. Put the blot in a small polythene bag, add color developer under
dim light and seal the bag.

9. Incubate the blot within the sealed bag (working in a dark

closet) by wrapping with an aluminium foil until desired level of
color signal is visible.

10. When adequate signal appears, take out the blot from the bag
and keep it in buffer - IV to stop reaction. Store blot in Buffer - IV
or in dry condition.

Notes
• Under optimal conditions of probe labeling, 0.1 pg of probe gives a detectable
signal within 30 mins.

Step-III :

Treatment of prepared slides

1. Dip the prepared slides in a freshly prepared 0.1% solution of

gelatin for 3 - 5 secs. Let the slides air dry (the gelatin coat
keeps the cells in good state by preventing the binding of the
probe).

2. Arrange the slides in a moist chamber having filter papers

soaked in 2 X SSC. Place 100 μl of RNase (100 μg/ml in 2 X
SSC) over the material and cover each preparation with 22 mm2
cover glasses to remove RNA. Incubate the slides at room
temperature for 2 hrs.

3. Dip the slides gently into a beaker containing 2 X SSC and let
the cover glasses fall in the solution.

 

 
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*$5* *$5* *(1(7,&6$1'%,27(&+12/2*<΁

4. Wash slides thrice in 2 X SSC for 5 PLQVHDFK  WZLFH LQ 
ethanol for 10 mins each and once in 95% ethanol for 5 mins.
Air dry and store as such, if required.

5. Keep slides in 0.07 N-NaOH for 3 mins to denature the

chromosomal DNA.

6. Finally, wash the preparations thrice in 70% ethanol for 10 mins

each and twice in 95% ethanol for 5 mins each. Air dry.

Hybridization mix
Formamide 500 μl
20 X SSC 250 μl
DIG labeled probe (per slide) 10-20 ng
H2O to make 1000 μl
*15-20 μl of hybridization mix is sufficient for one slide.

Step-IV :

Hybridization

1. Place the tubes in boiling waterbath for 10 mins to denature the

labeled probe DNA. Add required amount of denatured probe to
the hybridization mix.

2. Take 20 μl of hybridization mix containing 10 - 20 ng of labeled

probe. Place a cover glass over the hybridization mix and seal
the edges with DPX.

3. Incubate the slides at 37oC in a closed humid chamber for 12 -

14 hrs.

 

 
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*(1(7,&6$1'%,27(&+12/2*<΁ *$5* *$5*

4. Once the hybridization is complete, peel off DPX sealing with
the help of forceps. Remove the coverglass by dipping slides in
2 X SSC.

5. Wash the slides thrice in 1 X SSC for 15 mins each at 60oC.

Detection of Color

1. Rinse slides in Buffer - I for 1 min and in Buffer - II for 30 mins.

2. Rewash in Buffer - I, for a minute.

3. Incubate the slides in anti-Digoxigenin antibody alkaline

phosphatase conjugate (diluted 1:5000 in buffer - I) for 30 mins.

4. Now, wash twice in Buffer - I for 2 mins each and then rinse in
Buffer - III.

5. Put 20 - 30 ul of color reagent (freshly prepared) on the slide,

cover with a cover glass, seal with DPX and leave the slide in a
dark chamber at room temperature for 1 - 12 hrs depending on
the time required for the development of optimal signal.

6. Observe the slide under microscope and place the slides in

Buffer - IV to stop the reaction.

7. Air dry and counterstain with safranin for 5 - 10 secs and then
wash twice in distilled water.

8. Leave the preparations to dry in air and mount with Entellan (E.
Merk).

Observation

Hybridized probe appears as purpulish-blue color deposits at the site of

hybridization. The specific chromosome sites that show hybridization

 

 
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*$5* *$5* *(1(7,&6$1'%,27(&+12/2*<΁

signal can be recognized by referring to standard polytene chromosome
maps.

Notes
• Good quality chromosome preparations are essential for a strong hybridization
signal.

• Denaturation of chromosomes must be precisely controlled because prolonged

treatment would destroy the structural details while inadequate treatment may not
permit hybridization.

• The probe must be denatured just before application.

• Inadequate washing after hybridization and antibody binding may generate

undesirable background. Hence it must be done adequately to ensure that
excess probe and antibody are removed.

