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Submission of Project Booklet under Star College Scheme

STANDARD OPERATING PROCEDURE FOR AUTOCLAVE

Operating instructions:

1. Unscrew the nuts to access the chamber.

2. Fill the chamber with distilled water upto the top level of the heater covers stand placed at

the bottom of the chamber.

3. Load the material to be sterilized in the carriers.

4. Lower the lid on to the gasket. Tighten all the flynuts diagonally two at a time with equal

pressure to ensure uniform pressure on the rubber gasket. This will prevent any leaks

during the operation.

5. Turn the machine on using the mains switch or the MCB.

6. The vacuum breaker and the purge valve automatically vent the internal state air (purging).

You will see steam escaping from the same. Once the chamber is purged of all air, steam

pressure will close this valve. This will ensure proper sterilization using steam only.

7. The pressure will now steadily rise and stabilize between 15 and 16.5psi. The valve may

start leaking lightly from 12psi onwards, which is normal.

8. The safety valve is purposely set above 15psi inorder to avoid under sterilization.

9. Keep the material for 20 minutes or a long as required.

10. After the hold time has elapsed, turn the machine off using the mains switch or the mobs.

Remove all the steam through the exhaust valve, before opening ther flynuts. Never

prematurely open / loosen the flynuts, even at 1-2psi.

11. Unload the material.

12. Top up the chamber with distilled water, refer to instruments 2

Electric Shock:

In case of water spillage near the electric panel, there is a chance of electric shock.

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Pressure:

After completion of sterilization, lid should be opened only if pressure gauge shows 0 pressure.

Otherwise, there are chances of burns.

Injury/ Exposure Response Procedure:

First aider should immediately take care of injury, if any. The first aiders should seek

appropriate medical help if required.

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STANDARD OPERATING PROCEDURE FOR LAMINAR AIR FLOW

Name of procedure:

To perform aseptic work.

Procedure:

1. Swab the floor of LAF with spirit or 70% alcohol.

2. Put on the UV light for 20 minutes (LAF lid should be closed).

3. After 20 minutes switch off the UV light and switch on the light and air flow.

4. Again swab the floor and side wall of the LAF with spirit. Switch on the burner to do the

aseptic work.

5. After completion of work put off the burner, switch off the airflow and clean the hood,

swab it again with spirit or alcohol.

6. Close the lid. Switch off the light and airflow.

7. Switch on the UV light again for 20 minutes and then switch off.

Precaution:

1. Do not open lid of LAF while switching on the UV light as it acts as mutagen.

2. Do not talk while working in LAF.

Spill Response procedure:

In case of spillage, mop up with spill absorbent. Then clean up with soap solution.

Waste disposal procedure:

Waste material should be disposed in a waste disposal bin.

Protective Equipment:

Lab coat, Head gear, Face mask should be worn while working in LAF.

STANDARD OPERATING PROCEDURE FOR AGAROSE GEL ELECTROPHRESIS---------------------------------------------------------------------------------------------------------------------

|| Hindi Vidya Prachar Samiti’s RAMNIRANJAN JHUNJHUNWALA COLLEGE || Page 190 of 280


Operating instructions:

1. Clean the AGE assembly with ethanol and let it dry.

2. Place the gel pouring plate properly on the AGE apparatus.

3. Pour molten agarose and place the comb properly on it.

4. Allow the gel to set

5. Fill the tank buffer upto the brim. Remove the comb carefully.

6. Load the sample in the wells.

7. Connect the AC converter to the gel running unit by connecting anode to anode (red) and

cathode to cathode (black).

8. Switch on the power supply keep the voltage according to demand of the experiment

Precautions:

1. The table should be uniform for proper running of sample.

2. Make sure that the entire assembly is covered with lid.

3. Do not disturb the gel running assembly while running the sample.

4. Buffer level should be maintained throughout the experiment.

5. There should be no fluctuation in voltage supply.

6. High voltage should be avoided as it generates the heat which causes the evaporation of

buffer.

Hazard:

Ethidium bromide is carcinogenic hence it should be handled wearing gloves with care.


Lab coat and gloves should be worn while performing the experiment.

Waste Disposal procedure:

Used plastic tubes or micro-tips should be disposed into the respective bins. ---------------------------------------------------------------------------------------------------------------------



Gel should be treated with sodium hypochlorite before disposing.

Note:

1. Only the people trained by one of the personnel below are authorized to use the AGE

apparatus.

2. Once the usage is over please switch off the main switch.

3. Entry in log book is mandatory.



STANDARD OPERATING PROCEDURE FOR CENTRIFUGE

Procedure:

1. Turn ON the electrical power to the centrifuge.

2. Load the centrifuge tubes into the rotor.

3. Close the lid of the centrifuge.

4. Select the required time by adjusting the timer knob.

5. Select appropriate speed with the speed controller.

6. Wait next to the centrifuge until it has reached the desired speed and has not stopped due to

it being imbalanced.

7. Wait until the centrifuge run has being completed.

8. Unscrew the rotor lid and remove the samples.

9. Switch off the instrument after use.

Spill Response Procedure:

1. In case of spillage (water/ solution/ buffers), mop with absorbent.

2. Then clean up with soap solution.

3. Report spillage in the log book.

Precautions:

1. Tubes are to be filed to the sample level with the same density fluid.

2. Ensure the tubes to be centrifuged are matched in pairs or evenly distributed around the

rotor.

3. If centrifuge is vibrating a little too much, stop the rotor and wait until it has stopped

spinning and redistribute the tubes to make sure the rotor is completely balanced.

4. Entry in log book is mandatory



STANDARD OPERATING PROCEDURE FOR pH METER

Name of the procedure:

Measurement of pH

Operating Instruments:

1. Switch on the instrument.

2. Remove the electrode dipped in electrode buffer (3M KCI)

3. Rinse with distilled water.

4. Dry the outer surfaces of the electrode with a clean dry tissue.

5. Dip electrode in solution whose pH has to be measured.

6. After use rinse the electrode in distilled water, dry and dip the electrode back in the

electrode buffer.

7. Switch off the instrument after use.


Lab coat should be worn while using the instrument.

Waste disposal procedure:

All waste should be diluted and disposed in the sink.

Injury/ Exposure Response Procedure:

Steps to be taken in the event of an accident and appropriate first aid should be provided.

Spill Response Procedure:

In case of spillage (water/ solution/ buffers), mop with spill absorbent. Then clean up with soap

solution.

Note: ---------------------------------------------------------------------------------------------------------------------



1. Only the people trained by one of the personnel below are authorized to use the pH meter.

2. The electrode should always be immersed in the electrode buffer (Before and after use).

3. DO NOT rub the membrane of the electrode.

4. The instrument should not be used to measure pH of viscous solutions.

5. In case the instrument is nonfunctional, seek help from trained personal.




STANDARD OPERATING PROCEDURE FOR PAGE ELECTROPHRESIS

1. Clean the whole PAGE apparatus with ethanol and let it dry.

2. Arrange the apparatus by placing the ceramic plate behind glass plate, separated by

specific spacers.

