V – SOPS AND PRACTICAL KITS GENERATED

LAB MANUALS GENERATED FOR SMOOTH CONDUCTION OF PRACTICALS

I – LAB. MANUAL

AIM: Preparation of permanent stained different stages of chick embryo.

REQUIREMENTS: Egg fertilized having stage, needles, fixatives, grades of alcohol, DPX, glass slide, cover slip, microscope, dye.

PROCEDURE:

1. Take an egg and put in on side undisturbed for 2-3 minutes.

2. Note the blunt end of the egg and make a hole with needle.

3. Again put the egg undisturbed for 15 min so that stage of egg becomes stable on upper side.

4. Make a circle on a egg shell with pencil and then make window by removing the egg shell with forceps.

5. Note the stage of egg and put 2-3 drops of carnoys fixative on the material of egg to fix the stage. Take the fixed stage with the help of forceps and put it in 90% alcohol.

6. Follow the degrading in 90% -70%-50%-30% alcohols for 5 min each.

7. Then put it into dye stain (carmine) for 30 sec and do upgrading 30%-50%-70%-90%.

8. After that mount it in DPX and cover it by cover slip.

II - LAB. MANUAL

Preparation of permanent stained different stages of chick embryo.

Take an egg and put in on side undisturbed for 2-3 minutes.

Note the blunt end of the egg and make a hole with needle.

Put the egg undisturbed for 15 min.

Make a circle on a egg shell with pencil and then make window by

removing the egg shell with forceps.

Note the stage of chick embryo and put 2-3 drops of carnoys

fixative on the material of egg to fix the stage.

Follow the degrading in 90% -70%-50%-30% alcohol for 5 min

each.

Take the fixed stage with the help of forceps and put it in 90%

alcohol.

Then put it into dye stain caramine for 30 sec and do upgrading

30%-50%-70%-90%.

After that mount it in DPX and cover it by cover slip.

III - LAB. MANUAL

Experiment : Extraction and estimation of 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 lipids from

tissues. In biological materials, the lipids are generally bound to proteins and they are extracted either with a mixture of ethanol and diethyl ether or a

ethanol in the extraction medium help in breaking the bonds between the lipids and proteins.

Materials and reagents: Flasks, separatory funnel, oil seeds (sunflower, peanuts or soyabean), anhydrous sodium sulphate, chloroform: methanol

mixture (2:1, v/v), 1% sodium chloride.

Procedure:

1. Take 1g of the oil seed and grind it in presence of 5g 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 20ml of chloroform –methanol mixture to it and transfer to an air tight glass flask. Shake the content of the flask on a mechanical shaker for

one hour and then filter it through a glass sintered funnel .Repeat the extraction of the residue twice and pool the filtrates.

3. Remove the solvent from the residues by distilling under vaccum . Since the residue left after drying contains crude lipids, extract it again with

10 ml of chloroform – methanol mixture containing 1ml of 1% sodium chloride.

4. Take the pooled fractions in a separatory funnel , shake it thoroughlyand allow it to stand for 5min .The lipids will be recovered in the lower

chloroform layer while soap, glycerol and water insoluble impurities move to the upper layer.

5. Drain out the lower layer and treat the upper layer again 3-4 times with 5-10ml of chloroform – methanol mixture to extract any residual lipid

from it.

6. Collect the lipid containing fractions in a pre-weighed beaker.

7. Evaporate the solvent by keeping the beaker in warm water bath (50 oc) 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 the sample by subtracting the weight of empty beaker.

9. Express the results in terms of %crude lipids in the given sample of the oil seed.

Precautions:

1. The sample should not be exposed to high temperature or light as some lipids get polymerized or decomposed on exposure to light, heat and

oxygen.

Calculations:

Weight of oil seed taken (a) = 1gm

Weight of empty beaker = x gm

Weight of oil containing beaker = y gm

Weight of crude oil (z) = y – x gm

% of crude oil (z) = * 100

IV - LAB. MANUAL

Flow chart of Extraction and estimation of lipid contents in a given sample of oil seed.

Add 20ml of chloroform –methanol mixture in 2:1 ratio v:v.

