LABORATORY MANUAL

OF
B-Tech III semester (Old)
In
Biotechnology

DEPARTMENT OF BIOTECHNOLOGY
MOTILAL NEHRU NATIONAL INSTITUTE OF TECHNOLOGY ALLAHABAD
ALLAHABAD-211004 (U.P.)
Course name:

BT301 (BIOCHEMISTRY)
BT302 (MICROBIOLOGY)
BT303 (GENETICS)

Section 1: BT301 (BIOCHEMISTRY)

EXPERIMENT

S.No
1

Tests for carbohydrates

Tests for Proteins

Tests for lipids

4
5

To calculate the concentration & molarity of the given DNA

solution. Also verify whether the given DNA solution is pure or not.
Quantitative determination of individual bases in DNA.

Determination of malate dehydrogenase activity in plant tissues.

Section 2: BT302 (MICROBIOLOGY)

EXPERIMENT

S.No
1

Introduction to the tools and equipments used in microbiology

Examination of microorganisms by staining techniques.

Preparation of nutrient agar and nutrient broth media for

cultivation of microorganisms.

Isolation and enumeration of microorganisms from soil by serial

dilution agar plating method

To obtain pure culture of microorganisms by pour, spread and

streak plate method.

To assess biochemical activities viz., catalase, IMViC of given

culture of bacteria
6a. To detect catalase activity in the given bacterial culture

Assay of an antibiotic by zone-inhibition method using antibiotic

impregnated discs

Study of bacterial transformation and screening for recombinants

Section 3: BT303 (GENETICS)

S.No

EXPERIMENT

Chi Square Test for Monohybrid and dihybrid crosses

Probability and Pedigrees analysis

Study of chromosome morphology at different stages of cell

division.

Study of multiple alleles inheritance by ABO blood genotyping.

Study of genetic material transfer in two different strain bacteria by

conjugation method

Study of genetic markers in bacteria

Study of genetic polymorphism from diverse populations.

Study of allele frequency distribution in pool DNA sample

Identification of dominant F1 hybrid by DNA based marker

Section 1: BT301 (BIOCHEMISTRY)

EXPERIMENT No. 1
AIM-1a: To perform qualitative analysis of carbohydrate by molischs or anthrones test.
A) MOLISCHS TEST:
PRINCIPLE: Conc. H2SO4 hydrolyses glycosidic bonds to yield monosaccharides which in the
presence of acid get dehydrated to form furfural & its derivatives. These products react with
sulphonated -napthol to give a purple complex.
MATERIAL REQUIRED:
1. Conc.H2SO4
2. -napthol: 5% (w/v)in ethanol. Prepare fresh.
3. Test-sample- arabinose, xylose, fructose, galactose, mannose, glucose etc.
METHOD:
1. Add 2-3 drops of -napthol solution to 2ml of test solution.
2. Add 1ml Conc. H2SO4 along the side of test-tube so that to distinct layers is formed.
3. Appearance of purple colour indicates the presence of carbohydrate in the sample.
B) ANTHRONE TEST
PRINCIPLE: Conc.H2SO4 hydrolyses glycosidic bonds to yield monosaccharides which in the
presence of acid get dehydrated to form furfural & its derivatives. These products react with
anthrone to give a bluish-green complex.
MATERIAL REQUIRED:
1. Conc.H2SO4
2. anthrone: 0.2% (w/v) in Conc.H2SO4.. Prepare fresh.
3. Boiling H2O-Bath
4. test tube
METHOD:
1. Add 2ml of anthrone reagent to 1ml of test solution.
2. Keep the test-tube in boiling H2O-bath for 10 min.
3. Appearance of bluish-green colour indicates the presence of carbohydrate in the sample.

AIM-1b: To perform qualitative analysis of polysaccaharides by iodine test.

PRINCIPLE: Iodine forms coloured adsorption complex with polysaccaharides. Starch contain
polymer of -amylose and amylopectin which forms a complex with iodine to give the blue
black colour, while glycogen reacts to form reddish brown complex. A positive iodine test
indicates the presence of polysaccharides and a negative iodine test indicates the presence of
monosaccharides in the sample.
MATERIAL REQUIRED:
1. Iodine solution: prepare 0.005N iodine solution in 3% (w/v) potassium iodide solution.
2. 1% test solution of glucose, sucrose, starch, glycogen, cellulose, glucose, fructose etc.

METHOD:
1. Take 1ml of test solution test tube.
2. Add 4-5 drops of Iodine solution to it & mix the content gently.
3. Note the colour of product.
4.
AIM-1c: To perform qualitative analysis of reducing sugar by benedicts Test.
PRINCIPLE: The test is performed to know whether the given monosaccharides are reducing or
non reducing sugars. Depending on the sugar concentration yellow to green colour is developed.
All monosaccharides are reducing sugars; they all have a free reactive carbonyl group. Some
disaccharides like maltose have exposed carbonyl groups and are also reducing sugars but less
reactive than monosaccharides
MATERIAL REQUIRED:
1. Benadicts reagent: prepare by mixing solution (A) and Copper sulphate solution
a) Solution A: dissolve 173g of Na-citrate & 100g of anhy. Sodium carbonate
(Na2CO3) in 600ml of hot H2O. Dilute to 100ml with H2O.
b) Copper sulphate ( Cu2SO4.5H2O) solution: dissolve 17.3g of Cu2SO4.5H2O in
100ml hot H2O.
2. Boiling H2O-bath.
3. Test sample- monosaccharide (arabinose, xylose, fructose, galactose, mannose, glucose)
& disaccharide (lactose, maltose).
METHOD:
1. Add 2ml Benadicts reagent in 1ml test sample.
2. Keep the test tube in boiling H2O-bath for 5 minute.
3. Formation of red precipitate indicates the presence of reducing sugar.
AIM-1d: To perform qualitative analysis of monosaccharide by barfoeds test.
PRINCIPLE: The test is done to differentiate between mono and disaccharides. A biochemical
test to detect monosaccharide (reducing) sugars in solution. Barfoed's reagent, a mixture of
ethanoic (acetic) acid and copper (II) acetate, is added to the test solution and boiled. If any
reducing sugars are present a red precipitate of copper (II) oxide is formed. The reaction will be
negative in the presence of disaccharide sugars as they are weaker reducing agents. Since
Barfoeds reagent is weakly acidic, it is reduced only by monosaccharides.
MATERIAL REQUIRED:
1. Boiling H2O-bath
2. Barfoed's reagent: dissolve 13.3g copper-acetate in 200ml H2O & add 1.8ml glacial
acetic acid.
3. Test sample- arabinose, xylose, fructose, galactose, mannose, glucose etc.
4. Test-tubes

METHOD:
1. Add 2ml Barfoed's reagent into 1ml of test-sample.
2. Mix & warm in boiling H2O-bath for 1 minute.
3. Appearance of brick red colour indicates the presence of keto sugar in sample.
AIM-1e: To perform qualitative analysis of pentose & hexose by bials Test.
PRINCIPLE:Bials test is used to distinguish between pentoses and hexoses. They get
converted to furfural. In the presence of ferric ion orcinol and furfural condense to yield a
coloured product. Appearance of green colour or precipitate indicates the presence of pentoses
and formation of muddy brown precipitate shows the presence of hexoses.
MATERIAL REQUIRED:
1. Bials reagent: dissolve 1.5g of resorcinol in 100ml of conc. HCL & add 20-30 drops
of 10% ferric chloride (FeCL3) solution to it.
2. Boiling H2O-bath.
3. Test sample- pentose (arabinose, xylose) & hexose ( glucose)
4. Test-tubes
METHOD:
1. To 2ml of Bials reagent add 4-5 drops of test-solution.
2. Heat in a boiling H2O-bath for 10 minute.
3. Appearance of blue-green colour indicates the presence of pentose sugar.

AIM-1f: To perform qualitative analysis of ketoses & aldoses by Seliwanoffs Test.

PRINCIPLE: This test is used to distinguish aldoses from ketoses. The test reagent dehydrates
ketohexoses to form 5-hydroxymethylfurfural. 5-hydroxymethylfurfural further reacts with
resorcinol present in the test reagent to produce a red product within two minutes (reaction not
shown). Aldohexoses react to form the same product, but do so more slowly.
Prolonged heating will hydrolysed disaccaharides & other monosaccaharides will also
eventually give colour.
MATERIAL REQUIRED:
1. Seliwanoffs reagent: 0.05% (w/v) resorcinol in 3N HCL.
2. Boiling H2O-bath.
3. Test sample- fructose, galactose, mannose, glucose etc.
METHOD:
1. Add1ml of test solution to 2ml of Seliwanoffs reagent.
2. Mix & warm in boiling H2O-bath for 1 minute.
3. Appearance of deep red colour indicates the presence of keto sugar in sample.

EXPERIMENT No. 2
AIM-2a: To detect the presence of amino acids in a given sample by ninhydrin.
PRINCIPLE: This test is due to a reaction between amino acid and ninhydrin. Ninhydrin is a
powerful oxidising agent and in its presence, amino acids undergo oxidative deamination
liberating ammonia. Carbon dioxide, a corresponding aldehyde and reduced form of ninhydrin.
The ammonia formed from -amino group reacts ninhydrin and its reduced product to give a
blue substance diketohydrin (Ruhemanns purple). However, in case of imino acids like proline,
a different product having a bright yellow colour is formed. Asparagine which has a free amide
group reacts to give a brown coloured product. This test is also given by proteins and peptides.
MATERIALS AND REAGENTS:
1) Boiling water bath.
2) Ninhydrin : 0.2% solution prepared in acetone .
3) Test solution: prepare solution containing 50 g/ml of individual amino acids.
METHOD:
1) Add 2-5 drops of ninhydrin solution to 1 ml of test solution or sample preparation.
2) Mix and keep for 5 min in boiling water bath and observe the development of pink,
purple or violet blue colour.
3) Imino acids like proline and hydroxyproline give a yellow coloured complex.
AIM-2b: To detect the presence of aromatic amino acids in a given sample.
PPINCIPLE: Amino acids containing an aromatic nucleus (tyrosin, tryptophan, phenylalanine)
form yellow nitro derivatives on heating with conc. HNO 3. The salts of these derivatives are
orange in colour. Proteins containing these amino acids also give a positive response to this test.
MATERIALS AND REAGENTS:
1) Conc . HNO3 (Nitric acid).
2) NaOH solution (40% w/v): dissolve 40 gm of NaOH in water and make the final volume
to 100 ml.
3) Test solutions: prepare separate solutions containing 50 g/ml of amino acids like
tyrosine, glycine, tryptophan, phenylalanine, lysine, cysteine, leucine etc.
METHOD:
1) Add 1 ml conc . HNO3 into 1ml of test solution.
2) Mix the contents and keep in boiling water bath for 1 min.
3) Cool it and then slowly pipette NaOH till the solution becomes alkaline
4) Appearance of orange red colour denotes presence of aromatic amino acids.

