Lab in Industrial, Plant and Animal Biotechnology Student's Manual

PGPCAS/DBT/Lab in Industrial, Plant & Animal Biotechnology
Lab in Industrial, Plant and Animal

Biotechnology
Students Manual
Department of Biotechnology, PGP College of Arts & Science, Namakkal
INDEX
S.NO
DATE
PAGE
NO
DESCRIPTION
PREPARATION OF TISSUE CULTURE MEDIUM
PLANT TISSUE CULTURE- AN INTRODUCTION
MICROPROPAGATION
CALLUS INDUCTION
DIRECT ORGANOGENESIS SHOOT TIP CULTURE
EMBRYO CULTURE
ISOLATION OF PROTOPLASTS
ENZYMATIC METHOD
ANTHER CULTURE
10
CELL SUSPENSION CULTURE
11
INTRODUCTION FOR ANIMAL CELL CULTURE

LABORATORY
12
STERILIZATION TECHNIQUES
13
PREPARATION OF MEDIA
14
PRIMARY CELL CULTURE
15
CELL COUNTING AND VIABILITY
16
STAINING OF ANIMAL CELLS
17
CULTURE OF VIRUS IN CHICK EMBRYO
18
CELL CULTURE TERMNOLOGY
REMARKS
Ex.No:
Date:
PREPARATION OF TISSUE CULTURE MEDIUM

INTRODUCTION:
The basic nutritional requirements of cultured plant cells as well as plants are very
similar. However, the nutritional composition varies according to the cells, tissues, organs
and protoplasts and also with respect to particular plant species. The appropriate composition
of the medium largely determines the success of the culture. A wide variety of salt mixtures
have been reported in various media.
A nutrient medium is defined by its mineral salt composition, carbon source,
vitamins, growth regulators and other organic supplements. When referring to a particular
medium, the intention is to identify only the salt composition unless otherwise specified. Any
number and concentration of amino acids, vitamins, growth regulators and organic
supplements can be added in an infinite variety of compositions to a given salt composition in
order to achieve the desired results.
UNITS FOR SOLUTION PREPARATION
The concentration of a particular substance in the media can be expressed in various units
that are as follows :
Units in weight
It is represented as milligram per litre (mg/l) 10
-6
= 1.0 mg/l or 1 part per million (ppm) 10
-7
= 0.1 mg/l.
-8
10 = 0.001 mg/l or 1 g/l.

Molar concentration
A molar solution (M) contains the same number of grams of substance as is given by
molecular weight in total volume of one litre.
1 molar (M)
the molecular weight in g/l
1 mM
the molecular weight in mg/l or 10 M
1 M
-3
-6
-3
the molecular weight in g/l or 10 M or 10 mM.
Conversion from milli molar (mM) to mg/l

For example, molecular weight of auxin 2,4-D = 221.0 1M 2,4-D
solution consists of 221.0 g per litre
1 mM 2,4-D solution consists of 0.221 g per litre = 221.0 mg per litre 1 M 2,4D solution consists of 0.000221 g/l = 0.221 mg/l
Conversion from mg/l to mM
The molecular weight of CaCl2 - 2H2O
= 40.08 + 2 x 35.453 + 4 x 1.008 + 2 x 16 = 147.018
(the atomic weights of Ca, Cl, H and O being 40.08, 35.453, 1.008 and 16.0 respectively).
If, 440 mg/l of CaCl2 - 2H2O is to be converted into mM; then
The number of mM CaCl2 . 2H2O =
No. of mg CaCl2 - 2H2O
Molecular weight of CaCl2 . 2H2O
440
=
=2.99 mM
147.019
Thus, 440 mg/l CaCl2 - 2H2O = 2.99 mM

REQUIREMENTS
Glassware / plasticware / minor items
1. Aluminum foil
2. Beakers of different sizes from 50ml to 2000ml
Chemicals of Analar grade, depending upon the medium

Conical flasks (with wide mouth) of different capacities
3. (100ml, 150ml, 250ml, 11, 2.5l)
Culture tubes (25mm x 150 mm)

Funnels
4. Glass markers
5. Graduated cylinders of various capacities
6. Measuring cylinders of various capacities
7. Non-absorbent cotton and muslin/cheese cloth for cotton plugs
8. Petri dishes of different sizes (glass or sterilized plastic)
9. Pipettes (different capacities from 1ml to 10ml)
10. Sterile filtration assembly

11. Wrapping paper (brown sheet)
MEDIA COMPOSITION
A number of basic media are listed in Table 1. The salt composition of Murashige and Skoog
(1962) nutrient medium, referred to as MS medium, is very widely used in different culture
systems as it gives satisfactory results. But it must be remembered that it is not always the
best medium. Generally, in all the media, the nutritional milieu consists of inorganic
nutrients, carbon and energy sources, vitamins, growth regulators, and complex organic
supplements. It is desirable to choose a composition according to the knowledge of the
physiology of species vis-a-vis mineral nutrition.
INORGANIC NUTRIENTS
Mineral elements are very important in the life of a plant. Besides, C,H,N, and O, 12 other
elements are known to be essential for plant growth. According to the recommendations of
the International Association for Plant Physiology, the elements required by plants in
concentration greater than 0.5 mmol/l are referred to as macroelemetns or major elements and
those required in concentration less than the prescribed amount are microelements of minor
elements. A variety of salts supply the needed macro and micronutrients that are the same as
those required by the normal plant.
Major salts : The salts of potassium (K), nitrogen (N), calcium (Ca), magnesium (Mg),
phosphorus (P) and sulphur (S) are required in macro or millimole quantities. Nitrogen is
generally used as nitrate or ammonium salts, sulphur as sulphates and phosphorus as
phosphate.
Minor salts : The salts of iron (Fe), manganese (Mn), boron (B), copper (Cu), zinc (Zn),
iodine (I), molybdenum (Mo) and cobalt (Co) are required in micromolar concentrations and
are considered to be minor salts. These salts are essential for the growth of tissues and are
required in trace quantities.
To achieve the maximum growth rate, the optimum concentration of each nutrient can vary
considerably. The mineral composition of a culture medium is defined precisely by the
equilibrium of the concentrations of differention in a solution. When mineral salts are
dissolve in water, they undergo dissociation and ionization. The active factor in the mediums
is the ions of different types rather than the compounds. Therefore, a useful comparison
between the two media can be made by looking into the total concentrations of different types
of ons in them. To choose a mineral composition and then compare their differnet ionic
balances, one uses ionic concentrations expressed in milli equivalents per litre (Table 2). Any
success with a medium is in all probability due to the fact that the ratios as well as
concentrations most nearly match the optimum requirements for the cells or tissues for
growth and/or differentiation.
+
++
Cultured tissues have specific needs vis-a-vis the following ions: K , NO3 , NH4 , Ca ,
++
Mg . Phosphorus is often carried in low concentrations. The ions K , NO3 , and NH4
have a profound influence on the growth of tissues.

Tabel 1 : Composition of plant tissue culture media (values expressed as mg per
litre)
White
Murashige
& Gamborg's Chu
Constituent
(1963)
Skoog (1962) - MS (1968) - B6 (1978) N6
KCl
65
MgSO4 . 7H2O
720
370
250
185
NaH2PO4 . H2O
16.5
150
CaCl2 . 2H2O
440
150
166
KNO3
80
1900
2500
2830
Na2SO4
200
NH4NO3
1650
KH2PO4
170
400
Ca(NO3)2 . 4H2O
300
(NH4)2SO4
134
463
FeSO4 . 7H2O
27.8
27.8
MnSO4 . 4H2O
22.3
MnSO4 . H2O
Kl
0.75
0.83
10
0.75
3.3
0.8
CoCl2 . 6H2O
0.025
0.025
ZnSO4 . 7H2O
8.6
1.5
CuSO4 . 5H2O
0.025
0.025
H3BO3
1.5
6.2
1.6
Na2MoO4.2H2O
0.25
0.25
Fe2(SO4)3
EDTA disodium salt
EDTA-Na ferric salt
m-inositol
2.5
-
37.3
100
43
100
37.3
-
Thiamine HCl
Pyridoxine HCl
Nicotinic acid
Glycine
Cysteine
Sucrose
0.1
0.1
0.5
3
1.0
20,000
0.1
0.5
0.5
2
30,000
1.0
1.0
1.0
10
20,000
1.0
0.5
0.5
30,000
In place of FeSO4 . 7H2O (27.8 mg and EDTA - Disodium salt (37.3 mg), EDTANaferric salt (40) mg can be added.
Table 2 : Composition of mineral solutions used in vitro
Ions
Gautheret
Gamborg Murashige & Skoog
White
NO3
5.49
25.0
39.4
3.34
H2PO4
0.92
1.1
1.3
0.12
SO4
1.0
4.0
3.0
8.64
7.41
2.0
32.1
6.0
49.7
0.88
12.98
2.17
25.0
20.1
1.68
2.0
20.6
1.1
2.92
Ca
4.24
2.0
6.0
2.54
Mg++
Total cations (mEq/l)
Total anions + cations
1.0
7.41
11.70
2.0
32.1
60.2
3.0
49.7
93.3
5.84
12.98
17.45
Cl
Total anions (mEq/l)
+
NH4
Na
++
(10 M/I)
The role of different elements has been described below:
Nitrogen: Nitrogen is the major component supplied in the form of nitrates or
ammonium salts. Nitrogen is an important part of amino acids, proteins, nucleic acids.
Inorganic nitrogen is utilized in order to synthesize organic molecules. For most
purposes, a nutrient medium should contain from 25 to 60 mM inorganic nitrogen. The
cells may grow on nitrate alone, but often there is a distinct beneficial effect and
requirement for ammonium or another source of reduced nitrogen. Besides, nitrate alone
in the medium drifts the pH towards alkalinity. Adding a small amount of an ammonium
is the range of 25-40 mM and ammonium in the range of 2-20 mM. Nitrate cannot be
simply used to synthesize organic molecules but has to be reduced to ammonia first. The
response to ammonium varies from inhibitory to essential, depending upon the tissue and
the purpose of culture
e. In case of amounts in excess of 8 mM of ammonium or if grown solely on this source of

nitrogen, then citrate, malate, succinate or another TCA cycle acid should be present. Most
plants prefer nitrate to ammonium, although the opposite is also true in some cases.
Potassium: Potassium is required at concentrations of 2 to 26 mM. This element is generally
supplied as the nitrate or as the chloride form and cannot be substituted by sodium. It is a
monovalent cation with high mobility in the plant. Potassium salts have an important function
in the osmotic regulation of the cell. Potassium ion is essential for the activation of many
+
enzymes. In photosynthesis, K regulates the ion balance and pH of chloroplasts.

Calcium: Calcium is essential for cation-anion balance by counteracting organic and
inorganic anions. A concentration of 1-3 mM of calcium is usually adequate. Calcium is also
important for cell and root multiplication. Calcium, a component of the cell wall, is largely
bound to the cell wall and membrane. This is because of the large number of Ca
++
binding
places on the cell wall and limited mobility of calcium through the membrane into the
++
cytoplasm. The stability of cell membrane is highly influenced by Ca .

