International E – Publicationisca.co.in/BIO_SCI/lab_manual/ISBN 978-93-84648-30-5.pdf ·...

.

(A Reference Book)

By

Dr. S. Mohana Roopan & Mr. Ganesh Elango

International E – Publication

www.isca.me , www.isca.co.in

(A Reference Book)

By

Dr. S. Mohana Roopan

M.Sc., Ph.D., MBA (HR), F.I.S.C.A. Assistant Professor (Senior), Organic Chemistry Division, School of Advanced

Sciences, VIT University, Vellore 632014, Tamilnadu, India.

Email: [email protected] , [email protected]

&

Mr. G. Elango Chemistry Research Laboratory, Organic Chemistry Division, School of Advanced

Sciences, VIT University, Vellore 632014, Tamilnadu, India.

2014

International E - Publication

www.isca.me , www.isca.co.in

International E - Publication 427, Palhar Nagar, RAPTC, VIP-Road, Indore-452005 (MP) INDIA

Phone: +91-731-2616100, Mobile: +91-80570-83382

E-mail: [email protected], Website: www.isca.me, www.isca.co.in

© Copyright Reserved

2014

All rights reserved. No part of this publication may be reproduced, stored, in a

retrieval system or transmitted, in any form or by any means, electronic,

mechanical, photocopying, reordering or otherwise, without the prior permission

of the publisher.

ISBN: 978-93-84648-30-5

International Science Congress Association

www.isca.in , www.isca.co.in , www.isca.net.co , www.isca.net.in

Lab Manual for Biotechnology i

AUTHOR PREFACE

Biotechnology courses were one of the basic course subjects of school, college and

universities. This laboratory manual designed in such a way to make students to

understand the basic principle behind the biotechnology experiments. This Laboratory

manual was dedicated for undergraduate level laboratory sessions in biotechnology.

This manual contains several experiments that may help full for students to easily

pursue it. Before starting each lab session all the reagents should be prepared for

proceedings of the experiment. Some of the experiments can be processed each week

or may be it can also continue in the next week session. We advice the students to do

practical’s with interest and get full benefit out of this book.

- Dr. S. Mohana Roopan

&

Mr. G. Elango



Lab Manual for Biotechnology ii

Table of Content

Author Preface

General Instructions for using the laboratory 1

1. Degradation of different agricultural waste products (Orange Pomaces) by soil

streptomycetes 2

2. Screening for ethanol producing yeasts 4

3. Preparation of wine, beer and cheese from sour-cabbage 6

4. Preparation of competent cells of E.coli for harvesting plant transformation vector 8

5. Preparation of small scale plasmid from E.coli 10

6. Aseptic culture techniques for establishment and maintenance of cultures 13

7. Quantitative determination of DNA/RNS using spectrophotometer 15

8. Determination of the pH of the given samples by using pH meter 16

9. Determination of end-point of an acid base titration by using Conductivity meter 19

10. Determination of sulphate content by using Nephelometer 23

11. Determination of sodium using Flame photometer 27

12. Estimation of total dissolved solids (TDS) in water sample 31

13. Estimation of dissolved nitrate in a given sample 33

14. Estimation of Bovine Serum Albumin (BSA) by Bradford method 35

UV-visible spectrophotometery

15. Separation of sugars using paper chromatography 37

16. Separation of amino acids using paper chromatography 39

17. Estimation of dissolved oxygen concentration of water sample 41

18. Estimation of Biological oxygen demand (BOD) of given water sample 45

19. Estimation of Chemical oxygen demand (COD) of given water sample 50

20. Isolation of Xenobiotic degrading bacteria by using selective media 53

21. Isolation of hydrocarbon degrading microorganism 56

22. Isolation of degradative plasmids in microbes growing in polluted environment 58

23. Separation of DNA fragments by agarose gel electrophoresis 60



Lab Manual for Biotechnology iii

24. ISOLATION OF INDUSTRIALLY IMPORTANT MICROORGANISMS 62

25. GRAM STAINING 63

26. CAPSULE STAINING 66

27. SPORE STAINING 68

28. METHYL RED AND VOGES PASKAUR TEST 70

29. CITRATE UTILIZATION TEST 72

30. UREASE PRODUCTION TEST 73

31. OXIDASE TEST 74

32. ESTIMATION OF GLUCOSE BY GLUCOSE OXIDASE METHOD 75

33. TRANSFORMATION OF CELLS 77

34. ISOLATION OF DNA FROM HUMAN BLOOD SAMPLE 79

35. BLOOD GROUPING IN RH TYPING 81

36. ANTI-STREPTOLYSIN O ACTIVITY 83

Authors Profile 84



Lab Manual for Biotechnology 1

GENERAL INSTRUCTIONS FOR USING THE LABORATORY

Precautionary measure to be followed in Laboratory

• Never mouth-pipette any solution (acids, phenols, peroxides, organic solvents,

etc.)

• Wear a laboratory coat (use gloves when necessary).

• Do not, eat, drink or smoke inside the laboratory.

• Aseptic techniques should be followed rigorously at all times.

• Dispose all wastes properly. Radioactive wastes should be stored separately.

• No chatting, gossiping, and loud discussions inside the laboratory as it affect

your concentration on the work and that of your colleagues as well.

• Do not store food materials and drinks in refrigerator where other chemicals and

solvents are stored (Consumption of such stored food items may lead to cancer).

Standard operating procedure

• In accredited analytical laboratories, all methods and procedures must be in the

form of SOP. It will give written details of the protocol that must be followed for all

standard analyses.

• They should include the method of collecting and handling samples, performing

analyses, storing and retrieving data and preparing reports.

• They should contain instructions on the use, maintenance, servicing, calibration

and repair of equipments and instruments.

• The hazards associated with overall protocol are stated clearly and safe working

practices are specified.




Expt. No. 1

DEGRADATION OF DIFFERENT AGRICULTURAL WASTE PRODUCTS (ORANGE

POMACES) BY SOIL STREPTOMYCETES

AIM

To degrade agricultural waste products by the help of soil streptomycetes

PRINCIPLE

The major respiratory of microorganism which can develop antibiotics was soil. These

antibiotics can able to inhibit other microorganisms which are harmful. Most of the

antibiotics are isolated from four soil microorganisms namely Streptomycetes, Bacillus,

Penicillin and Cephalosporium. The Streptomycetes genus was first discovered in 1943

from soil actinomycetes known as Streptomyces griseus which was responsible for the

formation of more than 60 % of known antibiotics. It was one of the gram negative

bacteria which involves in protein synthesis. Most of Streptomycetes species can be

isolated from soil which contain pectinase enzyme in it for hydrolysis of glycosidic bonds

in pectic polymers. This experiment will processed clearly using streptomycetes species

on fruit peels to isolate pectinase enzyme to degrade agricultural waste products.

MATERIALS REQUIRED

• Streptomycetes organism

• Agricultural waste products (Orange pomaces media)

• 3, 5-dinitrosalicylic acid (DNS reagent)

• pH meter

PROCUDURE

Pectinase production in orange pomaces media

• Take 100 mL distilled water and add 1 gm of fruit pomaces.




• Then suspended it with 0.1 mL trace salt solution contains 1 mL/L of

FeSO4.7H2O, 0.1 g of MnCl2.4H2O and 0.1g of ZnSO4.7H2O.

• Before autoclaving the comprised samples adjust pH to 7.5.

• Take 1 mL of spore suspension and inoculate in orange pomaces media.

• Place in incubator for 8 days at 28 °C.

• And perform pectinase activity daily using standard 3, 5-dinitrosalicylic acid

(DNS) method as follows.

Pectinolytic assay

• Incubate a reaction mixture composed of 0.2 mL of crude enzyme solution.

• Add 1.8 mL of 1.0 % (w/v) citrus pectin in 50 mM sodium phosphate buffer (pH

7.0) at 37 °C in a shaker water bath for 30 min.

• Stop the reaction by adding DNS reagent

• Keep it in boiling water bath for 5 min and check it for any colour change.

• Using calorimeter check OD value at 575 nm against blank

• Blank= Reaction mixture – Crude enzyme

• Compare the results to controls inoculated with an inactive pectinolytic

streptomycetes isolate.

RESULT

The highest pectinase activity was at the time of .




Expt. No. 2

SCREENING FOR ETHANOL- PRODUCING YEASTS

AIM

To screen ethanol producing yeasts

INTRODUCTION

C2H5OH (Ethanol), it was used as colorless oxygenated hydrocarbon and also it can be

used as fuel for transport. Biotechnology contains several microorganisms which will

produce alcohols from sugars which can be used in several industries like brewing, wine

and spirits industries. There are several microorganism used in formation of

carbohydrates to ethanol like Saccharomyces cerevisiae, Zymomonas mobilis,

Clostridium thermocellum.

MATERIALS REQUIRED

• Yeast

• Edinburgh Minimal Medium agar

• Sodium phosphate buffer

PROCEDURE

• Collect different Yeast and maintain its growth on Edinburgh Minimal Medium

agar plates for 3-5 days at 28° C.

Add 5 mL of molten agar (10g/L) make up in sodium phosphate buffer 0.1 M of

pH 8.

• Add reaction mixture which contains 50 µL 0.05 M 2, 6-dichlorophenolindophenol

and 100 µL 0.15 M NAD solutions mix it gently.

• Place it in a room temperature for 30 min.




• Gently spread 3 mL of 0.005 M 5- methyl-phenzinium methyl sulfate over the

agar media of each plate and incubate in room temperature for 30 min.

• After incubation yellow color zone will appears and it denotes that presence of

ethanol.

RESULT

Ethanol was




Expt. No. 3

PREPARATION OF WINE, BEER AND CHEESE FROM SOUR-CABBAGE

AIM

To preparation of wine, beer and cheese from sour-cabbage

PRINCIPLE

The transfer occurs from cabbage to sour cabbage by two organisms like gram positive

bacilli (Lactobacillus) and cocci (Leuconostoc). These organisms inhibit the growth of

organism and enhance high salt preparation which will be help for preservation for long

time.

MATERIALS REQUIRED

• Cabbage

• Non-iodized salt

• Grape juice

• Malt extract broth

• Milk

• Rennin

PROCEDURE

• Take a cabbage and remove the core and outer cover leaves.