• Safranin gives better chromosome morphology and contrast so that the

hybridization signal is seen more distinctly. If safranin stain is not available, stain
the slides with 2% aceto-orcein for 5-6 mins, cover the preparations with cover
glass and rinse them in two quick changes of 70% alcohol. Staining of
chromosomes must be controlled so that the hybridization signal is not masked.

• No air bubble should be trapped while mounting cover glasses at any step since
the trapped bubble would hamper the reaction in local region and thereby prevent
the hybridization signal.

 

 
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Extraction of Genomic DNA

from Calotes
Stock
SE
Sodium chloride (75 mM) 1M 37.5 ml
EDTA (25 mM) 500 mM 25.0 ml
Distilled water 437.5 ml
Sodium acetate 3M
Sodium acetate 24.6 gm
Distilled water 100.0 ml
(adjust pH to 5.2 with glacial acetic acid)

TEN
Tris (pH 8.0) (10 mM) 1M 5.0 ml
Sodium chloride (100 mM) 1M 50 ml
EDTA (pH 8.0) (25 mM) 500 mM 25 ml
Distilled water 420 ml
TE
Tris (pH 8.0) (10 mM) 1M 1 ml
EDTA (1 mM) 500 mM 0.2 ml
Distilled water 98.8 ml
SDS 10% and Proteinase K (10 mg/ml)
Distilled and buffered saturated phenol
Phenol - Chloroform - Isoamyl alcohol (25:24:1)

Absolute alcohol
Ethanol 70%, prechilled at - 20oC

 

 
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*$5* *$5* *(1(7,&6$1'%,27(&+12/2*<΁

Laboratory ware
Homogenizer
Micropipettes
Pipette tips
Centrifuge tubes
Eppendorf tubes
Cheese clothe

Procedure

1. Take a Calotes embryo and homogenise it in SE at 4oC.

2. Centrifuge at 4000 rpm for 10 mins at the same temperature.

Discard the supernatant.

3. Resuspend the pellet in 20 ml volume of TEN at room

temperature.

4. Add proteinase K to a final concentration of 100 ug/ml and SDS

to a final concentration of 1%PL[JHQWO\

5. Incubate the contents overnight at 37oC.

6. Add equal volume of phenol, agitate the contents for 15 mins in

a vortex mixer and then spin at 12000 rpm for 10 mins.

7. Pour the aqeous phase into a fresh tube and mix an equal
volume of phenol - chloroform - isoamyl alcohol.

8. &HQWULIXJH WKH PDWHULDO DQG UHPRYH LWV RUJDQLF SKDVH UHSHDW

the step-7 once again.

 

 
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*(1(7,&6$1'%,27(&+12/2*<΁ *$5* *$5*

9. Mix the aqeous phase with an equal volume of chloroform-
isoamyl alcohol. Centrifuge and transfer the aqeous phase to a
fresh tube.

10. Add 1/30 volume of Sodium acetate (3M) and 2 volumes of

DEVROXWHHWKDQRONHHSDW- 20oC for 1 hr.

11. Transfer the precipitated DNA carefully with fine forceps to a

fresh eppendorf tube containing 70% ethanol (chilled at - 20oC).

12. Spin briefly, decant off the overlying alcohol as much as

possible, lypophilize the DNA and dissolve in TE.

Observations

1 gm of tissue yeilds approximately 1 mg of DNA.

 

 
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*$5* *$5* *(1(7,&6$1'%,27(&+12/2*<΁


Agarose Gel Electrophoresis of DNA

Electrophoresis allows the separation and localization of DNA, in an
agarose gel, by observing ethidium bromide stained flourescent bands, in
an ultraviolet light, at a wavelength of 254 nm /310 nm/ 354 nm, using a
transluminator. The electrophoretic mobility of DNA through agarose gel
is effected by molecular size of DNA, agarose concentration,
conformation of the DNA and applied current.

Stock
Tris-Borate-EDTA (TBE) stock solution (5X)
Tris base 54.0 gm
Boric acid 27.5 gm
EDTA (pH 8.0) 0.5 M 20.0 ml
Distilled water to make 1000.0 ml
Working Buffer : 1X or 0.5X TBE
Loading Buffer (10X)
Bromophenol blue 0.25 %
Xylene cyanol 0.25 %
Ficoll (type 400) 25.0 %
in distilled water
DNA Sample
DNA 150-200 ng
Distilled water 18 μl
10X loading buffer 2 μl

Prepare beforehand, a stock solution of 10 mg/ml ethidium bromide and

store in a colored glass tube/bottle at 4oC.