Phosphorus: Phosphorus is present in the plant in the form of inorganic phosphate (iP). A
concentration of 1-3 mM phosphate is usual adequate. The high-energy pyrophosphate bond
of phosphorus, when bound to another P atom as in ATP, is very important for the energy
metabolism in the cell. Phosphorous is an essential element in DNA and RNA nucleic acids.
In phospholipids, this element is very important for the energy metabolism of the plant in
form energy-rich phosphate esters.
Magnesium: A concentration of 1-3 magnesium is usually adequate. This element is an
essential component for many enzymes reactions and is very important in photosynthesis.
Magnesium is indispensable for the energy metabolism of the plant because of its importance
in the synthesis of ATP.
Sulphur: A concentration of 1-3 mM sulphate is usually adequate. These have to be reduced
first for the synthesis of sulphur containing compounds such as amino acids, proteins and
enzymes. Sulphur in its non-reduced form is incorporated in sulpholipids and
polysaccharides.
Boron: Boron is required for the synthesis of cell wall as well as in the stabilization of the
constituents of cell wall and cell membrane.
Chlorine: Chlorine is taken up as a chloride and is very mobile in the plant. The main
functions of the ions are in osmoregulation. Chlorides play a role in photosystem II during
the Hill reaction. Chlorine also regulates the opening and closing of stomata and is thus very
important in the regulation of the osmotic potential of vacuoles as also to turgor-related
processes.
Copper: Copper is taken up by the plant as Cu
++
or as a copper chelate complex. Within the
cell, copper is mostly part of the enzyme complexes and important in redox reactions
executed by these enzymes. It is useful in photosynthesis.
Cobalt: Cobalt is assumed to be important in nitrogen fixation. In higher plants the function
of this element is not very clear.
Manganese: Manganese is taken up by the plant as bivalent unbound Mn
++
ions. The
element is strongly bound to several metalloproteins. The ion is involved in the Hill reaction
of photosystem II in which water is split into oxygen and protons.
Molybdenum: Molybdenum is used as a cofactor in many enzymes, including nitrogenase
and nitrate reductase. It is also directly involved in the reduction of N2.
++
Zinc: Zinc is taken up by the roots as Zn . It is neither oxidized nor reduced in the plants. It
is an important component of a number of enzymes, e.g. alcohol dehydrogenase in the
meristem zone of the plant. Zinc is also very important for protein synthesis.
Iron: Iron is generally added as a chelate with ethylene diamine tetra acetic acid (EDTA). In
this form, iron remains available up to a pH of 8.0. It is mainly bound to chelators and
3+
complex compounds in plants. Most plants absorb only ferric ions (Fe ). The main function
of iron is to form iron chelates and two kinds of proteins: haeme proteins and iron sulphur
proteins. The most well-known haeme proteins are the cytochromes, functioning as
intermediates for electrons required for the reduction of nitrate to nitrite by the enzyme
nitrate reductase in nitrogen assimilation. The second group of iron-binding proteins are the
iron sulphur proteins. The iron is bound to a thiol group (-SH) of cystine and/or inorganic
sulphur. Ferridoxin is the most common iron sulphur protein. It functions as a carrier in the
electron transport reaction catalyzed by nitrate reductase, sulphate reductase, the synthesis of
+
NADP during photosynthesis and nitrogen reduction by nitrogenase complex. Iron is also
important in the biosynthesis of chlorophyll.

Carbon and energy source
The standard carbon source without exception is sucrose but plant tissues can utilize a variety
of carbohydrates such as glucose, fructose, lactose, maltose, galactose and starch. In the
cultured tissues or cells, photosynthesis is inhibited and thus carbohydrates are needed for
tissue growth in the medium. Sucrose, at a concentration of 2-5% in the medium, is widely
used. The autoclaving process does cause an alteration in the sugars by hydrolysis but
presents no drawbacks to the growth plan. Most media contain myo-inositol at a
concentration of 100-mg per litre, which improves cell growth.
VITAMINS
Normal plants synthesize the vitamins required for growth and development, but plant cells
in culture have an absolute requirement for vitamin B1 (thiamine), vitamin B (nicotinic acid)
and vitamin B6 (pyridoxine). Some media contain pantothenic acid, biotin, folic acid, pamino benzoic acid, choline chloride, riboflavine and ascorbic acid. The concentrations are in
the order of one mg/l. Myo-inositol is another vitamin used in the nutrient medium with a
concentration of the order of 10-100 mg/l.
GROWTH REGULATORS
Hormones now referred to as growth regulators are organic compounds that have been
naturally synthesized in higher plants, which influence growth and development. These are
usually active at different sites from where they are produced and are only present and active
in very small quantities. Two main classes of growth regulators of special importance in plant
tissue culture are the auxins and cytokinins, while others are of minor importance, viz.,
gibberellins, abscisic acid, ethylene, etc. Some of the naturally-occurring growth regulators
are indole acetic acid (IAA), an auxin and zeatin and isopentenyl adenine (2 iP) as cytokinins,
while others are synthetic growth regulators. Characteristics of growth regulators have been
shown in Table
Table Characteristics of growth regulators
Name
Chemical formula
p-Chlorophenoxy acetic C8H7O3Cl

acid
Molecular Solubility
weight
186.6
96% ethanol
10
2,4-Dichlorophenoxy
acetic acid
C8H6O3Cl
221.0
96% ethanol,
lightly
Indole-3 acetic acid

Indole-3 butyric acid
-Naphthalene acetic
acid
-Naphthoxy
acetic
acid
C10H9NO2
C12H13NO2
C12H10O2
175.2
203.2
186.2
1N NaOH/96% ethanol
1N NaOH/96% ethanol
1N NaOH/96% ethanol
C12H10O3
202.3
1N NaOH
Adenine
C5H5N5.3H2O
189.1
H2O
Adenine sulphate
Benzyl adenine 6
benzyl amino purine
(C5H5N5)2.H2SO4.2H2O 404.4
C12H11N5
225.2
H2O
1N NaOH
N-isopentenyladenine
(2 iP)
C10H13N5
203.3
1N NaOH
Kinetic
Zeatin
C10H9N5O
C10H13N5O
215.2
219.2
1N NaOH
1N NaOH/1N
heated lightly
Gibberellic acid
C19H22O6
C15H20O4
346.4
Ethanol
264.3
C22H25NO6
399.4
1N NaOH
H2O
Abscisic acid
Colchicine
Auxins
heated
HCl,
Auxin was discovered following experiments on the reaction of coleoptile curvature in

Gramineae. It owes its name to its effect on the elongation of cells (auxesis). Auxins have
an indole nucleus with the basic formula C10H9O2N.
A common feature of auxins is their property to induce cell division and cell elongation.
The stimulation of division of cells of cambial origin resulted in initial successes with in
vitro cultures. This effect leads to a number of cells, which further result in the formation
of callus. Auxin has a clear rhizogenic action, i.e. induction of
adventitious roots. It often inhibits adventitious and auxillary shoot formation. At low auxin
concentration, adventitious root formation predominates, whereas at high auxin
concentration, root formation fails to occur and callus formation takes place. All the plants
synthesize auxin that is modulated according to the stage of development. Auxin is present in
sufficient concentration in the growing shoot tips or flowering tips of plants to ensure cell
multiplication and elongation. Auxin circulates from the top towards the base of the organs
11
with a polarity strongly marked in young organs. The compounds most frequently used and
highly effective are 2,4-dichlorophenoxy acetic acid (2,4-D), naphthalene acetic acid (NAA),
indole acetic acid (IAA), indole butyric acid (IBA). Other auxins in use are 2,4,5trichlorophenoxy acetic acid (2,4,5-T), p-chlorophenoxy aceticacid (pCPA) and pichoram (4amino-3,5,6-trichloropicolinic acid).
Cytokinins
Cytokinins were discovered during in vitro culture studies. Coconut milk was known to have
a favourable effect on cellular multiplication and bud formation. Researchers discovered that
an active compound of a purine nature causes such an effect. This enabled Skoog in 1956 to
isolate an active substance from denatured RNA, which he called kinetin. Thus, cytokinins
are derivatives of adenine and have an important role in shoot induction. Cytokinins also
have a clear effect on cell division. Often used to stimulate growth and development, they
usually promote cell division if added together with an auxin. Auxins favour DNA
duplication and cytokinins enable the separation of chromosomes. Cytokinins have a clear
role in organogenesis where they stimulate bud formation. They are antagonistic to
rhizogenesis. At higher concentrations (1 to 10 mg/l), adventitious shoot formation is induced
but root formation is generally inhibited. Cytokinins promote axillary shoot formation by
decreasing apical dominance. The most frequently used compounds are kinetic, benzyl
adenine (BA) or 6-benzyl amino purine (BAP), zeatin, and isopentenyladenine (2 iP). Zeatin
and 2 iP are natural cytokinins. Zeatin, the first endogenous cytokinin, was identified in 1963
in immature embryos of maize, while 2 iP was discovered a little later from plants attacked
by the bacterium
Cornyebacterium fascines.
Stock solutions of IAA and kinetic are stored in amber bottles or bottles covered with a black
paper and kept in the dark since they are unstable in light.
Other Growth Regulators
Gibberellins, although found in all plants and fungi, are unevenly distributed. The sites of
synthesis are very young, unopened leaves, active buds, root tips and embryos. There are
various gibberellin compounds with a similar gibbane nucleus but differ in the quality and
position of the substituents of the nucleus. Gibberellic acid (GA3) is the most widely used
compound.
12
Gibberellins are normally used in plant regeneration. GA3 is essential for meristem culture of
some species. In general, gibberellins induce elongation of internodes and the growth of
meristems or buds in vitro. In its absence, the culture appears globular, due to the
accumulation of nodes. Gibberellins usually inhibit adventitious root as well as shoot
formation. During organogenesis, gibberellins are antagonistic. They seem to oppose the
phenomenon of dedifferentiation. Thus, in in vitro cultures, they cannot be used for this
purpose, but can be utilized for explants already organized (meristems, apices, buds).
Abscisic acid is an important growth regulator for induction of embryogenesis. Abscisic acid
was identified in 1965 and since then, has been found in all plants. This is a growth inhibitor,
which seems to be synthesized when a plant is under difficult conditions. It has a favourable
effect on abscission.
Ethylene is a gaseous compound identified a long time ago, but its functions as a growth
regulator were not evident. Ethylene is produced by cultured cells, but its role in cell and
organ culture is not known. It also influences fruit maturation, acceleration of the process of
leaf or fruit abscission, flowering induction and tuberization. The practical use of ethylene,
which is difficult in a gaseous state, made great progress after the discovery of 2chloroethane phosphoric acid. This product, when applied in a powder form, penetrates the
tissue where it liberates ethylene.
ORGANIC SUPPLEMENTS
Certain complex substances are also added in the media which supply organic nitrogen,
carbon or vitamins. Organic nitrogen in the form of casein hydrolysate (0.2-1 g/l) or certain
amino acids such as glutamine and asparagine, nucleotide as adenine are included in the
medium L-glutamine (upto 8 mM, i.e. 150 mg/l) may replace the casein hydrolysate. The
amino acids, when added, should be used with caution, since they can be inhibitory. The
other amino acids included in the media in mg/l include: glycine (2), aspargine (100),
tyrosine (100), arginine (10), cysteine (10), and aspartic acid, glutamic
acid and proline, etc. Only L-isomers are used, while D-isomers have proved to be
ineffective. Adenine or adenine sulphate (2-120 mg/l) is added to agar media for
morphogenesis. Addition of TCA cycle acids such as citrate, malate, succinate or fumarate
permits the growth of plant cells on ammonium as the sole nitrogen source. A variety of
extracts, viz., protein hydrolysate, yeast extract, malt extract, coconut milk, orange and
13
tomato juices have also been tested. With the exception of protein hydrolysate and coconut
milk, most of the others are used as a last resort. Coconut milk is commonly used at 2-15%
(v/v). The present trend is, however, towards fully defined media and the use of complex
mixtures is losing favour.
Antibrowning compounds: Many plants are rich in polyphenolic compounds. After tissue
injury during dissection, such compounds will be oxidized by polyphenol oxidases and the
tissue will turn brown or black. The oxidation products are known to inhibit enzyme activity,
kill the explants, and darken the tissues and culture media, a process which severely affects
the establishment of explants. Activated charcoal at concentrations of 0.2 to 3.0% (w/v) is
used where phenol-like compounds are a problem for in vitro growth of cultures. It can
adsorb toxic brown/black pigments and also stabilize pH. Besides activated charcoal,
polyvinylpyrolidone (250-1000 mg/l), citric acid and ascorbic acid (100 mg/l each), thiourea
or L-cysteine are also used to prevent oxidation of phenols.
Some of the procedures used by various workers to combat this problem of browning are: (i)
Adding antioxidants to culture medium, viz, ascorbic acid, citric acid, polyvinylpyrolidone
(PVP), dithiothreitol, bovine serum albumin, etc. (ii) Pre-soaking explants in antioxidant
before inoculating into the culture medium; (iii) incubating the initial period of primary
cultures in reduced light or darkness because it is known that phenolic oxidation products are
formed under illumination; and (iv) Frequently transferring explants into fresh medium
whenever browning of the medium is observed.
GELLING AGENTS
Agar, the most popular solidifying agent, is a seaweed derivative. Plant tissue culturists often
use Difco Bacto agar at a concentration of 0.6 to 1.0% (w/v), although other forms of agar
(agarose, phytoagar, flow agar, etc.) are also gaining popularity. Solubilized agar forms a gel
that can bind water and adsorb compounds. The higher the agar concentration, the stronger is
the water bound. With higher concentrations, the medium becomes hard and does not allow
the diffusion of nutrients into the tissues. Thus in vitro growth may be adversely affected if
the agar concentration is too high. Besides agar, the following alternatives can be used.
i.
Alginate can be used for plant protoplast culture.
ii.
Gelrite at 0.2% can be used for solidification of media. Gelrite gels are remarkably
clear in comparison to those formed by agar. Gelrite requires both a heating cycle and the
14