3. Pour 1% molten agarose on the bottom of the gel casting unit to seal the bottom of the glass

plates, so that the gel will not leak out.

4. Place the glass plate assembly vertically on the gel casting unit. Tighten it with the help of

screws.

5. Pour the gel in the gel casting unit, insert the comb and allow it to set.

6. Transfer the gel in the gel running unit and tighten it with the help of clamps.

7. Fill the tank buffer upto the brim. Then load the sample in the wells prepared by using a

comb.

8. Connenct the AC convertr to the gel running unit by connecting Anode to Anode (red) and

Cathode to Cathode ( black).

9. Switch on the power supply and keep the voltage at 80V.

Hazard:

Monomer of acrylamide is neurotoxic hence it should be handled wearing gloves with care.


Lab coat and gloves should be worn while performing the experiment.

Waste Disposal procedure:

Used plastic tubes or micro-tips should be disposed into the respective bins.

Gel should be wrapped in paper and disposed.

Note:

1. Once the usage is over please switch off the main switch.




STANDARD OPERATING PROCEDURE FOR BALANCE

1. Insert plug in socket.

2. Switch on the instrument at least 10minutes before use.

3. Adjust to zero.

4. Place the weighing paper on the pan and use tare to reset zero.

5. Weigh the required quantity of sample.

6. Remove the paper along with sample weighed.

7. Adjust to zero.

8. Switch off and clean the balance.

9. Make an entry in the log book.

10.



STANDARD OPERATING PROCEDURE FOR COLORIMETER

1. Insert plug in the socket.

2. Switch on the instrument at least 15 minutes before use.

3. Ensure to keep water blank before switching on the instrument.

4. Set the required wavelength.

5. Select %T using the knob and adjust to 100%.

6. Switch the knob to O.D which should be zero.

7. Clean the cuvettes after use.

8. Make an entry in the log book.



INDEX for EXTENSION PRACTICALS F.Y.B.SC

Sr. no Experiment

1 Hyperchromicity of DNA

2 Absorption Spectrum of Proteins

3 Identification of different sugars using TLC

4 Separation of Spinach pigments by using Column Chromatography

5 Separation of DNA fragments on Agarose gel electrophoresis

6 Culture of Paramecium

7 Observation of Succession of Microzoons in stagnant Pond Water



Hyperchromicity of DNA

Principle:

The absorption maximum of nucleic acids at 260 nm originates from their constituent aromatic

groups. High temperature causes DNA to melt. The hydrogen bonds connecting the bases start to

break and absorbance at 260 nm rises. This phenomenon is known as hyperchromicity. The

transition temperature is known as melting temperature (T m).

Requirements:

1. Isolated DNA

2. Sodium Chloride (0.9%): 0.9gm of NaCl dissolved in 100ml of distilled water.

3. Thermometer

Instruments:

1. UV-VIS spectrophotometer

2. Water bath

Ice bath

Method:

1. Dissolve the isolated DNA strands in 3ml saline. Place this solution in the

spectrophotometer and record the absorbance at room temperature. Prepare a distilled

water blank.

2. Transfer the DNA solution in a water bath with 55oC temperature and record the

absorbance at 260nm.

3. Increase the temperature of the water bath by 10oC and read the absorbance. Repeat this up

to 95oC temperature and read the absorbance. at 260nm

4. Plot the graph of absorbance (OD) against temperature.

Observation Table:---------------------------------------------------------------------------------------------------------------------



Sr. no Temperature oC Absorbance

1 55

2 65

3 75

4 85

5 95

Graph:

Result and Inference:



Absorption spectrum of proteins

Principle:

Amino acids with an aromatic group in their side chain have a peak in absorption at 280 nm.

Non-aromatic amino acids do not absorb at this wavelength, except for the low absorbance of

cystine (a pair of cysteines forming a disulfide bond). Absorption values calculated based on

amino acid composition generally fall close to values determined experimentally, making them

suitable for the determination of protein concentration. The side chains of tryptophan and, to a

lesser extent, that of tyrosine are also fluorescent. Tryptophan can be selectively excited at 295

nm. In the spectrum of proteins, absorption at 220 nm is due to the peptide bonds and the peak at

280 nm is caused by the absorption of aromatic amino acids. The latter is better suited for

concentration measurements because there are many other materials, including minor impurities

in solvents, that exhibit absorption at 220 nm.

Requirements:

1. Tyrosine (1mg/ml in 10% isopropanol)

2. Trytophan(1mg/ml in 10% isopropanol)

3. Cystine(1mg/ml in 10% isopropanol)

4. Sample: Bovine serum albumin( BSA)[ 1mg/ml in 10% isopropanol]

Instrument:

1. U.V-Spectrophotometer

2. Centrifuge

Method:

1. Read the absorbance of the amino acid solutions tyrosine, tryptophan and cystine at

220nm, 280nm and 295nm and record.

2. Centrifuge BSA and use the supernatant.

3. Read the absorbance of supernatant at 220nm, 280nm and 295nm and record.

4. Compare the absorbance of amino acids and egg albumin.---------------------------------------------------------------------------------------------------------------------



5. Prepare a distilled water blank for sample and 10% isopropanol blank for amino acids.

Observation Table:

Wavelength

(nm)

Absorbance

Tryptophan Tyrosine Cystine BSA

220

280

295

Inference:



Identification of different sugars using TLC

Aim:

To separate sugars using Thin layer Chromatography.

Principle:

Requirements:

1. Glass slides

2. Coupling jars

3. Silica gel-G

4. Choloroform

5. Glacial acetic acid

6. Distilled water

7. Diphenylamine

8. Aniline

9. Acetone

10. Orthophosphoric acid

11. Isopropanol

12. Fructose, Glucose

Preparation:

Solvent System: Chloroform, Acetic acid , water [3:3.5:0.5]

Spraying System: 1gm diphenylamine+1ml aniline in 100ml acetone. This mixture is mixed with

85% orthoposphoric acid prior to use (10:1 v/v)

Standard sugars: glucose, fructose 1% solution in 10% isopropanol

Fruit juices: citrus fruits, any available ripe fruit of the season



Sample preparation: Add 3ml of ethanol to 1ml to fruit juice and centrifuge to remove the

denature protein. Use the supernatant as the sample.

Method:

1. Activate the TLC plates in the oven at 100oC for 30minutes.

2. Load the standard sugars on separate TLC plates at about 1cm from the base of the plate.

3. Similarly load the fruit juice sample each on a separate TLC plate

4. Place the loaded TLC plate in the solvent system and allow the solvent to run upto 1cm

from the top.