 

 
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Procedure

Preparation of Agarose Gel

For analysis of restriction digested genomic DNA, usually a 0.8% gel is

made in 0.5X or 1X TBE. A higher or lower percentage of gel can be used
depending upon the size of DNA molecules to be analyzed.

1. Prepare the gel mold by sealing both the free ends of the gel
platform with plastic tapes. Place the comb on the gel platform
leaving a gap of 0.5 - 0.1 mm between the platform and the base
of the comb.

2. Add an appropriate amount of agarose (say 320 mg of agarose

in 40 ml of 0.5 X TBE), to a definite volume (about 40 ml. of 0.5
X TBE) of buffer.

3. Boil till the agarose dissolves.

4. Cool solution to 50oC and add ethidium bromide to a final

concentration of 0.5 μg/ml.

5. Pour the contents into the prepared mold and let the
temperature come down to normal (avoid vibrations while
polymerization goes on).

6. After 30 - 45 mins at room temperature, when the gel is set

completely, remove the comb carefully. Addition of
electrophoretic buffer, beforehand, can facilitate the removal of
the comb.

7. Remove the plastic tapes from both the ends of gel mould and
put the gel with its platform in a submarine electrophoresis. Add

 

 
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*$5* *$5* *(1(7,&6$1'%,27(&+12/2*<΁

sufficient electrophoresis buffer (1 X or 0.5 X TBE) to let the gel
submerge.

Loading of DNA

1. Mix the DNA sample and spin briefly before loading into the
wells.

2. Switch on the power supply between 50 to 150 volts depending

upon the size of the gel (@ 5 volts /cm).

3. After the run is over, examine the gel in UV light using a

transluminator. To evade harmful effects of UV radiations,
perspex shield or protective glasses can be worn.

Observations

In DNA agarose gel electrophoresis, Lambda DNA digested with Hind III
restriction enzyme is usually run to find out the molecular weight of the
experimental DNA. It shows 8 bands of different sizes :

23.132 Kb
09.420 Kb
06.555 Kb
04.360 Kb
02.333 Kb
02.039 Kb
00.566 Kb
00.124 Kb
Notes
Restriction enzymes digested with plasmid or phage DNA renders sharp bands of expected
molecular weight sizes. However, similarly digested genomic DNA shows a smear.

 

 
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*(1(7,&6$1'%,27(&+12/2*<΁ *$5* *$5*


Polymerase Chain Reaction

The polymerase chain reaction (PCR) is a technique, by which a very
small amount of DNA can be amplified, utilizing information of nucleotide
frequencies flanking the region of interest. It has manifold applications in
molecular biology, genetic engineering and DNA fingerprinting.

To replicate the parental DNA, a DNA polymerase needs a short primer

sequence which is complementary to a region in the template. Since all
DNA polymerases elongate the nascent chain 5’ - 3’ direction, a given
stretch of DNA is replicated in both directions using two different primers.
In the case of PCR, a specific set of two primers, each complimentary to
short sequences on either sides of the region, that has to be amplified,
are allowed to anneal and a DNA polymerase then elongates primer on
both ends to replicate the intervening region. This cycle of denaturation of
the template DNA, reannealing with primers and chain elongation goes on
and on producing a high yield of target DNA.

Stock
Purified genomic DNA of male & female Calotes
Stock solution of each primer 10 pM/μl
10X Taq Polymerase Buffer
KCl 500 mM
o
Tris-HCl (pH 8.4 at 26 C) 100 mM
MgCl2 15 mM
Gelatin 1 mg/ml
DNTPs mix 1.25 mM
(each of the 4 dNTPs)
Mineral Oil
 

 
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*$5* *$5* *(1(7,&6$1'%,27(&+12/2*<΁

1% Agarose gel

Laboratory ware

Thermal cycler/ 3 water baths set at 92oC, 72oC and 60oC.

Selection of DNA for amplification

For the purpose of amplification, two PCR primers should be synthesized

to match two regions of potential sequence conservation separated by a
DNA region of about 60 to 1000 nucleotides.