++
presence of divalent cations (Mg

iii.
++
or Ca ) for gelation to take place.
Synthetic polymer biogel P200 (polyacrylamide pellets) or a starch polymer can be
used.
iv.
Agargel: A mixture of agar and synthetic gel has been developed by Sigma Company.
It has the properties of both synthetic gel and agar.

Liquid media with a support can also be used instead of solid media. Such media include
i.
Liquid medium without agar using clean foam plastic, glasswool or rockwool as
support.
ii.
Filter paper-bridge, which is hung in a liquid medium.
iii.
Growth on a liquid medium containing glass beads.
iv.
Viscose sponge underneath the filter paper as a carrier for a liquid medium instead of
agar.
pH
pH determines many important aspects of the structure and activity of biological
macromolecules. pH is the negative logarithm of the concentration of hydrogen ions. Nutrient
medium pH ranges from 5.0 to 6.0 for suitable in vitro growth of explant. pH higher than 7.0
and lower than 4.5 generally stops the growth and development. The pH of the medium
changes during autoclaving. It generally falls by 0.3 to 0.5 units after autoclaving. If the pH
falls appreciably during plant tissue culture (the medium becomes liquid), then a fresh
medium should be prepared. One should know that a starting pH of 6.0 could often fall to 5.5
or even lower during growth. pH higher than 6.0 gives a fairly hard medium and a pH below
5.0 does not allow satisfactory gelling of the agar.
General methodology for preparation of medium (MS Medium)
Preparation of stock solutions: Since it is a time - consuming and tedious process to weigh
the necessary products each time a medium is prepared, concentrated solutions of the desired
composition of a medium are used which one dilutes adequately. These concentrated
solutions are called stock solutions. Simple stock solutions comprise only one constituent at a
time. Complex stock solutions comprise several chemicals. Stock solutions of macro and
micronutrients, vitamins and growth regulators are prepared in distilled or high purity
demineralized water. Chemicals should be of the highest grade.
i.
Macronutrient stock solution(s): Usually, the stock solution of macronutrients is
15
prepared as 10x. Dissolve all the macronutrients one by one except (CaCl2 for macronutrient
stock solution. The stock solution of CaCl2 should be prepared separately. Another way is to
dissolve the different macronutrients one after the other and CaCl2 is dissolved separately
and later added to the rest of the stock solution in order to avoid precipitation.
ii.
Micronutrient stock solution: A stock solution of all the micronutrients with 100x is
generally prepared. Since copper and cobalt are required in very small quantities, it is
preferable to first make a separate stock solution of these two salts (100x) and then an
appropriate volume can be pipetted and put into the main micronutrient stock solution. These
nutrient solutions can be dispensed in plastic bags with zipper seals and stored frozen (e.g.
10x macronutrient solution is dispensed into a bag containing 100ml of solution to prepare 1
litre medium).
iii.
Iron-EDTA: Iron EDTA should be added fresh. If stock solution (100x) is prepared,
then it should be stored after autoclaving in an amber bottle or a bottle covered with an
aluminium foil.
iv.
Vitamins and growth regulators stock solutions: These are simple stock solutions.
All the growth regulators are not soluble in water. Solubility of different growth regulators is
given in Table 4.3. The compound should be dissolved in a few ml of solvent and then water
is slowly added to make the requisite volume. Concentrations of compounds can be taken as
mg/l or in molarity.
Concentration in mg/l - It is preferable to dissolve 50 mg / 100 ml to give a concentration of
0.5 mg/ml or 100 mg/100 ml in order to give a concentration of 1 mg/ml.
Concentration in mM - The growth regulator solutions can be prepared as 1 mM for 100ml.
If a culture medium is to contain 10 M of the growth regulator (e.g. 2,4-D M.W. = 221.0),
then
1M = 221 g/l
1 mM = 221 mg/l or 0.221 mg/ml
The amount in 100 ml stock solution = 0.221 x 100ml = 22.1
mg 10 M = 2210 g or 2.210 mg
The required volume of stock solution to be added = 10ml of this stock (22.1 / 10 = 2.210
mg).
16
1.
Media chart is prepared as shown in Table 5. In a sequence, different components are
added into a beaker according to the list : nutrients, iron-EDTA, vitamins, myo-inositol,
growth regulators (if thermostable and autoclavable), organic supplements, sucrose etc., by
using the correctly sized graduated cylinders or pipettes or balance.
2. Water is added to just below the final volume (e.g. 800 ml volume for one litre medium)
3. pH of the medium is adjusted to the required value (e.g. pH 5.8 for MS) by adding
dropwise while stirring 1N KOH or 1N HCl.
4. Required quantity of agar or any other gelling agent is added while the medium is being
stirred.
5. The solution to brought to the final volume, i,e 1 Litre and heated with continuous
stirring until all the agar is dissolved and the solution becomes transparent.
6. The medium is dispensed in glass or polypropylene vessels and plugged with cotton
plugs.
o
7. Culture medium is sterilized in an autoclave for 20 min at 121 C at 15 psi (105 kPa).
8. If the medium contains heat-labile substances :F
a.
steps 1- 5 are followed except for the addition of heat labile substances.
b.
Culture medium is sterilized as such in a big Erienmeyes flask without dispensing in

o
vessels in an autoclave for 20-25 min at 121 C at 15 psi (105 kPa).

c.
the thermolabile compound solutions are filter sterilized using millipore or any other
filter assembly using 0.22 m filter.

d.
After autoclaving, the medium is kept in a laminar airflow hood and allowed to cool to a
o
temperature of around 50 C. The requisite quantity of the compound is added to the medium
with the help of micropipettes while the mediuim is being stirred.
e.
the medium is dispensed into sterile containers (generally sterile petri dishes) under the
hood of laminar airflow, provided the neck of the Erlenmeyer flask is passed over a flame
before the medium is poured from it.
1 Medium is allowed to cool and solidify in a laminar airflow hood.
2.
Prepation of the commercial medium:

1. The commercial medium which is obtained contains the nutritional components, agar
and sucrose. But it is devoid of CaCl2 and the growth factors. These components have to
17
be prepared as stock solution and added in the medium during preparation.

2. The obtained medium should be disolved in 1L of water, if the amount of the the total
content is mentioned it should be weighed and dissolved in the appropriate amount of
water and should be heated for complete dissolving of agar. The medium while heating
should be strried continuously inorder to prevent charing of agar.
3. After dissolving it the required amount is dispensed into culture tubes and kept for
autoclaving.
4. After sterilization the medium is cooled to the room temperature.
5. Medium is allowed to cool and solidity in a laminar airflow hood.
STORAGE OF MEDIA
o
After cooling, the media containers are stored preferably at 4-10 C but that is not absolutely
necessary. Medium should be used after 3-4 days of preparation, so that it medium is not
properly sterilized, contamination will start to appear.
Table 4 Preparation of stock solutions of Murashige and Skoog (MS) medium
Concentration in
Volume to
Constituent
Concentration in
the stock solution
taken/litre
MS medium (mg/l)
(mg/l)
medium
Macronutrients (10x) Stock solution I
NH4NO3
1650
16500
100 ml
KNO3
1900
19000
MgSO4 . 7H2O
370
3700
KH2PO4
170
1700
Macronutrient (10x) Stock solution II

CaCl2 2H2O
440
Micronutrients (100x) Stock solution III
H3BO3
6.2
4400
100 ml
620
10 ml
MnSO4 . 4H2O
22.3
2230
ZnSO4 . 7H2O
Kl
Na2MoO4.2H2O
8.6
0.83
860
83
0.25
25
CuSO45H2O
0.025
2.5
CoCl2 . 6H2O
Iron source
0.025
2.5
18
be
of
Fe EDTA Na salt
40
Added fresh
Vitamins
Nicotinic acid
0.5
50 mg/100 ml
1 ml
Thiamine HCl
0.1
50 mg/100 ml
0.2 ml
Pyridoxine HCl
0.5
50 mg/100 ml
1 ml
Myo-inositol
100
Added fresh
Others
Glycine
2.0
50 mg/100 ml
4 ml
Sucrose
30,000
Added fresh
Agar
8000
Added fresh
pH 5.8
Nutrientmedium chart for preparation of culture medium
Quantity required
Stock
for
volume
of
Quantity
Constituents
solution
medium
under Remarks
required for 1 L preparation
(conc.)
(e.g. 500ml)
Macro
stock 10x
100ml
50 ml
solution I
Macro
stock 10x
100 ml
50 ml
solution II (CaCl2)
Micro
stock 100x
10 ml
5 ml
solution III
Iron-EDTA
Na Added fresh
40 mg
20 mg
salt
Vitamins
Nicotinic acid
Thiamine HCl
Pyridoxine HCl
Myo-inositol
Others
Glycine
Growth
regulators
Sucrose
Agar
pH
50 mg/100 ml
50 mg/100 ml
50 mg/100 ml
Added fresh
0.5 mg/l = 1 ml
0.1 mg/1 = 0.2ml
0.5 mg/l = 1ml
100 mg
0.5 ml
0.1 ml
0.5 ml
50 mg
50 mg/100 ml
2 mg/l = 4ml
2.0 ml
Added fresh
Added fresh
30 g
8g
15 g
4g
19
Ex.No:
Date:
PLANT TISSUE CULTURE- AN INTRODUCTION