AIM-2c: Specterophotometric estimation of protein by Lowry method.

PRINCIPLE: It is the most commonly used method for determination of proteins in cell free
extracts because of its high sensitivity and quantities as low as 20 g of proteins can be
measured. The -CO-NH (peptide bonds) in polypeptide chain reacts with copper sulphate in an
alkaline medium to give a blue coloured complex. In addition, tyrosine and tryptophan residues
of proteins cause reduction of phosphomolybdate and phosphotungstate components of the
Fehlings reagent to give bluish products which contribute towards enhancing the sensitivity of
this method. It is however , important to remember that several compounds like EDTA, Tris,
carbohydrates, NH4+, k+, Mg2+ , ions thiol reagent, phenol etc interfere with the colour
development and it should be ensured that such substances are not present in sample
preparations.
MATERIALS AND REAGENTS:
1) Phosphate buffer(0.1M, pH7.6 ):
A) 0.2M monobasic sodium phosphate- dissolve 27.8g sodium phosphate in 1 lt distilled
water.
B) 0.2M dibasic sodium phosphate- dissolve 53.75g Na2HPO4.7H2O in1lt distill water.
Prepare by- 16 ml of solution A+84 ml of solution B. Set the pH 7.5 & make the
total volume of 200ml.
2) Alkaline sodium carbonate reagent: dissolve 2 gm sodium carbonate in 0.1N sodium
hydroxide and make up the volume to 100 ml with 0.1N sodium hydroxide.
3) Copper sulphate reagent: prepare 0.5 % copper sulphate in 1% sodium potassium
tartarate solution.
4) Alkaline copper sulphate reagent: add 1ml of reagent 3 + 50 ml of reagent 2. Prepare
fresh.
5) Folins reagent: dilute the reagent to 1 N.
6) 20% (w/v) Trichloro Acetic Acid (TCA): dissolve 20 gm of trichloro acetic acid in water
and make up the volume to 100 ml.
7) Acetone
8) 0.1N sodium hydroxide
9) Bovine Serum Albumin (BSA):100 g per ml solution in distilled water.
METHOD :
1) Sample extract: weigh 1 gm of sample (germinating seeds, leaves, bacterial cells),
macerate the sample in pestle and mortar in 5 ml of phosphate buffer and transfer the
material to centrifuge tubes. Centrifuge the homogenate at 8000 rpm for 20 min. Collect
the supernatant and repeat the extraction 4-5 times. Combine the supernatant and make
the volume to 50 ml with phosphate buffer.
2) Take 1 ml of above extract and 1 ml of TCA. Keep it for 30 min and centrifuge at 8000
rpm for 20 min. Wash the pellet with acetone twice and again centrifuge it. Discard the
supernatant.
3) Dissolve the pellet in 5 ml of 1 N sodium hydroxide and mix well till it gets dissolve.

4) Take suitable liquid aliquot (1ml) of above solution and add to it 5 ml of freshly prepared
alkaline copper sulphate reagent. Mix properly and after 10 min add 0.5ml of Folins
reagent.
5) Record the absorbance at 660nm after setting the instrumentation with the reagent blank,
which contain 1ml of 0.1N NaOH instead of sample aliquot.
6) In other set of tube take suitable aliquot of BSA solution & make the volume to 1 ml with
0.1N NaOH & develop the colour as describe in step 4 & step 5.
7) Draw the absorbance curve at 660nm versus per g of BSA. From this standard curve
determine the amount of protein in the sample tube.

AIM-2d: Spectrophotometric estimation of protein by Bradford method.

PRINCIPLE: This is rapid, simple & sensitive method for estimation of proteins in a sample
extract. This method relies on the binding of the dye Coomassie Brilliant Blue to protein. At low
pH the free dye has absorption maxima at 470nm but when bound to protein has absorption
maxima at 595nm.
The amount of dye binding appears to vary with the content of basic amino acids arginine
& lysine in the protein.
MATERIAL REQUIRED:
Bradford reagent: dissolve 100mg of Coomassie Brilliant Blue G250 in 50ml of ethanol
(C2H5OH). Add 100ml of 85% of phosphoric acid & make the volume to 1lt with water.
0.1M Phosphate buffer(pH 7.5): prepare as mention in experiment 3.
Standard protein solution: dissolve 5mg 0f bovine serum albumin in 50 ml of 0.1M
Phosphate buffer. This solution contains 100g protein/ml.
Protein sample
Specterophotometer, test-tube.
METHOD:
1. Take 0.1ml of protein sample & Standard protein solution in separate test-tubes & make
the volume to 1ml with 0.1M Phosphate buffer.
2. Add 5ml of Bradford reagent in all the test tubes.
3. Mix thoroughly & incubate in H2O-bath for 10 minute.
4. Record the absorbance at 595nm against the reagent blank.
5. Plot the standard curve of A595 versus g of protein in the standards.
6. Determine the protein in the sample extract from the standard curve.

EXPERIMENT No 3
AIM-3a: To detect the lipid in a given sample by acrolein formation test.
PRINCIPLE: When glycerol, either in free form or as an ester of fatty acids, is heated with
potassium hydrogen sulphate it gets dehydrated to an unsaturated aldehyde called acrolein.
Acrolein can be identified by its characteristic pungent smell.
MATERIALS AND REAGENTS:
1) Lipid sample (butter , olive oil , stearic acid , glycerol )
2) Anhydrous potassium hydrogen sulphate
METHOD:
1) Take 1.5 gm potassium hydrogen sulphate in a test tube and add 5 drops of the liquid test
sample or an approx equivalent weight of the test sample if it is solid.
2) Cover the test sample completely by adding more of solid potassium hydrogen sulphate on
top of it.
3) Heat the test tube slowly on burner and note the odour of the fumes evolved from the tube.
AIM-3b: To estimate the presence of fatty acids in a sample qualitatively by titrimetric method.
PRINCIPLE: The presence of non esterified fatty acids in a given sample can be determined
by titrating it with an alkali using phenolphthalein as an indicator.
MATERIAL AND REAGENTS:
1) Lipid samples (butter, olive oil, stearic acid dissolved in 50% alcohol)
2) Phenolphathalein: prepare 1% solution in ethanol
3) 0.1N NaOH
4) Erlenmeyer flasks
METHOD:
1) Take 10 ml of 0.1 N NaOH in an Erlenmeyer flask and add a drop of phenolphthalein
solution which will give permanent pink colour.
2) To this, with pipette add the test solution drop by drop with constant shaking of the flask.
3) Disappearance of the pink colour indicates the presences of free fatty acids in the given
test compound.
4) Repeat this with other lipid samples.

AIM-3c: To test the unsaturation of fatty acids in lipid samples

PRINCIPLE: The fatty acids present in animal fats are usually saturated whereas those
occurring in vegetable oils contain one or more double bonds in their hydrocarbon chain. A semi
quantitative estimate about the degree of unsaturation of lipid samples can be deduced since
halogens are readily added across the double bonds and this reaction results in decolourization of
bromine water or iodine solution.
MATERIAL AND REAGENTS:
1) Test solutions (olive oil , corn oil , coconut oil , oleic acid , stearic acid )

2) Bromine water: add 5 ml of bromine to 100 ml of water. Shake the mixture and keep it in
dark bottle.
METHOD:
1) Take approximately 5 ml of test solution in a test tube and slowly add bromine water
drop wise and shake the tube after each addition.
2) Keep on adding bromine water till it fails to get decolorized and retains its colour.
3) Note the amount of bromine water added.
4) Repeat the experiment with other test solutions.

AIM-3d: To extract and estimate the total lipid content in the given sample of oil seed.
PRINCIPLE: Lipids are soluble in some organic solvents. This property of specific solubility in
non polar solvents is utilized for extracting lipid from tissues. In biological materials the lipids
are generally bound to proteins and they are, therefore extracted either with a mixture of ethanol
and diethyl ether or a mixture of chloroform and methanol. Inclusion of methanol or ethanol in
the extraction medium helps in breaking the bonds between the lipids and proteins.
MATERIALS AND REAGENTS:
1)
Iodometric flasks
2)
Separatory funnel
3)
Oil seeds ( sunflower , peanuts , or soybean )
4)
Anhydrous sodium sulphate
5)
Chloroform methanol mixture (2:1, v/v)
6)
1% sodium chloride
METHOD:
1) Take 1 gm of the oil seed and grind it in presence of 5 gm of anhydrous sodium sulphate in a
pestle and mortar. A small amount of acid washed sand may be used as an abrasive if the seed
material is tough.
2) Add 20 ml of chloroform methanol mixture to it and transfer to an air tight stoppered
iodometric flask .Shake the content of the flask on a mechanical shaker for one hour and then
filter it through a glass funnel. Repeat the extraction of the residue twice and pool the filtrates.
3) Remove the solvent from the residue by distilling under vacuum. Extract it again for any left
crude lipid residues , extract it again with 10 ml of chloroform methanol mixture containing 1
ml of 1% sodium chloride .
4) Take the pooled fractions in a separatory funnel, shake it thoroughly and allow it to stand for 5
min. The lipids will be recovered in the lower chloroform layer while soap, glycerol and other
water insoluble impurities move into the upper layer.
5) Drain out the lower layer and treat the upper layer again 3-4 times with 5-10 ml of chloroformmethanol mixture.
6) Collect the lipid containing fractions in a pre-weighed beaker.