Grind 1gm of oil seed with 5g of anhydrous sodium sulphate in a

pestle and mortar.

Shake a mechanical shaker at 1000rpm

Extract lipid by adding chloroform-methanol and 1L Nacl

solution.

Transfer to separating funnel, shake and keep undisturbed.

Collect the lower layer 3-4 times in pre weighed beaker.

Evaporate the solvent on hot water bath.

Weigh the beaker containing crude oil

Calculate the amount and % of crude oil.

V- LAB. MANUAL

Experiment: SDS-Polyacrylamide gel electrophoresis of proteins (SDS-PAGE)

PRINCIPLE: In SDS-PAGE ,a thiol reagent such as β-mercaptoethanol is included which cleaves the disulphide bonds .hence polypeptides in a native protein which still remain associated even in the

SDS get separated .So in this method ,protein sample is boiled in presence of excess SDS and β-mercaptoethanol to denature proteins to their individual polypeptides .SDS polypeptides complexes (1.4

SDS :1 protein) have net negative charge and migrate towards anode at rate based solely on size or mol wt of the polypeptides. This system of electrophoresis is used for determination of mol wt of the

protein under investigation.

Materials and reagents:

1. Vertical slab gel electrophoresis apparatus :

a. 2 glass plates ,with 1 glass plate is notched & having vertical glass spacers of 1.5mm

b. Gel casting assembly

c. Tygon tubing , comb with teeth

d. Electrophoresis tank with upper and lower buffer reservoirs

e. Electrophoresis leads

2. Power pack (0-500 volts)

3. Reagents :

a. Acrylamide – bisacrylamide stock solution :

Acrylamide 30.0g

Bisacrylamide 0.8 g

Dissolve in water and make final volume to 100ml .Filter the solution through Whatman no.1 filter paper and store in brown bottle at 0- 4 .This solution is stable for one month.

b. Stacking gel buffer stock (Tris-HCL,pH6.8):

Tris 6.0g

1M HCL 48.0ml

Adjust pH to 6.8 and make final volume to 100ml with water. Filter the solution through Whatman no.1 filter paper and store in brown bottle at 0- 4 .

c. Resolving gel buffer stock: (Tris-HCL,pH8.8):

Tris 36.3g

1M HCL 48.0ml

Adjust pH to 8.8 and make final volume to 100ml with water. Filter the solution through Whatman no.1 filter paper and store in brown bottle at 0- 4 .

d. 1.5 %( w/v) Ammonium persulphate (APS) in water: prepare by dissolving 0.15g of APS in 10 ml water. This reagent should be prepared fresh just before use.

e. N,N,N’,N’-Tetramethyl ethylene diamine (TEMED

f. Reservoir buffer or electrode buffer: (Tris-glycine pH8.3):

Tris 3.0g

Glycine 14.4g

SDS 1.0g

Adjust pH to 8.3 and make final volume to 100ml with water.

g. Staining solution:

Coomassie brilliant blue R-250 1.25g

Methanol 200ml

Glacial acetic acid 35ml

Make final volume to 500ml with water. Filter the solution.& store it at room temperature.

h. Destaining solution:

Methanol 50ml

Glacial acetic acid 75ml

Mix the above components and make final volume to 1L with water.

4. SDS (10%, w/v): Dissolve 1g SDS in 10ml of distilled water. Store the reagent at room temp.

5. Sample buffer 2x: the sample preparation buffer which contains a two fod concenteration of various components is prepared as below:

1M Tris-HCL, pH6.8 12.5ml

SDS 4.0g

Β-mercaptoethanol 10.0ml

Glycerol 20.0ml

1% Bromophenol 4.0ml

Make final volume to 100ml with water.

6. Standard molecular weight marker proteins: the standard molecular weight proteins kits, having different mol wts ranges available.

Protein Molecular weight

β-Lactoglobulin

Trypsinogen

Carbonic anhydrase

Pepsin

Egg albumin

Bovine serum albumin

18.4

24.0

29.0

34.7

45.0

66.0

Dissolve 1mg of each of the above proteins in 1ml of the sample buffer .Dilute with water in ratio of 1:1 .About 20µl of this mixture should be loaded into one of the wells.