Plant tissue culture is the science or art of growing plant cells, tissues or organs isolated from the
mother plant, on artificial media. The culture initiation and plant regeneration are still
accomplished ampirically by varying conditions until the desired response is obtained. Scheiden
and Schwanns suggestion that the totipotency of cells was the foundation of the plant cell and
the tissue culture. However it was Haberlandt who look the first real steps in in vitro cultures.
Since then many investigate have studied plant regeneration under in vitro conditions and have
obtained further understanding of cell totipotency.
Tissue culture is a collective term commonly used to describe all kinds of in vitro plant cultures.
The tissue cultures can be cultures of an organized tissue, viz., callus cultures, suspension or cell
cultures, protoplat cultures, pollen cultures etc., or cultures of organized structure like meristem
cultures, shoot and root cultures, embryo cultures, inflorescence cultures, ovule cultures, etc..
Plant tissue culture has direct commercial applications as well as value in basic research into cell
biology, genetics and biochemistry.
Prepartion of glassware:
Cleaning:
It is important to use clean glassware for the growth of tissue in vitro. In our laboratory these
include:
1. Boil all glassware in washing soda solution (10%) for 1 hour.
2. Rinse thoroughly with tap water and leave in HCl (1N) for 2 hours.
3. Remove traces of acid by thorough washing with tap water.
4. Rinse glassware with double-distilled water.
5. Allow glassware to dry overnight at room temperature.
Sterilization:
The objective of sterilization is to make media, glassware and instruments free from
microorganisms. It is accomplished by wet heat, dry heat or filteration.
1) Wet-heat sterilization:
1. Plug glassware such as conical flasks, test tubes, etc. with non-absorbent cotton.
20
2. Wrap petriplates dishes with wrapping paper or aluminium foil.

3. Place forceps and scalpels in test tubes. Plug the tubes with non-absorbent cotton and cover
with brown paper.
4. Plug the mouth end of graduated pipettes (1, 2, 5 and 10ml) with cotton. wrap them
individually in wrapping paper or aluminium foil.
5. Autoclave glassware and instruments at 1210C for 1 hour.
2) Dry-heat sterilization:
Steps 1-4 are similar as wet-heat sterilization. But the final step involves placing the instruments
in an oven at 1400-1600C for 2 hours.
3) Filter sterilization:
Aminoacids, vitamins, phytohormones,etc.. may get destroyed during autoclaving. So such as
chemicals are therefore sterilized by filtration through a Seitz or Millipore filtration assembly
using filter membranes of 0.45 or 0.22m porosity.
1. Plug 500 or 1000ml receiver flask with cotton.
2. Assemble Millipore filtration unit with bacteriological membrane filter (0.45 or 0.22 m).
3. Wrap filtration until with wrapping paper
4. Autoclave receiver flask and filtration unit at 121C for 1 hour (do not sterilize in dry heat as
membrane filters get damaged)
5. Fix filtration unit to the receiver flask in a sterile cabinet.
6. Pour solution to be sterilized into the filtration.
7. Apply slight air pressure to commence filtration (don not exceed air pressure by 7.03
Kg/cm2).
8. Transfer under aseptic conditions to sterile flasks.
9. Using a sterile pipette and filter sterilized solution to the autoclaved medium, shake well and
dispense into sterile cultures tubes or flasks under aseptic conditions.
Preparation of media:
The appropriate composition of the medium largely determines the success of the cultures. Plant
materials do vary in their nutritional requirements and therefore it is often necessary to modify
the medium to suit a particular tissue. Initially tissues and organs from a wide variety of plant
species were cultures on the nutrient salt solutions formulated by Gautheret and White. However,
21
these were found inadequate for sustaining growth of many plant tissues. This led to the
formation of several basal nutrient media (Hildebrandt et al., 1946; Burkholder & Nickel,1949;
Heller,1953; Murashige and Skoog,1962; Gamborg et al., 1968). In general, the medium
contains i) inorganic salts, ii)vitamins, iii)growth regulators, iv)carbon source and v) organic
supplements.
Inorganic salts:
These are divided into two groups: major and minor salts.
Major salts: The salts of potassium, nitrogen, calcium, magnesium, phosphorous and sulphur
constitute the major salts. Nitrogen is generally used as nitrate or ammonium salts, sulphur as
sulphates and phosphorus as phosphates.
Minor salts: The salts of iron, zinc, manganese, boron, copper, cobalt, molybdenum, iodine,
etc.. make up the minor salts. These salts are essential for the growth of tissues and are required
in trace quantities.
Vitamins:
The B-vitamins play an important role in growth of tissues. Thiamine, nicotinic acid and
pyridoxine are generally incorporated in all media, although pantothenic acid, folic acid, biotin,
riboflavin, etc..have also been used.
Growth regulators:
Growth as well as differentiation of tissues in vitro is controlled by various growth regulators,
auxins, cytokinins, gibberellins, ethylene, abscisic acid, etc..
Auxins:
Indole acetic acid, indole butyric acid, naphthalene acetic acid, 2,4-dichlorophenoxy acetic acid
are the frequently used auxins. These are generally used at 0.1 to 10mg/l concentration in plant
tissue culture media. Naphthalene acetic acid and 2,4-dichlorophenoxy acetic acid are
thermostable and do not lose their activity on autoclaving. Whereas, Indole acetic acid is
thermolabile and loses most of their activity on autoclaving. Hence, it is sterilized by filtration.
Cytokinins:
Cytokinins have a profound effect on cell division and cell differentiation. Kinetin, zeatin and 6benzylamino purine, the commonly used cytokinins, are used in 0.1-10mg/l concentration.
Others:
22
Gibberellic acid, ethylene releasing compounds and abscisic acid are also incorporated in the
media.
Carbon sources:
Plant tissues in culture can utilize a variety of carbohydrates-sucrose, glucose, fructose, starch
and maltose. Sucrose, at 2-5% concentrations in the nutrient media, however, remains the most
widely used carbohydrate source.
Organic supplements:
Complex substance such as yeast extract, malt-extract and casein hydrolysates are also added
(0.1- 1% w/v).
Among various plant extracts, liquid endosperm of immature (coconut water) is widely used (520%v/v)
General methodology for media preparation:
Stock solutions of major salts, minor salts and vitamins are prepared to be used in the
preparation of media are stored in a refrigerator. For preparing 1 litre of the medium:
Transfer appropriate amounts of stock solution of salts to 1 litre flasks.
1. Add vitamins, auxins, cytokinins and inorganic supplements.

2. Add a carbohydrate source such as sucrose (2-5%)
3. Adjust the pH to 5.6-5.8 using a pH meter.
4. Make up the volume to 1 litre with double distilled water.
5. Add powdered agar 8g/l for making the medium semisolid.
6. Cover the flask with paper of aluminium foil and keep in a steamer or water-bath at 1000C for
dissolving the agar.
7. Shake the flask well for the uniform distribution of agar.
8. Dispense the medium into culture tubes or flasks as the case may be.
9. Autoclave tubes or flasks containing medium at 1210C for 20 minutes.
10. Again steam the medium for 20 minutes on the following day (i.e the day after it is
prepared).
Surface sterilization of plant material:
Before inoculating the medium with the explants, it is necessary to surface sterilize it. There are
many sterilizing agents such as calcium hypochorite, chlorine water, bleaching powder, mercuric
23
chloride, hydrogen peroxide and ethylene oxide. The concentration of the sterilizing agent and
the duration of treatment varies with the plant material. These should be no damage to the
explants in the process of surface sterilization. The plant material should then be thoroughly
washed with sterile distilled water before transferring it to the nutrient medium.
a)
Sterilization of seeds:
1. About 15-20 healthy seeds were selected and wash them thoroughly with water.
2. Now added 2-3 drops of liquid detergent 100ml of water and shake well for 5 minutes.
3. Pour off the detergent solution and wash thoroughly to remove any traces of the detergent.
4. Rinse seeds in 50ml of 70% ethanol for 1 minutes.
5. Wash thoroughly 3-4 times with single distilled water.
6. Transfer seeds in a sterile 250ml flask and add mercuric chloride solution (0.1%).
7. Treat for 20 minutes, shake the flask occasionally. All the operations thereafter should be
carried out under sterile conditions.
8. Transfer the flask to a sterile cabinet, decant the mercuric chloride solution and wash the
seeds thoroughly with sterile water to remove any traces of mercuric chloride.
b)
Sterilization of leaves:
1. Collect the apical portion about 20cm long from 6-8 month old plants.
2. Remove the outer green sheathing leaves with a scalpel till the inner thin and white leaves are
exposed.
3. Dip 2-3 cm long segments of this portion in 70%alcohol for 1 minute. Then follow the steps 18 as in sterilization of seeds.
4. Using sterile forceps, place segments in a sterile petridish and remove 1-2 outer leaves with
scalpel.
5. Cut the inner leaves into 5mm segments.
6. Transfer 1-2 leaf segments to each tube containing 20ml of semisolid medium.
24
Ex.No:
Date:
MICROPROPAGATION
Tissue culture is particularly useful for multiplication of plants which are slow growing
(turmeric, ginger, cardamom); cross- pollinated (coconut, teak, eucaluptus, cashew, mango and
those which show wide variation in the progeny); male-sterile lines (cotton, sorghum, pearlmillet); newly produced varieties (normally vegetatively propagated); and for multiplication of
virus free plants by meristem cultures (sugarcane, potatoes, tapioca, etc..).
Tissue culture is now being commonly used for clonal propagation of a large number
of horticultural plants. Crop plants and also for forest trees (Murashige;1974; Conger,1981).
The success of clonal multiplication in higher plants depends generally on 3 main stages:
STAGE 1: ESTABLISHMENT OF AN ASEPTIC CULTURE
The explants taken from the plant has first to be made free of microorganisms which would
outgrow the plant tissue when placed on a nutrient medium. This would result in the death of the
explants. These surface contaminants, e.g. bacteria, fungi and yeast are removed by surface
sterilization prior to culture, but without killing the plant tissue.
STAGE 2: MULTIPLICATION
The surface sterilized material when inoculated on sterile nutrient media and incubated at
252C with a definite photoperiod and light intensity grows to form large nuber of shoots.
STAGE 3: ROOTING AND HARDENING OF PLANTS
The shoots obtained are carefully excised and transferred to a rooting medium,
preferably a liquid medium,containing an auxin and supported on a filterpaper platform in
order to obtain rooting in these shoots.
These plants which have rooted and have developed secondary roots with root hairs
can be
transferred to pots containing soil:vermiculite mixture (1:1). This mixture is
preautoclaved for 1 hour at 15 psi and steamed for 3 days successively and cooled. The
potted plants can be transferred to the field where the first new leaf emerges.
MULTIPLICATION BY SUBCULTURE AT STAGE 2
However, excised shoot tips can be inoculated on the same medium used in stage 2 instead
of the rooting media. By regular repetition of this subculture procedure, high rates of
multiplication can be achieved.
Vegetative multiplication of plants depends on various factors as nutrient medium, agar
25
concentration, photoperiod and light intensity, hydrogen ion concentration, size and source of the
explants (Conger,1983).
Explants source:
Requirements:
a) Equipments
Conical flasks (100ml capacity) Test tubes (25mm*150mm) Petridishes (80mm diameter)
Pair of forceps and scalpel (15 cm long), Environmental growth cabinets adjusted to 252C with
18hr photoperiod and 1500lux intensity and 152 and 600 lux light intensity, Shaker with
120rpm and 1000 lux light intensity.
b) Culture media, washing solutions, sterilizing agents, Glass distilled water, Sterile glass distilled
water 0.5%HgCl solution Detergent, Medium
c) Source tissue
Procedure
a. Sterilization of glassware
b. Preparation and sterilization of media
c. Explants collection:
1. Select a twig (60-90 cm long, 10-15mm wide) from mature elite trees and cut, making sure
that the twig contains many young axillary buds. The length is important in selecting twig
that do not wither before being brought to the laboratory.
2. Bring the twigs containing axillary bud to the laboratory, remove the leaves and cut them
into small pieces of about 5-8 cm.
3. Transfer the buds to a sterile 250ml conical flask and surface sterilize the explants.
d. Culture of buds:
1. Keep sterile petridishes, scalpel, forceps and medium inside a sterile cabinet along with the
flask containing surface-sterilized explants.
2. Transfer these explants into sterile petri dishes with the help of a pair of sterile forceps and
cut these explants into small pieces of 10-15 mm each containing atleast one axillary bud.
3. Inoculate 2 pieces to each tube containing medium.
4. Incubate the tubes in an environment growth cabinet at 152 and 500 lux light intensity for
72 hours.
5. Transfer the cultures after 72hr to another incubator maintained at 252C with 16hr
26
photoperiod and 1500lux intensity.