7) Evaporate the solvent by keeping the beaker in warm water bath (50 C) with a constant blowing
of a slow stream of nitrogen gas over the surface.
8) Record the weight of beaker and determine the amount of crude lipids in sample by subtracting
the weight of empty beaker.
9) Express the results in terms of terms % crude lipids in the given sample of the oil seeds.

EXPERIMENT No. 4
AIM: To calculate the concentration & molarity of the given DNA solution. Also verify whether
the given DNA solution is pure or not.
PRINCIPLE: Due to the presence of conjugated double bond in purine & pyrimidine, DNA
absorbs the UV light. They show absorption maxima at 260nm. Proteins are the main
contaminants in nucleic acid extracts & have absorption maxima at 280nm.
So, the ratio of absorption maxima at 260nm & 280nm determine the purity of DNA.
If A260/A280=1.8, DNA is pure.
If A260/A280<1.8, protein & phenol contamination is present.
If A260/A280>1.8, RNA is present.
A solution of DNA with a concentration of 50 g/ml will have an absobance at 260nm
equal to 1
i.e. 1( A260) unit of ds DNA = 50 g DNA/ml
According to Beers law, the concentration of DNA can be measure by following
formula:
A=CL
Where, A= A260, C= concentration, L= path length in cm,
= molar absorbance coefficient (260 of DNA=20)
Molarity of DNA can also be calculated by comparing result that,
260 of 1Mm ds DNA= 6.7
MATERIAL REQUIRED:
Specterophotometer, Cuvette, pipette, DNA sample, distill H2O
METHOD:
1. Place the DNA sample in cuvette & take absorbance at 260nm & 280nm in
Specterophotometer.
2. If optical density (OD) is high, dilute the sample with distill H20.

EXPERIMENT No 5
AIM: Quantitative determination of individual bases in DNA.
PRINCIPAL:
DNA is hydrolysed to constituent bases by heating with 72% perchloric acid for 1 h. The
resulting bases are than separated by paper chromatography and located by viewing the
chromatogram under UV light. After eluting them from the filter paper, the amount of different
bases is determined from their absorbance values obtained at appropriate wavelengths.
MATERIALS AND REAGENTS:
1. Paper chromatography chamber
2. Whatman no.1 filter paper
3. Micropipette (20l)
4. Ultraviolet lamp
5. UV-spectrophotometer
6. Deoxyribonucleic acid: Commercially available DNA sample may be used
7. Perchloric acid
8. Iso-propanol
9. Cons HCL
10. Standard bases: Prepare separately the aqueous solutions of adenine, guanine, cytosine
and thymine to contain 10 ug of the base per 50 ul of the solution
11. 0.1 N HCL
12. SOLVENT SYSTEM: Prepare the solvent system for separation of base by mixing isoprapanol, water and conc HCL in ratio of 130:37:33
METHOD:
1. Dissolve 10 mg of DNA in ml of 72% perchloric acid and heat on a boiling water bath for
1 h.
2. Use the clear supernatant for separation of bases by paper chromatography.
3. Apply 50 l spot of the supernatant & each of the bases as the reference standards about
2.5 c apart from each other on the starting line of whattman filter paper.
4. Allow the spot to dry completely and using iso-propanol: water: conc HCL solvent
system, develop the chromatogram in the chromatographic chamber.
5. Identify each spot by comparing its Rf value (see Section 9.3.3) with the standard bases.
6. For quantitation, cut the area of the chromatogram containing individual spots and
transfer each piece into a separate tube containing 0 1 N HCL. Allow the tubes to stand
overnight for elution of the spot. Next morning, drain out the liquid from the tubes and
note its absorbance on UV spectrophotometer at a wavelength corresponding to the
absorbance maxima of each base.
7. Further identify the base by measuring and comparing their absorption spectra with the
standard base
Table: Absorption of purine and pyrimidine bases in 0.1 N HCL
Nucleotide base
Absorption maxima
Millimolar extianction

(nm)
Adenine
Guanine
Thymine
Cytosine
Uracil

260
250
265
275
260

Coefficient*
13.3
11.2
7.9
10.5
7.9

*Absorbance VALUE OF 1 mM solution of the compound when measured in a cuvtte of

1 cm length path.
CALCULATION:
Suppose A260 for adenine is y OD units
So, moles of adenine/ mg of DNA sample= y/13.3 * v2/v1 * v3/w
Where, V1= volume in ml of hydrolysate spotted on chromatogram
V2= total volume of supernatant in step 2
V3= ml of 0.1N HCLused for elution of spot from chromatogram
W= mg of DNA taken for hydrolysis
Similarly, calculate the value of other bases.

EXPERIMENT No 6
AIM: Determination of malate dehydrogenase activity in plant tissues.
PRINCIPAL: Malate dehydrogenase (MDH) in an important enzyme of TCA cycle and
catalyses the following reaction which is readily reversible.
Oxaloacetate +NADH + H
Malate + NADH
In addition to mitochondria, the enzyme also occurs in cytosol. Another process in which the
enzyme is involved is CO assimilation in C4 plants where it utilizes NADPH rather than NADH
as the coenzyme.
MDH is, thus, an oxidoreductase which utilizes the reduced from of nicotinamide adenine
dinucleotide (NADH) for reduction of oxaloacetate to L-malate. During this reaction NADH is
oxidized to NAD. NADH exhibits absorbance maxima at 340 nm whereas at this wavelength
NAD has a negligible absorbance. Hence the decrease in absorbance at 340 nm due to utilization
of NADH provides a convenient parameter for following the progress of the reaction catalysed
by MDH.
MATERIALS AND REAGENTS:
1. Plant material: Freshly harvested leaves of any plants like wheat, pea, rise etc.
2. Acid washed river sand: Suspend the sand in 1N HCL and keep it for several hours with
occasional stirring. Drain out the acid and wash it thoroughly with distilled water till it
completely free of the acid.
3. Refrigerated centrifuge.
4. Spectrophotometer.
5. Extraction buffer: Prepare 50mM imidazole-HCL buffer containing 10Mm dithiothreitol,
20mM MgCl2 and 2mM EDTA (refer to section 1.3.8 for preparation of imidazole buffer).
6. PVP (polyvinyl pyrrolidone).
7. 50Mm Tris-HCL buffer (pH8.0):
A) 0.2M Tris (hydroxymethyl) aminomethane- 24.2g / lt
B) 0.2M HCL
Prepare by- 50ml of A + 26.8 ml of B. Set the (pH8.0)& make the total volume upto
200ml.
8. Assay buffer: Make 50 mM Tris-HCL buffer (pH8.0) containing 1 mM EDTA.
9. NADH solution: Make 6mM NADH solution n 50 Mm Tris-HCL (pH 8.0). Store this
solution in ice.
10. Oxaloaceatate solution: Prepare 0.3M solution of oxaloacetate in water and neutralize it
by adding solid NaHCO3 till gas bubbles cease to evolve
METHOD:
1. Prepare the enzyme extract by homogenizing 0.5 g of leaves at 0-4C in chilled pestle and
mortar using a small amount of acid washed sand as an abrasive in presence of
1%(w/v)PVP and 10 ml of the ice-cold extraction buffer which should be added in small
amount at a time.

2. Centrifuge the leaf homogenate at 10,000*g for 20 min in a refrigerated centrifuge at 04C.
3. Use supernatant as the enzyme preparation. Preparation in ice till the enzyme assays is
performed.
4. Spectrophotometer set the wavelength of 340 nm. Take 1.5ml of the assay buffer and 0.1
ml of the enzyme extract in a quartz cuvette (glass cuvette should not be used since it
absorbs UV light)and adjust 100%transmission with it
5. Take out the cuvette and keep it in water bath at 37C for about 2-3 min Add 0.05 ml of
NADH and mix the contents by inverting the cuvette and place it immediately in
spectrophotometer and note the initial reading.
6. Record the decrease in absorbance at intervals of 15 sec upto 3min. this serves as a
blank.
7. Start the reaction by adding 0.05 ml of oxaloacetate solution and note the decrease in
absorbance upto 3 mn at regular intervals of 15 sec.
8. Prepare a standard curve of NADH over a range of 0-0.3 u moles. Make sure that total
volume of the solution in the cuvette for preparation of the standard curve is the same as
during the assay (i.e. 1.7 ml) and this is done by adding calculated volume of assay
buffer.
CALCULATION:

The enzyme activity is generally expressed as umoles of malate formed/g tissue/min and
is calculated as follows.
Suppose
Weight of the taken = 0.5 g
Final volume of enzyme extract = 10ml
Enzyme extract taken for assay = 0.1ml
Decrease in A340 in blank = 0.01 O.D. units/min
Decrease in A340 in simple = 0.06 O.D. units/min
Decrease
in
A340
due
to
MDH
activity=0.060.01
= 0.05 O.D. units/min

Let it be assumed that from the standard curve, O.D of 0.12 corresponds to 40n moles of
NADH.
Then,
0.12 O.D. units = 40 n moles of NADH
0.05 O.D. units = 40/.12 into .05 = 16.66 n moles of NADH

In this reaction one molecule of NADH is oxidized for conversion of one molecule of
oxaloacetate to give one molecule of malate. Therefore, the number of molecules of
malate produced will be equale to the numberof NADH molecules oxidized.
Hence,
Total enzyme activity as n moles/min
In10 ml of enzyme preparation = 16.66/0.1*10
Total enzyme activity in 1 g of tissue = 16.66/0.1 * 10 * (1.0/0.5)
=3332 nmoles/min
If one unit of enzyme activity is defined as the amount of enzyme which forms 1 u moles/
of malate/min. then the tissue contains 3.332 units of MDH activity/g fresh weight.

Section 2: BT302 (MICROBIOLOGY)

EXPERIMENT NO. 1
1.