• Shred the cabbage and mix it with the Non-iodized salt.

• Pack the cabbage in a sterile container for anaerobic conditions which was

necessary for fermentation at 30 ºC for 1 to 2 weeks.

• Observe the cabbage at regular intervals and maintain the accumulation of fluid

is drawn out from cabbage by salt.




• At various intervals collect some fluid samples and perform gram staining

• After incubation take some water for tasting and record your observations

Fermentation of Wine and Beer

• Take some malt extract broth and sterile grape juice containing 5 % sucrose

covered with cotton plugs.

• The cotton plugs will help to release CO2

• Using inoculation loop inoculate Saccharomyces cerevisiae.

• Incubate tubes approximately 30 ºC for 1 week.

• Shake the tubes for formation of foaming and presence of CO2

• Smell and taste the product to determine its quality.

Preparation of Cheese

• Place warm heat milk and add a rennin tablet. This will indicate the formation of

curd.

• Squeeze as much liquid from curds as possible and mix a small piece of blue

cheese (Penicillium Roquefort)

• Now add flavor and replace the cheese cloth covering

• Place the cheese in a plastic container, cover tightly and incubate in cool

temperature

• Taste the cheese and note the characteristic flavor and aroma.

RESULT

The preparation of brewing and cheese was .




Expt. No. 4

PREPARATION OF COMPETENT CELLS OF E. COLI FOR HARVESTING PLANT

TRANSFORMATION VECTOR

AIM

Preparation of competent cells of E. coli for harvesting plant transformation vector

PRINCIPLE

In several species of bacteria including E.coli will take up only limited amount of DNA.

For efficient uptake they need some physical or chemical treatment to increase the DNA

content. For this purpose E.coli cells are soaked in CaCl2 to make competent E.coli

cells.

MATERIALS REQUIRED

• Lysogeny broth medium

• 100 mM CaCl2

• 250 mL conical flask,

• 1.5 mL centrifuge tube,

• Micro tips and sterile polypropylene tubes

PROCEDURE

• Take 2 mL of LB broth and inoculate E.coli at 37°C for overnight at 180 rpm.

• Take 30 mL of LB broth and inoculate 300 µL of overnight culture and incubate in

room temperature for 3 to 4 hrs until it reaches an optical density value of 0.5 to

0.6 at 600 nm.

• Transfer in a tube and incubate in ice for 30 min.

• Using centrifuge rotate it to 5000 rpm at 40 °C for 5 min.




• Remove the supernatant and suspend the pellet in 30 mL of ice cold 100 mM

CaCl2 gently and incubate in ice for 30 min.

• Repeat the fourth and fifth step and in addition with 100 mM CaCl2 and cells

become fragile after CaCl2 treatment.

• Store cells and cool it for 30 min before use.

RESULT

Competent cells of E. coli for harvesting plant transformation vector




Expt. No. 5

PREPARATION OF SMALL SCALE PLASMID FROM E. COLI

AIM

Small scale plasmid preparation from E. coli

PRINCIPLE

Birnboim and Doly in 1979 they developed a procedure for isolation of plasmid in purest

form. The small extra chromosomal super coiled DNA molecules were known as

plasmids. Under alkaline conditions nucleic acids and proteins was denature and when

solution was neutralized by potassium acetate super coiled are denatured.

Chromosomal DNA is precipitated out because the structure is too big to denature

correctly; hence plasmid DNA is extracted efficiently in the solution.

MATERIALS REQUIRED

• Bacterial culture

• Eppendorf tubes

• Micro tips

• Micropipette

• RNAse

• Solution I, II and III

• Phenol

• Chloroform: isoamylalcohol (24:1)

• Isopropanol

• Sodium acetate

• Ethanol

• TE buffer




REAGENTS

Solution I: 100 mL

(Mol. Wt) (For 100 mL)

Tris (25 mM) 121.1 0.303 gm

EDTA (10 mM) 372.0 0.372 gm

Glucose (50 mM) 180.16 0.901 gm

Dissolve all the ingredients in 80 mL of water and adjust to pH 8.0 using 1 N HCL and

make up the volume to 100 mL and store it in a room temperature.

Solution II: (prepare fresh each time)

NaOH 0.2 M

SDS 1.0 %

Prepare 0.4 normality NaOH and store it in a separate reagent bottle and 0.2 % of SDS

and autoclave it and mix it in 1:1 ratio.

Solution III

Take potassium acetate and weigh 29.4 gm and mix it in a 30 mL of distilled water and

using pH meter adjust its pH to 5.5pH and make it to 100 mL and store it for further use

RNAse

At a concentration of 10 mg/mL dissolve the RNAse in 10mM of Tris NaCl and heat it at

high temperature for denaturing of DNA strands and allow it to cool and store it for

further experiments

Phenol

Distillate the phenol without the water circulation and collect the phenol between 160°C

and 182 °C




TE Buffer

Take Tris HCl (1mM) of 100 µL from 1 M stock (pH 8.0) and EDTA (0.1 M) of 20 µL from

0.5 M stock (pH 8.0) and makeup it with 98.8 mL of distilled water.

3M Sodium acetate (pH 5.2) 100 mL

Take sodium acetate of 24.61 gm and dissolve it in 80 mL. Make up the volume to 100

mL by adjusting pH by glacial acetic acid.

PROCEDURE

• Grow some 2 mL of antibiotic culture in a shaker for 4-5 hrs at 37 °C

• Take 1.5 mL of culture and store it in eppendorf for centrifugation and discard

supernatant and rest of the culture for centrifugation in same eppendorf and

discard the supernatant by 1000 rpm for 2 min.

• Mix the cells with solution 1 and vortex it and add 200 µL of freshly prepared

Solution II and mix well and add 150 µL of Solution III further centrifuge it for

12,000 rpm for 15 min.

• Transfer it and add 5 µL of RNAse to each tube and incubate at 37 °C for 1 hr

• Add equal amount of Chloroform and Isoamylalcohol by vortexing and Spin at

12,000 rpm for 15 min.

• Transfer supernatant in eppendorf and add equal volume of Propan –2– ol and

then sodium acetate and keep inversed overnight at –20 °C.

• Discard supernatant and add 200 µL of ice cold 70 % ethanol and centrifuge it for

12000 rpm, 40 °C for 5 min.

• Discard the supernatant and dry the pellet and dissolve it in TE buffer and store

in -20 °C.

RESULT

Plasmid was .




Expt. No. 6

ASEPTIC CULTURE TECHNIQUES FOR ESTABLISHMENT AND MAINTENANCE

OF CULTURES

AIM

Aseptic culture techniques for establishment and maintenance of cultures

PRINCIPLE

Maintenance of aseptic environment

All the laboratory materials should be sterilized and importance to keep air surface free

of dust. All experiments should be carried out in laminar air flow which was a sterile

cabinet and sterilization should be done in autoclave, preparation of plant growth media

regulators by filter sterilization. Aseptic working condition and mainly explants used in

chemical sterilents like NaOCl.

STERILIZATION TECHNIQUES

Steam sterilization by Autoclaving

Using pressure cooker we can sterilize by super heated vacuum under pressure. The

size can be used as small or large in quantities of liters. Most of the media like nutrient

media sterilized in autoclave machine upto 115-135 °C. To achieve sterility conditions

for autoclaving has a temperature of 121 °C and a pressure of 15 psi (Pounds per

square inch) for 15 min. The time taken for reach this sterility was depends upon the

volume of media and quantity of vessel. The most efficiency wave to check autoclave by

using autoclave tape by indicating dark diagonal strips.

Precautions

• Level of water should be maintained correctly at the bottom of the autoclave

• The lids of autoclave was shuttled tightly




• Ensure the air exhaust should work correctly

• Excessive quantity of autoclave should be avoided

• Bottles should not be tightened at the time of autoclave

Filter Sterilization

Amino acids and vitamins are some growth regulators which are heat liable and get

destroyed while autoclaving. Therefore it was sterilized by the filter sterilization

technique using filter membranes of 0.22 µm to 0.45 µm size.

Irradiation

The process can be carried out only under UV radiation. This UV radiation will kill

microorganisms which are present in the room or work benches so if any other work

was going on we should not turn on UV lamp and also prolonged exposure will lead to

damage of skin and eye.

Laminar air flow cabinet

This was one of the primary equipment for sterilization. This was equipped with gas

corks, gas burners and HEPA filter to flow the air in direct lines. Before starting any

experiments that are mandatory to clean the working area with 70 % of ethanol to avoid

further contamination of microorganisms and mainly this HEPA filters should be cleaned

periodically.




Expt. No. 7

QUANTITATIVE DETERMINATION OF DNA /RNA USING SPECTROPHOTOMETER

AIM

To detect DNA and RNA using Spectrophotometer

PRINCIPLE

These both DNA/RNA exhibit strong UV lights due to strong purine and pyrimidine

bases and have an absorption maximum peak at 260 nm and optical density value of

2.0 to clarify the purity of RNA/DNA this spectrometric technique was performed.

Proteins and nucleic acids were the major contaminants of DNA/RNA which has

absorption peak at 280 nm. This shows the purified DNA/RNA or it contains some

contaminants. In optical density value less than 0.8 confirms it contains impurities like

proteins.

MATERIALS REQUIRED

SSC solution- Saline Sodium Citrate: Prepare 0.015 M solution of sodium citrate (pH

7.0) and dissolve NaCl so that its final concentration in sodium is 0.15M.

PROCUDURE

Take SSC solution as blank make 260 nm has zero absorbance in spectrophotometer

Take the provided sample and check the absorbance. If optical density value is high

again dilute the sample solution of SSC and take the reading again

CALCULATION

For double stranded DNA

Concentration of DNA in sample solution (µg/mL) = 50 x A260 x Dilution factor




For RNA

Concentration of RNA in sample solution (µg/mL) = 40 x A260 x Dilution factor

Expt. No. 8

DETRMINATION OF pH OF THE GIVEN SAMPLES USING pH METER

AIM

To determine the pH of the given unknown sample

PRINCIPLE

pH is a measure of hydrogen ion concentration which will convert into a logarithmic

scale so that the values are linear and can be remembered easily. pH meter is nothing

but a potentiometer which measures the voltage between two electrodes placed in a

solution. The two electrodes used are a calomel electrode and a glass electrode. The

calomel electrode is the external reference electrode whose electric potential is always

constant whereas the glass electrode is the standard test electrode whose electric

potential will depends on the test solution of pH. The electromotive form (EMF) of the

complete cell (E) is given by E = Eref - Eglass.