6. After 25 days, the young buds start sprouting.
7. When the sprouts are 10-15mm long, transfer them to liquid medium in 100 ml
Erlenmeyer flasks.
8. Incubate the flasks on a rotatory shaker at 120 rpm and 500 lux light intensity.
9. Observe the formation of multiple shoots after 10-15 days.
e. Multiplication by subculture:
1. Transfer the multiple shoots from the flask to a sterile petridish aseptically.
2. Incubate the cultures in an environmental growth cabinet at 252C and at 1000 lux light
intensity (12 hr photo periods) and observe the cultures regularly.
3. Observe the explants produces multiple shoots within 15 days.
4. Separate these shoots again aseptically and transfer the tubes containing medium for
shoot formation.
f. Transfer of plants to pots:
1. Remove the rooted plantlet from the tube and wash the roots gently with tap water to
remove any traces of medium.
2. Transfer the plantlets to soil: vermiculite (1:1) sterile mixture in a pot.
3. Irrigate with about 20 ml of tap water.
4. Keep the pots in a growth cabinet at 252C and at 1000 lux light intensity and water
them.
5. Transfer the plants to the field after 8 days of hardening in which 70-80% plants survive.
Result:
S.No.
Number of bottles
Number of explants
Number of explants
inoculated
inoculated per bottle
developed per bottle
1.
27
Ex.No:
Date:
CALLUS INDUCTION
Callus is an actively-dividing non-organized mass of undifferentiated and
differentiated cells often developing either from injury (Wounding) or in tissue culture in
the presence of growth regulators. Explants from both mature and immature organs can
be induced to form callus. However, explants with mitotically active cells (young,
juvenile cells) are generally good for callus initiation. Callus is produced on explants in
vitro from peripheral layers as a result of wounding and in response to growth regulators,
either endogenous or exogenously supplied in the medium. The season of the year, donor
conditions of the plant, the age and physiological state of the parent plant contribute to
the success of organogenesis in cell cultures.
Growth regulator concentration in the culture medium is critical for morphogenesis.
Auxin, at a moderate to high concentration, is the primary hormone used to produce
callus. In some species, a high concentration of auxin and a low concentration of
cytokinin in the medium promotes abundant cell proliferation with the formation of
callus. Callus may be serially subcultured and grown for extended periods, but its
composition and structure may change with time as certain cells are favoured by the
medium and come to dominate the culture.
Callus tissue from different plants species may be different in structure and growth habit:
white or coloured, soft (watery) or hard, friable (easy to separate in to cells) or compact.
The callus growth within a plant species is dependent on various factors such as the
original position of the explant within the plant, and the growth conditions.
Although the callus remains unorganized, with increasing growth, some kinds of
specialized cells may be formed again. Such differentiation can appear to take place at
random, but may be associated with centers of morphogenesis, which can give rise to
organs such as roots, shoots and embryos.
AIM:
To induce callus from the explants of Phaseolus mungo (Green Gram)
Reagents and other requirements
1. Culture tubes or conical flasks containing media
2. Sterile Petri dishes
28
3. Scalpel, blades, forceps and steel dissecting needles

4. Sterile distilled water
5. Alcohol
6. Detergent (Tween 20, Teepol, etc.)
7. Sterilants HgCl2, Sodium. Hypochlorite
8. Nutrition medium reagents MS basic salts and
vitamins Growth regulators 2, 4-D
Plant material Green gram
Media
Seed Germination: MS Medium
Callus Induction: MS + 2, 4-D (2mg/lL)
I. Seed Germination
1. The seeds washed by submerging in water with a few drops of detergent in a
beaker with vigorous shaking.
2. The seeds were submerge in 70% alcohol for 40 s after which the alcohol was
decanted.
3. The seeds were transfer to a flask containing 20% commercial sodium
hypochlorite solution and left there for 20 min for surface sterilization. Later they
were rinsed thrice with sterile distilled water.
o
4. 2-3 seeds were placed on the surface of MS medium and incubated at 25 C for 16
2
h photoperiod with 250 E/m / s light intensity for 2 weeks.

5. Observe regularly for germination. If need be, transfer the individual plantlets to
half MS medium.
II. Callus Induction
1.
The leaves were removed from in vitro germinated seeds 2 weeks were cut into
pieces and placed on the MS mediu.As a control measure, some explants should be
inoculated on MS medium without hormones.
2.
The cultures were incubated in dark at 25 C. Callus started appearing within 2
weeks and good callus growth can be observed in 3-4 weeks.

3.
Callus can be subcultured after the 4
th
week on fresh medium with the same

29
composition.
Result:
The undifferentiated mass of cells was formed from the inoculated leaf explant.
Callus induction from the explant of Brassica
30
Ex.No:
Date:
DIRECT ORGANOGENESIS SHOOT TIP CULTURE
Plant production through organogenesis can be achieved by two modes: (i)

Organogenesis through callus formation with de novo origin; and (ii) Emergence of
adventitious organs directly from the explant. In this exercise, organogenesis through
callus formation has been discussed.
By varying the growth regulator levels, i.e. lowering the auxin and increasing the
cytokinin concentration is traditionally performed to induce shoots from the explant. The
next phase involves the induction of roots from the shoots developed. IAA or IBA auxins,
either alone or in combination with a low concentration of cytokinin, are important in the
induction of root primordia. Thus organ formation is determined by quantitative
interaction, i.e. ratios rather than absolute concentrations of substances participating in
growth and development.
Aim :
To perform regeneration of the plant from shoot tip of Bougainvillea
Material Required :
1.
Bougainvillea shoot tips
2.
M.S Medium
3.
Growth regulators: Auxin 2,4 D (0.5mg/l)
4.
Cytokinin BAP (1.5mg/l)
Procedure:
1. M.S medium was amended with required concentration of growth hormones and
sterilized
2. Shoot tips of Bougainvillea were obtained from 5 year old plants growing outside
in the lawn campus. One cm long shoot tips of Bougainvillea
3. The apical portions of the plant were collected and the shoot tip was carefully cut
using a sterile surgical blade.
4. It is washed under running tap water for 30 mins to remove dust particles and then
surface sterilized in alcohol for 30s followed by sodium hypochloride for 3 mins and
rinsed thoroughly in sterile distilled water.
31
5. The explant was then air dried and inoculated in the medium and incubated at
o
25 C for 16 h photoperiod with 250 E/m / s light intensity for 2 weeks.

Result:
Shoot proliferation was observed after 2 weeks from the explants.
Shoot tip culture Bougainvilla
32
Ex.No:
Date:
EMBRYO CULTURE
Aim:
To isolate embryos of Cicer aertinum and perform in vitro culture
Requirement:
1.
Sterilants - alcohol, HgCl2, sodium hypochlorite
2.
Nutrition medium reagents - MS basic salts and vitamins
3.
Growth regulators usually not required for embryogenesis
4.
Plant Material- Embryo of Cicer auritinum
5.
Culture tubes containing media
6.
Sterile Petri dishes
7.
Scalpel, blades, forceps knives and steel-dissecting needles
8.
Sterile distilled water
Procedure:
1. The seeds were washed by submerging them in water with a few drops of
detergent in a beaker and shake them by hand.
2. The embryo was teased and collected without any damage
3. It was washed with distilled water and then treated with 70% alcohol for 30
seconds.
4. This was followed by rinsing completely with distilled water and then transferred
to 20% sodium hypochlorite, where it was left for 0 minutes.
5. Then the embryo was thoroughly rinsed with distilled water for 3 times and dried
using the autoclaved tissue paper and inoculated in the culture tubes containing the
MS medium.
o
6. The culture tubes were incubated at 25 C under 16 h photoperiod for 2 to 3