INTRODUCTION

TO

THE

TOOLS

AND

EQUIPMENTS

USED

IN

MICROBIOLOGY
1a. LAMINAR AIR FLOW: PRINCIPLE: It is an apparatus consists of an air blower in the rear side of the chamber which can produce air
flow with uniform velocity along parallel flow lines. There is a special filter system of high
efficiency particulate air filter (HEPA) which can remove particles as small as 0.3 nm.
WORKING: 1.) In front of the blower, there lies a mechanism through which air blown from the blower
produces air velocity along parallel flow lines.
2.) Inside the chamber one fluorescent tube and the other UV tube are fitted. Due to uniform
velocity and parallel flow of air current, pouring of media, plating, slant preparations,
streaking etc. without any kind of contamination are performed.
3.) Initially, dust particles are removed from the surface of the laminar flow with the help of
smooth cloth containing alcohol. Switch on the UV light for a period of 30 minutes so as
to kill the germs, if any present in the area of working space.
4.) All the work related to pouring, plating, streaking etc. are to be carried out in the flame
zone of the burner or spirit lamp.
PRECAUTIONS: Put off the shoes before entering to operate the apparatus. Wash the hands with detergent or
soap. One should not talk inside the chamber while performing microbial culture transfer, failing
which chances of contamination may be more which may come either through mouth, sneezing
or air.

Fig: Laminar Air Flow

1b. AUTOCLAVE: PRINCIPLE: The killing action of heat on the organisms can be done by using increase in the stream in a
closed system. The water molecules become aggregated resulting in increase in their penetration.
The water boils at 1000C and the steam accumulates in a closed container resulting in increase in
pressure.
WORKING: 1.) The autoclave is usually of pressure cooker type made up of gun metal sheets which is
supported in an iron case.
2.) It is closed by swing door which is fastened by radical bolts tightly.
In microbiology laboratory, system jacketed horizontal type autoclave is necessary.
3.) The steam passes from below at the base. The side walls are heated by the steam jacket. It
has a provision to record the pressure.
4.) There is a possibility to regulate the pressure using pressure meter. It consists of safety
valve that guards against the accidents. It is based on moist heat that used sterilisation.
5.) The autoclave is usually operated at 15 lb/inch2 steam pressure for 30min. As
demonstrated in the table. The temperature for 30min. is enough to kill all the spores and
cells of micro organisms.
PRECAUTIONS
The level of water should be checked before operating. The air should be completely evacuated
and the steam must have access to the materials to be sterilised. The cotton or glass beads must
be sterilised in a glass container closed with foil. The heat sensitiser substances should not be
sterilised by autoclaving.

Fig: Autoclave

1c. INCUBATOR
PRINCIPLE: Consists of copper/steel chamber, around which warm water or air is circulated by electric
current or by means of small gas flame.
WORKING:1.) The temperature of incubator is kept constant due to its control by using thermostat.
2.) It is made up of double walled chamber adjusted to a desired temperature. It is done by
using an external knob controlling the thermostat system. The gap between two walls is
insulated to check heat conduction. A thermometer is inserted from the top for recording
the temperature.
3.) It is operated to allow the microbial growth on a suitable medium under proper
temperature. The temperature variation should not be more then 1.
4.) If a lower temperature than the room is required, the water is circulated around the
chamber to pass through an ice chest.
PRECAUTIONS:The door of the incubator should be opened only when necessary. If the tubes are to be incubated
for a long time or at a higher temperature, the medium may become too dry due to excessive
evaporation. In such cases cotton plug should be pushed inside the neck of the tube. The tube
should be covered by a rubber cap so as to cover the plug.
LABORATORY OVEN
PRINCIPLE:It is generally used for sterilisation of glassware, metal devices and other articles which are
spoiled by autoclaving. For such purpose dry heat sterilisation is used. Ovens based on their
principle. Although sterilisation can be made by autoclaving but glassware require dry heat
instead of moist heat. It kills the microbes by oxidising their chemical constituents. It is less
effective as compared to moist heat.
WORKING:1.) The ovens generally consists of double walled chamber, the gap between two walls is
insulated. It is heated from below by using electric current and heating elements are
arranged in a number to heat the inside chamber uniformly.
2.) There is a in-built thermostat when required, it helps in regulating the temperature. The
calibration knob sets the desired temperature.
3.) For sterilisation, the holding time depends upon the temperature. For temperature of
160C, holding time should be 1 hr and at 180C it is 30min.
PRECAUTIONS:The glass materials should be wiped and dried before keeping inside the chamber, otherwise it
may break. After the holding time is over, the glassware should not be taken out immediately
rather, the temp should go down so as the cool air from outer environment could not damage or
break the glassware. The air, within the oven, should be circulated by a fan to ensure that all
parts are kept at required temperature, the articles should be placed properly so as not to obstruct
the flow of air.

1d. THE pH METER

PRINCIPLE:In 1909, Sorenson used the term pH to denote the hydrogen ion concentration of the solution.
pH= -log10(H+) = 7
pH is the degree of acidity and alkalinity of a solution on a scale 1 to 14. pH acid is proton donor
and base is base acceptor (acid = base + H+) i.e. acid dissociates and produces H+.
WORKING:1.) pH is measured by using a pH meter which is an instrument calibrated against a series of
buffers of known pH values.
2.) A standard pH meter has two electrodes, one glass electrode and the second mercury
mercurous chloride (calomel) or silver-silver chloride reference electrode. The reference
electrode is emerge in saturated KCl solution.
3.) A normal pH meter consists of a glass electrode made up of a thin glass membrane which
is selectively permeable to H+.
4.) Thus, the fixation of electrode is to form conductive bridge between the metallic element
and the sample solution in which two electrodes are placed.
PRECAUTIONS:Should be handled with care so that its electrode may not be broken. The electrodes should
always be kept inside distilled water so that they may not be non-functional.
1e. CENTRIFUGE MACHINE
PRINCIPLE: Instruments which operates through the centrifugation technique. This technique is based on
molecular mass, shape and density of the particles. The rate or velocity at which particles
sediment is proportional to force rate and velocity of sedimentation applied forces. The
application of larger force than the earths gravitational force, thus increase the rate of
sedimentation of particles. The different particles sediment separately due to variation in their
density, shape or size.
There are two techniques of centrifugation:
i). Preparative and ;
ii). Analytical
The separation, isolation and purification of whole cell, plasma membrane, nucleic acids and
vercises are carried out by preparative technique, whereas pure macromolecules or particles are
separated by analytical technique.
PRINCIPLE OF SEDIMENTATION:The centrifugal force is applied which induces the gravitational force of the materials present in a
solution. The centrifugal force acts in a direction away from the centre of the axis. If the speed of
rotation is faster, the force will be higher accordingly. The rate of sedimentation depends upon
the applied centrifugal field (G). The G is the function of square of the angular velocity of the
rotor and the radial distance of the particles present from the axis of rotation.
1 rev.= 2 radians
rev/min

=
The centrifugal force is expressed as relative centrifugal field (RCF) in g units. (g=9.8 m/sec-2)
1.11 X 10-5 (rev/min)2r
The sedimentation rate or velocity (v) of particles varies according to shape, size and density
coefficient S Suedberg unit.

TYPES OF CENTRIFUGE: a).Small bench centrifuge

b).Refrigerated centrifuge of large capacity
c).High speed refrigerated centrifuge
d).Ultracentrifuge
1f. SPECTROPHOTOMETER (COLORIMETER)
PRINCIPLE: It is an instrument that utilises light as a source of radiation and measures changes in optical
density or absorbance. It has three basic principles i). the source of radiation, ii). a unit for
dispersing radiation at different wavelength and iii). a device that detect the amount of radiation
at different wavelengths.
WORKING: 1.) Spectrophotometer uses monochromatic (narrow wavelengths) radiation, whereas
colorimeter uses broad wavelength bands. There are several atoms and their electron
clouds in every chromophore (a molecule or part of it). Due to changes in energy contents
of the electrons, the shape of the chromophore is changed. These changes in energy level
can be recorded by any spectrophotometer.
2.) For quantitative estimation the absorbance of different concentration of a substance can
be measured by following Beers law (absorbance is directly proportional to
concentration of a substance i.e. when a ray of monochromatic light passes through an
absorbing medium, its mathematical expression of Beers law is
=
I0= original intensity of light beam
I= intensity of beam passing through a solution
C= conc. of the solution in moles/l
K=constant A plot of absorbance (Y-axis) versus conc. (x-axis) yields a calibration curve. This
shows the range of Beers law. It may be used for Quantitative estimation.
3.) The spectrophotometer is of many types such as
a).visible range spectrophotometer (400 to 1000 nm)
b).UV visible spectrophotometer (200 to 1000 nm)
c).IR spectrophotometer (1000-3000 nm) wavelength.
1g. PETRIDISH
PRINCIPLE:-