Design of PCR primers

Once conserved protein regions have been selected, degenerate

oligonucleotide primers must be designed to match the potential DNA
sequences which could encode the amino acids. A mixture of forward
primers (more 5’ in the gene) are synthesized to represent DNA
sequences potentially coding for more N - WHUPLQDOSURWHLQSDWFKUHYHUVH
primers (more 3’ in the gene) are made to match the inverse compliment
of DNA sequences which could encode more C-terminal protein patch.
These primers can be designed as follows :

1. Each degenerate primer should roughly match the potential

encoding DNA sequences for atleast 15 to 20 nucleotides.

2. Except for the extreme 3’ position, degeneracy should be

introduced into the 3’ half (at 6 - 10 nucleotide positions of each
primer sequence) in such a way that all possible codons which
could code for the conserved amino acids are represented. The
5’ half of each primer need not have degeneracies.

3. The 3’ terminal base of the forward primer should match either

the second codon position for an amino acid encoded by 2, 3 or
 

 
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*(1(7,&6$1'%,27(&+12/2*<΁ *$5* *$5*

4 codons (i.e, excluding Arg, Leu, Ser) or the third position of a
Met or Trp codon. The 3’ terminal base of the reverse primer
should compliment the first base of a codon specifying an
amino acid encoded by few alternative codons (e.g, Met, Cys,
Trp, His....). Primers with C as the 3’ nucleotide are often most
reliable.

4. Degeneracy should be kept low (upto 1024 fold) by proper

selection of priming sites.

5. Primers sequences should not have mono or dinucleotide

polymers and should not end on three G or C residues at the 3’
end.

6. If cloning genes are more divergent than the species you have
sequence data for, design primers around regions rich in Cys,
His, Pro....... They are unlikely to be substituted whereas, avoid
Ser, Ala, Asp, Glu, Phe, Tyr, Arg....... .

7. If the amplified DNA fragment becomes longer than 600 base

pairs, the 5’ end of each primer should be altered in such a way
that a six-cutter restriction site is present. Use Bam’II, EcoRI or
Xbal since all enzymes do not work well near the ends of
primers.

8. Using a compatible software, make it sure that the designed

primer sequences do not form stable hybrids with each other or
amongst degenerate primer mixes. Avoid complementarity of
more than three contiguous bases if these lie at the extreme 3’
end of a primer.

 

 
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*$5* *$5* *(1(7,&6$1'%,27(&+12/2*<΁

Procedure

1. Take two clean eppendorf tubes of 1.5 ml each (one for male
and another for female genomic '1$DQGDGG
10x Polymerase Buffer 5 μl
dNTPs mix 8 μl
Primers (10pM/ul) 3 μl each
Genomic DNA (male & female) 100 ng each
(in respective tubes)
1 U
Taq polymerase
Distilled water to make 50 μl
Mix and overlay with 50 ul of mineral oil.

2. Denature the contents in the tube at 92oC for 5 mins.

3. Now keep the tubes at :

50oC for 2 mins (annealing)

72oC for 2 mins (elongation)

92oC for 1 mins (denaturation)

4. Repeat the cycle for 35 times.

5. During the 35th cycle, extend the synthesis period (72oC) upto 5
mins to falicitate completion of elongating chains. Terminate the
reaction by chilling the tube on ice.

6. Take 20 μl each of the reaction contents, mix them with the

loading dye buffer and run on a 1% Agarose gel. Use Hind - I
digested pUC DNA as marker.

7. After the run, observe the amplified DNA in ultraviolet light using
a transluminator.
 

 
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*(1(7,&6$1'%,27(&+12/2*<΁ *$5* *$5*

Observation

A 600 bp elongated band is possible.

 

 
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*$5* *$5* *(1(7,&6$1'%,27(&+12/2*<΁


RT-PCR : Reverse Transcriptase -

Poly Chain Reaction
In order to observe the expression of gene during development, there are
generally two motives :
i) ZKHWKHUWKHSDUWLFXODUJHQHLVH[SUHVVHGLQWKHJLYHQHPEU\R
and
ii) at which specific region of the embryo is the gene expressed.

Though the techniques including extraction of RNA and susequent

localization by northern blotting successfully demonstrate the expression
in the embryo yet to analyse the site of expression, in situ hybridization
has to be employed.