weeks.
Result:
The plant was developed from inoculated embryo.
33
Induction of plant let from the embryo of Cicer aertinum
34
Ex.No:
Date:
ISOLATION OF PROTOPLASTS
Protoplast is the living material of the cell where as an isolated protoplast is the cell from
which the cell wall is removed. In plant breeding programme many desirable
combination of characters could not be transmitted through the conventional method of
genetic manipulation. higher plants that could lead to the genetic process involving fusion
between the subsequent developments of a product to a hybrid plant is known as somatic
hybridization.
Plant protoplasts can be isolated from cells by two methods:
1. Mechanical method
2. Enzymatic method.
MECHANICAL METHOD
Aim:
To isolate protoplast by mechanical method
Principle:
Protoplast can be isolated from almost all plant parts: roots, leaves, fruits, tuber, root
nodules, pollen mother cell etc. Protoplast isolated by mechanical is a crude and tedious
procedure.
Cells are plasmolysed causing the protoplast to shrink from the cell wall. The protoplast
obtained from this method is then cultured on suitable culture medium. The principle
deficiency of this approach is that the protoplast released is few in number. Mechanical
isolation was that of only historical event now.
Materials Required:
1. Plant leaves Duranta repens
2. Mortar and pestle
3. Phosphate buffer pH-7.0
4. 0.3 M sorbitol
5. 0.3 M mannitol
6. Glass slides
7. Microscope.
35
Procedure:
1. Young leaves were obtained from plants growing out doors and initially washed
with tap water to remove any dust particles.
2. The leaves were washed with phosphate buffer and homogenized gently with the
mortar and pestle.
3. The crude protoplast suspension was centrifuged at very low 50-100 rpm for 10
minutes.
4. The supernatant containing intact protoplast was carefully pipetted out and the
pellet containing cell debris and other cell organelles were discarded.
5. Small volume of supernatant was placed in the slides and covered with coverslip.
6. The slide was observed in light microscope to find out viable protoplast
Result:
The spherical shaped protoplasts were observed using the microscope.
36
Ex.No:
Date:
ENZYMATIC METHOD
AIM:
To isolate protoplasts by enzymatic method
PRINCIPLE:
Protoplasts are isolated by treating tissues with a mixture of cell wall degrading enzyme
in solution, which contain osmotic stabilizer. A most suitable source of protoplasts is
mesophyll tissue from fully expanded leaves of young plants or new shoots. The release
of protoplast is very much dependent on the nature and composition of enzymes used to
digest the cell wall. There are three primary components of the cell wall which have been
identified as cellulose, hemicellulase and pectin substance. Pectinase (macrozyme)
mainly degrades the middle lamella while cellulose and hemicellulase degrades the
cellulose and hemicellulosic components of the cell wall. During this enzymatic
treatment, the protoplast obtained should be stabilized because the mechanical barrier of
the cell wall which offered support has been broken. For this reason an osmoticm is
added which prevents the protoplast from bursting.
MATERIALS REQUIRED:
1. Young leaves
2. 70% ethanol
3. 2% cellulose
4. 13% mannitol
5. 0.5% macrozyme
6. CPW salt solution:
KH2PO4 - 27.2mg/l
KNO3
- 101mg/l
CaCl2
- 1480mg/l
MgSo4 - 246mg/l
KI
- 0.16mg/l
CaSo4
- 0,026mg/l
pH
- 5.8.
37
PROCEDURE:
1. The young leaves were collected and washed in sterile distilled water thrice.
2. The leaves were cut into small bits.
3. Then the leaves were kept immersed in 13% mannitol for 1 h for pre-plasmolysis.
4. Mannitol was removed after incubation ant sterilized enzyme mixture (Cellulase +
macerozyme) was added and incubated at 25C in a shaker for 12 h
5. The filtrate was centrifuged at 100g for 5 min to sediment the protoplast.
6. The supernatant was removed and the protoplast pellet was suspended in 10ml of
CPW +21% sucrose solution.
7. The mixture was centrifuged at 100g for 5 min. The viable protoplast will float to
the surface of the sucrose solution.
8. The supernatant was collected and viewed under microscope.
9. The protoplasts were visualized in microscope.
RESULT:
Protoplasts were isolated by enzymatic method and viewed under the microscope
38
Ex.No:
Date:
ANTHER CULTURE
AIM:
To isolate and inoculate anthers for haploid production.
PRINCIPLE:
Haploids refer to those plants which possess a gametophytic number of chromosomes in
their sporophytes. Haploids may be grouped into two broad categories:
(a)
monoploids which possess half the number of chromosomes from a diploid
species.
(b)
Polyhaploids which possess half the number of chromosomes from a polyploidy
species.
Haploid production through anther culture has been referred to as androgenesis while
gynogenesis is the production of haploid plants from ovary or ovule culture where the
female gamete or gametophyte is triggered to sporophytic development.
MATERIALS REQUIRED:1.
Anthers from Hibiscus
2.
MS medium
3.
growth factors
4.
70% ethanol
5.
2% mercuric chloride
6.
Meso inositol
7.
Scissors
8.
Scalples
9.
Petriplates
10.
Forceps.
PROCEDURE:
1. Flower buds of Hibiscus were collected.
2. The flower buds are surface sterilized by immersing in 70% ethanol for 60 sec
followed by immersing in 2% sodium hypochloride solution for 1 min or in mercuric
39
chloride.
3. The buds were washed four or five times with sterile distilled water.
5. The buds were transferred to a sterile Petridish.

6. The buds were split open using a blade and the anthers were removed without
damage and the filaments were removed.
7. The anthers were placed horizontally on the MS medium supplemented with
different concentration of plant growth regulators or mesoinositol.
8. The Petriplates were sealed and incubated in dark at 28C.
9. The Petriplates were examined for the germination of anthers.
RESULT:
The anther underwent germination leading to the formation of haploid plantlets.
Anthers inoculated on the MS medium
40
Ex.No:
Date:
CELL SUSPENSION CULTURE

INTRODUCTION:
A cell suspension culture consists of cell aggregates dispersed and growing in a moving liquid
media. It is normally initiated by transferring pieces of undifferentiated and friable callus to a
liquid medium, which is continuously agitated by a suitable device. Suspension cultures can
also be started from sterile seedlings or imbibed embryos or leaves by the mechanical method.
Leaves or the other tissues (e.g. hypocotyl, cotyledon, etc.) can be gently grinded or soft
tissues can be broken up in a hand-operated glass homogenizer. This homogenate, containing
intact living cells, dead cells and cell debris is cleared by filtration and centrifugation and then
transferred to moving liquid medium.
For general purpose, the objective with cell suspension cultures is to achieve rapid growth
rates, along with uniform cells, all the cells being viable. Cells should be subcultured at weekly
intervals or less if they are to be used for experimental purposes. Optimally, suspension
cultures passage length is 1-2 weeks, but the exact time and the dilution required must be
determined for each cell line. Dilutions of 1:4 after 1 week or 1:10 after 2 weeks are commonly
used. It is recommended that a small sample should be withdrawn and the cell density
determined before sub culturing.
Equipments:
Shaker
Centrifuge
Microscope
Haemocytometer
Reagents and other requirements
Sterilants alcohol, HgCl2, sodium hypochlorite
Nutrient medium reagents -MS basic salts and vitamins
Growth regulators - 2, 4-D
Seedlings of Phaseolus mungo
41
Media
MS + (1.5mg/l) 2, 4-D
PROCEDURE
1. Seeds are germinated in vitro.
2. Seedlings are collected when the cotyledons are fully expanded and the epicotyl is
beginning to emerge (2-weeks). Each seedling is place on a sterile slide/Petri dish and
crush the expalnt gently.
3. MS liquid medium is prepared. For experiment purposes, 15 ml medium in a 125 ml
Erlenmeyer flask is taken. Sterilize the opening of a flask with the flame of a burner in the
hood.
4. Pieces of the gently crushed plant material are transferred to sterile petri dish to the
liquid media using pipette.
5. The culture is incubated on a gyratory shaker set at 125 rpm under controlled
conditions of temperature.
6. An aliquot from the flask on a glass slide while maintaining sterile conditions.
CELL COUNT
The cell number in very finely divided suspensions may be counted directly in a
haemocytometer. However, most cultures usually contain aggregates and it is difficult to count
the number of cells in each clump. Thus clumps are generally broken and then the cell number
is counted.
1.
Place the haemocytometer and focus the center 25 squares.
2.
Place the coverslip and Pipette out 100 l of suspension into a sterile
haemocytometer
3.
The cells were counted in 4 corner squares and the center one is counted.
4.
The number and the average are counted.
RESULT:
Suspension culture containing clumps was observed
42
Ex.No:
Date:
INTRODUCTION FOR ANIMAL CELL CULTURE LABORATORY
Cell culture is an indispensable technique for understanding the structure and function of cells, in recent
times it has very good implications in biotechnology. Cultured animal cells are commercially used for the
production of interferon, vaccines and clinical materials like growth hormones and urokinase. In the process
of learning the techniques of cell culture and gene transfer you will become familiar with several
terminologies and hypotheses.
Make yourself familiar with the equipments (incubators, centrifuges and microscope etc.,). If you have
problems, approach the faculty members.
Good laboratory work habits will help you a grand success. Follow the following guide lines very strictly.
These will protect you and your experiments.
1.
No eating and drinking in the lab.
2.
No storage of food materials in the lab.
3.
No mouth pipetting. Use appropriate pipetting aids that are available.
4.
Wear gloves for certain experiments whenever it is necessary.
5.
Work cleanly in an organized manner. Wipe your tissue culture hood bench with 70%
ethanol.
6.
Most important, label every thing you use with your Reg. number, date and name of the
reagent, buffer or medium.

Handling Methods
Use sterile glassware and pipettes.
When you open a glass bottle, before and after use flame the mouth in a flame.
Flame glass pipettes.
Use rubber bulbs to control pipettes. DO NOT MOUTH PIPETTE. This is to protect both you and the
cells from contamination
Prewarm medium and serum to 37C in water bath.
Wipe bottles with filter paper and transfer to the tissue culture hood (Laminar Air Flow).
Open bottles, flame tops, replace caps loosely but so they wont fall off.
Transfer, desired quantity of serum to the medium, or mix in separate (sterile) container.
Using sterile but NOT plugged, Pasteur pipette, aspirate the medium. Use a separate pipette for different
cells.
Using sterile pipette, transfer desired amount of medium and serum to the dish. Do not reuse the pipette.
Return dishes to incubator without shaking to avoid spill-over of medium.
Reflame tops of bottles and close tightly using aluminium foil. Close pipette cans.
43

Work only in designated tissue culture area and Wipe surfaces with ethanol before starting.
Work cleanly, mopping spills immediately.
Discard used medium, and especially contaminated plates/TC bottles carefully. Otherwise, they serve as
sources of contamination.
Label bottles and dishes with Reg. No., cell type, date etc. When you open a new bottle of medium or
serum, write the date on it and indicate how much has been removed.
If you add anything to medium, indicate this on the bottle. If there is only a small amount left in a
bottle, discard it.
After you have finished, remove your belongings; discard properly. Eventually, wipe the working
surface with ethanol.
A sterile hood is available for your use. Keep UV, lamp on for at least 10 mins before use. Turn off the
UV lamp while you work.
Sterile glassware
Sterile pipetes
Autoclaved
Sterile medium
Sterile serum
Millipore filtered (0.45u)
44
Ex.No:
Date:
STERILIZATION TECHNIQUES
AIM : To prepare the materials required for various cell culture practices in sterile condition.
INTRODUCTION
The term control as used here refers to the reduction in numbers and or activity of the total microbial flora.
The principal reasons for controlling microorganisms and to prevent transmission of disease and infection, to
prevent contamination by or growth of undesirable microorganisms and to prevent deterioration and spoilage
of materials by microorganisms.
Microorganisms can be removed , inhibited or killed by various physical agents, physical processes or
chemical agents. A variety of techniques and agents are available, they act in many different ways and each
has kits own limits of applications.
Steam under pressure: Heat in the form of saturated steam under pressure is the most practical and
dependable agent for sterilization. Steam under pressure provides temperatures above those obtainable by
boiling as shown in Table 22-5. In addition, it has the advantages of rapid heating, penetration, and moisture
in abundance, which facilitates the coagulation of proteins.
TYPES OF STERLISATION TECHNIQUES
AUTOCLAVE
The laboratory apparatus designed to use steam under regulated pressure is called an autoclave. The
autoclave is an essential unit of equipment in every microbiology or cell culture laboratory. Many media,
solutions, discarded cultures, and contaminated materials are routinely sterilized with this apparatus.
2
Generally, but not always, the autoclave is operated at a pressure of approximately 15lb/in (121 C). The
time of operation to achieve sterility depends on the nature of the material being sterilized, the type of the
container, and the volume. For example, 1000test tubes containing 10ml each of a liquid medium can be
0
sterilized in 10 to 15 min at 121 C, 10 litres of the same medium contained in a single container would
require 1hr or more at the same temperature to ensure sterilization..
BOILING WATER
Contaminated materials exposed to boiling water cannot be sterilized with certainty. It is true that all
vegetative cells will be destroyed within minutes by exposure to boiling water, but some bacterial spores can
withstand this condition for many hours. The practice of exposing instruments for short periods of time in
boiling water is more likely to bring about disinfection(destruction of vegetative cells of disease producing
microorganisms) rather than sterilization. Boiling water cannot be used in the laboratory as a method of
sterilization..
DRY HEAT OR HOT AIR OVEN
Dry hear or hot air sterilization is recommended where it is either undesirable or unlikely that steam under
45