CHEMICALS REQUIRED

1. pH 4.0 buffer

Mix 0.0084 M of acetic acid and 0.0015 M of sodium acetate

2. pH 7.0 buffer

To 1 L of distilled water add 0.78 g (5mM) of monobasic sodium phosphate and

1.79 g (5mM) of dibasic sodium phosphate.

3. pH 9.2 Buffer

4. Unknown buffers

MATERIALS REQUIRED

pH meter, beakers, Tissue paper, double distilled water, wash bottle.




OPERATION OF INSTRUMENT

New or dry electrodes should be soaked in water or in a buffer of pH 6-7 overnight. The

electrode can also be achieved by soaking in 0.1 M HCL for 12 to 24 hours. Electrodes

should always be stored in distilled water or as per the manufacturer’s recommendation.

CALIBRION OF pH METER

1. Keep the controls as follows: Temp comp @ 30°C, pH/mv switch in pressed pH

position, check/read switch in pressed position (check position).

2. Display should be pH 7.00. If not adjust to 7.00. Set preset to do so. This reading

should be stable.

3. Wash the electrode with distilled water. Wipe off the moisture with tissue paper,

dip electrode in freshly prepared buffer of pH 7.00

4. After 30 s release to check/read switch to Read mode

5. Display should read pH 7.00 if not wait until fluctuations stabilize, then adjust

SET BUFFER control to do so.

6. Keep the switch in checked mode. Wash the probe thoroughly with distilled

water. Now, dip the probe in freshly prepared buffer of pH 4.00 and subsequently

to pH 9.20 buffers to calibrate the instrument.

7. After 30 s, de-press the check/read switch to Read mode. The reading on the

display should be 4.00 and pH 9.20 respectively. If not, adjust SET BUFFER

control to do so. The instrument is now calibrated.

8. Dip the electrode in to unknown sample, whose pH is to be finding out. Release

the check/read switch to read the pH value of the unknown sample.

9. After finding out the pH, wash and store the electrode in distilled water.

10. Switch off the instrument when not in use.




OBSERVATION

Sl. No

Unknown

sample

(1)

Unknown

sample

(2)

Unknown

sample

(3)

1

2

3

4

5

PRECAUTION

• Always keep the electrode in distilled water dipped upto at least 3 cm.

• Don’t allow probe to dry.

• Every time wash the probe with distilled water and rinsed with tissue paper.




RESULT: The pH of the given unknown sample is

Expt. No. 9

DETERMINATION OF END-POINT OF AN ACID-BASE TITRATION BY

USING THE CONDUCTIVITY METER

AIM

To determine the end-point of an acid-base titration by using the conductivity meter

PRINCIPLE

Electrolytic conductivity is a measure of the ability of a solution to carry an electric

current, due to the migration of ions. The electrical conductance is the reciprocal of

resistance, and its basic unit is the Siemens, formerly called mho. In the case of acid-

base titrations, the variation of electrical conductivity during the course of titration is

followed. The measured conductance is a linear function of the concentration of ions

present. The titrant is introduced by means of a burette and the conductance of ions

present. The titrant is introduced by means of a burette and the conductance readings

corresponding to various increments of titrant are plotted against the latter. The falling

branch represents the conductance of one species along with the salt formed due to the

neutralization. The rising branch represents the conductance of the opposite species

with the salts present. The point of intersection of the two branches gives the end-point.

The steepness of the branches depends upon the ionic conductance of the replaceable

and the replaced ions. The acuteness of the angle at the point of intersection of the two

branches will be a measure of the individual ionic conductance of the reactants. The

titrant should be at least ten times as concentrated as the solution being titrated in order

to keep the volume change is small.

CHEMICALS REQUIRED

a) 0.1 N HCL




b) 0.1 N H2SO4

c) 0.1 N NAOH

d) 0.1 N KOH

MATERIALS REQUIRED

Conductivity meter, beakers, tissue paper, double distilled water, wash bottle

OPERATION OF THE INSTRUMENT

Before use soak the conductivity electrode in distilled or deionized water for 5 to 10 min.

Connect the conductivity cell to the conductivity meter and follow the meter manual

instructions for standardizing the cell for use at a given temperature. Rinse the

conductivity cell sensing elements with distilled or deionized water between samples.

Note: Each conductivity cell has a cell constant which is predetermined by the

manufacturer and often indicated on the electrode upon shipment. The cell constant

may change slightly during shipping and storage should be measured on the user

conductivity meter before initial use. Measure the cell constant according to the meter

instruction manual. Because temperature has a large effect on conductivity

measurements allow probe to sit in solution until a stable temperature reading is

obtained before taking measurements.

CALIBRATION OF CONDUCTIVITY METER

1. Take 0.1 N KCL in a beaker and check the conductivity reading display in

conductance at 25°C and in 20 mS mode.

2. The reading should be 12.88, else adjust the reading for the same. Now the

instrument is calibrated and ready for use.

PROCEDURE

1. Prepare 0.1 N of the acids and bases.

2. Pipette out 50 mL of acid in a 250 mL of a beaker.

3. Transfer the base solution to the burette.

4. Dip the conductivity meter probe into the beaker containing 0.1 N acids.

5. Note the conductivity of the acid solution prior to titration.




6. Titrate the acid against base by noting the conductance after the addition of

every 2 mL of base.

7. Note the titer readings between which the conductance readings show an upturn.

8. Repeat the titration again this time nothing the conductance of the solution for

every 0.2 mL added at the mentioned range.

9. Plot a graph of conductance against the volume of base added.

10. Determine the end-point graphically.

PRECAUTION

Cleaning

The single most important requirement of accurate and reproducible results in

conductivity measurement is a clean cell. A dirty cell will contaminate the solution and

cause the conductivity to change, grease, oil, fingerprints and other contaminants on the

sensing elements can cause erroneous measurements and sporadic responses. For

most applications, hot water with domestic cleaning detergent can be used for cleaning

Storage

It is best to store cells so that the electrodes are immersed in deionized water. Any cell

that has been stored dry should be soaked in distilled water for 5 to 10 min before use

to assure complete wetting of the electrodes. Some platinum conductivity cells use to

assure complete wetting of electrodes. Some platinum conductivity cells are coated with

platinum black before calibration. This coating is extremely important to cell operation,

especially in solutions of high conductivity. Electrodes are platinized to avoid errors due

to polarization. Cells should be inspected periodically and after each cleaning. If the

black coating appears to be wearing or flaking off the electrodes or if the cell constant

has changed by 50 %, the cell should be cleaned and the electrodes replatinized.

Conductivity cells

Most conductivity meters have a two-electrode cell. The electrode surface is usually

platinum, titanium, gold-plated nickel or graphite. The four-electrode cell uses a

reference voltage to compensate for any polarization or fouling of the electrode plates.




The reference voltage ensures that measurements indicate actual conductivity

independent of electrode condition, resulting in higher accuracy for measuring pure

water.

OBSERVATION AND CALCULATION

Volume of base

added (mL)

Conductance

(mmho)

GRAPH

RESULT

The end point of the given acid-base titration is




Expt. No. 10

DETERMINATION OF SULPHATE CONTENT BY USING NEPHELOMETER

AIM

To estimate the concentration of sulphate in given unknown sample using

Nephelometer

PRINCIPLE

This method is based on a comparison of the intensity of light scattered by the sample

under defined conditions with the intensity of light scattered by a standard reference

suspension under the same conditions. The higher the intensity of scattered light, the

higher is the turbidity. Formazin polymer is used as the primary standard reference

suspension. Nephelometer consists of a light source (tungsten filament lamp) for

illuminating the sample and one or more photometric detectors with a readout device to

indicate intensity of light scattered at 90° to the path of incident light.

Keep sample cells scrupulously clean both inside and out. If it scratched or etched

discard it. Never handle them where the instruments light beam will strike them. Use

tubes with sufficient extra length, or with a protective case, so that they may be handled

properly. Fill cells with samples and standards that have been agitated thoroughly and

allow sufficient time for bubbles to escape. Clean samples cells by thoroughly washing

with laboratory soap inside and out by multiple rinses with distilled or deionized water.

A more popular term for this instrument in water quality testing is a turbidimeter.

However there can be differences between models of turbidimeter, depending upon the

arrangement (geometry) of the source beam and the detector. A Nephelometric

turbidimeter always monitors light reflected off the particles and not attenuation due to




cloudiness. The most common units of turbidity in the United States are called

Nephelometric turbity Units (NTU).

CHEMICALS REQUIRED

1. Standard sulphate solution

Dissolve 1.814 g of dry analytical R quality of potassium sulphate in distilled water

and dilute to one litre. This solution contains 1000 mg/L of sulphate.

2. Sodium chloride- hydrochloric acid reagent

Dissolve 60 g of analytical R quality sodium chloride in 200 mL of distilled water and

then add 5 mL of pure concentrated hydrochloric acid and dilute it to 250 mL.

3. Barium chloride

4. Glycerol-Ethanol solution

Dissolve one volume of pure glycerol in two volumes of absolute ethanol.

5. Formazin solution

The particles of formazin are uniform in size and shape. The stock standard of 4000

NTU is prepared as per the following procedure:

(i) Take 5 gm of reagent grade Hydrazine sulphate and dissolve in 400 mL of

distilled water. This is solution A.

(ii) Next dissolve 50 gm of pure Hexamethyl tetramine in 400 mL of distilled

water. This is solution B.

(iii) Mix solution A and B and make it up to one litre by adding distilled water and

allow this mixture to settle for 48 hours at normal room temperature.

This is stock solution of 4000 NTU strength of formazin. Working standards can be

prepared from this stock solution.

MATERIALS REQUIRED

Nephelometers, beakers, tissue papers, double distilled water, wash bottle, standard

volumetric flask and pipettes.


(I) Switch on the instrument ON and allow 10-15 min warm-up.




(II) Select the appropriate range

(III) Set the CALIB control to maximum clockwise position

(IV) Insert the test tube with distilled water into cell holder and cover with light

shield.

(V) Adjust SET ZERO controls to get zero on display

(VI) Remove the test tube and replace with the test tube containing standard

formazin solution.