To understand the role of genes involved in the developmental process,

the expression pattern of a gene is studied. But owing to very low levels
of expression, it becomes difficult to perform northern blotting, RNase
protection assay or in situ hybridization. Therefore transcripts of low
abundance are amplified using an RT- PCR.

PCR amplification of RNA sequences requires conversion of mRNA

template to cDNA by reverse transcriptase (RT) and amplification of
cDNA by PCR. Reverse Transcriptase - Poly Chain Reaction (RT - PCR) is
used for quali-quantitative analysis of the transcripts as well as cDNA
cloning.

Stock
Stock solution - D
Guanidium isothiocyanate 10 gm

 

 
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*(1(7,&6$1'%,27(&+12/2*<΁ *$5* *$5*

Water 11.72 ml
Sodium citrate (pH 7.4) 0.704 ml
10% Sacrocyl 1.05 ml
Working solution - D
Stock solution - D 1 ml
2 -mercaptoethanol 7.5 ml
Sodium citrate (pH 7.0) 0.75 M
10% N-lauryl sarcosine
Water saturated phenol
Chloroform
Isopropanol
Ethanol
10 X DNase I reaction buffer
Sodium acetate 1 M
MgSO4 1 M
DNase I (RNase free)
RNase inhibitor
EDTA 20 mM
Oligo dT 0.5 μg/ml
dNTP mix 10 mM
DTT 0.1 M
10 X Synthesis buffer
Reverse transcriptase
Primers
Taq DNA polymerase
Agarose
Solutions for RNA extraction should be DEPC treated

 

 
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*$5* *$5* *(1(7,&6$1'%,27(&+12/2*<΁

*Glassware should be acid treated autoclaved and baked at 300oC for 4
hrs and plasticwares should be rinsed with chloroform to inactivate
RNase.

Procedure

Step-1 :

Isolation of RNA by AGPC Technique

1. Take out 10 mg of tissue in chilled PBS.

2. Mince the tissue on an ice-slab and homogenize with 100 μl

solution D. Transfer the contents into a test tube.

3. Add 10 μl of 2M Sodium acetate (pH 4.0), 100 μl of phenol and

200 μl of chloroform. Agitate in a cyclomixer and cool on ice for
15 mins.

4. Centrifuge the sample at 10 K for 20 mins.

5. Carefully collect the aqeous phase (RNA) without disturbing the

organic and interphase (proteins and DNA).

6. Add an equal volume of isopropanol and store at - 70oC for 4

hrs.

7. Spin the RNA at 10 K for 20 mins. Decant off the supernatant

and dissolve the pellet in 25 μl of solution- D (1/4th volume).

8. Transfer the suspension to an eppendorf tube and precipitate

with 1 volume of isopropanol or 2 volume of ethanol at - 20oC for
4 hrs.

9. Centrifuge at 15 K for 10 mins, maintaining the temperature at

4oC.

 

 
 ΀5(6($5&+7(&+1,48(6,102/(&8/$5%,2/2*<
*(1(7,&6$1'%,27(&+12/2*<΁ *$5* *$5*

10. Wash the pellet in 80% ethanol, sediment and dry.

11. Treat the extract in DEPC water at 65oC for 10 mins, if required,
otherwise dissolve in 0.5% SDS.

Step - 2 :

DNase I Treatment to RNA samples

1. Take 1 μg of RNA to an RNase free microcentrifuge tube (0.5

ml) and place it on an ice slab.

2. Add 1 μl of 10 X DNase I reaction buffer and 1 μl of RNase

inhibitor.

3. Add 1 unit of amplification grade DNase I

4. Bring the volume to 10 μl with DEPC treated water and incubate

at 37oC for 15 mins.

5. Inactivate DNase I by treating the reaction mixture with 1 μl of

20 mM EDTA and heating at 65oC for 10 mins.

6. Extract first with phenol : chloroform and then with chloroform

only. Now, precipitate by adding 2 volumes of alcohol.

7. Wash in 80% alcohol, dry and dissolve in DEPC treated water.

Step - III :

Preparation of Ist cDNA strand

1. To an autoclaved microcentrifuge tube, add 1 - 5 ug of total RNA

in 13 μl of DEPC treated water.