pressure will make direct and complete contact with the materials to be sterilized. This is true of certain
items of laboratory glass wares, such as Petri dishes and pipettes, as well as oils, powders and similar
substances. The apparatus employed for this t7ype of sterilization may be a special electric or gas oven or
0
even the kitchen dry oven. For laboratory glassware, a 2h exposure to a temperature of 160 C is sufficient
for sterilization.
ULTRAVIOLET LIGHT
The ultraviolet portion of spectrum includes all radiation from 150 to 3900 A. Wavelengths around 2650A
have the highest bactericidal efficiency. Although the radiant energy of sunlight partly composed of
ultraviolet light, most of the shorter wavelengths of this type are filtered out by the earths atmosphere.
Consequently, the ultraviolet radiation at the surface of the earth is restricted to the span from about 2670A
to 3900A. From this, we may conclude that sunlight under certain conditions, has microbicidal capacity, but
to a limited degree. An important practical consideration in using this means of destroying microorganisms
is that ultraviolet light has very little ability to penetrate matter. Even as thin layer of glass filters off a large
percentage of the light. Thus, only the microorganisms on the surface of an object where they are exposed
directly to the ultraviolet light are susceptible to destruction.
FILTRATION
For many years a variety of filters have been available to the microbiologist which can remove
microorganisms from liquids or gases. These filters are made of different materials an asbestos pad in the
Seitz filter, diatomaceous earth in the Berkeloid filter, porcelain in the Chamberland-Pasteur filter and
sintered glass disks in other filters.The mean pore diameter in these bacteriological filters ranges from
approximately one to several microfilters; most filters are available in several grades based on the average
size of the pores. However, it should be understand that these filters do not act as mere mechanical sieves;
porosity alone is not the only factor preventing the passage of organisms. Other factors, such as the electric
charge of the filter, the electric charge carried by the organisms, and the nature of the fluid being filtered,
can influence the efficiency of filtration.
In recent years a new type of filter termed the membrane or molecular filter has been developed whose pores
are of a uniform and specific predetermined size. Membrane or molecular filters are composed of
biologically inert cellulose esters. They are prepared as circular membranes of about 150m thickness and
contain million of microscopic pores of very uniform diameter. Filters of this type can be produced with
known porosities ranging from approximately 0.01 to 10m. Membrane filters are used extensively in the
laboratory and in industry to sterilize fluid materials. They have been adapted to microbiological procedures
for the identification and enumeration of microorganisms from water samples and other materials. It is
customary to form the fluid through the filter by applying a negative pressure to the filter flask by use of a
vacuum or water pump or to impose a positive pressure above the fluid in the filter chamber, thus forcing it
through. Upon completion of filtration, precautions must be taken to prevent contamination of the filtered
material.
46
Ex.No:
Date:
PREPARATION OF MEDIA
AIM : To prepare desired medium for the given Animal cell culture.
PRINCIPLE
All the Animal cells can be grown in a liquid culture medium consisting of a mixture of vitamins, salts,
glucose, amino acids and growth factors. Moreover, Calf serum is an easily available source of growth and
attachment factors. Antibiotics are added to prevent the growth of bacteria. Under these conditions cells will
grow at physiological pH (7.4) and at body temperature (37C) to form a monolayer on the culture vessels.
MATERIALS REQUIRED
Medium
Adult bovine serum
Membrane filter (Millipore 0.45)
Sterilize
Double distilled water 1000 ml
1 litre measuring cylinder
100 ml measuring cylinder
1 litre filtration flask
Medium storage bottles
Other Glasswares
Method:
Sterilize the laminar air flow by UV irradiation for 45 minutes before using it.
1.
Take 500ml of sterile double distilled water in a 1000 ml measuring cylinder.
2.
Transfer the contents of the powdered medium into 1 litre measuring cylinder add 3.7
gms of NaHCO3 in the absence of CO2 incubator.

3.
Mix thoroughly to dissolve the powdered medium, and add penicillin
/streptomycin/gentamycin.
4.
Fill the cylinder with1 litre double distilled water mix and transfer to sterile 2 litre flask
and mix. Pinkish red color of the medium indicates normal pH range.
5.
Assemble the filter sterilization set-up and carry out the filtration under negative
pressure.
6.
Prepare 400 ml of medium containing 10% Adult bovine serum using 100 ml
measuring cylinder and store in a 500 ml sera lab bottle.

7.
Transfer the remaining medium without serum into big glass bottles.
47
8.
Store the medium in refrigerator, dispose the used membrane and immerse the used
glassware in water for washing.

9.
Different types of medium is used for various kind of Experiments.
0.
The components of different types of medium is given in the following tables.
EAGLES MEDIUM AND DERIVATIVES (mg/l)
Components
BME
Inorganic salts (mg/l)

6800.00
NaCl
400.00
KCl
200.00
CaCl2
--
CaCl2.2H2O
--
MgCl2.6H2O
200.00
MgSO4.7H2O
150.00
NaH2PO4.2H2O
2200.00
NaHCO3
L-Amino acids (mg/l)
-Alanine
21.00
Arginine--HCl
--
Asparagine.H2O
--
Aspartic acid
--
Cysteine-HCl.H2O
12.01
Cysteine
--
Cysteine-diNa
--
Glutamic acid
10.50
Histidine-HCl.H2O
26.23
Isoleucine
26.23
Leucine
36.52
Lysine-HCl
7.46
Methionine
16.51
Phenylalanine
--
48

Proline
--
Serine
23.82
Threonine
4.02
Tryptophan
18.11
Tyrosine
--
Tyrosine-diNa
23.43
Valine
Vitamins/cofactors(mg/l)
Ascorbic acid
1.00
Biotin
1.00
Choline-Cl
1.00
Folic acid
--
Inositol
1.00
Nicotinamide
1.00
Pantothenate-Ca
1.00
Pyridoxal-HCl
0.10
Riboflavin
1.00
Thiamine-HCl
--
Vitamin B112
Nucleosides and ribonucleosides(mg/l)
Adenosine
--
Cytidine
--
2-Deoxyadenosine
--
2-Deoxycytidine
--
2-Deoxyguanosine
--
Guanosine
--
Thymidine
Uridine
Other components(mg/l)
10.00
1000.00
--
49
Ex.No:
Date:
PRIMARY CELL CULTURE

Aim :
To perform primary cell culture technique using chick embryo under aseptic condition.
Introduction:
Development of techniques for the in vitro culture of animal cells has proven valuable for the study
of structure and function of cells under controlled conditions. Further, cultured cells find important
applications in vaccine production, hybridoma production and in chromosome karyotyping. Almost any
tissue can be cultured, if it is appropriately dispersed, though high rates of success in cell culture is often
recorded with embryonic and tumor tissues rather than normal adult tissues.
Cultures started fresh from tissues are called primary cultures. A method for the propagation of primary
cultures of mouse embryo cells is given below which can be adopted for the culture of other embryonic
tissues derived from different species. Often primary and secondary cultures derived from normal tissue
have finite life span similar to their in vitro life. However, some cells out of a large population are secondary
cultures by pass this definite life span and get immortalized with a capacity to divide indefinitely and these
are called cell lines. Many cancer cells have a capability to divide indefinitely in culture. Normal cells
transformed by viruses and chemical carcinogens also become continues cell lines.
Principle
Primary cultures are usually prepared from large tissue masses. Thus, these cultures contains a variety of
differentiated cells. Embryonic tissues are preferred for primary cultures due to that the embryonic cells can
be disaggregated easily and yield more viable cells. The quantity of cells used in the primary culture should
be higher since their survival rate is substantially lower.
Materials Required:
1. 13 14 days pregnant mouse / 8-10 days old embryonic eggs. 100 ml beaker 1
2. Pairs of scissors
3. A pair of bent scissors
4. 2 big forceps
5. Petriplates
6. 2 pairs 100 ml conical flasks
7. Small funnel covered with cheese cloth 1
8. 10 ml testubes cotton plugged 4 Trypsinization flask 1
9. Growth medium (M.199 with 10% ox serum
10. Calcium, Magnesium free phosphate buffered saline (PBS).
50

METHODS
MOUSE EMBRYO FIBROBLASTS
Mouse embryos of age 13-15 days are needed for culture. To get these, Swiss mice were kept for
mating and the gestation period was times by designating the day of finding the genital plug as the
first day of development.
1. Sacrifice the pregnant mouse by cervical dislocation. Place the animal in supine position.
2. Swab the abdomen with 70% ethanol and cut open along the midventral line.
3. Remove uterine horns and transfer into a beaker containing PBS. Transfer into flow hood
immediately.
4. Transfer the uterine horns into petriplates containing PBS inside the flowhood and cut open
the uterine horns to remove embryos.
5. Wash the embryos with PBS and remove head, visceral organs and appendages.
6. Transfer the remains of the embryos to another petriplate containing small amount of PBS
and mince thoroughly with a pair of bent scissors.
7. Transfer the minced tissue to the trypsinization flask containing 40 ml of 0.25% trypsin in
PBS.
8. Stir the contents at 37C for 30-60 mins.
9. At the end of the above period add 5ml of medium containing serum and stir the contents for
2 more minutes to inactivate the action of trypsin.
10. Filter the cell suspension through sterile cheese cloth and collect the filtrate into a 100 ml
conical flask.
11. Centrifuge the filtrate at ~ 1000 rpm for 10mins.
12. Pour out the supernatant and resuspend the pellet in 5 ml of medium. Distribute equally to all
2
the 120 cm culture bottles and incubate at 37C.