(VII) Adjust CALIB CONTROL such that the display indicates as follows

Range Standard solution Display

0-1 NTU 1 NTU 1.00

0-10 NTU 10 NTU 10.0

0-100 NTU 100 NTU 100.0

0-1000 NTU 500 NTU 500

Preparation of standard solutions from Formazin stock solution (4000 NTU)

Working Standard For 50 mL

1 NTU 12.5 µL

10 NTU 125 µL

100 NTU 1250 µL

500 NTU 6250 µL

PROCEDURE

1. Pipette out 0.5, 1.0, 1.5, 2.5 and 3.0 mL of the standard potassium sulphate

solution from a burette into separate 100 mL volumetric flasks.

2. To each flask add 10 mL of the sodium chloride- hydrochloric acid reagent and

20 mL of the glycerol-ethanol solution.

3. Dilute to 100 mL with turbidity free distilled water.

4. About 0.3 g of sieved barium chloride to each flask.

5. Stopper and mix the contents of each flask for one minute by inverting and

making upright each flask once in a second.

6. The value of turbidity of the samples will be displayed on the Nephelometer.




7. The concentration of the sulphate in the sample is calculated by plotting a

standard graph of Nephelometer readings against concentration of standard

sulphate.


S.No Test samples Turbidity (NTU)

1

2

3

4

PRECAUTIONS

• Spillage on the sample holder is not advisable.

• Proper pipette out is highly required.

• Before reading the sample mix thoroughly.

RESULT

The given unknown concentration of the sample is




Expt. No. 11

DETERMINATION OF SODIUM USING FLAME PHOTOMETER

AIM

To determine the sodium content in the given sample

INTRODUCTION

The major cation of the extracellular fluid is sodium. The typically daily diet contains

130-280 Mmol (8-15 g) sodium chloride. The body requirement is for 1-2 Mmol per day,

so the excess is excreted by the kidneys in the urine. Hyponatraemia (lowered plasma

sodium) hypernatremia (raised plasma sodium) are associated with a variety of

diseases and illnesses and the accurate measurement of sodium in body fluids is an

important diagnostic aid, A traditional and simple method for determining sodium and

potassium in biological fluids involves the technique of emission flame photometry.

PRINCIPLE

This relies on the principle that an alkali metal salt drawn into a non-luminous flame will

ionize, absorb energy from the flame and then emit light of a characteristic wavelength

as the excited atoms decay to the unexcited ground state. The intensity of emission is

proportional to the concentration of the element in the solution. You are probably

familiar with the fact that if you sprinkle table salt (NACL) into a gas flame then it glows

bright orange (KCL gives purple colour). This is the basic principle of flame photometry.

A photocell detects the emitted light and converts it to voltage, which can be recorded.

Since sodium and potassium emit light of different wavelengths (Na: 589 nm, Ca: 622

nm, Li: 677 nm, K: 768 nm) by using appropriate coloured filters the emission due to

sodium and potassium can be specifically measured in the same sample. One

drawback of flame photometers, however, is that they respond linearly to ion

concentrations over a rather narrow concentration range so suitable solutions usually

have to be prepared. They also rather complex and relatively expensive machines, as

you will see.




A flame photometer can also be used to measure the element lithium in serum or

plasma in order to determine the correct dosage of lithium carbonate, a drug used to

treat certain mental disturbances, such as manic-depressive illness (bipolar disorder).

CHEMICALS REQUIRED

Standard Sodium solutions

Weigh accurately 2.5416 gm of Anal R quality of sodium chloride (NaCl) and transfer it

to 1 L standard flask and dissolve the crystals and dissolve the crystals using distilled

water and make up the solution to the mark. The stock standard solution contains 1000

mg/L sodium (Na). The stock solution is successively diluted further with double distilled

water to have a series of working standard solution.

Dilute the standard solution into 25, 50, 75 and 100 ppm.

MATERIALS REQUIRED

Flame photometer, beakers, tissue paper, double distilled water, wash bottle, pipettes.


1. Switch on the flame photometer, Digital display will turn on.

2. Ensure that the air tube, gas tube and the drain tube were properly connected.

3. Switch on the compressor. Ensure that the output pressure is close to 0.45

Kg/cm2 and is stable.

4. Dip the atomizer capillary tube in distilled water. Ensure that the regular droplets

fall in drain cup and drains out.

5. Set the fuel gas fine adjustment at ignite position

6. Switch on the fuel supply from the fuel source LPG cylinder and immediately

ignite the flame with the ignition through the ignition window.

7. Watch the flame through the flame view window. Do the fine adjustment of fuel

flow with the help of fuel gas fine adjustment valve to get a stable flame having

well defined cones.

8. Adjust the gas regulator to get a maximum height non-luminous blue flame.

9. Aspired distilled water to the atomizer and wait atleast for 30 seconds.




10. Aspirate distilled water into the flame and adjust emission reading to zero.

11. Using the highest concentration of working standard solution, the emission

reading is adjusted to 100.

12. Then the series of working standard solution in decreasing order aspirated into

the flame and the emission readings are noted.

13. The unknown sample is fed into the flame and the emission reading measured.

14. A standard graph is plotted using concentration of sodium Vs emission reading.

15. From the standard graph the concentration of sodium present in the unknown

sample is calculated.

OBSERVATIONS

S.No Concentration of

Sodium

(mg/ L)

Emission Reading

1

2

3

4

5

PRECAUTION

Cleaning of various units in a flame photometer

Atomizer and capillary tube

Flushing with copious amount of distilled water is adequate, if blockage occurs, remove

the atomizer from seating and flush with dry air or clean it using a thin wire, if cleaning

of atomizer is done with a wire before and after using it, blockage will rarely occur. If all

the above fails, a new atomizer to be fixed.




Mixing chamber

Flushing with distilled water is adequate. Do not use detergent or soap solution because

it will remain inside if washing is not done properly out and will give erratic due to the

presence of sodium/ potassium in the soap solution.

RESULT

The concentration of sodium present in the given unknown sample is found to be .




Expt. No. 12

ESTIMATION OF TOTAL DISSOLVED SOLID (TDS) IN WATER SAMPLE

AIM

To estimate the concentration of dissolved solids present in given water sample.

SIGNIFICANCE

• TDS concentration is the sum of cations and anions and thus provides a

quantitative measure of the amount of dissolved ions.

• TDS is used as an indicator test to determine the general water quality.

• By estimating the TDS concentration one can control the adverse effects.

• Water containing TDS concentrations below 1000mg/liter is usually acceptable to

consumers, although acceptability may vary according to circumstances.

However, the presence of high levels of TDS in water may be objectionable to

consumers owing to resulting taste and to excessive scaling in water pipes,

heaters, boilers, and household appliances. Water with extremely low

concentrations of TDS may also be unacceptable to consumers because of its

flat, insipid taste; it is also often corrosive to water-supply systems.

PRINCIPLE

A well-mixed measured portion of sample is filtered through a standard filter (Whattman

paper) to remove suspended solids and the filtrate is evaporated to dryness in a hot air

oven to give the amount of total dissolved solids.

MATERIAL REQUIRED

• Evaporating dish

• Balance

• Whattman filter paper

• Measuring cylinder

• Hot air oven




PROCEDURE

1. A clean dry evaporating dish of suitable size (to hold 100 mL of sample) was

taken and its initial weight (A) was noted.

2. 100 mL of water sample was filtered through the Whattman filter paper and the

filtrate was taken in the evaporating dish.

3. The sample (filtrate) was evaporated in hot air oven at 100ºC completely, cooled

and finally weighed (B).

FORMULA

Total dissolved solids (TDS) in mg/l =��

� � 1000

Where,

B – Weight of container after drying

A – Weight of container

V – Volume of sample taken.

Normal range of TDS in mainstream for discharge: 30mg/l.

CALCULATION

Water sample collected from one of the drains in VIT

A= B= V= 100 mL

Using the above formula TDS mg/l =

RESULT

The TDS present in 100 mL of sample is ______________




Expt. No. 13

ESTIMATION OF DISSOLVED NITRATE IN A GIVEN SAMPLE

AIM

To estimate the concentration of dissolved nitrate present in the given sample by

spectroscopic methods.

PRINCIPLE

Dissolved nitrate present in the water sample can be estimated by UV spectroscopy.

Nitrate and other dissolved salts absorb UV light at 220 nm. Other salts present in water

can also absorb UV light at 275 nm. Therefore, 220 nm can be used as a reading filter

and 275 nm as reference filter to estimate the exact concentration of nitrate present in

water sample.

MATERIALS REQUIRED

• Stock nitrate solution: (KNO3 – 0.7218 mg in100 mL distilled water )

• 1 N HCL

• Water sample

PROCEDURE

1. 8 test tubes where taken and labeled as B1, S1 - S5 and test1, test2.

2. As per the table, required amount of the stock solution was transferred to the

tubes marked as S1, S2, S3, S4 and S5.

3. Make the volumes of all the test tubes to 10 mL with distilled water.

4. 10 mL of test sample added to the ‘test 1’ and 200 µL of conc. HCl is added.

5. 10 mL of unknown concentration of KNO3 provided was taken in ‘test 2’.

6. OD was measured at 220nm and 275nm and exact OD was calculated.

7. Standard graph was plotted and concentration of nitrate present in the test

sample was determined from the standard graph.




8. Concentration of nitrate present in the sample was calculated using the given

formula

FORMULA

Total dissolved nitrate (mg/l) = Conc. of nitrate (mg)

× 1000

Conc. of sample (mL)

OBSERVATION

CONTENTS B S1 S2 S3 S4 S5 TEST 1 TEST2

Volume of KNO3(mL) - 2 4 6 8 10 - -

Volume of H2 O (mL) 10 8 6 4 2 - - -

Concentration

(µg/mL) - 2000 4000 6000 8000 10000 - -

Volume of test (mL) - - - - - - 10 10

Volume of HCL (µL) - - - - - - 200 -

O.D. at 220 nm

O.D. at 275 nm

Difference in O.D

Observation from the Graph

Concentration of nitrate =

RESULT

Amount of dissolved nitrate (mg/ mL) in the given sample is




Expt. No. 14

ESTIMATION OF BOVINE SERUM ALBUMIN BY BRADFORD METHOD

UV-VISBLE SPECTROPHOTOMETREY

AIM

To estimate the protein content by Bradford method

PRINCIPLE

The assay is based on the ability of proteins to bind to Coomassie brilliant blue G250

and from a complex whose extinction coefficient is much greater than that of free dye.