It consists of two shallow glass dishes, the upper half a lid and the lower half or bottom half.
WORKING:1.) For isolation and cultivation of different types of microorganisms these dishes are used in
all microbiological lab.
2.) According to the requirement, its diameter varies. Molten agar medium is aseptically
poured on the bottom half of the sterilised Petridish and covered with the upper half.
3.) The Petridishes are sterilised by putting them in a Petridish container and in turn in an
oven/ autoclave.
4.) Now, disposable sterile plastic Petridishes are also available for the same purpose.
1h. INOCULATION LOOP:
PRINCIPLE:It is made up of a long platinum wire fixed into a metallic rod.
WORKING:1.) It has a handle with steel screw shaft in which nichrome or platinum wire is to be fitted.
2.) The wire is to be wrapped around a small round object such as pencil etc. To form a loop
by twisting it mechanically. The loop should be such so as to retain a small circular film
in it by dipping into solution. For this a proper size (5-7 cm) of the wire is recommended.
3.) The straight wire or straight needle has a wire in place of loop. The open or free end of
which is blunt. Both loop and straight wire are to be sterilised either by using Bunsen
burner or hot heating coil wire till the needle or loop become red hot. After loop or wire
is cooled these are generally used in transferring culture from liquid broth.
i. The needle straight wire is used for transferring culture from solid medium.
ii. The loop is also for picking small quantities of solid materials from a microbial colony,
and can be used to inoculate either a liquid or a solid medium. Both the loop and straight
wire must be flamed immediately after use so that contamination is avoided.
1i. MICROSCOPE:PRINCIPLE:The basic microscope consists of eye piece(10X magnification)that is fitted into the
microscope tube. The magnification varies in ocular eye piece. For identification another low
power lenses called objective are used which have varying powers(10X, 40X). For high
magnification an oil immersion (100X) is also used. The Abbe type condenser is required
while using oil immersion objective because of providing light. It has an iris diaphragm and a
filter that is moved below the objective lens. The condenser consists of aplanatic lenses, has a
numerical aperture of 1.3 to 1.4 under high resolution.
Two kinds of preparations, wet mount and stained smear on the slide surface under cover
glass, are used for simple microscopy. The wet mount preparation is used with objective (10X,
40X) while for detailed observations, oil immersion objective is recommended. A stained smear

is used to locate the specimen with dry objectives (10X, 40X) whereas smear covered with
immersion oil provides a clear and translucent image of bacteria/ microorganism.
WORKING:1.) Place smear on stage and focus the image.
2.) To observe a specimen with aid of eyepiece and objective lens, during the initial stage
keep the condenser up to the level of stage of microscope.
3.) Completely open the aperture diaphragm of the condenser and with tilting the mirror
focus the optical axis.
4.) Observe the image of the specimen with a sharp focus by using the coarse control of the
objective lens.
i. Most of the time flat mirror is used, for lower power concave mirror is used.
ii. Adjust the position of condenser best fitted for focussing smear by removing the eyepiece
and look down into the tube at the visible illumination. Tilting the mirror helps in
illumination.
iii. Reduce the scattered light from lens by closing the aperture diaphragm of the condenser
iv.
It is suggested that before placing a slide on the stage apply oil drop on the smear. It is
also convenient to apply the oil drop to the slide with the nosepiece half rotated to the
correct objective and before swing the oil
immersion objective into position.

Fig: Compound Microscope

EXPERIMENT NO. 2
Aim: Examination of microorganisms by staining techniques.
2a. To perform Gram staining of the given bacterial strain
Principle: Gram staining, a differential staining technique was developed by Dr. Hans Christian
Gram in 1884. It is a very useful stain for identifying and classifying bacteria into two major
groups: Gram-positive and Gram-negative. Gram-negative bacterial cell wall is thin, complex,
multilayered structure and contains relatively a high lipid contents, in addition to protein and
mucopeptides. The higher amount of lipid is readily dissolved by alcohol, resulting in the
formation of large pores in the cell wall which do not close appreciably on dehydration of cellwall proteins, thus facilitating the leakage of crystal violet-iodine complex (CV-I) and resulting
in the decolorization of the bacterium which later takes the counter stain and appears red. In
contrast, the gram-positive cell walls are thick and chemically simple, composed mainly of
protein and cross-linked mucopeptides. When treated with alcohol, it causes dehydration and
closure of cell wall pores, thereby not allowing the loss of (CV-I) complex and cell remains
purple.
Procedure:

Take a fresh clean slide and mark the smear area on the underside of the slide with a marking
pencil.

Put a looopful of sterile distilled water on a clean slide.

Aseptically transfer a small amount of bacterial growth or colony into the sterile distilled
water droplet with the inoculating loop.

Spread the bacterial suspension thinly to form a smear

Firstly allow the smear to air dry.

Heat fix the smear by passing it rapidly through the tip of the blue portion of the Bunsen
burner flame for four to five times.

Cover the smear with crystal violet for 30 seconds.

Wash slide with DW for a few seconds,using wash bottle.

Cover smear with grams iodine solution for 60 seconds.

Wash off the iodine solution.

Add ethyl alcohol for few seconds (10-20 seconds) until no more colour flows from the
smear.

Wash the slides with distilled water and drain.

Apply (counter stain) safranin to smears for 30 seconds.

Wash with DW and blot dry with absorbent paper.

Examine the slides microscopially under oil-immersion objective.

Observations

Those bacteria that appear purple are referred to as Gram-positive and those that appear pink
are Gram-negative.

2b. To perform Negative staining for the given bacterial strain

Principle: In a negative staining technique, a simple stain is used that does not stain the bacteria
but stains the background. Acidic stains like (nigrosin or India ink) carry a negative charge on
their surface and bacterial cells also carry negative charge and thus they do not take up the stain
and appear transparent and unstained upon examination. Negative staining is advantageous for
two reasons 1. Cells appear less distorted because no heat fixing is done, 2. Capsulated bacteria
that are difficult to stain can be observed by this technique.
Procedure:
Place one drop of nigrosin at one end of a clean glass slide.
Transfer a loopful of the inoculum from the broth culture in the drop of stain and mix gently
with the loop.
Take another clean slide, place it against the drop of suspended organism at an angle of 30 0
and allow the droplet to spread across the edge of the top slide.
Allow the smear to air dry.
Examine the preparation under oil-immersion objective.
Observations
Spherical cells occurring singly as well as in clusters appear colourless (transparent) against a
blue background.
2c. To perform Capsule staining by negative staining technique.
Principle: Some bacterial cells are surrounded by a mucilaginous substance forming a viscous
coat around the cell. This structure is referred to as a capsule. In negative staining the
background but not the bacteria is stained by the use of an acidic stain which carries a negative

charge on its surface and is repelled by the bacteria that too carry a negative charge on their
surface. Capsules are clearly visible in the light microscope when negative stains and special
capsule stains are employed.
Procedure:

Place one drop of nigrosin at one end of a clean glass slide.

Transfer a loopful of the inoculum from the broth culture in the drop of stain and mix gently
with the loop.

Take another clean slide, place it against the drop of suspended organism at an angle of 30 0
and allow the droplet to spread across the edge of the top slide.

Allow the smear to air dry. Do not heat dry.

Examine the preparation under oil-immersion objective.

Observations
Spherical cells occurring singly as well as in clusters appear colourless (transparent) against a
blue background.
2d. To perform endospore staining by Schaeffer- Fulton method.
Principle: Some bacteria are capable of changing into dormant structures that are metabolically
inactive and do not grow or reproduce. Since these structures are formed inside the cells, they are
called endospores. A German botanist Fertinand Cohn discovered the existence of endospore in
the bacteria these are remarkably resistant to heat, radiation, chemicals and other agents that are
typically lethal to the organism. The heat resistant of spores has been linked to their high content
of calcium and dipicolenic acid. During sporulation, vegetative cells gives rise to a new,
intracellular structure termed as endospore, that is surrounded by impermeable layers are called
spore coats. An endospore develops in a characteristic position with in a cell that is central,
subterminal or terminal.
Endospores are extremely resistant due to their thick wall, the spore coat. The spore coat does
not stain easily. Malachite green, however, penetrates the spore coat of endospore after
considerable heating. Once stained, the endospore does not decolourizes easily hence appears
green even after washing. In contrast, the counter stains entering the endospore but stains rest of
the cell content that appears red.
Procedure:

Take a fresh clean slide and mark the smear area on the underside of the slide with a marking
Pencil.
Put a looopful of sterile distilled water on a clean slide.
Make a smear of the bacterial culture on a clean slide.
Air dry & heat fix the smear.
Flood the smears with malachite green.
Heat the slides to steaming and steam for 5 minutes, adding more stain to the smear from
time to time.
Wash the slides under slowly running tap water.
Counterstain with safranin for 30 seconds.
Wash smear with DW.
Blot dry slides with blotting paper.
Observations
Endospores stain green and the vegetative cells stain red...
2e. Staining of Fungi using Lactophenol cotton blue.
Principle: Lactophenol cotton blue is a stain commonly used for microscopic preparations of
fungi. It stains the fungal cytoplasm and provides a light blue background, against which the
walls of hyphae can readily be seen. It contains four constituents: phenol, which serves as a
fungicide; lactic acid, which acts as a clearing agent; cotton blue, which stains of the cytoplasm
of the fungus; and glycerine, which gives a semi-permanent preparation.
Procedure:
Place a drop of lactophenol cotton blue on a clean slide.

Transfer a small tuft off the fungus, preferably with spores and spore bearing structures, into
the drop, using a flamed, cooled needle.

Gently tease the material using the two needles.

Mix gently the stain with the mold structures.

Observe the slide under low and high power objectives of a microscope.

Observations:
Observe the types of conidia, conidiophores, hyphae and their arrangement.

EXPERIMENT NO. 3
Aim: Preparation of nutrient agar and nutrient broth media for cultivation of
microorganisms.
MATERIALS REQUIRED:Peptones, Beef extract, NaCl, Agar, distilled water, Electron balance, test tube, measuring
cylinder, pH meter, heater, autoclave.
Nutrient Agar Medium Composition: 100 ml distilled water
Peptone- 5g/l
Beef extract-3g/l
NaCl-5g/l
pH- 7.0 0.02
Nutrient Broth Medium Composition: 100ml distilled water
Peptone- 5g/l
Beef extract-3g/l
NaCl-5g/l
pH- 7.0 0.02
PROCEDURE:1.) Weigh all the components of media using electronic balance (peptones, beef extract and
Nacl) and then mix with 100ml distilled water in a glass beaker.
2.) Check pH of the solution and maintain the pH- 7.0 0.02 by adding acid or base.
3.) Add agar to the mixture and heat.
4.) Autoclave at 121 C for 20 minutes.
5.) Cool the medium and pour in petri-dishes/ test tubes.
6.) For nutrient broth medium all the components are added to distilled water in the same
way except agar-agar powder.
Nutrient agar and nutrient broth media is ready for culture growth.
PRECAUTIONS:1.) Handle the electronic balance carefully.
2.) Handle the pH meter and other instruments of laboratory carefully.
3.) Proper handling of the chemical components should be done.