5. Dry the slide and spray with the spraying system. Observe the spot and calculate the Rf

value for standard as well as sample.

6. Compare the Rf of samples and Rf of standard sugars.

Observation:

Plates can also be observed under UV light.

Calculation:

Rf= Distance travelled by solute

Distance travelled by solvent

Sr. no Rf

1 Glucose

2 Fructose

3

4

5

Inference:



Separation of Spinach Pigments by Column Chromatography

Aim:

To separate the spinach pigments by column chromatography.

Principle:

The technique in which the various solutes of a solution are allowed to travel down a column, the

individual components being adsorbed by the stationary phase. The most strongly adsorbed

component will remain near the top of the column; the other components will pass to positions

farther and farther down the column according to their affinity for the adsorbent. If the individual

components are naturally colored, they will form a series of colored bands or zones. The mobile

phase of chromatography refers to the fluid that carries the mixture of substances in the sample

through the adsorptive material. The phases are chosen such that components of the sample have

differing solubilities in each phase.

The spinach extract separates into two main bands on the column. The fastest-moving band

(yellow) contains the carotenes and the slower-moving band (green) contains the chlorophylls.

Equipment:

10ml plastic/glass Syringe Funnel

Erlenmeyer flask 50 mL * 6 Pipette 10 mL * 4

Mortar and pestle Beaker 400 mL

Beaker 100 mL Filtrol paper

Stand

Chemicals:

Silica Gel 40 Acetone, CH3COCH3

Petroleum ether Sodium chloride, NaCl

Calcium carbonate, CaCO3 Sodium sulfate, Na2SO4

Procedure:---------------------------------------------------------------------------------------------------------------------



Preparation of the column

1. Place the filtrol paper at the bottom of the Syringe in which column is to be prepared.

2. Weigh 10 g silica gel 40 in a 50 mL erlenmeyer flask, add some amount of petroleum ether

acetone solution slowly while mixing the slurry with a glass rod.

3. Place the slurry in the column with a pipette continuously, then place an erlenmeyer flask

under the column and allow the excess solvent to drain. Fill the syringe upto 7ml and leave

space for loading of sample and solvents. Be careful : Never let silica to dry in the column.

4. Collected excess solvent can be reused to place the column. Plug the bottom outlet of the

syringe with cello tape.

Preparation of spinach sample

1. Put the spinach into a mortar; add 22 mL acetone, 3 mL petroleum ether and spatula tip of

calcium carbonate, CaCO3.

2. Grind the leaves until the pieces are too small to be seen and then filter the pigment extract

if needed.

3. Put the filtrate into a separatory funnel and add 20 mL petroleum ether and 20 mL 10%

NaCl solution.

4. Shake the separatory funnel and drain the lower layer into a beaker.

5. Wash the upper layer twice with 5 mL distilled water.

6. Then put the extract into a 50 mL erlenmeyer flask and add a spatula tip of sodium sulfate,

Na2SO4, then decant the solution in another 50 mL erlenmeyer flask. Evaporate the excess

solvent if needed.

Performing the column chromatography:

1. Using a pipette, add 2 mL leaf extract to the top of the syringe column.---------------------------------------------------------------------------------------------------------------------

|| Hindi Vidya Prachar Samiti’s RAMNIRANJAN JHUNJHUNWALA COLLEGE || 7 of 280


2. Open the bottom outlet of the syringe column, let the elute pass through the column slowly.

As the eluent passes down the column, the sample is developed.

3. Observe the separation on the column and note the fndings.

Observation:



Separation of DNA using Agarose Gel Electrophoresis

Aim:

To separate genomic DNA and mechanically fragmented DNA on Agarose gel electrophoresis.

1. Understand the mechanism by which DNA fragments are separated within a gel matrix.

2. Understand how conformation of the DNA molecule will determine its mobility through a

gel matrix.

3. Prepare an agarose gel for electrophoresis of DNA samples

4. Set up the gel electrophoresis apparatus and power supply

5. Select an appropriate voltage for the separation of DNA fragments

6. Understand the mechanism by which ethidium bromide allows for the visualization of

DNA bands

Principle:

Agarose gel electrophoresis is the most effective way of separating DNA fragments of varying

sizes ranging from 100 bp to 25 kb1. To separate DNA using agarose gel electrophoresis, the

DNA is loaded into pre-cast wells in the gel and a current applied. The phosphate backbone of

the DNA (and RNA) molecule is negatively charged, therefore when placed in an electric field,

DNA fragments will migrate to the positively charged anode. DNA molecules are separated by

size within an agarose gel. The rate of migration of a DNA molecule through a gel is determined

by the following: 1) size of DNA molecule; 2) agarose concentration; 3) DNA conformation 4)

voltage applied. After separation, the DNA molecules can be visualized under UV trans

illuminator.

Requirements:

1. Horizontal electrophoresis assembly

2. UV trans illuminator

3. Sonicator



Chemicals:

1. Agarose

2. TAE buffer (40 mM Tris-acetate, 1 mM EDTA).

3. Gel loading Buffer ( 0.25% bromophenol blue + 1% glycerol)

Protocol:

Preparation of the Gel

1. Make 1% Agarose gel in TAE buffer

2. Add ethidium bromide (EtBr) to a concentration of 0.5 μg/ml.

3. Pour the molten agarose into the gel mold ane place the comb. Allow the agarose to set at

room temperature. Remove the comb and place the gel in the gel box. 2. Setting up of Gel

Apparatus and Separation of DNA Fragments

Preparation of Sample

1. Isolate the DNA from onion. Dissolve the strands of DNA in saline label as sample 1.

2. Prepare another sample DNA sample by dissolving in saline and subject it to sonication for

15minutes. Label this as sample 2.

3. Add gel loading dye to sample 1 and 2.

Program the power supply to desired voltage (80v between electrodes).

1. Add TAE buffer to cover the surface of the gel.

2. Attach the leads of the gel box to the power supply.

3. Turn on the power supply and verify that both gel box and power supply are working.

4. Remove the lid. Slowly and carefully load the DNA sample(s) into the wells.

5. Replace the lid to the gel box. The cathode (black leads) should be closer the wells than

the anode (red leads).

6. Turn on the power supply. Run the gel until the dye has migrated to an appropriate

distance.



Observing Separated DNA fragments:

1. When electrophoresis has completed, turn off the power supply and remove the lid of the

gel box.

2. Remove the gel from the gel tray and expose the gel to UV light and observe under UV

trans illuminator. DNA bands should show up as orange fluorescent bands. Take a picture

of the gel.