Chick Embryo Fibroblasts
The procedure for culturing CHF is the same as for MEF. Briefly remove embryos from 8-10 day old
embryos, break the shell with the help of foreceps and transfer the embryo into petriplates containing
PBS and follow steps 5 to 12 of mouse embryo fibroblasts culture (see above). Although the chick
embryo cells will grow in the same medium, they will grow better if 1% chicken serum is also added.
Instead of calf serum you can substitute Millipore-filtered goat serum (10%) for the culturing of above
cells.
51
Ex.No:
Date:
CELL COUNTING AND VIABILITY

AIM : To ensure the population of cells required for the culture works by cell counting method and its
viability by vital staining methods
Introduction
Haemocytometer (also known as hemocytometer) is a glass slide with two counting chambers etched
in a surface area of 9mm square. Each chamber is divided into nine 1.0mm square. It has raised sides
which keep the cover slip 0.1mm above the chamber floor so that the total volume of each square
becomes 0.0001ml(1.0mm x 0.1mm or 0.1mm or 10 cm, L x W x H ).
Principle
Staining of cells identifies viable cells. Stains generally used are Trypan Blue, Erythrosin B and
Nigrosin. Nuclei of damaged or dead cells take up the stain whereas the viable cells do not do
so.
Requirements
1. Cell suspension
2. Spirit lamp
3. Hemocytometer
4. Microscope
5. Micropipette
6. PBS
7. Tryphan Blue 0.4%
Methodology
1.
Take the Hemocytometer and place it on the flat surface of the work bench. Place
the cover slip on the counting chamber.

2.
Mix 20l cells that have been well mixed prior to sampling with an equal volume of
tryphan blue.
3. Apply to a hemocytometer by pipetting from the edge of the cover slip and permitting
diffusion by capillary action.
4. Make sure that there is no air bubble and there is no overfilling beyond the ruled area.
5. Leave the counting chamber on the bench for 2-3 minutes to allow the cells to settle.
6. Place the counting chamber on the stage of the microscope between the clips to the hold
slide so that the counting chamber can be moved (if the microscope is provided with a moving
stage).
7. Switch to low power (10x) objective, adjust the light (less light needed, hence close the
aperture or lower the condenser) and focus on the wall of the counting chamber.
52

8. Then slowly move the stage towards the middle of the slide until the ruling area visible,
sharpen the focus and locate the large square in the centre.
9. Locate the large square in the centre with 25 small squares. Place in the middle of the
field of vision and examine the distribution of viable cells on the entire area. It must be
uniform orelse refill the chamber with cell suspension.
10. Carefully switch to high power objective (40 x) and move the chamber so that the
smaller upper left corner square (with 16 smaller squares) is completely in the field of
vision.
11. Count the number of unstained cells seen on the small square (0.2x0.2=0.04sq mm) of
the upper left corner which is divided into 16 smaller squares to facilitate counting.
12. Repeat the counting with three other corner squares.
13. Make a total of all the cells counted in 4 squares. Repeat the same on the other side of
the chamber and make an average of the two chambers.
Result
The concentration of cells in the original suspension in

cells/ml = No. of cells counted
------------------------ X10 x 2
No. of Grids counted
-----------------------/ml
53
Ex.No:
Date:
STAINING OF ANIMAL CELLS
Aim :
To ensure the differentiation of live cells from dead cells by giemsa stain method.
Introduction
A number of vital staining procedures have been developed to have quick quantitation of living
cells. Quite often, when tissues are dispersed to obtain cells, a substantial proportion of cells are killed. So
before proceeding further, it is necessary to know the percentage of living cells. Some staining procedures
depend on the metabolic activity of cells (e.g. staining with methylene blue) while others using trypan blue
or erythrosine B depend on the membrane transport properties of cells. Living cells exclude (do not take up)
these stains. These vital staining procedures are very arbitrary and should be used with reservations. The
best way to determine viability is to plate (propagate) the cells and to estimate the plating efficiency later by
counting the cells or nuclei. This method is based on the fact that only the viable cells will replicate.
Vital Staining
1.
Keep 0.5 ml of dilute cell suspension in a tube and add 0.1 ml of 0.4% erythrosin B or
Trypan Blue
2.
Observe a drop under the microscope. Dead cells are stained red with erythrosin B or
Trypan Blue.
Preparation of Erythrosin B Stain
Dissolve 0.4 g of Erythrosin B in 100ml sterile PBS.
Giemsa staining
Preparation of 10X Giemsa Stain
1.
Add 20ml glycerol to 0.3g Giemsa powder and keep in a water bath at 56C, 2 hours.
2.
Then add 20ml of methanol and mix well. Leave it for 7 days at RT then filter.
Staining Method
1.
Rinse Monolayer with PBS
2.
Fix with 10ml of 3:1 Methanol : acetic acid or ethanol
3.
Drain fixer and air dry
4.
Dilute Giesma 1 to 10 (10ml + 90ml H2O) and use 20 ml of diluted stain for staining for 30 min.
5.
Discard stain, rinse several times with water, finally with tap water, air dry and look
under microscope.
Observation
The viable cells appears unstained and the dead cells looks red colour, this results because of
manual disturbance (chopping) or chemical disturbance (tripsinisation). The cell membrane gets damage by
disaggregation so the stain penetrates into the damaged cells not in normal cell.
54
Ex.No:
Date:
CULTURE OF VIRUS IN CHICK EMBRYO
Aim:
To adapt and propagate New castle disease virus in chicken embryo.
Principle
Virus is an obligate endoparasite which can grow inside the host. It is effectively grown in
embryonated egg i.e. viral suspension is inoculated into the egg allantoic membrane, where it infects
the embryo by replication or multiplication. The infected embryo forms lesion on it.
Materials required
1. Eighth day embyonated egg
2. Iodine-alchol disinfectant Syringe
3. Scotch tape Incubator
4. NDV suspension culture
Method
1. Locate the position of air sac by holding the long axis of egg horizontally in front of a light source
and mark the position.
2. Sterilize the needle by dipping in alcohol and then flaming it.
3. Use this needle to make a small hole or puncture the shell over the air sac. The membrane at base
should not be punctured.
4. Inject 0.2ml of dilute viral suspension into the allantoic cavity. Seal the hole in the shell with
scotch tape.
5. Maintain an inoculated control by injecting 0.2ml of sterile saline and incubate at 37C in
incubator.
Observation
Examine the egg periodically in front of light source for embryo death (usually takes 3-4 days). This is
indicated by cessation of movement and disappearance of veins from egg shell. Once embryo death has been
confirmed then crack the shell and collect the contents in a Petridish. Repeat the procedure with control egg.
Compare the two embryos the infected embryo will have lesions on it. Virus can be separated from the
infected tissue and purified.
55
CELL CULTURE TERMNOLOGY

Anchorage dependence: Requiring attachment to a solid substratum for survival and
growth. Cell Density: The number of cells per unit area of the monolayer.
Cell culture: in vitro growth of the cells that in vivo no longer get organized in to tissue of their natural
origin.
Cell line: A cell line is a populations of cells derive from animal tissue and grow in vitro by serial
subcultivations for indefinite periods of time with a departure from the chromosome under characterizing its
source.
Cell strain: Cell strain is a population of cells derived from animal tissue, subcultivated more than once in
vitro ,and lacking the property of indefinite serial passage while preserving the chromosomal karyotype
characterizing the tissue of origin.
Clone: A population of cells derived from a single parent cell by mitosis.
Confluence: The culture situation where all the cells are contact all around their periphery with other cells
and no available substratum is left uncovered.
Contact inhibition: Inhibition of cell membrane ruffling and cell motility when cells are in complete
contact with other adjacent cells as in confluent culture. Often precedes cessation of cell proliferation but not
necessarily related.
Density limitation of growth: Inhibition of mitotic cell division correlated with an increase in cell density
to a particular number. This number varies from one cell line to another.
Doubling time: Doubling time generation time is the time it takes for an entire culture to double in number.
If all cells are dividing then this time is a measure of overall metabolic efficiency of the population.
Epithelial cell: Cells of epithelial layer origin which in turn is derived from the embryonic endoderm and
ectoderm. Often used more loosly to describe cells of a polygonal shape with clear sharp boundaries
between cells. Pavement like appearance at confluent state. More correct, this should be termed epitheloid or
epithelial like.
Established cell line/continuous cell line: A cell line that has the potential to be subcultured indefinitely in
vivo.
Explant cells: Cells/tissue/organs removed from its normal environment (in vivo) and transferred to an
artificial medium (in vitro) for growth.
Fibroblast cell: A proliferating precursor cell of the matured differentiated fibrocyte. Often used to describe
any cells derived from differentiated mesoderm; usually spindle shaped (bipolar) or stellate (multipolar) and
arranged in parallel arrays at confluence if contact inhibited. More correctly this should be termed
fibroblastic or fibroblast-like.
Finite cell line: A culture which has been propagated by subculture but is only capable of limited number of
cell generation in vitro before dying out.
Growth factor: A specific substance that must be present in growth medium to permit a cell to
multiply. Growth curve: The change in the number of cells in growing culture as a function of time.
Hybridoma cell: A single mononucleated (synkaryonic) hybrid cell derived through fusion of an antibody
forming lymphocyte and a myeloma cell. When this newly created hybrid cell is subcultured, the clones are
referred to as a hybridoma cell line.
Idiogram:The arrangement of(incase of genetic analysis of a cell)the chromosome in order by size and
morphology so that the karyotype may be studied.
Karyotype:The distinctive chromosomal complement of a cell strain.
Microcarriers: These are different types of beads coated with charged groups to which anchorage
dependent cells get attached and grow in the suspension of beads.
Mitogens:Substance which provoke cell division (mitosis).
56

Monolayer:A single layer of cells growing on a substratum (Petridish, culture flask
etc). Neoplasia:A new, unnecessary proliferation of cells giving rise to a tumor.
Organ culture: The maintenance of growth of organ primordial, or the whole or parts of an organ in vitro
specifically designed to allow differentiation and maintenance of the original architecture and function.
Passage: The process of transferring cells from one culture vessel to another. This term is synonymous with
subculture.
Passage number:The number of times a culture has been subcultured.
Plating Efficiency:Plating Efficiency is the number of cells that grow up into colonies per 100 cells
inoculated, when the inoculum is sparse enough to permit separate colonies.
Plasmids: Cytoplasmic, autonomously replicating chromosomal elements found in bacteria. Primary
culture:The first in vitro culture started from any explant taken directly from an organism.
2
Saturation Density:Maximum number of cells present per cm (monolayer culture)or per ml (suspension
culture) under specified culture conditions.
Secondary culture:Cells of the first few (4-6) passages derived from the primary culture.
Split Ratio: The divisor of the dilution ratio of a cell culture at subculture eg, cells from one dish or bottle
divided into four (split ratio of 1-4).
Subconfluence:Less then confluent; all of the available substratum is not covered.
Superconfluence:When a monolayer culture in which the cells proliferate suspended in medium, without
being attached to the substratum of the vessel.
Tissue culture: A general term that encompasses and is concerned with the study of cells, tissues and
organs maintained or grown in vitro for more than 24hrs. Three main branches of tissue culture are: (1) Cell
culture ;(2) Tissue culture and(3) Organ culture.
Transformation:A process by which a normal eukaryotic cells is altered irreversibly into transformed cell.
Transformed cell:A transformed cell is one that has undergone a stable, heritable change by exposure from
certain viruses, chemicals, irradiation, as well as spontaneous mutations. Transformation is often
characterized (but not defined) by the emergence of an established cell line from a primary explant, an
alteration in typical morphology, loss of contact inhibition, abnormal karyotype, changes in viral
susceptibility, changes in antigenic properties, neoplastic properties and the ability to grow in suspensio
57

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