EXPERIMENT NO. 4
Aim: Isolation and enumeration of microorganisms from soil by serial dilution agar plating
method
Principle: The serial dilution agar plating method or viable plate count method is one of the
commonly used procedures for the isolation and enumeration of fungi, bacteria and
actinomycetes which are most prevalent microorganisms. This method is based upon the
principle that when material containing microorganisms is cultured each viable microorganism
will develop into a colony; hence the number of colonies appearing on the plates represents the
number of living organisms present in the sample.

Suspend one gram soil in the test tube containing 9 ml sterile distilled water and shake the
tube on a rotary shaker.

Perform Serial dilution technique upto 10-1, 10-2, 10-3, 10-4, 10-5, 10-6 and 10-7 dilution.

Vigorously shake the dilution on a rotary shaker to obtain uniform suspension of

microorganisms.

Transfer aliquots of 0.1 ml suspension from 10-5 dilution blank on sterilized petri dishes.

Add approximately 15-20 ml of the sterilized nutrient agar in the petri dishes. (Note: the
nutrient agar should be cool enough so that it should not kill the microorganisms in the
sample, simultaneously it should be warm enough to be in liquid state).

Mix the the contents of the petri plates by slow rotary movement.

Prepare such plates in triplicates for each sample.

This is pour plate technique.

Incubate all the plates in an inverted position at 280.1 C for 24-48h

Fig. Serial dilution of sample for pour plate

Calculations:
The colony forming units will be calculated by the formula: (Johnson and Curl, 1972)
CFUg-1 dry soil = Average number of colonies
Dry weight of soil taken

X Dilution factor

EXPERIMENT NO. 5
Aim: To obtain pure culture of microorganisms by pour, spread and streak plate method.
5a. Spread Plate Technique
In this technique, the number of bacteria per unit volume of sample is reduced
by serial dilution before the sample is spread on the surface of an agar plate.
1. Prepare serial dilutions of the broth culture as shown below. Be sure to mix the nutrient broth
tubes before each serial transfer. Transfer 0.1 ml of the final three dilutions (10-5, 10-6, 10-7) to
each of three nutrient agar plates, and label the plates.
2. Position the beaker of alcohol containing the L shaped glass spreader away from the
flame.
3. Remove the spreader and very carefully pass it over the flame just once. This will ignite the
excess alcohol on the spreader and effectively sterilize it.
4. Spread the 0.1 ml inoculum evenly over the entire surface of one of the nutrient agar plates
until the medium no longer appears moist. Return the spreader to the alcohol.
4. Repeat the flaming and spreading for each of the remaining two plates.
5. Invert the three plates and incubate at 30 C for 24 hours.
5b. Streak Plate Technique
The streak plating technique isolates individual bacterial cells (colony-forming units) on
the surface of an agar plate using a wire loop. The streaking patterns shown in the figure below
result in continuous dilution of the inoculum to give well separated surface colonies. Once again,
the idea is to obtain isolated colonies after incubation of the plate.

Fig. Streak plate technique

2. Sterilize inoculating loop by heating it over Bunsen burner until it is red hot. Let the loop cool
down
3. Take a nutrient agar plate in your left hand and loop in right hand.
4. Using the sterilized loop mark streaks on nutrient agar plates in 4 quadrants in such a way that
first quadrant is densely streaked and fourth one is sparsely streaked (as shown in figure). You
may use 10-1 dilution as inoculum.
5. Invert the plates and incubate at 30 C for 24 hours.
5c. Pour Plate Technique
Usually, before isolation of microorganisms by pour plate method sample is serially diluted and
then poured onto petridishes.
1. Add 1ml of appropriately diluted sample in sterilized petri dish.
2. Add approximately 15-20 ml of the sterilized and cooled nutrient agar medium (45 C) in the
petri dishes. (Note: the nutrient agar should be cool enough so that it should not kill the
microorganisms in the sample, simultaneously it should be warm enough to be in liquid state).

Mix the the contents of the petri plates by slow rotary movement.

Prepare such plates in triplicates for each sample.

This is pour plate technique.

Incubate all the plates in an inverted position at 280.1 C for 24-48h

EXPERIMENT NO. 6
Aim: To assess biochemical activities viz., catalase, IMViC of given culture of bacteria
6a. To detect catalase activity in the given bacterial culture
Principle:
Some bacteria contain flavoproteins that reduce oxygen (O2) during aerobic respiration, resulting
in the production of hydrogen peroxide (H2O2). Accumulation of hydrogen peroxide can result in
death of the organism as it is a powerful oxidizing agent and destroy cellular constituents very
rapidly unless enzymatically degraded. A bacterium must be able to protect itself against such
O2 products or it will be killed. These bacteria have enzymes like catalase or peroxidase, which
catalyze the destruction of hydrogen peroxide as follows:

Procedure:

Pick up a colony of the bacteria from a plate.

And transfer the colony on a microscope glass slide in a drop of water.

Place a few drops of 3% H2O2 over the culture.

Observe culture for the appearance or absence of gas bubbles.

Observation
Catalase positive culture will produce bubbles of oxygen within one minute after addition of
H2O2.
6b. Study of biochemical characteristics of bacterial strains by IMViC test
Introduction:
The IMViC tests are a group of individual tests used in microbiology lab testing to identify and
distinguish members of Enterobacteriaceae. Except for the lowercase "i", which is added for ease
of pronunciation, each of the letters in "IMViC" stands for one of these tests. "I" is for indole
test; "M" is for methyl red test; "V" is for Voges-Proskauer test, and "C" is for citrate test.
(i) Indole production test
Principle: Tryptophan is an essential amino acid, is oxidized by bacteria by the enzyme
tryptophanase resulting in the formation of indole, pyruvic acid and ammonia. The indole

produced during the reaction is detected by adding Kovacs reagent which produces a cherry-red
reagent layer.
Tryptophan is an amino acid that can undergo deamination and hydrolysis by bacteria that
express

tryptophanase

enzyme.

Tryptophan + water = indole + pyruvic acid + ammonia.

Procedure
Preparation of 1% tryptone/peptone broth
Composition of peptone broth (g/litre)

Peptone: 10.0

Sodium chloride: 5.0

pH:

Dispensed the medium in 5 ml aliquots in 10 ml vials and sterilized at 121 C for 15 min.

Sterilize peptone broth in the autoclave at 15psi (1210C) for 15 minutes.

Inoculate it with bacterial culture and keep one tube as an uninoculated comparative control.

Incubate inoculated and uninoculated tube at 370C for 48 hours.

After 48 hour of incubation, add 1 ml of Kovacs reagent) to each tube including control.

Allow the tubes to stand to permit the reagent to come to the top.

7.00.2

Kovacs reagent:

Isoamyl alcohol : 150.0 ml

p-dimethylaminobenzaldehyde : 10.0 g

Hydrochloric acid : 50.0 ml

Dissolved the aldehyde in alcohol and slowly added acid. Shaken gently before use.

Observation
Development of a cherry (deep) red colour in the top layer of the tube is indole positive.

(ii) MR-VP test

Principle: MR-VP tests are used to differentiate two major types of facultatively anaerobic
enteric bacteria that produce large amounts of acid and those that produce the neutral product
acetoin as end product. Both are performed simultaneously because they are physiologically

related and are performed on the same medium. Opposite results are usually obtained for the
MR-VP tests i.e. MR+, VP- or MR-, VP+.
Procedure:
Preparation of MR-VP broth tubes
Composition
Peptone: 7.0 g
Dextrose/Glucose: 5.0 g
Potassium Phosphate: 5.0 g
Distilled water: 1000.0 ml
pH : 6.9

Pour the 5ml broth in each tube and sterilize by autoclaving at 12lb pressure for 15 minutes.

Inoculate the tubes with bacterial culture and keep one tube as an uninoculated comparative
control.

Incubate inoculated and uninoculated tube at 370C for 48 hours.

Methy Red test

Principle: Methyl red is used in Methyl Red (MR) Test, to identify bacteria producing stable
acids by mechanisms of mixed acid fermentation of glucose. The methyl red test is used to
identify enteric bacteria based on their pattern of glucose metabolism. All enterics initially
produce pyruvic acid from glucose metabolism. Some enteric subsequently use the mixed acid
pathway to metabolize pyruvic acid to other acids, such as lactic, acetic, and formic acids. These
bacteria are called methyl-red positive and include Escherichia coli and Proteus vulgaris.
Procedure:
After 48 hour of incubation, add 3-4 drops of methyl-red indicator to each tube including control.
Observation
Development of yellow to red colour is positive test.
Voges Proskauer test
Principle: VP is a test used to detect acetoin in a bacterial broth culture. The test is performed by
adding alpha-naphthol and potassium hydroxide to the Voges-Proskauer broth which has been
inoculated with bacteria. A cherry red colour indicates a positive result, while a yellow-brown
colour indicates a negative result.

The test depends on the digestion of glucose to acetyl methyl carbinol. If glucose is being broken
down to form acetyl methyl carbinol, it will react with alpha-naphthol and potassium hydroxide
to form a red color. Alpha-naphthol and potassium hydroxide are chemicals that detect acetoin.
Procedure:
Add 3ml of 5% - naphthol in absolute ethanol and 1 ml of 40% KOH in each tube including
control.
Shake the tubes gently for 30 seconds to ensure aeration.
Observations
Appearance of strong red colour which changed to crimson in about 15-20 min is indication of
positive VP test.
(iii) Citrate utilization test
Principle: Citrate test is used to differentiate among enteric bacteria on the basis of their ability
to utilize/ferment citrate as the the sole carbon source. The utilization of citrate depends on the
presence of enzyme Citrase produced by the organism , that breaks down the citrate to
oxaloacetic acid and acetic acid and these products are later converted to pyruvic acid and carbon
dioxide by enzymatically.
Citric acid = oxaloacetic acid + acetic acid
Oxaloacetic acid + acetic acid = pyruvic acid + CO2
In citrate test, Bromothymol blue is used as an indicator.When the citric acid is metabolized , the
CO2 generated combines with sodium and water to form sodium carbonate an alkaline product ,
which changes the color of the indicator from green to blue and this constitutes a positive test.
Simmons citrate agar
Sodium chloride: 5.0 g
Magnesium sulphate: 0.2 g
Ammonium dihydrogen phosphate: 1.0 g
Potassium dihydrogen phosphate: 1.0 g
Sodium citrate: 5.0 g
Bromothymol blue: 40 ml
De-ionized water: up to 1000 ml
Agar-agar: 18.0 g
Procedure

Prepare Simmons citrate agar slants.