Binding of dye to the protein and the subsequent color formation is probably due to its

interaction with arginine residues which results in a shift of the wavelength maxima from

465 to 495 nm.

REQUIREMENTS

a) BSA (Bovine Serum Albumin) solution (1 mg/mL)

Dissolve 100 mg of BSA in 100 mL of phosphate buffer (0.2 M Na2HPO4 / Nah2PO4)

of pH 7.0

b) Phosphate buffer-

Solution A- Dissolve 53.65 g of Na2HPO4 in 1000 mL distilled water

Solution B- Dissolve 27.8 g of NaH2PO4 in 1000 mL of distilled water

Mix 61 mL of solution A and 31 mL of solution B and make up to 200 mL with

distilled water.

c) Coomassie Brilliant Blue G 250 (Bradford reagent) (CBB)

(i) Stock solution: Dissolve 100 mg of CBB in 50 mL of 95 % ethanol. Add 100 mL of

concentrated phosphoric acid. Add distilled water to a final

volume of 200 mL

(ii) Working solution: 1 volume of the stock is diluted 4 volumes of distilled water filter

if necessary using Whattman filter paper No. 1.

d) Spectrophotometer




PROCEDURE

1. Pipette out a series of standards of varying concentration (40-200 µg) using a

automatic dispenser. Volume pipette out is form 40 µL to 200 µL.

2. Make up to 200 µL using phosphate buffer of pH 7.0

3. Add 5 mL of Bradford reagent and mix well

4. Incubate for 5 min at room temperature

5. Measure the absorbance at 595 nm after 30 min.

6. Prepare a blank using the buffer under similar conditions

7. Plot a graph of absorbance at 595 nm against the concentration of BSA

8. Subject the unknown sample to a similar procedure


S.No Concentration of

BSA (µg/mL)

Absorbance

(nm)

RESULT

The protein content in the given sample is .




Expt. No. 15

SEPERATION OF SUGARS USING PAPER CHROMATOGRAPHY

AIM

To separate the given mixture of sugar samples using paper chromatography.

PRINCIPLE

Paper chromatography has been one of the most popular chromatography procedures

as it is the simplest one. It is a form of partition chromatography where a mixture of

solutes is separated on a sheet of paper. While one solvent is stationary and held in the

fibers of the paper (Stationary phase), the other solvent moves along the paper and is

called mobile phase. The components of the mixture to be separated migrate at the

different rates depending on their solubility in either phases and appear as spots at

different points on the paper. The distance moved by the individual solutes is denoted

as Retention front (Rf). Rf denotes the relative fronts of the solute and the solvent. This

is defined as the ratio of the distance moved by the solute front to that of the solvent

front. For a given developing solutions, the Rf is characteristic of the solute.

Identification of sugars using spraying agents is based on the principle that treatment of

sugars with acid/alkali at high temperatures lead to the cyclisation of the sugars to

furfural derivatives, which then condense with the dye giving characteristic colours.

CHEMICALS REQUIRED

1. Standard sugar solutions (2 % w/v) in water

2. Developing agent: n-butanol acid-water (4:1:1 v/v)

3. Spray reagent: Aniline or Diphenylamine phthalate- Dissolve 1g of either aniline or

diphenylamine and add 5 mL of HCL + 1 g of phthalic acid make it upto 100 mL with

ethanol.




PROCEDURE

1. Cut out a strip of Whattman No. 1filter paper 12 cm * 10 cm in size (Whattman No.1

for larger quantities is used).

2. Draw a thin line in pencil approximately 2 cm from one end of the paper

3. Using a capillary tube spot the given sugar sample, Keeping a distance of at least

1.5 cm between individual spots.

4. Ensure also that the first and the last spots are atleast 1 cm away from the either

ends.

5. The size of the spot should be as small as possible

6. In a tall beaker pour developing solvent to about 0.5 cm deep

7. Suspend the paper strip in the beaker in such a way that the edge of the paper is

just below the solvent level.

8. The paper should not be kept in a staining position nor should it touch the sides

9. Cover the beaker with a glass plate and leave the set-up undisturbed till the solvent

rises up and reaches almost the top edge.

10. Once the solvent reaches almost the top-edge, remove the paper and mark the

solvent front quickly.

11. The paper is then air dried for 10-15 min

12. The chromatogram is sprayed with the visualizing agent (Aniline or Diphenylamine

phthalate).

13. After spraying with this mixture, the chromatogram is kept at 100 °C for a few min,

when the individual sugars appear coloured spots on the paper.

Determine the Rf of the spots:

Rf = Distance moved by the solute front

Distance moved by the solvent front

OBSERVATION AND CALCULATIONS

S.No Sugar samples Fraction Decimal Rf

RESULTS

The relative front of the given sugars sample is .




Expt. No. 16

SEPERATION OF AMINO ACIDS USING PAPER CHROMATOGRAPHY

AIM

To separate the given mixture of amino acids using paper chromatography

PRINCIPLE

Paper chromatography has been one of the most popular chromatography procedures

as it is the simplest one. It is a form of partition chromatography where a mixture of

solutes is separated on a sheet of paper. While one solvent is stationary and held in the

fibers of the paper (Stationary phase), the other solvent moves along the paper and is

called mobile phase. The components of the mixture to be separated migrate at the

different rates depending on their solubility in either phases and appear as spots at

different points on the paper. The distance moved by the individual solutes is denoted

as Retention front (Rf). Rf denotes the relative fronts of the solute and the solvent. This

is defined as the ratio of the distance moved by the solute front to that of the solvent

front. For a given developing solutions, the Rf is characteristic of the solute.

The colour reaction is a condensation of individual amino acids with the highly reactive

central carboxy group of a 1, 2, 3-triketone (Ninhydrin). The reaction involves

condensation of the amino acid with a molecule of ninhydrin, decarboxylation and

hydrolysis of the condensed amino acid and further condensation of this complex with

one or more molecule of ninhydrin.

CHEMICALS REQUIRED

1. Standard amino acid solutions (1 % w/v) in water

2. Developing agent: n-butanol acid-water (4:1:1 v/v)

3. Spray reagent: Ninhydrin




PROCEDURE

1. Cut out a strip of Whattman No. 1filter paper 12 cm * 10 cm in size (Whattman No.1

for larger quantities is used).

2. Draw a thin line in pencil approximately 2 cm from one end of the paper

3. Using a capillary tube spot the given amino acid sample, keeping a distance of at

least 1.5 cm between individual spots.

4. Ensure also that the first and the last spots are atleast 1 cm away from the either

ends.

5. The size of the spot should be as small as possible

6. In a tall beaker pour developing solvent to about 0.5 cm deep

7. Suspend the paper strip in the beaker in such a way that the edge of the paper is

just below the solvent level.

8. The paper should not be kept in a staining position nor should it touch the sides

9. Cover the beaker with a glass plate and leave the set-up undisturbed till the solvent

rises up and reaches almost the top edge.

10. Once the solvent reaches almost the top-edge, remove the paper and mark the

solvent front quickly.

11. The chromatogram is sprayed with the visualizing agent (ninhydrin).

12. After spraying with the mixture the chromatograms are kept at 100° C for a few min,

when the individual amino acid appears purple spots.

13. Determine the Rf of the spots:

Rf = Distance moved by the solute front

Distance moved by the solvent front

CALCULATION OF THE RF VALUE:

S. No Amino acid Retention Factor

RESULT:

The relative front of the given amino acid sample is .




Expt. No. 17

ESTIMATION OF DISSOLVED OXYGEN CONCENTRATION OF

WATER SAMPLE

AIM

To estimate the dissolved oxygen (DO) content of given water sample by Azide

modification of Winkler’s method.

SIGNIFICANCE

1. To evaluate pollution strength of domestic and industrial effluents

2. To control rate of aeration during biological treatment of effluents

3. To determine rate of biological oxidation

4. To maintain desirable aquatic life. As dissolved oxygen drops, aquatic life are

threatened and in extreme cases killed

5. To maintain portability of water. As dissolved oxygen falls then undesirable

odor, taste and colour develop and reduces its acceptability

PRINCIPLE

Estimation of dissolved oxygen by azide modification method of Winkler's is an indirect

titrimetric method. Oxygen present in the sample oxidizes the divalent higher valence

which precipitates as brown hydrate, oxidized after the addition of NaOH and KI. Upon

acidification manganese reverts to its divalent state and liberates I2 from KI equivalent to

dissolve oxygen content in the sample. The liberated iodine is titrated against Na2S2O3

using starch as indicator. Disappearance of blue colour is the end point of titration. The

series of reaction take place can be summarized as:

The azide modification of Winkler test is used to determine the concentration of

dissolved oxygen in water samples.

In the first step, manganese (II) sulfate is added to an environmental water sample.

Manganese sulphate reacts with sodium hydroxide to give a white precipitate of

manganese hydroxide. In the presence of Oxygen brown manganese hydroxide is

formed.




MnSO4+2NaOH---Mn (OH)2{White precipitate}+K2SO4

4Mn (OH)2+O2+2H2O---4Mn(OH)3{reddish brown}

The second part of the Winkler test reduces (acidifies) the solution. Addition of sulphuric

acid dissolves the brown manganic sulphate which reacts instantly with Potassium

Iodide to yield Iodine.

2Mn (OH) 3+2H2SO4---2Mn (SO4)2{reddish brown}+6H2O

Mn (SO4)2+2KI---MnSO4+K2SO4+I2

In effect, Oxygen oxidizes Mn2+ to Mn4+and theMn4+oxidizes I- to I2. Iodine is then

determined titrametrically via titration with Sodium Thiosulphate with starch as an

endpoint indicator (deep blue).

Thiosulfate is used, with a starch indicator, to titrate the iodine. The amount of

dissolved oxygen is directly proportional to the titration value.

4Na2S2O3 (aq) + 2I2---- 2Na2S4O6 (aq) + 2NaI(aq)

REAGENTS

1. Concentrated Sulphuric Acid

2.0.025 N Sodium Thiosulfate

(Sodium Thiosulfate solution: Prepare daily. Dissolve 6.205 g sodium sulphite up

to one litre distilled water).