7. Stacking and resolving gels :

Stock solution Stacking gel(2.5%) Resolving gel(12.5%)

(ml) (ml)

1. Acrylamide – bisacrylamide(30:0.8)

2. Stacking gel buffer stock solution (Tris-HCL,pH6.8)

3. Resolving gel buffer stock solution (Tris-HCL,pH8.8)

4. 10%SDS

5. 1.5% APS

6. Water

7. TEMED

2.50

5.00

0.20

1.00

11.30

0.015

12.50

3.75

0.30

1.50

11.95

0.015

Total volume 20.00 30.00

Procedure:

1. Sample preparation : mix the sample with equal volume of sample buffer .Boil the mixture for 3 min in a boiling water bath and cool to temperature .If protein sample is too dilute , precipitate

the proteins with 10% TCA (incubate in Ice for 30 min) , centrifuge at 10,000* g for 5 min.Wash the precipitate with ethanol-ether(1:1) to remove TCA. Dissolve the precipitate in the sample

buffer diluted with water in 1:1 ratio, boil for 3min and cool.

2. Preparation of slab gels:

a. Clean and dry the glass plates and assemble them in gel casting assembly. Seal the two glass plates with the help of Tygon tubing, clamp them and place the whole assembly in an upright

position.

b. Mix the various components of resolving gel except for SDS, APS, TEMED .Degas the solution for 1min using a water pump and then add the above remaining components of the gel.

c. Mix gently and pour the gel solution into the mould in b/w the clamped glass plates taking care to avoid entrapment of any air bubbles .overlay distilled water on the top as gently as possible

and leave for 30min for stacking gel buffer. Remove the water layer and rinse with stacking buffer.

d. Mix the stacking gel components.

e. Pour the stacking gel and insert the supplied plastic comb in the stacking gel no air bubbles should be entrapped. Allow the gel to polymerize for about 20min.

f. After the stacking gel has polymerized, remove the comb without damaging the wells. Clean the wells by flushing with electrode buffer using syringe.

g. Remove the Tygon tubing and install the gel plate assembly into the electrophoretic apparatus. Pour reservoir buffer in the lower and upper chambers. Remove any bubbles at bottom of gel.

3. Electrophoresis of sample:

a. Load 10-20µl sample (100-200µg) in the sample well. Also load molecular weight marker proteins in one or two wells.

b. Switch on the current maintain at 10-15mA for initial 10-15 min until the sample have travelled through stacking gel. Then increase the current to 30mA until the bromophenol blue dye

reaches near the bottom of gel slab. This requires 3-4 hrs.

c. After the electrophoresis is complete turn off and disconnect the power supply and carefully remove the gel slab from in between the glass plate.

d. Place the gel in a trough containing staining solution for 3-4 hrs or it can be kept overnight. Destain the gel with destaining solution till a clear background of the gel is obtained.

e. Record the distance travelled by the dye and the various protein bands and calculate Rm values.

f. Plot a graph between log10 molecular weight vs. Rm values of standard molecular weight marker. A straight would be obtained. From the Rm values of the sample polypeptides determine

their molecular weights using the above standard calibration curve.

VI - LAB. MANUAL

Experiment: SDS-Polyacrylamide gel electrophoresis of proteins.(SDS-PAGE)

Mix the sample with buffer .Boil for 3 min and cool it. If sample is dilute,

precipitate with 10% TCA then centrifuge at 10,000* g for 5 min.

Remove the water layer and rinse with stacking buffer. Mix the stacking

gel components.

Wash with ethanol-ether (1:1) & Dissolve in sample buffer diluted with

water in 1:1 ratio, boil for 3min and cool

Set the whole assembly in an upright position.

Mix the components of resolving gel except for SDS, APS, TEMED .Degas it

for 1min then add the remaining components.

Mix and pour the gel solution in b/w the glass plates .Overlay distilled

water and leave for 30min for stacking gel buffer.

Pour it & and insert comb in stacking gel & leave for 20 min.

Remove the comb &clean the wells by electrode buffer

Install the gel plate assembly & Pour reservoir buffer

Load 10-20µl sample in well. Load molecular weight marker proteins in

one or two wells.