3. Properly dispose of the gel as per the SOP.

Result:



Paramecium Culture

Aim:

Preparation of Paramecium culture

Requirements:

1. Culture plates

2. Hay

3. Pond Water

4. Wheat grains

5. NaOH

6. Black soil

Method of Preparation:

Hay Infusion

1. Boil 10 g of chopped hay in one liter of pond water for about 30 minutes.

2. Filter the boiled hay mixture through several layers of cheesecloth. Allow the solution to

cool and then add 2 drops of 1 M NaOH and a pinch of black soil.

3. Pour 200 mL of this liquid medium into each culture dish (stacking culture dishes work

best) and place it at room temperature for 24 hours uncovered.

4. Add cooked, crushed wheat seeds to the liquid media.

Observation:

Observe under 100X and 400X magnification.

The same culture can be used to study the phenomenon of cyclosis and osmoregulation in

paramecium.



Observation of Succession of Microzoons in stagnant Pond Water

Aim:

To observe the population changes in stagnant pond water.

Method:

1. Keep about a litre of freshly collected pond water in a glass bowl.

2. Remove 10ml of water and dispense 5ml each in two centrifuge tubes. Spin at 100rpm for

10 minutes and collect the debris from the base of the centrifuge tube.

3. Place a small quantity under coverslip and observe for different microorganisms.

4. Draw different forms and tabulate the observation. Allow the water in the bowl to remain

stagnant without cover.

5. Repeat centrifugation and observation at 3 day interval for a period of 2 weeks.

6. Note the changes in the populations of microorganism in the pond water. Tabulate your

observations and draw the process of succession in populations of microorganisms.

Result:

Typically one may observe microalgae, ciliates, flagellates, rotifers and worm occurring over a

period of observation.



INDEX for EXTENSION PRACTICALS S.Y.B.SC

Sr. no Experiment

1 Estimation of Total Lipids (FeCl3 Method)

2 Determination of adulteration in milk (Starch & Urea).

3 Estimation of protein (Folin-Lowry method)

4 Antibacterial effects of honey

5 Preparation of probiotic yogurt



ESTIMATION OF TOTAL LIPIDS (FeCl3 METHOD).

Aim:

To determine the total lipid concentration in different varieties of milk.

Background Information:

Milk plays a major role in human nutrition and the milk produced by different farm animals is

used for human consumption. Therefore, the total fat content of the milk consumed by human

beings is of major concern for the proper health. Excessive fat from the milk adds to the body

burden, which will reflect in late age by atherosclerosis or cardiac ailments. Hence it is essential

to know the fat content of milk produced by different farm mammals. The milk produced by

different farm mammal should contain different levels of total fat in their milk.

Requirement:

1. Stock standard lipid: 1 % of commercial DALDA prepared in chloroform.

2. Lipid reagent: 10 g FeCl3 dissolved in 100 ml of 0.1 N HCL.

3. 14% NaOH: 14 gms of NaOH in 100 ml of water.

4. 14% Hydroxylamine hydrochloride prepared freshly in distilled water.

5. 2:1 chloroform methanol mixture or 3:1 ethanol ether mixture (60-80°C boiling point)

6. Working standard lipid: Dilute 1 ml of stock standard in 20ml of chloroform (if 1%

Dalda is used then the conc. of std is 500mg or 0.5gm.

7. 33% HCI :33ml of conc. HCl in 100ml of water.

8. Different varieties of milk



Principle:

Hydroxylamine in alkaline solution reacts with esters of fatty acids to give hydroxamic acids,

which produces red to violet colour with ferric chlorides.

Procedure:

1. Take two test tubes of 20 ml capacity and add 1.0 ml of two different varieties of milk into

two separate tubes.

2. Add 9ml of chloroform: methanol mixture or ethanol: ether mixture and close the mouth of

the test tube with cotton plug. Keep the test tubes in a water bath maintained at 600C for 5

min. Filter the sample through Whatman no. 1 filter paper.

3. Take 0.1 ml of filtrate and add 2ml of chloroform methanol mixture or ethanol ether

mixture and shake it well.

4. Add 0.5 ml of 14% hydroxylamine hydrochloride, shake well and add 0.5ml of 14% NaOH

shake again. Allow the tubes to stand at room temperature for twenty minutes. Add 0.6 ml

of

5. 33% HCI and 0.5ml 10% FeCl3.

6. In another test tube add 1 ml of working standard, to which 2ml of chloroform methanol

mixture or ethanol ether mixture is added. Add the reagents as mentioned in step 4 and

allow the tubes to remain for twenty minutes.

7. To prepare a blank, take 3ml of chloroform methanol:mixture or ethanol: ether mixture and

add reagents as mentioned in step 4.

8. Read the absorbance of standard and test sample at 540nm against the blank.



Calculation: Absorbance of the sample X Conc. Of std (35mg or 500 mg)

Absorbance of std

= _________________mg of lipid /0.1 ml of filtrate

= _________________ x 10.mg of lipid / ml of sample.

Result:

Compare the values of fat in different varieties of milk and conclude.

Conclusion:



DETERMINATION OF ADULTERATION IN MILK (STARCH & UREA).

Aim:

To detect the starch, glucose and urea adulterations in milk.

Back ground Information:

Milk sold loose in the local market is sometimes adulterated with starch, urea, ammonium sulfate

or glucose to increase the thickness of the milk and to sweeten it. It is adulterated with water.

Starch and urea are used to increase the specific gravity of milk so that lactometer fails to detect

the adulteration of milk with water. Following test can be performed to detect the presence of

starch and urea in milk.

A) Detection of starch as adulterant:

Principle: Since starch reacts with iodine to form a blue coloured complex, Lugol’s iodine

can be used as detecting agent.

Requirement: Lugol’s iodine, 5% starch solution, Pure sealed packet of milk of two

different sources/Brands

Procedure:

1) Take two test tubes;

2) In test tube 1 take 2 to 5ml of sample 1 and in test tube 2 take sample 2.

3) To each test tube add few drops of Lugol’s iodine and shake the test tube. If needed

add few more drops of Lugol’s iodine.

Result: lf the milk turns blue; it indicates that the milk is adulterated with starch. If the

milk remains white it is unadulterated.



B) Detection of Urea as an adulterant.

Principle : Since Urea is highly soluble in water and cools the water, when it is added to

milk it not only maintains the temperature of the milk cold during transport by acting as a

refrigerant and preservative but also helps in maintaining the specific gravity of milk when

adulterated with- water. To test whether the milk is adulterated with urea, following tests

can be performed.

Requirement: Conc. HCI, 1% NaNO2. Acetate buffer pH 4.6, 2% NaOH, 2% sodium

hypochlorite, 5% phenol, Pure milk sample (sealed packet) (Sample No .1), Milk sample

adulterated with urea (1 to 2% urea solution 75:25) (Sample No.2)

Procedure: Deproteinize the milk by adding 10ml of acetate buffer pH 4.6 to 20ml of

milk. Filter the sample to get the filtrate.

a) Test for Ammonia release: Take two test tubes. In both tubes add 2 to 5ml filtrate of milk

To each add 1 ml of 2% NaOH and boil the contents, smell the vapour.