3. Starch indicator

4. Potassium Iodide/ Azide Reagent

(Alkali Azide solution: In a 1 litre volumetric, dissolve 500 grams sodium

hydroxide pellets with 150 grams potassium iodide. When dissolution is

complete, add an additional 40 mL of distilled water with 10 grams sodium Azide.

(Caution must be taken when handling this solution)

5. Manganese sulphate

(Manganese Sulphate Solution: Dissolve 364 grams manganese sulphate up to

one litre distilled water. Slight heating and filtration may be necessary).




MATERIALS/EQUIPMENTS

1. 300 mL BOD bottles with ground glass stoppers, and caps

2. Incubator set at 20 degrees Celsius

3. Series of pipettes (0.2 mL-10 mL)

4.100 mL burette

5. Conical flasks

PROCEDURE

The sample must be incubated within 48hours of its original sampling time. Analysis

after this point will have significant effects on the oxygen concentration within the

sample and may often lead to less than accurate results. Usually, the DO of the sample

will tend to decrease.

When the sample is first brought in for analysis, it must be maintained at a temperature

of approximately 4 degrees Celsius. Once in the lab, the pH of the sample must be

adjusted for analysis. The desired pH for this procedure is between 6.5 and 7.5 where

bacterial growth is possible. This is to ensure the fact that the oxygen concentration will

remain constant and also inhibit the further growth of organisms.

1. Carefully fill two 300 mL glass Biological Oxygen Demand (BOD) stopper'

bottles brim-full with sample water avoiding air bubble.

2. Immediately add 1 mL of manganese sulphate to the collection bottle by

inserting the calibrated pipette just below the surface of the liquid (If the

reagent is added above the sample surface you will introduce the oxygen in to

the sample). Care is taken that no bubbles are introduced via the pipette.

3. Add 1 mL of potassium-iodide reagent in the same manner.

4. Stopper the bottle with care to be sure no air is introduced. Mix the sample by

inverting the several times. Check for air bubbles; discard the sample and start

over if any are seen. If oxygen is present, a brownish-orange cloud of

precipitate or flocs will appear. When these flocs have settle to the bottom, mix

the sample by turning it upside down several times and let it settle again.

5. Add 1 mL of concentrated sulphuric acid via a pipette held just above the

surface of the sample. Carefully stopper and invert several times to dissolve the




flocs. At this point the sample is “fixed” and can be stored for up to 8 hours if

kept in cool, dark place. AS an added precaution, squirt distilled water along the

stopper, and cap the bottle with aluminum foil and a rubber band during the

storage period.

6. In a glass flask, titrate the volume of sample which corresponds to 100 mL of

the original sample with sodium thiosulphate to a pale straw color. Titrate by

slowly dropping titrant solution from calibrated pipette into the flask and

continually stirring or swirling the sample water.

Volume of sample to titrate = 100 x300/ (300-2) =100.67 mL.

7. Add small amount of starch solution so a blue colour forms.

*Note: If a solution has no reddish brown colour or only slightly coloured, add a

small quantity of starch indicator. If no blue colour develops, there is no

dissolved oxygen.

8. Continue slowly titrating until the sample turns clear.

9. The concentration of dissolved oxygen in the sample is equivalent to the

number of milliliters of titrant used. Each mL of sodium thiosulfate added in

steps 6 and 8 equals 1 mg/L dissolved oxygen.

Observation

S.No Titrate

value

Normality of

Titrant

Volume

of sample

Dissolved

Oxygen

1

Calculation

A. Calculating the DO in the sample using the formula-

DO [mg/ mL] = (mL) titrant value x Normality of titrant x8000

Volume of sample (mL)

RESULTS

The value of dissolved oxygen (DO) content of given water sample _________




Expt. No. 18

DETERMINATION OF BIOLOGICAL OXYGEN DEMAND OF GIVEN WATER

SAMPLE

AIM

Determination of BOD of the water sample using Azide modification of Winkler's Method

PRINCIPLE

Biochemical Oxygen Demand is a common environmental procedure for determining

the extent to which oxygen within a sample can support microbial life. B.O.D. is the

amount of dissolved oxygen needed by aerobic biological organisms in a body of water

to break down organic material present in a given water sample at certain temperature

over a specific time period. The term also refers to chemical procedure for determining

this amount it is widely used as an indicator of the organic quality of water. The BOD

value is most commonly expressed in milligrams of oxygen consumed per litre of

sample during 5 days of incubation at 20°C in BOD incubator providing suitable

environment for bacterial growth.

The test for Biochemical Oxygen Demand is especially important in waste water

treatment, food manufacturing and filtration facilities where the concentration of oxygen

is crucial to the overall process and end products. High concentrations of dissolved

oxygen (DO) predict that oxygen uptake by microorganisms is low along the required

break down of nutrient sources in the medium (sample). On the other hand, low DO

readings signify high oxygen demand from microorganisms, and can lead to possible

sources of contamination depending on the process.

REACTION MECHANISM

The Azide modification of Winkler test is used to determine the concentration of

dissolved oxygen in waste samples.

In the first step, manganese (II) sulphate is added to an environmental water sample.

Manganese Sulphate reacts with sodium hydroxide to give a white precipitate of




manganous hydroxide. In the presence of Oxygen brown manganic hydroxide is

formed.

MnSO4 +2NaOH---Mn(OH)2 {white precipitate} +K2SO4

4Mn (OH)2 +O2 +2H2O---4Mn(OH)3{reddish brown}

The second part of the Winkler test reduces (acidifies) the solution. Addition of

Sulphuric acid dissolves the brown Manganic Sulphate which reacts with instantly with

Potassium Iodide to yield Iodine.

2Mn (OH)3 +2H2SO4---2Mn(SO4)2{reddish brown)}+6 H2O

Mn (SO4)2 +2KI---MnSO4+K2SO4+I2

In effect, Oxygen oxidizes Mn2+ to Mn4+ and the Mn4+ oxidizes I- to I2. Iodine is then

determined titrametrically via titration with Sodium Thiosulphate with starch as an

endpoint indicator (deep blue).

Thiosulphate is used, with a starch indicator, to titrate the iodine. The amount of

dissolved oxygen is directly proportional to the titration value.

2Na2S2O3 (aq) +2I2→→→→2Na2S4O6(aq) + 4NaI(aq)

REAGENTS

1. Concentrated Sulphuric Acid.

2. 0.025 N Sodium Thiosulphate

(Sodium Thiosulfate solution: Prepare daily. Dissolve 6.205 g of Sodium sulphite

up to one litre distilled water).

3. Starch indicator.

4. Potassium Iodide/Azide Reagent:

(Alkali Azide solution: In a 1 litre volumetric, dissolve 500 gram sodium hydroxide

pellets with 150grams potassium iodide. When dissolution is complete, add

additional 40 mL distilled water with 10 grams sodium Azide. Caution must be

taken when handling this solution)

5. Manganese sulphate

(Manganese Sulphate solution: Dissolve 364 grams manganese sulphate up to

one litre distilled water. Slight heating and filtration may be necessary).




MATERIALS / EQUIPMENTS

1.300 mL BOD bottles with ground glass stoppers, and caps

2. Incubator set at 20 degrees Celsius

3. Series of pipettes (0.2 mL-10 mL)

4. 100 mL burette

5. Conical flasks

PROCEDURE

The sample must be incubated within 48hours of its original sampling time. Analysis

after this point will have significant effects on the oxygen concentration within the

sample and may often lead to less than accurate results. Usually, the DO of the sample

will tend to decrease.

When the sample is first brought in for analysis, it must be maintained at a temperature

of approximately 4 degrees Celsius. Once in the lab, the pH of the sample must be

adjusted for analysis. The desired pH for this procedure is between 6.5 and 7.5 where

bacterial growth is possible. This is to ensure the fact that the oxygen concentration will

remain constant and will also inhibit the further growth of organisms.

1. Carefully fill two 300 mL glass Biological Oxygen Demand (BOD) stopper' bottles

brim-full with sample water avoiding air bubble.

2. One bottle is kept in the incubator for 3 days. The other bottle is used to determine

the Dissolved Oxygen using Azide modification of Winkler Method.

3. Immediately add 1 mL of manganese sulphate to the collection bottle by inserting

the calibrated pipette just below the surface of the liquid (If the reagent is added

above the sample surface, you will introduce oxygen into the sample). Care is taken

that no bubbles are introduced via the pipette.

4. Add 1 mL of alkali-iodide-azide reagent in the same manner.

5. Stopper the bottle with care to be sure no air is introduced.Mix the sample by

inverting several times. Check for air bubbles; discard the sample and start over if

any are seen. If oxygen is present, a brownish-orange cloud of precipitate or flocs




will appear. When these flocs have settle to the bottom, mix the sample by turning it

upside down several times and let it settle again.

6. Add 1 mL of concentrated sulphuric acid via a pipette held just above the surface of

the sample. Carefully stopper and invert several times to dissolve the flocs. At this

point, the sample is “fixed” and can be stored for upto 8hours if kept in a cool, dark

place. As an added precaution, squirt distilled water along the stopper, and cap the

bottle with aluminum foil and a rubber band during the storage period.

7. In a glass, titrate the volume of sample which corresponds to 100 mL of the original

sample with sodium thiosulphate to a pale straw colour. Titrate by slowly dropping

titrant solution from a calibrated pipette into the flask and continually stirring or

swirling the sample water.

This volume is calculated as

Volume of sample to titrate = 100x 300/(300-2) =100.67 mL

8. Add small amount of starch solution so a blue colour forms.

*Note: If a solution has no reddish brown colour or only slightly coloured, add a

small quantity of starch indicator. If no blue colour develops, there is no dissolved

oxygen.

9. Continue slowly titrating until the sample turns clear. As this experiment reaches the

endpoint, it will take only one drop of the titrant to eliminate the blue colour. Be

especially careful that each drop is fully mixed into the sample before adding the

next. It is sometimes helpful to hold the flask up to a white sheet of paper to check

for absence of the blue colour.

10. The concentration of dissolved oxygen in the sample is equivalent to the number of

milliliters of the titrant used. Each mL of sodium thiosulphate added in steps 6and

8equals 1mg/L dissolved oxygen.