Switch on current at 10-15mA for 10-15 min until Then to 30mA until dye.

This requires 3-4 hrs.

VII- LAB. MANUAL

Experiment: Estimation of protein by Lowry’s 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 mug of proteins can be

measured the -CO-NH- (PEPTIDE BOND) in polypeptide chain react with copper sulphate in an alkaline medium to give a blue coloured complex .In addition ,tyrosine and tryptophan

residues of proteins cause reduction of the phosphomolybdate and phototungstate components of the Folin-ciocalteau 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 reagents , phenols ,

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(0.1,Ph 7.6):

Stock solution:

A) 0.2M monobasic sodium phosphate (27.8g in 1L)

B) 0.2M Dibasic sodium phosphate (53.65g of Na2HPO4. 7H2O or 71.7g Na2HPO4.12H2O in 1L.)

X ml of A + y ml of B, diluted to a total volume of 200ml.

2. Alkaline Na2CO3 reagent: Dissolve the 2 g Na2CO3 in 0.1 N NaOH and make the vol to 100ml with 0.1 N NaOH.

3. Copper sulphate reagent: prepare 0.5% CuSO4.5H2O IN 1% sodium potassium tartarate solution.

4. Alkaline Copper sulphate reagent: Add 1ml of reagent 3 to 50ml of reagent 2 .This mixture is unstable and should be prepared fresh.

5. Folin’s reagent: Dilute the reagent appropriately so that it is 1N in respect of its acid content.

6. 20% (w/v) TCA: Dissolve 20g of trichloroacetic acid in water and make the vol to 100ml.

7. Acetone

8. 0.1 N NaOH

9. Bovine serum albumin (BSA): 100 mug/ml solution in distilled water.

Procedure:

1. Sample extract: weigh 1g sample (germinating seeds, leaves, bacterial cells), macerate the sample in pestle mortar in 5ml of phosphate buffer and transfer the material to

centrifuge tubes. Centrifuge the homogenate at 8000 rpm for 20 min .combine the supernatants and make the vol to 50ml with phosphate buffer.

2. Take 1ml of the above extract and add 1ml of 20%TCA .keep it for half an hour and centrifuge at 8000rpm for 20 min .wash the pellet with acetone twice and again centrifuge

it .Discard the supernatant.

3. Dissolved the pellet in 5ml of 0.1 N NaOH and mix well till it gets dissolved.

4. Take suitable aliquot (1ml) of above solution and add to it 5ml of freshly prepared alkaline copper sulphate reagent .Mix properly and after 10min add 0.5 ml of Folin’s

reagent. Mix the contents instantaneously .Allow the colour to develop for 30min.

5. Record the absorbance at 660nm after setting the instrument with reagent blank which contains 1ml of 0.1 N NaOH instead of the aliquot .

6. In another set of tubes take suitable aliquots of BSA solution (in a range of 0-100µ)make the total volume to 1ml with 0.1 N NaOH and develop the colour .Draw a standard

curve of absorbance at 660nm versus mug of BSA.From this standard curve determine the amount of protein in the sample tube.

7. Calculate the amount of protein per g of the sample.

Precautions:

1. It is important that contents of the tube be mixed immediately after the addition of Folin-ciocalteau reagent proper colour development will be affected if this step is unduly

delayed.

2. Compounds like tris, EDTA, ions interfere in this procedure of protein estimation.

VII- MANUAL

Flow chart for Estimation of protein by Lowry’s method.

\

Take 1gm sample in pestle and mortar in 5ml of phosphate buffer and

Centrifuge the upto a 8000 rpm for 20 min .

Repeat the above for 4-5 times. Combine the supernatants and make the vol

to 50ml with phosphate buffer.

Take 1ml of the above extract and add 1ml of 20%TCA .keep it for half

an hour and centrifuge at 8000rpm for 20 min

Wash the pellet with acetone twice and again centrifuge it. Dissolved

the pellet in 5ml of 0.1 N NaOH.