Inoculate Simmons citrate agar slants with bacterial culture by means of streak inoculation
and keep one tube as an uninoculated comparative control.

Incubate all the slants at 370C for 48 hours.

Observation
Colour of the medium changes from green to blue is indication of citrate positive test.

EXPERIMENT NO. 7
Aim: Assay of an antibiotic by zone-inhibition method using antibiotic impregnated discs
Principle: Zone-inhibition testing method is a fast, qualitative means to measure the ability of an
antimicrobial agent to inhibit the growth of microorganisms. It is also known as Kirby-Bauer
Test. It is used clinically to measure antibiotic resistance and industrially to test the ability of
solids and textiles to inhibit microbial growth. About a million cells from a single strain are
spread over the agar plate using a swab or s glass spreader, then incubated in the presence of the
antimicrobial disc (eg. methicillin, ampicillin, chloremphenicol, streptomycin etc.).
Procedure:

A bacterial or fungal strain of interest is grown in pure culture.

Using a sterile swab or a sterile glass spreader, a suspension of the pure culture is spread
evenly over the face of a sterile agar plate.

The antimicrobial agent is applied to the centre of the agar plate (in a fashion such that the
antimicrobial doesn't spread out from the centre).

The agar plate is incubated for 18-24 hours, at a temperature suitable for the test
microorganism.

If antimicrobial agent leaches from the object into the agar and then exerts a growthinhibiting effect, then a clear zone (the zone of inhibition) appears around the test product.

Observations
If the bacterial or fungal strain is susceptible to the antimicrobial agent, then a zone of
inhibition appears on the agar plate. If it is resistant to the antimicrobial agent, then no zone is
evident. The size of the zone of inhibition is usually related to the level of antimicrobial activity
present in the sample or product - a larger zone of inhibition usually means that the antimicrobial
is more potent.
Precautions:
Be careful while sterilizing the glass spreader using ethyl alcohol.
Ensure that the spreading of microbial culture etc are done within the zone of asepsis around
the Bunsen burner flame.

EXPERIMENT NO. 8
Study of bacterial transformation and screening for recombinants
Aim:- To make bacterial competent cell uptake foreign DNA followed by screening of
transformed colonies using X-Gal and IPTG.
TRANSFORMATION
Supplies and Solutions:
Ligated DNA
Competent cells
LB medium
LB agar plates with antibiotic.
Protocol:
1. Take 200 l cell suspension (competent cells) in 1.5 ml EP tube placed on ice.
2. Add the ligated DNA (50-100 ng) in the EP tube.
3. Leave it on ice for 30 min.
4. Transfer the tube at 420C (Water bath) and incubate for exactly 2 min without shaking.
5. Quickly transfer the tube on ice for 2 min.
6. Add 800 l LB medium. Incubate at 370C for 45-60 min in water bath.
7. Transfer 300 l transformed competent cells onto LB agar containing the appropriate
antibiotic.
8. Leave the plate at room temperature until liquid has been absorbed.
9. Invert the plate and incubate overnight at 370
2. SCREENING OF TRANSFORMED CELLS
Supplies and solutions:
Transformed colonies
X-gal (20mg/ml)
IPTG (200mg/ML)
LB plates
Ampicillin solution
Sterile tooth picks.
Protocol:
1. Prepare LB-agar plates with 60 l/ml ampicillin.
2. Add 40 l of X-gal and 4 l of IPTG.
3. Spread the solution over surface of the plate. Incubate the plate at 370C until all the
fluid disappears.
4. Transfer each individual transformed colony with one tooth pick onto the plate. Up to
100 colonies can be transferred onto a 90 mm plate.
5. Incubate the plate in an inverted position for 12 to 16 hours at 370C.
6. Observe the blue/white color of the colonies; the white colonies contain recombinant
plasmids, white blue colonies contain non-recombinant plasmids.

Section 1: BT303 (GENETICS)

EXPERIMENT NO.-1
Objective: Chi Square Test for Monohybrid and dihybrid crosses
Ques. 1 A geneticist crossed wild-grey colored mice with white mice. All the progeny were grey.
These progeny were intercrossed to produce F2 which consisted of 198 grey and 72 white mice.
Propose a hypothesis to explain the results and compare the results with prediction of hypothesis.
Ques. 2

F2 phenotype
1.
Red
2.
Pink
3.
White

A single gene is regulated into two alleles.

Observed Number
62
131
57

EXPERIMENT NO.-2
Objective: Probability and Pedigrees analysis
Ques. 1 Genes a, b and c assort independently and are recessive to their respective alleles A, B
and C. Two triply heterozygous (Aa, Bb and Cc) individuals are crossed.
a. What is the probability that a given offspring will be phenotypically ABC that is will
exhibit all three dominant traits?
b. What is the probability that a given offspring will be homozygous for all three dominant
alleles.

Ques. 2 Consider the following pedigree, in which all the alleles responsible for the trait (a) is
recessive to the normal Allales (A)

Generation I

II

a.
b.
c.
d.

What is the genotype of mother?

What is the genotype of father?
What are the genotypes of the children?
Given the mechanism of inheritance involved, does the ratio of children with the trait to
children without the trail match with what would be expected?

EXPERIMENT NO.-3
Objective: Study of chromosome morphology at different stages of cell division.
Materials:
Onion root tip
Beaker, slide
Cover slip
Microscope
Feulgen reagent
Carnoy's solution
Procedure
1. Advance preparation: Take an ordinary yellow onion. Cut off any old root growth. Place
the onion in a cup of water so that only the root portion is under water. To do this, push
toothpicks into the side of the onion which extend outward and hold it on the rim of the
cup. New roots should grow within two days.
2. Cut off .5-1 cm of growth at the root tip
3. Transfer immediately to Carnoy's solution. After 24 hours, roots should be stored in 70%
ethanol in a refrigerator. This stops cell division.
4. Obtain a root tip.
5. After obtaining the root tip, pour off the fixative and replace it with 2-5 ml distill water.
Solutions may be poured into a beaker or down the drain.
6. After 1 minute remove the water with a pipette and add 2-5 ml 6M HCl.
7. After 3 minutes carefully remove the acid and wash tissue off with distilled water.
Agitate the vial for 1-2 minutes. Discard the water.
8. Use forceps to transfer the tissue to a vial containing 1-2 ml Feulgen reagent. The
reagent may be added to this vial if desired. (CAUTION: this dye will stain hands and
clothes permanently.) After 20 minutes use forceps to transfer the tissue to a vial
containing 5 ml 45% acetic acid.
9. Place 1-2 drops of acetic acid onto a microscope slide and transfer the tissue to the drop.
Using dissecting pins and razor blades tease and macerate the tissue into tiny pieces.
10. Place a covers lip over the macerated tissue trying not to get air bubbles under the cover
slip. Press down firmly onto the cover slip with a small cork or pencil eraser to spread
the cells in a very thin layer. Push down in a perpendicular direction and the cover slip
should not break.
11. Once the slide has been prepared or obtained from the teacher, observe it and draw all the
different views of cells present under high power. Be careful to observe the nucleus and
chromosomes since this is what was not observed previously.

EXPERIMENT NO. 4
Objective: Study of multiple alleles inheritance by ABO blood genotyping.
Theory: Human blood is classified according to te presence and absence of certain antigens on
the surfaces of RBCs. ABO blood typing grouping is due of the most common blood type tests.
The ABO blood group type test classifies peoples blood into four types A,B, AB andO. Also
apart from these four they can additionally be classified as rhesus positive or negative. Giving
them as the name as A+or A- or B+ or B- similarly as others.
Antigen
Antibody
BloodGroup
A
B
A
B
A
B
AB
NONE
AB
O
AB
O
The Rh blood group type classifies peoples blood into two blood type Rh+ Or Rh-. depending on
whether the Rh antigen is present or not.
Now
Rh- antigen
Blood group
Present
Rh- positive
Present
Rh-positive
Materials: blood collection needle,glass slides, cotton, 75% alcohol, Standard A serum,
Standard B serum and standard D serum ( for Rh antigen)
Procedure: 1- Divide the glass slides into three parts. Mark the parts as A,B and D.
2- Drop standard serum into each respective part on glass slide.ie Standard A serum on part A
and so on.
3-Cleaned the fingertip with cotton dipped in alchohol and punctures into te wiped area with
needle.
4-Collect the blood sample and mix parts oh it with with each standard solution.
5-Wait for some time and then observe if phenomenon of aggregation and precipitation across on
region of sides.
6-conclude the blood type from the observation.

Precautions:
1- Do not mix the standard serum with one another.
2- Do not share the blood collection needle.
3- If aggregation is not found after 10 minutes, fully mix the standard serum again.