2. Add 1 μl of oligo dT (0.5 mg/ml) to the tube and mix gently.

 

 
 ΀5(6($5&+7(&+1,48(6,102/(&8/$5%,2/2*<
*$5* *$5* *(1(7,&6$1'%,27(&+12/2*<΁

3. Heat the mixture to 70oC for 10 mins and incubate on ice for a
minute.

4. Then add :
10 X synthesis buffer 2 μl
dNTP mix 10 mM 1 μl
DTT 0.1 M 2 μl
RTase (200 U/μl) 1 μl

5. Mix all the components gently and collect the reaction mixture
after a brief spinning.

6. Incubate the mixture at room temperature for 10 mins.

7. Transfer the tube to a water bath preset at 42oC and stand as

such for 50 mins.

8. Terminate the reaction by increasing the temperature upto 70oC

for 15 mins, followed by cooling on ice.

Step - IV :

PCR

1. Take a little sample of 1st strand cDNA and add :

10 X synthesis buffer 8 μl
Primer 1 10 μM 1 μl
Primer 2 10 μM 1 μl
Taq DNA polymerase 5U/μl 1 μl
Water to make 80 μl

2. Stir the reaction gently and overlay 2 drops (~ 100 μl) of mineral
oil.

 

 
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*(1(7,&6$1'%,27(&+12/2*<΁ *$5* *$5*

3. Heat the mixture at 94oC for 5 mins (to denature DNA).

4. Repeat the following cycle 15 - 30 times :

Denature at 94oC for 1 min

Anneal at 50oC for ½ min

Synthesize at 72oC for 1 min

5. Analyze 10 - 12 μl of the amplified DNA on agarose gel.

Notes
• Optimize the concentration of Mg++ for different PCR conditions.

 

 
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*$5* *$5* *(1(7,&6$1'%,27(&+12/2*<΁


Production of Pectinase by
Solid State Fermentation (SSF)
Plants and animals produce a wide range of enzymes and fungi, in
particular, are extensively used for the production of industrial enzymes
since they are easy to be manouvred. Fungal amylolytic, cellulolytic and
proteolytic enzymes are commercially produced in many countries and
are marketed under different trade names.

Stock
Wheat bran
Hydrochloric acid
Zinc sulphate (ZnSO4. 7H2O)
Copper sulphate (CuSO4. 5H2O)
Tween-80

Glassware
Conical flask - 500 ml
Inoculating needle
Sterile graduated pipettes - 2 to 5 ml

Procedure

A. carbonarius (CFTRI 1047) : The culture is maintained on PDA

slants.

Preparation of PDA slants

1. Boil 200 gm of peeled potatoes in one litre of tap water until

they become soft. Decant off the extract and make volume upto
1 litre.
 

 
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*(1(7,&6$1'%,27(&+12/2*<΁ *$5* *$5*

2. To this add 20 gm of agar-agar powder and boil till the agar
dissolves.

3. Now, add 20 gm of dextrose and mix well. Pour the hot soup in
the test - tube (8 - 10 ml tube) and plug them with cotton wool.

4. Sterilize the tubes in an autoclave at 15 psi for 20 min. Prepare

the slants and store them in a refrigerator at 4oC.

5. To use, inoculate them with the master culture at ambient

temperature (25- 30oC) for 4- 5 days.

The master culture should be sub-cultured every 3 - 4 months.

Spore suspension

1. Take 8 - 10 ml of sterile water containing a drop of Tween - 80.

Add this to a fully sporulated A. niger slant culture (raised on
PDA slants) by means of a sterile pipette.

2. Scrap the spores by using inoculating needle under strict

asceptic conditions.

3. Transfer 2 - 3 ml of spore suspension to each flask containing

sterile wheat bran medium.

Composition of mineral solution

ZnSO4. 7H2O 0.007 gm
FeSO4. 7H2O 0.007 gm
CuSO4. 5H2O 0.007 gm
HCl (0.2 N) 100 ml

 

 
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*$5* *$5* *(1(7,&6$1'%,27(&+12/2*<΁

Preparation of medium

1. Take 40 gm of wheat bran and mix with 20 - 25 ml of mineral

solution.

2. Transfer the mixture to 500 ml capacity conical flasks. Sterilize

the contents at 15 psi for 45- 60 min.

3. Allow to cool at room temperature, then inoculate each flask

with 2 - 3 ml of spore suspension.