Result: If the vapours arising from the test tube smells of ammonia, it indicates that the

sample is adulterated with urea.

b) Test for Nitrogen release: Take two test tubes and add 2 to 5 ml of filtrate of milk in one

test tube and in other test tube take 2 to 5ml filtrate of sample no.2.To each add 1 ml of

conc. HCI and 1ml of 1% NaNO2 shake the contents. Observe the reaction.

Result: If the bubbles of nitrogen arise from the sample, the sample is adulterated with

urea.

c) Colour test for urea: Take two test tubes and add 2 to 5 ml of filtrate of pure milk of two

sample. To each add 1 ml of 2% NaOH + 0.5 ml of 2 % sodium hypochlorite solution + 0.5

ml of 5% phenol solution. Shake the contents and heat. Observe the colour. The colour is



stable for 12 hrs and can detect urea as low as 0.1 %. Result: Blue or bluish green colour

develops, which indicates the milk is adulterated with urea.

d) Test for ammonium sulfate: The test is very similar to colour test for urea except that

there is no need to obtain protein free filtrate from milk. As in the case of urea, a bluish

colour forms on heating for 20 seconds.

Result: The bluish colour turns deep blue subsequently and indicates the presence of

ammonium sulfate.



ESTIMATION OF PROTEIN (FOLIN-LOWRY METHOD)

Aim:

To determine the protein concentration in milk by Folin-Lowry method.

Requirements:

1. Standard protein solution of BSA (bovine serum albumin): 0.2 mg/ml.

2. Alkaline copper sulfate reagent (Reagent A - 0.5 gm of CuSO4 in 100ml, Reagent B-1gm

of Na-K tartarate in 100 ml DW, Reagent C-2gm of Na2CO3 dissolved in 100 ml of

0.1 N NaOH). Mix 1: 1 proportion of reagent A and B and take 1ml of this mixture & add

it to 50ml of reagent C to get final alkaline copper reagent. Calculate the required amount

of alkaline copper reagent and prepare the reagent accordingly in the proportion mentioned

above. Prepared fresh before the experiment.

3. Prepare 1:2 dilution of Folin ciocaltaeu reagent in DW.

4. Milk sample

Principle:

When this protein solution is treated with alkaline CuSO4 it forms Biuret complex with 20 or

more peptide bonds. On addition of a reagent called Colin-Ciocalteau, which contains

phosphomolybdic acid it forms blue coloured complex due to reduction of MO-6 to MO-4 by the

amino acids like tyrosine and tryptophan present in the protein. This intense blue colour is

estimated colorimetrically at 620 nm. Intensity of colour varies with the concentration of protein

in the sample.



Procedure:

1. Take four test tubes; Blank, Sample (2) and std.

2. In test tube 1, take 1 ml of DW (Blank).

3. In test tube 2, & 3 take 0.5ml of diluted egg white solution (sample).

4. In test tube 4 take 1 ml of standard protein solution (Std).

5. To each test tube add 5ml of alkaline CuSO4 & mix it well allow the mixture to remain

for 15 min.

6. After 15min, add 0.5 ml of 1:2 diluted Folin-ciocaltaeu reagent to each tube, mix it well

& wait for 45 min.

7. Read the absorbance of standard & unknown at 620nm against the blank and tabulate the

readings.

8. Calculate the concentration of protein by using the formula.

Calculation:

Absorbance of the sample X 40(dilution factor) 0.2 (Conc. Of std)

Absorbance of std X 0.5 (volume of sample)

= __________________________ mg/ml of protein

Result:

Compare the values of protein in the two different varieties of milk and conclude the experiment.

Conclusion:



ANTIBACTERIAL EFFECTS OF HONEY

Honey is antibacterial and can prevent growth of most types of bacteria. This experiment

compares the effect of different types of honey on bacteria growing on agar plates.

The antibacterial properties of honey should be examined on non-pathogenic bacteria, such

as E. coli.

Background

Honey has several properties that help to kill bacteria. Honey has a high sugar content, which has

an osmotic effect, drawing water from bacterial cells and dehydrating them. Honey produces

small quantities of hydrogen peroxide, which kills many bacteria. It is also acidic, killing

bacteria by denaturing their enzymes.

Get information sheet: How honey heals wounds

Mānuka honey also has an additional factor, called the Unique Mānuka Factor or UMF, which

makes it particularly effective at killing bacteria.

Aim

To compare the antibacterial effect of different honey types.

Equipment

4 different honey samples

Antibacterial cream (for example, Savlon)

Squeezy pipette or eye-dropper

Agar plates

E. coli bacteria

Bunsen burner

Cork borer


http://www.biotechlearn.org.nz/focus_stories/honey_to_heal/how_honey_heals_wounds


Ruler

Incubator at 30ºC

Method

1. Inoculate the full surface of the agar plate with non-pathogenic bacteria. Protect the

surface of the agar from other microbes by keeping the lid on the Petri dish as much as

possible.

2. Sterilise the cork borer in the flame of the Bunsen burner, cool, then use to cut five wells

in each agar plate (see diagram below):

3. The holes on the bottom of the plate and fill each of them with either a control solution,

like antibacterial cream, or a honey sample using a clean pipette or eye-dropper.

4. Cover, seal with tape and place the dishes in an incubator (with the lid on top) at 30ºC for

two days, or at 37ºC overnight.


http://www.biotechlearn.org.nz/focus_stories/honey_to_heal/images/agar_plate


Yoghurt: whole full cream milk. Organic milk. Yoghurt maker

Procedure:

Take 2 pints of whole full cream milk and and organic milk . Heat in enclosed pan till it boils.

Turn off gas and let it cool down till warm. Take some amount in wooden spoon and put in

yoghurt maker jar. Add vegan or traditional flavor yoghurt starter culture in it. Stir well. Now

pour all the solution in jar and cap it or lid it airtight. Put jar in yoghurt maker for 8 hours.

Yoghurt is ready preserve in refrigerator in cups or bowls. Note: For lactose intolerant people

leave the yoghurt in maker for 24 hours for health benefit.

Strawberry Probiotic Yoghurt through yoghurt maker or homemade: For probiotic fruit flavoured

yoghurt crush the fruits or strawberry in crusher and strain out add to the heated milk.

For homemade: Whisk fresh cream 200 ounces and put one fourth of its sugar in it. Again

whisk. Add any colour essence in it. Add yoghurt to it or dahi. Add fruit pieces or strawberry to

it and crush it and whisk all the mixture. Put in box or cups and refrigerate it airtight. Remove

after some refrigeration. Stir yoghurt with spoon to remove air and again refrigerate it. Yoghurt

is ready.