EXPERIMENT NO.-5
Objective: Study of genetic material transfer in two different strain bacteria by
conjugation method
Materials:
Alcohol , LB broth; LB agar ; Nalidixic acid and a selectable marker: kanamycin, A bottle of
sterile tooth-picks; 1.5-ml micro centrifuge tubes; A micropipette; TE buffer (300 l), pH 8.0;
Agarose gel (0.8%) and Agarose gel electrophoresis; Ethidium bromide (0.5 lg/ml); 0.53 TBE
buffer ; 1-kb DNA ladder ; Incubator shaker or water bath shaker; Vortex mixer; UV-Tran
illuminator
Procedure
1. The overnight cultures of donor and recipient strains are serially diluted in 10fold (by adding 10 l of culture to 90 l sterile water).
2. Three dilutions of the donor and the recipient are selected to grown on LB agar plates.
After growing overnight at 370C, colonies on the plates are counted, and the colony
forming units calculated (e.g., colony forming units/mL).
3. The overnight cultures of donor strains are diluted serially 10-fold to 10-3. About 100
lof10-2 and 10-3 ml-1 of donor cells are mixed with 100 l of undiluted
recipient cells.
4. The mixtures are incubated at 370C for 30 minutes with very gentle shaking.
5. After incubation to terminate the conjugal transfer and then spread on LB agar
containing nalidixic acid and a selectable marker: kanamycin or ampicillin.
6. All plates are incubated overnight at 370C. Colonies grown on the plates are counted and
the efficiency of conjugation calculated, which is estimated as the number of
transconjugants per donor cell.
7. Three to ve colonies of transconjugants are selected to conrm plasmid carriage by
agarose gel electrophoresis.
8. Each transconjugant colony from LB agar plate is picked with a sterile tooth pick and
dipped into a micro centrifuge tube containing 30l of TE buffer. The tooth pick is
agitated to disperse the cell clumps.
9. Fifteen micro liters of this culture suspension is vortex mixed for 3 min, remixed with 1
l of loading dye and run on 0.8% agarose gel in0.5X TBE buffer.
10. Observe the bands on UV- transilluminaor.

EXPERIMENT NO. 6
Objective: Study of genetic markers in bacteria
Materials
Micropipettes - 0-20 L, 20-200 L, and 100-1000 L
Sterile tips for micropipettes
Markers for labeling
Vortexes
Micro centrifuge, 14,000rpm max speed, rotor holds 1.5 mL tubes
2 water baths
Thermocycler
37 C incubator with shaker
Micro centrifuge tubes (1.5 mL and 0.2 mL) and racks
Agarose gel
DNA isolation kit
Electrophoresis unit
Bacterial strains
Procedure
1. Genomic DNA will have to be isolated from strains using genomic DNA extraction kit.
2. DNA integrity and quality will have to be electrophoresed 0.8 %( w/v) agarose gels in
0.5X Tris-borate-EDTA and quantified by using spectrophotometer.
3. DNA sample adjusted to concentration of 10 ng l-1 with sterile double-distilled water
and stored at 20C until use.
4. RAPD analysis performed using random primers (10-mer oligonucleotides with
arbitrary sequences).
5. Initial optimization performed with random primers. That generated reproducible
fingerprints then used in the RAPD analysis.
6. PCR amplifications carried out in a 12.5 l reaction volume that contained 1.25 l of 10X
PCR buffer 2 l MgCl2 (2.5 mM), 1.25 l of each primer (100 M), 1.25 mM dNTPs, 20
ng of DNA and 0.5 units of Taq polymerase
7. A negative control containing all components except the DNA extract included in each
PCR reaction. PCRs performed in a 96-well Eppendorf thermocycler.
8. The entire PCR amplification product run on 1.2% agarose gel in 0.5X Trisborate- EDTA
for at least 120 min at 90 V. To determine molecular sizes of fragments generated for a
comparative analysis, a 100 bp DNA ladder was concurrently run in each gel.
9. Bands generated by the RAPD analysis were visually scored as present (1) or absent (0)
to generate binary data.
10. Gel fingerprints considered to be similar when all visible DNA bands had the same
migration distance. Only clear unambiguous and reproducible bands recorded.

EXPERIMENT NO. 7
Objective: Study of genetic polymorphism from diverse populations.
Materials
Populations
Throughout the natural distribution area of the species
Total DNA extraction kit
Agarose gel
Electrophoresis unit
Procedure
1. Total DNA of samples isolated by using DNA isolation kit.
2. About 0.1 g of samples ground in a mortar with nitrogen and incubated for 1 h at 65C in
950 l of CTAB isolation buffer, using the following concentration: 2% CTAB, 50 mM
Tris-HCl, pH 8.0, 0.7 M NaCl, 10 mM EDTA, pH 8, 20 mM mercaptoethanal and 1.5%
PVP. DNA extracted with 950 l of chloroform-isoamyl alcohol (CI, 24: 1).
3. After being mixed by inversion for 5 min, the mixture centrifuged at 5000 g for 10 min
at 10C, and the supernatant mixed with a 2/3 vol of ice-cold isopropanol.
4. The DNA was recovered as a pellet by centrifugation at 6000 g for 10 min at 4C,
washed twice, each with 300 l of 75% ethanol, air-dried at room temperature and resuspended in 200 l of 1 TE; 2 l of 10 g/l of RNAase was added and the solution was
incubated for 1 h at 37C.
5. Then 200 l of CI added to extracted DNA; after being mixed by inversion for 5 min the
mixture centrifuged at 5000 g for 10 min at 10C, and the supernatant was mixed with 2
vol of cold absolute ethanol and a 1/10 vol of 3 M NaAc, pH 5.2.
6. The DNA recovered as a pellet by centrifugation at 6000 g for 10 min at 4C, washed
twice, each with 300 l of 75% ethanol, air-dried at room temperature and re-suspended
in 200 l of 0.1 TE, and then stored at 20C.
7. DNA amplification performed in a programmed PCR for an initial 120 s at 94C, 10 s at
35C, 20 s at 72C for two cycles, followed by 40 cycles of 0 s at 94C, 0 s at 35C, and
60 s at 72C, and a final 7 min at 72C.
8. 8.Reactions carried out in a volume of 10 l containing 1X buffer, 2 mM of MgCl2, 200
M of dNTP, 1 M of primer, 50 ng of DNA template, and 0.5 U of Taq polymerase.
9. Amplification products analyzed by electrophoresis on 1.5% agarose gels stained with
ethidium bromide, and photographed under ultraviolet light.
10. Molecular weights estimated using a 100-bp DNA ladder.

EXPERIMENT NO.-8
Objective: Study of allele frequency distribution in pool DNA sample
Material: Genomic DNA from different individuals, UV spectrophotometer, Thermocycler,
PCR amplification reagents, GENESCAN software or other analysis software
Procedure:
1. DNA samples from unrelated parents: Parents were selected on the basis of morphological
character and DNA sample will be collected from unrelated parents.
2. Construction of DNA Pool: The concentration of each DNA sample estimated by UV
spectrophotometer. Each sample diluted to a concentration of 10g/ml and reread to confirm
the DNA concentration. Equal amounts of DNA from each sample constituting a pool (from
different individuals) will manually combined prior to PCR amplification. DNA pool divided
in different groups according to classes of diverse samples.
3. PCR Amplification and Quantitation: PCR amplification will be perform according to nature
of primers and genomic DNA. In generally each sample was amplified with 50 ng of pooled
DNA in a 25-l reaction volume containing 1.5 mM MgCl2, 10 mM Tris, 50 mM KCl, 200
M of each dNTP, 5 pmoles of each and 0.6 units of Taq DNA polymerase; PCR
amplification was performed for 30 cycles in a thermocycler. PCR products will diluted 1:5 to
1:10 prior to loading onto 4.25% polyacrylamide/ 1.2 % agarose gels and run up to
appropriate time.
4. Estimation of Allele Frequencies: Following electrophoresis, the GENESCAN output will be
use to estimate peak height for each allele. Allele frequencies were estimated from the relative
proportion of the peak height for each allele versus the sum of peak heights for all alleles.
5. Tests of Accuracy of Allele Frequencies in DNA Pools: To quantitate the difference between
the estimated allele frequencies from DNA pools (denoted yi for the ith allele at any locus) to
that obtained by direct allele counts (denoted xi for the ith allele at the same locus), both the
standard product moment correlation (rxy or r) and the RMSE calculated as
RMSE =
where k is the number of alleles at the marker locus. RMSE is an estimate of the average
difference in the two frequencies per allele. To study the relationship between y and x values,
standard e linear regression analysis using the model y = ax + b + performed, where is
random error. Null hypothesis tested to know if x values differ only by random measurement
error.
Observation: Homomorphic or heteromorphic band visualized.
Result: This is the accurate, quantitative data on allele frequencies, suitable for identifying
markers for complex traits that can be identified from pooled DNA samples.
Experiment No.-9
Objective: Identification of dominant F1 hybrid by DNA based marker
Materials: Genomic DNA from different plant material, primers, PCR primers, PCR reagent,
Electrophoresis system and Gel documentation systems.

Procedure:
1. Plant material and DNA Extraction: Take some F1 hybrid of plant and their
correspondent parents for analysis. Ten to 20 plants of each parent were used to validate
the homozygosity of the inbred lines. DNA will be isolate from both of the parent and
F1 hybrid s lines from young leaves.
DNA from several genotypes will test against eight ten-base oligonucleotide primers.
Most of the tested primer/parent plant combinations yielded polymorphic PCR products
(RAPD markers). Selected primers will then use in PCR reactions with template DNA
isolated from offspring in plant diallel crosses among the same parental lines.
2. PCR conditions: PCR condition will be use according to nature of primers. in generally
RAPD reactions will performe as reaction volumes of 20 l will prepare containing:25 ng
genomic DNA, 25 ng primer, 0.5 mM MgCl2, 0.125 mM of each dNTPs and 1.25 units
of Taq DNA polymerase.Reactions will buffered by addition of 1/10 of 100 mM of TrisHCl (pH 8.3), 500mM of KCl, 15 mM of MgCl2 and 0.1% gelatin and overlaid with
mineral oil.
3. The amplification program will the following: 45 cycles of 10 s at 94 0C, 10 s at 35 0C, 1
min and 10 s at 72 0C, with an initial denaturation step at 94 0C for 1 min and a final
extension step at 72 0C for 2 min.
4. PCR products were analyzed after separation on 2% agarose gels .
5. Gels will be stain with 0.2 mg/ml of ethidium bromide and photographed on a UV light
transilluminator.
Result: The diallel study confirmed the appropriate inheritance of RAPD markers in the F1
generation. The value of these dominant RAPD markers for genetic linkage mapping in trees
will be established from both theoretical and applied perspectives. Results demonstrated that
AFLP method detected more polymorphic markers in comparison to the SSR technique.
Accordingly, it is suggested that the AFLP method can be used as a very useful tool for
germplasm screening in plant.