11. The above method to determine the DO of water is repeated again for the other

sample kept in the BOD incubator after three days.




Calculation

A. Calculating the DO in the sample using the formula-

DO [mg/mL] = (mL) titrate value x Normality of titrant x800/Volume of sample (mL)

B. The BOD of the sample is calculated by subtracting the final DO from the initial DO

and multiplying this number by the dilution factor:

BOD Sample = (DO1-DO3) X Dilution factor per 300 mL

Observation

S.NO

Titrate

value

Normality

of Titrant

Volume of

Sample

Dissolved

Oxygen

DO1

DO3

BOD =

RESULTS

The value of BOD of the water sample ____________




Expt. No. 19

DETERMINATION OF CHEMICAL OXYGEN DEMAND OF GIVEN WATER SAMPLE

AIM

To estimate the chemical oxygen demand of the given water sample.

SIGNIFICANCE

• Indication of pollution in water

• Determination of the amount of chemically oxidizable substance present in water

• When used in conjugation with BOD test context is helpful in indicating the

presence of biologically resistant organic substance

• Determination of the strength of domestic and industrial waste in terms of oxygen

required

• Like BOD, COD is also used to design waste water treatment process

• COD is one of the standards for effluents discharge

• COD depends on the presence of biologically inert substance present in water.

• COD determines an advantage over the BOD determination in which the result

can be obtained in about 3hours when compared to 3-5days required for BOD

• The test is relatively easy which gives reproducible results and is not affected by

interferences as in BOD test

PRINCIPLE

The organic matter and oxidizable inorganic matter present in the water sample gets

oxidizable completely by the chemical oxidant K2Cr2O7which liberates O2in the

presence of an acid .The excess O2 is allowed to react with H2SO4 at 100 °C. Because

of its unique chemical properties, the dichromate ion is the specified oxidant; it is

required to chromic ion in this test.




MATERIALS REQUIRED

• Glass wares

• Conical flask

• Burette

• pipette

REAGENTS REQUIRED

• Potassium Dichromate solution

• Conc. H2SO4

• Ferroin indicator

• 0.1 N Ferrous Ammonium sulphate

PROCEDURE

1. Take two clean test tubes

2. Add 2.5 mL of sample water in one test tube and 2.5 mL of distilled water in other

test tubes and mark respectively

3. Add 1.5 mL of K2Cr2O7and keep in a water bath at 100°C

4. Add 3.5 mL of conc.H2SO4 in both the test tubes. Mix well

5. Keep in water bath for about for 2hours at 100°C

6. Cool to room temperature

7. Transfer the contents to conical flask

8. Add 2 drops of Ferroin indicator and titrate using ferrous ammonium sulphate

9. The titration is continued till the bluish green turns to reddish brown.

CALCULATION

Working formula:

COD (mg/L) = (A-B) x N x 8000/ mL of sample

Where, A = mL of FAS for blank; B = mL of FAS for sample,

N= Normality of FAS; S= Sample taken




OBSERVATION

RESULT: The value COD for the given water sample is_________




Expt. No. 20

ISOLATION OF XENOBIOTIC DEGRADING BACTERIA BY USING SELECTIVE

MEDIA

AIM

To isolate the Xenobiotic degrading bacteria from the given sample using selective

media

INTRODUCTION

Xenobiotic compounds are human made chemicals that are present in environment at

naturally high concentration. The Xenobiotic compounds are either not produced

generally or produced at lower concentration. Microorganisms have the capability of

degrading all naturally occurring compounds. This is known as principle of microbial

infallibility proposed by Alexander in 1965. Microorganisms are also able to degrade

many of Xenobiotic compounds, but they are unable to degrade many others.

Compounds which resist biodegradation and thereby persist in the environment are

called recalcitrant.

Few common examples of the types of recalcitrant compounds:

1. Halogenated hydrocarbon

2. Polychlorinated biphenyls

3. Synthetic polymer

4. Alkyl benzyl sulfonate

5. Oil mixture

PRINCIPLE

Xenotypic compounds include chlorinated solutions, herbicide, pesticides, etc. They

often carry uncommon chemical structures side chain or functional groups which can

either have toxic effects on bacteria or provide specific carbon source which can be

metabolized by specialized group of microorganism which have their metabolism to

degrade Xenobiotic compound. Microorganism play important side role in degradation




of Xenobiotic and maintaining steady state concentration of chemicals in the

environment. Complete biodegradation of an organic molecule to its inorganic

components that can be used in oxidative cycle removes its potential toxicity from the

environment.

MATERIALS REQUIRED

• Petri plates

• Micropipette

• Test tubes

• Xenobiotic compound – chlorpyrifos (0.01mg/mL)

• M9 Minimal media

• Conical flask

• Sample: A Serially diluted soil sample collected from one of the gardens in VIT,

usually treated with chemical pesticides.

PROCEDURE

1. One gram of soil sample was serially diluted using sterile distilled water till 10-4

dilution factor.

2. Five Petri plates were taken and marked as:

a. Control with Xenobiotic compound

b. Control without Xenobiotic compound

c. Three dilutions used 10-1, 10-3 and 10-4.

• The sterilized M9 Minimal Media without Xenobiotic compound was poured in

one control

• 100 µL of Xenobiotic compound was then added to the remaining media and

poured in all the four plates

• The plates are inoculated with the respective serially diluted sample using spread

plate technique

• The plates were carefully sealed with the paraffin tape and incubated at 37° C




OBSERVATION

RESULT




Expt. No. 21

ISOLATION OF HYDROCARBON DEGRADING MICROORGANISM

AIM

To isolate microorganism from the soil sample using hydrocarbon as one of the

substance component in the media

PRINCIPLE

In pure cultures studies, it has been demonstrated that when regular carbon source in

synthetic media(such as glucose, sucrose, etc.) are replaced by hydrocarbon such as

paraffin, kerosene, gasoline, and lubricating oil, there is growth of certain microorganism

utilizing hydrocarbons. It is known that both saturated and unsaturated molecules are

degraded by certain microbes, the latter being more vulnerable. Many fungi, bacteria,

Actinomycetes are known to degrade hydrocarbon; for instance: paraffin is metabolized

by Mycobacterium, Nocarida, Streptomyces, Pseudomonas, Flavobacterium, and

several fungi. High molecular weight hydrocarbons are also degraded some of the same

group of organism.

MATERIALS REQUIRED

• Petri plates

• Micropipette

• Test tubes

• Nutrient media

• Any hydrocarbon(here 0.1 % benzene was used)

• Conical flask

• Sample: A serially diluted soil sample collected from the petrol pump near VIT

was used for inoculation.




PROCEDURE

1. One gram of soil sample was serially diluted using sterile distilled water till 10-4

dilution factor.

2. Five Petri plates were taken and marked as:

a. control with hydrocarbon

b. control without hydrocarbon

c. Three dilutions used 10-1, 10-3 and 10-4.

3. The sterilized nutrient agar without benzene was poured in one control.

4. 100 µL of benzene was then added to the remaining media and poured in all the

four plates.

5. The plates were inoculated with the respective serially diluted sample using

spread plate technique.

6. The plates were carefully sealed with the paraffin tape and incubated at 37° C

OBSERVATION

RESULT




Expt. No. 22

ISOLATION OF DEGRADATIVE PLASMIDS IN MICROBES GROWING IN

POLLUTED ENVIORNMENT

AIM

Isolation of plasmids DNA from bacteria by alkaline Lysis method

PRINCIPLE

The basis to this technique is that there is a narrow pH range at which non super coiled

DNA is denatured whereas super coiled plasmids are not. If sodium hydroxide is added

to cell extract so that the pH is adjusted to 12.0-12.5, then the hydrogen bonding in non-

super coiled DNA molecules will be broken, causing the double helix to unwind and the

two polynucleotide chain to separate. If acid is now added, these denatured bacterial

DNA strands will aggregate into a tangled mass. Centrifugation will pellet the insoluble

network leaving pure plasmid DNA in the supernatant, Addition of isopropanol further

precipitate the plasmid DNA.

REQUIREMENTS

The 24 h LB broth of the bacterial strain expected to contain plasmid, Glucose, Tris

chloride, EDTA, Sodium hydroxide, SDS, Potassium acetate, Glacial acetic acid,

isopropanol, absolute alcohol, conical flask, cotton plugs, aluminum foil, centrifuge,

paper towel, ice flakes, etc.

PREPARATION OF REAGENTS

1. Solution 1

Glucose 50 mM

Trischloride 25 mM

EDTA 10mM

2. Solution II

NaOH 0.2 N

SDS 1 %




3. Solution III

Potassium Acetate (5M) - 60 mL

Glacial acetic acid - 11.5 mL

Autoclave water - 28.5 mL

4. 75 % Ethanol

PROCEDURE

1. Hundred milliliters of autoclave LB media was taken in a conical flask of 250 mL.

2. To the media was added 100 µL of inoculums.

3. Culture was incubated at 37 °C overnight in a shaker at 70 revolution/min.

4. 1.5 mL of the bacteria culture was taken in a 2 mL Eppendorf tube.

5. Harvesting of the bacteria was done by subjecting the culture at 3000 rpm for 10 min

at 4 °C.

6. Supernatant was removed and then pellet was collected.

7. To the pellet was added 30 µL of solution I. It was then mixed nicely by vortexing

and then mixing with pipette. Further it was incubated in ice for 10 min.

8. Tube was then taken from ice and to it was added 60 µL of freshly prepared solution

II. It was mixed gently and then kept at room temperature for 10 min.

9. To the same tube was added 45 µL of solution III. It was then kept in ice for 10 min.

10. Centrifuge the bacterial lysates for 30min at 8000-12000rpm

11. Carefully decant the supernatant.

12. 0.6 volume of isopropanol was added to the supernatant. Mix it and then keep it at

room temperature for an hour.