Butter: Firstly take 1200g fresh cream, 10g salt and 10g extra virgin olive oil.

Procedure:

1. Put fresh cream in mixer for 3 minutes till it gets whipped. Some part of buttermilk will get

separated from whipped cream. Strain the whole mixture in a strainer and not through a

muslin cloth.

2. put the separated whipped cream again in mixer and whip it for another 2-3 minutes and

strain. Now put the solid whipped cream in mixer and add 10g salt and 10g extravirgin



olive oil. Add essence in it fruit, vanilla etc for the flavour specific. Leave it for 3-5

minutes.

3. Through a spatula put the whole lot on plate. Now put transparent plastic paper on it and

covering all sides and edges of butter press it till it becomes rectangular form. Put in

refrigerator for 2-3 hours. Butter is prepared.

Cheese: Whole raw milk, Heavy raw cream.

Procedure:

1. Take 2 cups whole raw milk in jar or jug add 2 cups heavy raw cream. To it add little bit

mesophilic starter culture. Mix with shaker for even blend. add two drops of animal rennet.

Mix in jug for 30 sec. Now put in glass bowl and put lid on it ensuring it airtight.

2. Keep in oven for 70 degree celsius overnight. Now take another bowl and put cheese cloth

on it and pour the whole mixture in that cheese cloth bowl. Hold it in sink and put plate on

it for 8 hours.

3. After that solid mixture residue in the cheese cloth in another bowl. To it add garlic

crushed, little bit salt and seasoning of your choice. spread and mix gently with spatula in

whatever form you want though the paper transparent enclosing all ends. Put in refrigerator

for 4-5 days. Cheese is ready.

Note:

Take proportions in the same ratio described for both products. If less or more then adjust

accordingly.



INDEX for EXTENSION PRACTICALS T.Y.B.SC

Sr. no Experiment

1 Fragility test of RBC

2 Sickling Test

3 Electrophoresis of hemoglobin

4 Viability Count of cells

5 PAGE separation of LDH isozymes



Fragility Test of RBC

Aim:

To determine the osmotic fragility of Red Blood Corpuscles.

Principle:

The test measures the resistance of RBC to hemolysis when exposed to hypotonic solutions. In

this test, RBC are exposed to a series of saline (NaCl) solutions with increasing dilution. In

hypotonic solution, water enters the RBC and leads their swelling. In case of spherocytes due to

their small size cannot absorb much of the extracellular fluid and break away easily. The time

required for hemolysis is inversely proportional to osmotic fragility of

Application:

Osmotic fragility test is performed to diagnose two hereditary conditions: thalassemia and

hereditary spherocytosis.

Requirements:

1. Normal saline [0.9%]: 0.9g of NaCl in 100ml distilled water.

2. Distilled water, 0.1%, 0.2%, 0.3%, 04%,0.5%,0.6%,0.7% , 0.8% NaCl solution.

Procedure:

1. Make a series of test tubes with 1ml of Distilled water, 0.1%, 0.2%, 0.3%, 04%,

0.5%,0.6%,0.7% , 0.8% NaCl solution.

2. Add 0.2ml of freshly collected blood to these tubes. Allow it to stay for 30minutes at room

temperature.

3. Centrifuge to remove the cell debris and estimate the quantity of hemolobin release due to

lysis of RBC.



4. Estimate haemoglobin using standard method.

5. Tabulate the results with NaCl concentration as variable.

6. Plot a graph with Haemoglobin release as a function of sodium chloride concentration

(NaCl).



Sickling Test

Introduction:

This test can be used to screen individuals for presence and absence of sickle shaped anemia, an

inherited condition more prevalent in certain belts of Vidharbha.

Principle:

The principle of sickling test was based on microscopical observation of sickling of red blood

cells when exposed to a low oxygen tension.

Requirements:

1. Blood

2. 2% Sodium metabisulphate ( 2g in 100ml distilled water)

3. Compound Microscope

Procedure:

1. Twenty micro litres of each blood sample, was mixed with 20 micro liters of 2% sodium

metabisulphite on a cover slip.

2. A slide was then gently pressed onto the cover slip and after inversion; the coverslip was

ringed with candle wax.

3. The slide preparations were left in a humidified chamber for 15 minutes at room

temperature and then examined under the microscope (x10).

4. Further observations were taken after 30 minutes, 1 and 2 hours. In each test, an average of

5 films were randomly selected and wholly examined.

5. The proportion of the number of red blood cells that were sickled was then expressed as

percentage and results were considered positive when more than 25% of the red blood cells

sickled.

Observation and Inference:

Electrophoresis of Hemoglobin---------------------------------------------------------------------------------------------------------------------



Use the technique of agarose gel electrophoresis to separate human hemoglobin molecules

according to their electrical charge, size, and/or shape. Relate the migration of hemoglobin

molecules on the gel (phenotype) to the alleles possessed by individuals in the scenario

(genotypes). In this experiment the human haemoglobin samples are electrophorese on Agarose

gel and the gel is stained with Coomassie blue stain and analysed for separation of different types

of hemoglobins.

Requirements:

1. Horizontal electrophoresis chamber, casting tray, comb

2. Power supply

3. Micropipette, disposable tips to load the gel

4. Staining dish, disposable gloves

5. Tris Glycine buffer(1X Tris-Glycine) : pH 9.2

Tris base 8.0 gm

Glycine 3.6 gm

distilled water 500.0 mL

6. 1.5% Agarose: 1.5g agarose in 100ml Tris –glycine buffer

7. Samples containing normal adult hemoglobin, sickle hemoglobin, and a mixture of the two

types

8. Coomassie blue stain : 40.0% methanol +10.0% acetic acid+0.5% Coomassie blue+ 49.5%

distilled water

9. Destaining solution:10% methanol

10% acetic acid

80% distilled water

Procedure:

Preparation of the Gel---------------------------------------------------------------------------------------------------------------------



1. Make 1.5% Agarose gel in Tris- glycine buffer

2. Pour the molten agarose into the gel mold ane place the comb. Allow the agarose to set at

room temperature. Remove the comb and place the gel in the gel box.

Preparation of Sample

Prepare haemoglobin lysate from the sample blood. Centrifuge to remove debris and use the

supernatant for analysis. Mix lysate with gel loading buffer.

Preparation of Sample buffer

glycerol 8.00 mL

1.5M Tris, pH=9.2 4.00 mL

deionized water 28.00 mL

bromophenol blue 0.01 gm

Program the power supply to desired voltage (80v between electrodes).