Take 1 ml of above extract & add to it 5ml of freshly prepared alkaline copper

sulphate reagent .Mix properly and after 10min add 0.5 ml of folin’s reagent.

Mix the contents instantaneously .Allow the colour to develop for 30min .

Record the absorbance at 660nm after setting the instrument with reagent

blank.

In another set of tubes take suitable aliquots of BSA solution & make the

total volume to 1ml with 0.1 N NaOH and develop the colour

Draw a standard curve of absorbance & calculate the amount of protein per

g of the sample.

VIII - LAB. MANUAL

Circular Paper Chromatography

1. Prepare (BAW) n-Butanol : Acetic acid : Water ; 40:10:15

2. Extract Amino acids in 20% isopropanol in N/10 molar HCl

A. Spotting of amino acid

B. Set of equipment for Paper chromatography

D.

Chromatogram showing spots of Amino-acids after drying C. Aspirator for spray of ninhydrin

IX - LAB. MANUAL

VARIOUS KINDS OF EQUIPMENTS REQUIRED FOR COLLECTION AND PRESERVATION OF ANIMALS

There are various ways to collect animals. For this various techniques are employed for collecting animals.Piechooki gave a good account for many

techniques for the collection of macroscopic specimens like mist nets for birds, ultra violet damps for insect collection different kinds of baits are also

used.

INSECTS: Insects can be collected by sweeping, light traps, baits, bait traps, different types of traps for flying insects, example: Malaise traps.

A) NETS:

1. Dipnets-Aquatic insects and other arthropods are best collected by dipnets.

2. Plankton net-for aquatic insects and arthropods.

3. Insect net-It consists of a wire ring, a bag and a handle. Ring should be 12-15 inches in diameter ,made up of about 3mm iron or steel wire. Ends of

wire are straight which can be fit into grooves of handle. If captured animal is large, the handle is twisted, lapping the bag over the rim and the specimen

is then enclosed in the bottom of bag.

B) ASPIRATOR: It is a simple suction apparatus used for collecting small insects and arachnids. There are several designs in which a vial of glass or

preferably transparent plastic is used.

In some cases, vial is opened at one end only while in another, it is opened at both ends. Open ends are provided with tightly fitting rubber stoppers to

avoid the pushing of small insects. A rubber tube is attached to outer end of the suction tube for sucking purpose. Other end of this tube is covered with a

piece of fine muslin cloth to prevent insects and arachnid from entering the tube. Second tube is opened at both ends.

BULB ASPIRATOR: It is useful for the collection of mites, small insects, spiders. The vial is of transparent plastic or heavy duty glass whose both

ends are provided with tightly fitting stoppers.

C) BERLESE FUNNEL: It is quite useful in extracting insects & others small arthropods from organic soils and leaf litter. It can also be used to extract

organisms from loose bark, rotting wood, fungi, mosses, flowers, stored food products. Manure and the material from nests of birds, mammals, social

insects.

It is a simple apparatus consisting of a metal or plastic funnel having a wire mesh on its bottom for holding the sample. The narrow end of funnel is

received in a beaker or any other container, having 70% alcohol with few drops of glycine to avoid desication.The funnel is covered with a lid having a

hole in middle for an electric bulb. As upper end dries, organisms avoid heat, start migrating deeper into funnel and are collected.

D) FLOTATION METHOD: It is used to extract insects, mites, other arthropods from soil, matted vegetation. It is also good collecting eggs and pupae

of insects. Sample is broken in base having MgSO4:H20 in 1:3 ratio and stirred gently. Organisms start floating and are collected on sieve or filter paper.

E) KILLING AGENTS/BOTTLES: The best ones are those which kill the insects immediately without affecting their colors. The most common are:

1: Cyanide bottle: It consists of a wide mouthed bottle or jar with a well filtered cork or lid. A layer of granulated KCN (10mm thick) is spread at the

bottom. Then, powered dry plaster of paris and water are added and is kept open for 20-30 hours to dry up plaster of paris then blotting paper is spread

for absorbing moisture to avoid direct contact of the specimen with killing agent.