4. Mix the medium thoroughly by gently tapping the flasks and

keeping them in slanting position at ambient temperature for 5- 7
days.

Preparation of 1% pectin solution

Dissolve 10 gm of powdered pectin (AR grade) in citrate buffer (pH 4.0)

by heating in a water bath. Make up the volume to 1 litre and store in
refrigerator with a toluene layer.

Procedure

• Reduction in viscosity of 1% pectin solution can be used as the

basis for the assay of enzyme activity.

1. Take 24 ml of 1% pectin solution in an Erlinmeyer’s flask and

add 1 ml of 1:10 diluted solution of the above enzyme to it.

2. Keep the contents in a water bath at 40oC for 30 mins.

Simultaneously, run a blank, with 24 ml of 1% pectin solution
and 1 ml of distilled water.

The reduction in viscosity of the pectin solution can be measured by using

Oswald No. 2 Viscometer.
 

 
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*(1(7,&6$1'%,27(&+12/2*<΁ *$5* *$5*

T1-T2
% Reduction in viscosity of 1% pectin solution = X 100
T1

where, T1 = time taken for the blank

T2 = time taken for the sample

(fifty percent reduction in viscosity under the above condition is defined as one unit)

Observation

Note the nature, color and type of growth. Record the pectinase activity of
the enzyme solution extracted using some other bran diluted with water in
the ratio of 1: 10.

 

 
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*$5* *$5* *(1(7,&6$1'%,27(&+12/2*<΁


Preparation of Stains & Reagents

1. Cornoy’s fluid
Absolute ethanol 6 parts 30 ml
Glacial Acetic acid 1 part 5 ml
or 2 parts 10 ml
Chloroform 3 parts 15 ml

2. Schiff’s reagent
Basic fuchsin 0.5 gm
Distilled water 100 ml
o
Bring to the boiling state and cool to 50 C. Add 1 gm of sodium or potassium metabisulphite
solution and 5 ml of 1N HCl. Allow to stand overnight. The solution decolourizes to watery
pink. Add ½ norite (activated carbon) and shake. Filter through filter paper (Whatman No. 1).
A clear solution is obtained.

3. Brilliant Cresyl Blue ( Alcoholic)

Brilliant Cresyl Blue 0.3 gm
Absolute alcohol or Methanol AR 100 ml
o
Heat on waterbath at 60 C, cool and filter.

4. Brilliant Cresyl Blue (Aqueous)

Brilliant Cresyl Blue 1 gm
Sodium chloride solution (0.9%) 100 ml
o
Heat on waterbath at 60 C, cool and filter.

5. Field’s stain A
Methylene Blue 1.6 gm
Azure I 1 gm

 

 
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*(1(7,&6$1'%,27(&+12/2*<΁ *$5* *$5*

Disodium hydrogen-orthophosphate 2.6 gm
(anhydrous)
Potassium di hydrogen- 2.6 gm
orthophosphate
Distilled water 1 litre
Heat on a waterbath for an hour. Allow to stand for 24 hrs at room temperature and filter.

6. Field’s stain B
Eosin (water soluble) 2 gm
Disodium hydrogen - orthophosphate 2.6 gm
(anhydrous)
Potassium di hydrogen - 2.6 gm
orthophosphate
Distilled water 1 litre
Heat on a waterbath for an hour. Allow to stand for 24 hrs at room temperature and filter.

7. Jenner’s Stain
Jenner’s Stain 0.5 gm
Methanol AR 100 ml
Stir vigorously and filter.

8. Leishman’s Stain
Leishman’s Stain 0.15 gm
Methanol AR 100 ml
Heat at 40oC for 1 hr, leave it to stand at room temperature for 72 hrs and filter.

9. Haemotoxylin Stain
Haematoxylin 1 mg
Ethanol (100%) 10 ml

 

 
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*$5* *$5* *(1(7,&6$1'%,27(&+12/2*<΁

Distilled water X 100
Take 25 ml of this solution and add a drop of saturated solution of lithium carbonate (color
changes from yellow to wine red). The stain is ready for use.

10. Wright’s stain

Wright’s stain 0.25 gm
Methanol AR 100 ml
Shake vigorously for 2-3 hrs and heat at 40oC for 1 hr. Allow to stand at room temperature for
3 days.