1. Add Tris- glycine buffer to cover the surface of the gel.

2. Attach the leads of the gel box to the power supply.

3. Turn on the power supply and verify that both gel box and power supply are working.

4. Remove the lid. Slowly and carefully load the 20µl of haemoglobin sample(s) into the

wells.

5. Replace the lid to the gel box. The cathode (black leads) should be closer the wells than

the anode (red leads).

6. Turn on the power supply. Run the gel until the dye has migrated to an appropriate

distance.

Staining of Gel

Remove the casting tray and slide the gel into the staining dish.



Pour Coomassie blue stain over the gel and stain for 1 hour.

Destaining of Gel

Replace the stain with destaining solution. For best results, change the destaining solution

several times. Destain the gel until the "bands" of hemoglobin are clearly visible on the gel.

Observation:

Make a photographic record of your observation.

Viability Count



This practical is an extension of trypsinization of kidney. The viability count of the cells is

estimated using a trypan blue, vital stain. The dead cells get stained by trypan blue whereas the

live cell membrane expels the dye and remain unstained.

Requirements:

Hemocytometer

Compound Microscope

Trypan blue

Procedure:

I. Preparing haemocytometer

Ensure the haemocytometer is clean using 70% ethanol.

II. Preparation of cell suspension

1. Make sure the cell suspension to be counted is well mixed by either gentle

agitation of the flask containing the cells.

2. Take out about 1 ml of cell suspension using a serological pipette and place in an

Eppendorf tube.

3. Mix the cell suspension with trypan blue in ration of 1:1

III. Counting

1. Using pipette, draw up some cell suspension containing trypan blue.

2. Carefully fill the haemocytometer by gently resting the end of the tip at the edge

of the chambers. Take care not to overfill the chamber. Allow the sample to be

drawn out of the pipette by capillary action, the fluid should run to the edges of

the grooves only. Re-load the pipette and fill the second chamber if required.

3. Focus on the grid lines of the haemocytometer using the 10X objective of the

microscope. Focus on one set of 16 corner square as indicated by the circle in the

diagram below:



4. Using a hand tally counter, count the number of cells in this area of 16 squares.

When counting, always count only live cells that look healthy (unstained by

trypan blue). Count cells that are within the square and any positioned on the right

hand or bottom boundary line.

5. Dead cells stained blue with trypan blue can be counted separately for a viability

count.

6. Move the haemocytometer to another set of 16 corner squares and carry on

counting until all 4 sets of 16 corner squares are counted.

7. The haemocytometer is designed so that the number of cells in one set of 16

corner squares is equivalent to the number of cells x 104 / ml .

Calculation:

Therefore, to obtain the count:

The total count from 4 sets of 16 corner = (cells / ml x104) x4 squares from one

haemocytometer grid

1. Divide the count by 4

2. Then multiply by 2 to adjust for the 1:2 dilution in trypan blue

These two steps are equivalent to dividing the cell count by 2



As an example:

If the total cell count is 145

The cell density is

145

2= 72.5 x 104 / ml

Viability: To check the viability

Live cell count (not including trypan blue cells)

Total cell count including those stained with trypan blue.

Live cell count

Total cell count = percentage viability

Results:

PAGE separation of LDH isoenzymes---------------------------------------------------------------------------------------------------------------------



Aim:

Determining the serum level of lactate dehydrogenase (LDH) activity.

Background theory:

The LDH molecule itself is a tetramer, built from two types of subunits coded by different genes.

From the linear combination of the H (heart) and M(muscle) type of subunits, five combinations

can be obtained: H4 (LDH-1),H3M1 (LDH-2), H2M2 (LDH-3), H3M3 (LDH-4) and M4 (LDH-

5).

While LDH-1 and LDH-2 can be found mainly in the heart muscle and in erythrocites, the liver

and smooth muscle tissue contain mostly the LDH-5 isoenzyme. All isoenzymes catalyze the

following reversible reaction: Pyruvate + NADH+H+ == Lactate + NAD+

Materials:

1. premade 6.5% polyacrylamide gel slabs

2. tank buffer (2.4 g Tris base, 11.6 g glycine/liter)

3. samples made from liver, heart muscle, and kidney in sample loading buffer

4. 1 M Tris- HCl, pH 8.0

5. NAD, 10 mM

6. tetrazolium-blue, 1 mg/ml

7. phenazine-methosulphate, 1.6 mg/ml

8. 1 M Na-lactate

Methodology:

In this experiment premade 6.5% polyacrylamide slab gels will be used. The sample slots are

rinsed out

with the tank buffer and the gels with the glass plates together are clamped to the buffer tank

using bulldog clips. Load 40 µl from each sample into different slots of the gel. Carefully cover

the samples with tank buffer. Pour tank buffer into the reservoirs and connect the electric cables.

Turn the power source on and set the voltage. Proceed for two hours. Turn off electricity, ---------------------------------------------------------------------------------------------------------------------



separate the two glass plates by prying them apart with a spatula and place the gel into the

developing chamber which already contains the developer solution ( H2O 18.4 ml, 1 M Tris 4

ml, tetrazolium-blue 12 ml, phenazinemethosulphate 4 ml, Na-lactate 4 ml and NAD 1.3 ml).

Incubate at 45 °C to develop color reaction for 20 minutes. In the color reaction NAD and lactate

serve as substrates, phenazine-methosulphate is the electron acceptor and tetrazolium-blue is the

final electron acceptor. Wash the gel with water and dry the gel between paper sheets.

Observation:

Make aphotographic record of your observation.

Interpretation:



List of Current F.Y. Practicals

1. Levels of Organisation in Animal kingdom

2. Animal Diversity from Protozoa to Vertebrates

3. Study of Food vacuole, Contractile of Paramecium

4. Nutritional apparatus in Animals

5. Study of effect of pH, temperature on amylase activity

6. Study of gills, lungs, hearts, kidney, brain.

7. Determination of heart beat of Daphnia

8. Population density of Daphnia

9. Safety measures in laboratory

10. Study of common laboratory equipments

11. Study of Asceptic techniques

12. Paper chromatography (amino acid separation)

13. TLC ( lipids separation)

14. Adsorption chromatography ( using chalk)

15. Qualitative test for proteins, carbohydrates and lipids

16. Preparation of beads

17. Immobilization of yeast cells

18. Fermentation of sugarcane juice

19. Effect of papain as a meat tenderizer

20. Human Pedigree analysis

21. Detection of normal and abnormal constituents of urine

22. Extraction and qualitative estimation of DNA( diphenyl method)

23. Extraction and qualitative estimation of RNA(orcinol method)

24. Gram Staining of bacteria

25. Study of reproductive system

26. Study of mimicry

27. Study of evidences of evolution



List of Current S.Y. PracticalsPractical-I

1. Mountings of foraminiferan shells

2. Study of slides and specimen-I

3. Study of slides and specimen-II

4. Study of crustacean larvae

5. Study of metamorphosis in insects

6. Study of shells in Molluscs

7. Study of echinoderm larvae.

8. Study of ascidian tadpole

9. Swim Bladder(in-situ)

10. Parental care

11. Study of neoteny

12. Adaptive radiation in reptiles

13. Venomous snakes

14. Mammals

15. Study of different types of eggs

16. Study of blastulae

17. Study of gastrulae

18. Quantitative estimation of DO of water

19. Quantitative estimation of phosphate-phosphorus

20. Quantitative estimation of free CO2

21. Quantitative estimation of salinity

22. Determination of total hardness of water

23. Determination of pH of soil

24. Determination of moisture content of soil

25. Determination of texture of soil.



Practical-II

1. Study of pH meter

2. Preparation of buffers of different pH using H-H equation

3. Preparation of titration curve

4. Determination of pKa of weak acid

5. Study of colorimeter

6. Selection of filter

7. Determination of concentration

8. Study of osmosis using RBCs

9. Electron micrograph study

10. Chromosome morphology

11. Study of polytene chromosome

12. Study of Barr body

13. Mimicry and warning colours

14. Estimation of glucose:

a) Glucometer

b) GOD-POD method

15. Problems in genetics (supplement)

16. Problems in Biotechnology(supplement)

17. Bioinformatics(Assignment)



Practical-III

1. Parasitology-types and adaptations:

a) Protozoan

b) Helminth

2. Honey bee-life history, bee hive, mouthparts

3. Honey bee-sting apparatus

4. Honey bee-mounting of legs

5. Silk moth-life history

6. Insects of economic importance

7. Fresh water and marine fishery

8. Non-fish fishery

9. Study of crafts

10. Study of gears

11. Animal husbandry

12. Paneer preparation from milk

13. Estimation of protein-Lowry method

14. Estimation of total lipids-FeCl3 method

15. Determination of adulterants in milk(starch and urea)

16. Extraction of casein from milk

17. Determination of density of milk by lactometry.



List of Current T.Y. Practicals

Hematology:

1. Colorimetric estimation of total plasma proteins

2. Separation of plasma proteins by electrophoresis on agarose/PAGE.

3. Estimation of blood glucose by O-Toluidine method

4. Estimation of serum/plasma total cholesterol by FeCl3 method.

5. Estimation of serum/plasma total triglycerides by Phosphovanillin method.

6. Enumeration of erythrocytes-total count

7. Enumeration of leucocytes-total and differential count.

8. Estimation of haemoglobin by Sahlis acid haematin method.

9. Study of Lymphoid organs: Lymph node, Thymus and Spleen.

10. Study of Leukemic cells from permanent slide

11. Observation of bone marrow cells.

(Students are expected to know the preparation of blood report)

Physiology:

1. Study of Acid phosphatase activity:

a) Effect of substrate concentration

b) Effect of pH variation

c) Effect of enzyme concentration

d) Effect of inhibitor

2. To determine specific activity of Acid phosphatase

3. Separation of LDH isozyme by electrophoresis on agarose / PAGE

4. Study of vaginal smear to identify stages of oestrous cycle.

5. Study of human ECG: Normal, Sinus tachycardia, Sinus bradycardia,

Ventricular fibrillation.

6. Mounting of nerve cells and neurosecretory cells from cockroach



Immunology:

1. Latex agglutination test [any available / Rheumatoid Arthritis]

2. Determination of blood group and Rh factor – RA test serum

Molecular Biology

1. Estimation of RNA Orcinol method.

2. Estimation of DNA Diphenylamine method.

3. Isolation of E. coli genomic DNA and checking its purity by horizontal gel

electrophoresis.

Biotechnology:

1. Use of autoaclave for sterilization of equipment for tissue culture.

2. To prepare cells for culture from mammalian kidney. spleen / chick embryo using

trypsin.

3. Problems in Biotechnology and Bioinformatics.

Genetics:

1. Karyotype analysis for the following syndromes with comments on numerical and

structural variations in chromosomes. : Turner’s, Klinefelter’s, Down’s, Cri-du-chat, D-G

translocation, Edwards’, and Patau’s syndrome.

Toxicology

1. Determination of LC50 for a suitable pollutant (any one salt of heavy metal dissolved in

water) on Daphnia, Probit analysis.

2. Effect of salt of heavy metal on the heart beat of Daphnia.



PRACTICALS

Natural Resources and Their Management

1. Rapid field tests for sulfate and nitrate contents as well as base deficiency of soil samples.

2. Determination of carbonates in soil by rapid titration.

3. Analysis of a community by working out ecological indices (frequency/ importance probability-,

rarity-, fidelity-, constancy-, species diversity- and Shannon-Weiner Indices):

a. using transect method

b. using quadrat method

4. Physical Properties of water:

a) Turbidity

b) Conductivity

5. Chemical Properties of water:

a) Total Acidity

b) Total Alkalinity

c) COD

d) Heavy metal- Copper

e) Nitrate and nitrite nitrogen

Environmental Biology

1. Study of interaction between organisms: Social organization in Honey bee, termite, hanuman

languor, and Asian elephant.

2. Study of present status, distribution, habitats and reasons for decline in India of the following

animals. Gaur, hangul, rhino, musk deer, wild ass, brow-antlered deer, blackbuck, elephant, tiger,

lion, snow leopard, red panda, reticulated python, pink headed duck, white-winged wood duck,

mountain quail, and great Indian bustard.

Zoogeography---------------------------------------------------------------------------------------------------------------------



1. Study of fauna of different zoogeographic regions (with the help of photographs, models or

sketches):

a) Palaearctic- Giant Panda, Japanese snow monkey

b) Nearctic- Virginia opossum, sea otter, raccoon

c) Neotropical- South American lung fish, low land tapir/ Brazilian Tapir, llama,

d) Oriental – flying frog, gharial, flying lizard, Asian elephant, Indian porcupine, and Great

Indian one horned rhino

e) Ethiopean- African lung fish, ostrich, African elephant

f) Australasian – duck billed platypus, spiny ant-eater, Australian lung fish, red kangaroo

g) Antarctic – penguins.

Epidemiology

1. Temporary preparation of head and mouthparts of mosquito.

2. Identification: Permanent slides/ specimens of – Plasmodium, Ascaris, Wuchereria

Biostatistics

1. From the given data make frequency distribution table.

2. From the given data plot frequency polygon/ histogram.

3. From the given data derive mean and standard deviation.

4. From the given data plot bar diagram/ pie diagram.

5. Application of Z test

6. Application of t-test

7. Application of chi-square test of significance

a. to test goodness of fit of observed and expected proportion

b. to test association between two events.

8. Use of spreadsheet programme in biostatistics.


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