2: Ethyl acetate bottle: It is used for insects, beetles, hymenopterns. Any glass bottle can be used, cotton soaked in this killing agent is placed at the

bottom, and then covered by piece of blotting paper. Few drops of killing agent may be added. Other agents are tetrachloroethane, carbon tetrachloride,

ether, chloroform, benzene, ammonia and ethylchloride. Insects should not be left for long time in bottles and there should be no overcrowding.

PROCEDURES OF COLLECTION AND PRESERVATION:

1. COLLECTION OF FAUNA: It is the 1st step in taxonomy. Some of the collection methods are:

A) Mammalian fauna: They are only photographed. But some traps like bait traps. Booby traps, made up of wire are used.Zig saw teethed, paw holders

are also employed for larger fauna.

B) Birds: Various types of aerial nets are used. These are kept in routine flight area. Tented nets, with various chambers and galleries are used. Mist nets

are also common.

C) Reptilian fauna: Snakes are captured by snake ranglers.Crocodilian fauna is captured by baits and rapes.

D) Amphibian fauna: Fish nets are used. Tongs are used for poisonous frog.

E) Echinoderms: They are captured directly with hands.

F) Insects:

� Aquatic: by dipnets

� Diurnal : by sweeping methods

� Soil insects: using tongs

� Nocturnal: Light traps and berlese funnel are used.

� Mosquitoes & house flies: Waxy traps glaced with sugary solution Aspirators are

also used.

� Organisms from soil and vegetation are collected by floatation, larval stages, immature stages, and eggs from mated vegetation.

2) PRESERVATION OF FAUNA: for this, killing of organism is must. In this, ammonia, chloroform, ethyl-acetate is used.

A) Wet preservation: Snakes, amphibians, fishes are wet preserved.40% formalin is injected into organs of multiple sides. Muscles, eyes, brain are

preserved.

• FOR MOUNT PREPARATION: Glass plate is used. All structures of animal should be visible, animal is tied with thread.

• FOR SEALING: Glass plate containing specimen is placed in jar, half filled with formalin. It is then covered with lid & sealed.

B) Whole mounts: These are prepared for soft bodied animals.2 Methods are used:

EPOXY SLABES: Jelly fishes,flukes,tapworms,round worms are prepared .Organisms are stained with eosine,borax carmine & then mounted in

mixture of arabite,epoxy resins making sure that all parts are visible.

• SIMPLE SLIDES: Whole mounts after upgradation can be stained, mounted in DPX, CB on slides.Trematodes, cestodes, thysanura are

prepared like this.

C) DRY PRESERVATION: Reptiles, birds, mammals, preserved by this method.

After killing, fore and hind limbs tarsal are kept along with skin in case of mammals and reptiles. Whereas in birds, humorous, femur are removed.

Animal is then disinfected with DDT, powder mixed in arsenic & alum. Eyes, brain is also removed .Mesh wire & small wires are inserted to reconstruct

the endoskeleton.

DRY PRESERVATION OF INSECTS:

It involves following steps:

1. PINNING: Pins should be inserted vertically downwards with perpendicular to long axis of body. Various body parts are used for pinning like:

� Orthoptera,dicteoptera, elytra : behind thorax

� Beetles : right elytron

� Diptra : mesothorax

� Lepidopterns, Odonata : middle of mesothorax

� Hemiptera : scutellum

�

Method of pinning in different orders of insects

2. SPREADING: After pinning, an even scud groove is prepared in therma-coal sheet. Pinned specimen is mested into groove vertically

downwards. Special care is taken for labial palps, legs. Forewings are placed in such a way that hindwings and anal margin is perpendicular to

long axis of body. Hindwings are fully spread, antennae are crossed pinned.such a pinned specimen is allowed to dry 3-5 days.

Method of stretching of insect (Moth)

3. DOUBLE MOUNTING: Double mounting is done for small insects. First pin is on stage/support & other on large entomological pin.

4. POINTING: For small dried insects, by attaching specimen to tip of small triangle of thin card, supported on large entomological pin.

5. CARDING: Carding is used for beetles, insect is glued to the card from ventral surface.

6. STORAGE: Information about place, date of collection should be written. For storage, wooden cabinets with glass on sides are

employed.Nephthlalene balls must be kept in.