11. Giemsa stain

Giemsa stain’s powder 0.8 gm
Glycerine LR 50 ml
Warm in a waterbath at 60o&ZLWKFRQVWDQWVWLUULQJWKHQDGG
Methanol AR 50 ml
Cool, filter and stock at 2oC upto a maximum of 2 months.

12. Enzyme solution

Pectinase 500 mg
Cellulase 500 mg
Distilled water 10 ml
1 N HCl 5-6 drops
Keep the solution in a corked tube inside a refrigerator at 2oC for a minimum of 24 days to 1-2
months.

13. Barium hydroxide

Barium hydroxide 5 gm
Distilled water 10 ml
Shake well and keep in a stoppered bottle. Use fresh solution everytime.

 

 
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*(1(7,&6$1'%,27(&+12/2*<΁ *$5* *$5*

14. Saline Sodium Citrate
Sodium chloride 8.716 gm
Sodium citrate 4.410 gm
1 N HCl 6 drops
Distilled water 500 ml
Heat at 60oC before use. Keep the stock solution at 2oC for a maximum of 2 weeks.

15. Citrate buffer

A Na2HPO4 0.06 M 14.2 gm
Water 500 ml
B Citric acid 0.06 M 2.1 gm
Water 10 ml
Mix A 4.55 ml
B 15.45 ml

16. Trypsin (for sister chromatid exchanges)

Trypsin 1 mg
Distilled water 100 ml

17. Trypsin (for G-banding)

Trypsin 5 mg
NaCl 0.89% 100 ml

18. Phosphate Buffer

A KH2PO4 9.08 gm
Distilled water 1000 ml
B Na2HPO4.2H2O 11.88 gm
Distilled water 1000 ml
 

 
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*$5* *$5* *(1(7,&6$1'%,27(&+12/2*<΁

KH2PO4 Na2HPO4.2H2O pH
98.8 1.2 5.0
87.7 12.3 6.0
81.4 18.6 6.4
68.7 31.3 6.5
57.0 43.0 6.7
50.8 49.2 6.8
39.2 60.8 7.0
28.5 71.5 7.2

19. Bromodeoxyuridine (5BudR)

Prepare in sterile water and store in a dark bottle.

20. Newcomer’s Fluid (Fixative)

Isopropyl alcohol 6 parts
Propionic acid 2 parts
Petroleum Ether 1 part
Acetone 1 part
Dioxane 1 part

21. Mordant (Lang)

4% iron alum (Ferric ammonium 500 ml
sulphate)
Glacial acetic acid 6 ml
Conc. H2SO4 0.6 ml

Sterilization
Put fresh cover glass in a petridish containing absolute alcohol and wipe them dry with a linen
cloth. Dip the cover glasses in 1% siliconizing fluid, wash in running tap water for 1-2 hrs.
Rinse in distilled water and dry in oven.
 

 
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*(1(7,&6$1'%,27(&+12/2*<΁ *$5* *$5*


References
• Aguilar G & Huitorn C (1987) Stimulation of the production of
extracellular pectinolytic activities of Aspergillus species by galacturonic
acid and glucose addition. Enzyme Microbiology & Technology 9: 690-
696.

• Arrighi FE & Hsu TC (1971) Localization of heterochromatin in human

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• Beg QK, Bhushan B, Kapoor M & Hoondal GS (2000) Effect of amino

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• Beg QK, Bhushan B, Kapoor M & Hoondal GS (2004) Production and

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• Carr, DH and Walker, JE (1986) Stain Tech. 36 : 233-236.

• Caspersson T, Zech L Johansson & Modest EJ (1970) Identification of

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• Ford JH, Callen FD, Adriame B, Jahuke B & Roberts CG (1982) Within
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• Gummadi SN & Panda T (2003) Purification and biochemical properties
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• Hours RA, Vogrt CE & Ertola RJ (1988) Apple pomace as raw material
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conditions on xylanase production by Streptomyces species. (strain lb
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its potential as a source of antibiotics active against antibiotic-resistant
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• Saadoun I & Wahiby L (2008) Recovery of soil streptomycetes from arid
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• Sreenath HK, Kogel F & Radola BJ (1986) Macerating Properties of a

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• Sumner AT (1972) A simple technique for demonstrating centromeric

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