13. Recover the plasmid by centrifuging it at 3000rpm for 30min.

14. Decant the supernatant.

15. Collect the pellet.

16. Wash the pellet with 75 % of ethanol.

17. Dry the pellet by keeping it in desiccators for 30 min or drying it under fan.

18. Dissolve it in 29 µL of autoclave triple distilled water.

19. Run it on 1 % agarose gel.

RESULT




Expt. No. 23

SEPARATION OF DNA FRAGMENTS BY AGAROSE GEL ELECTROPHORESIS

AIM

To analyze the given DNA sample using agarose gel electrophoresis

PRINCIPLE

Separation of molecules on the basis of their net electric charge is called

electrophoresis is used as a method to separate bio-molecules based on the charge

size and shape under the influence of electric current. Agarose gel is a polysaccharide

derivative of agar. It contains microscopic pores which acts a molecular sieve. The

sieving properties of gel influence the rate at which the molecular migrates. The rate of

migration of molecules depends on the two factors its shape and electric charge. The

smaller molecules move through the spores more easily than the larger ones. Gel

electrophoresis separate DNA molecules according to their size. The dye ethidium

bromide interchelates between the bases of DNA of give fluorescence bromide orange

colour when irradiated with UV.

MATERIALS REQUIRED

• Electrophoresis tank

• Power pack

• Insulated gloves

• Micropipette

• Cellophane tape

• Agarose

• Ethidium bromide

REAGENTS REQUIRED

• Gel electrophoresis buffer-TBE (Tris chloride, Boric acid)

• Gel loading dye (Bromophenol blue, Xylenecynol, Glycerol)




• Agarose 0.8 g in 100 mL of 1x TBE buffer

• Ethidium bromide

PROCEDURE

• Wipe the gel platform with cotton piece saturated with absolute ethanol and seal

the open ends with cello tape

• Dissolve 0.8 g of agarose in 80 mL of triple distilled water and boil it under

microwave oven for 2 min till the agarose get dissolved

• Add 20 mL of 5x TBE buffer so that the final concentration will be 1x

• Allow the flask to cool at 50°C and add 5µl of Ethidium bromide

• Place the comb in notched end of the platform and pore agarose solution

• Allow the solution to solidify

• Remove the comb as well as cello tape and place the platform in the

electrophoresis tank fixed with 1x buffer

• Mix DNA sample with loading dye and pour the sample into the coasted well

• Connect the electrode to the power supply and maintained at 100 Volt

• Run the gel for 1 hr depending on the size of the gel

• Disconnect the power supply and remove the gel from gel platform place it on the

transmit illuminator and view for the fluorescent bands

RESULT




Expt. No. 24

ISOLATION OF INDUSTRIALLY IMPORTANT MICROORGANISMS

AIM

To isolate industrially important microorganisms

INTRODUCTION

Most of the microorganisms were used to make or produce products which are useful to

human beings. These microorganisms stated to be industrial microorganism such as

Yeasts, Actinomycetes and Bacteria. In this experiment we are going to isolate

streptomycetes from soil because these streptomycetes doesn’t have any pathogens

and also it play a role in antibiotics. It was one of the best genus in family

Streptomycetaceae majorly found in the soil by method of zone of inhibition.

PROCEDURE

• Collect the soil samples from different locations in and around Vellore by

removing soil surface and collect sample from 10 cm depth

• Place them in a air tight polythene bag and keep in a refrigerator

• Mix the soil samples and dilute it to the ratio of 1gm in 100 mL of distilled water

on a water bath shaker.

• And make serial dilution up to 10-6 and spread (0.1 mL) over the surface of

glycerol nitrate casein agar by sterile L shaped rod.

• Incubate plates at room temperature and count the number of colonies after 10

days by colony counter

• Select some clear colonies and purify them by repeated streaking

RESULT

Industrially important microorganisms were .




Expt. No. 25

GRAM STAINING

AIM

To perform gram staining to differentiate bacteria into positive and negative categories

PRINCIPLE

Differential staining requires the use of atleast these chemical reagents that are applied

sequeantly to heat fixed reagent

PRIMARY STAIN

Crystal violet function was important to all the cells which acts have a colouring agent. It

was followed by an iodine solution acting as a mordant.

DECOLOURISING AGENT

Based on the chemical composition of cellular constituents, the decolorizing agent may

or may not remove the primary stain for the entire cell structure.

COUNTERSTAIN

It has different colour than primary stain post decolourization. Only if the primary stains

is washed out. Safranin was generally used for the counter stain to absorb different

colour for cells. The gram positive bacteria have a thick peptidoglycan layer which

presents the decolorizer from washing away the primary stain. Whereas for gram

negative bacteria with a thin peptidoglycan and considerable lipid content the primary

stain is washed off by the lipid soluble decolorizer and the counter stain gets in. Thus

gram positive bacteria remains one colour (purple), while the gram negative bacteria

(pink).

MATERIALS REQUIRED

• Broth cultures of klebsiella pneumoniae




• E.coli

• S.aureus

REAGENTS

• Crystal violet

• Gram`s decolorizer

• Safranin iodine wordant

• Slide

• Marker

• Staining rack

• Microscopes

PROCEDURE

• Heat fixed smears of E.coli, S.aureus are prepared on clear glass marked glass

slides

• Slide were placed on a staining rack

• Smears are flooded with crystal violet and let stand for 30 sec to 1 min

• It was rinsed with water 5 sec

• The slide was then covered with grams iodine for 1 min

• It was decolorized with 95% ethanol for 15 to 30 sec but not for too long. It was

added drop by drop till crystal violet washer of completely

• Alternatively, smears may be decolorized for 30-60 sec with a mixture of

isopropanol acetone

• Then rinsed with water for 5 sec

• The slide was counterstained with Safranin for about 30 sec

• It was again rinsed with water for 5 sec

• The slide was blotted dry with bibulous paper and examined under oil immersion




OBSERVATION

RESULTS




Expt. No. 26

CAPSULE STAINING

AIM

To distinguish capsule forming bacteria by Anthony’s procedure of capsule stain

PRINCIPLE

Capsule is a gelatinous outer layer surrounding by bacteria and adhering to the cell

wall. Capsule composition as well as thickness varies with individual bacterial species,

depending upon its relative content of polysaccharides, polypeptides and glycoprotein.

Generally capsulated bacteria are more virulent or pathogenic than others. But one

cannot determine the presence of capsule using procedures for simple staining

Anthony`s procedure is used crystal violet is the primary stain that adheres to the cell

structure. Copper sulphate as a decolorizing agent removed excess stain as well as a

counter stain and is absorbed by the capsules to give a lighter shade. Capsular

materials are water soluble and may be dislodged due to vigorous washing.

MATERIALS REQUIRED

• Broth of Klebsiella pneumonia

• Clean glass slide

• Inoculating loop

• CuSO4

• Crystal violet

PROCEDURE

• Clean glass slide was taken and marked for identification

• Bacterial smear was prepared by aseptically transferring loop full of culture using

an inoculating loop and air dried. Air dried without heat fixing. Smears should not

be heat fixed because the resultant cell shrinkage may give illusion of capsule




• After placing the slide on a staining tray it was flooded with crystal violet and

allowed to stand for 4-7 min

• The slide was washed with 20% CuSO4

• The slide was gently blot dried with bibulous paper and observed under 10x, 40x

and oil immersion.

OBSERVATION

RESULT




Expt. No. 27

SPORE STAINING

AIM

To distinguish bacterial endospores using Schaeffer-Fulton`s staining technique.

PRINCIPLE

Certain bacteria such as bacillus and clostridium produce a resistant, metabolically in

active structure, capable of surviving in unfavorable environments for long periods. The

structure is called endospores as it develops within the cell. They are generally

spherical, elliptical with characteristic position in the cell with calcium dupicolinic acid

and number of layer in its cell wall

The primary stain malachite green is applied to a heat fixed smear and heated to

enhance the penetration of dye by steaming into relatively impermeable super coat.

They are strongly stained by malachite green while the rest of the cell is counter stain

with a light red Safranin

MATERIALS REQUIRED

• 48-72 hr broth culture of bacillus reagents

• Safranin

• Malachite green

• Inoculating needle

• Boiling water bath

• Glass slide

PROCEDURE

• The smear was prepared by aseptically transferring a loop full of bacteria to the

slide and it was spread out air dried and heat fixed

• Smear was flooded with malachite green placed over a water bath for 2-3 min in

the ensuring steam




• Stain should not allowed to evaporate and should be replenished if needed, stain

should be presented from boiling

• Slides are removed cooled and washed under running tap water

• Smear was counterstained with Safranin for 60 sec and gently washed under tap

water.

• Smear was blot dried with bibulous paper and is examined under 10 x, 40 x and

finally oil emersion

OBSERVATION

RESULT




Expt. No. 28

METHYL RED AND VOGES PASKAUR TEST

AIM

To distinguish between mixed acid fermentors and butane diol fermentors by MR and

VP tests.

PRINCIPLE

May bacteria especially enterobacteria can metabolize pyruate to formic acid and

fermentation to H2O and CO2 is called formic acid and fermentation. These are two

types

Mixed acid fermentation

Butane diol fermentation

MR TEST

The bases of this test is the fact that mixed acid fermentor procedure 4 times more

acidic acid products than butane and pathway, acidifying media, such that pH drops

between 4.4 and methyl red indicator turns red instead of yellow.

VP TEST

Organism’s futualing butane diol pathway produce acetanin and 2,3-butanediol as end

products when treated with and napthol (Burette A) and 40 % of KOH (Burette B) end

products are oxidized and a pink complex is formed. Development of deep rose colour

15 min following the addition of burette indicator positive for VP.

REQUIREMENTS

• 24 - 48 hrs culture of E.coli and K. pneumonia

• MR-VP broth

• Test tubes and inoculating loop

• Methyl red indicator




PROCEDURE

• MR-VP broth was prepared and dispensed equally into 2 sets of test tubes

• Test tubes were aseptically inoculated with E.coli and K. pneumonia respectively

for VP test

• 0.5 mL of MR reagent was added to the remaining test tubes, shaken and

inoculated with test organisms

• Incubated for 24 hrs at 37°C

• VP test tubes were then treated with Burette A and Burette B

• Then results was observed

OBSERVATION

RESULT




Expt. No. 29

CITRATE UTILIZATION TEST

AIM

To demonstrate the ability of an organisms to utilize citrate as a sole carbon source

PRINCIPLE

Some organism possess citrate permease enzyme which exhibits them to use citrate as

sole carbon source. In these organisms citrate is acid on by citrase producing acetate

finally CO2 and pyruvic acid. Carbon that reacts with sodium and water to form sodium

carbonate an alkaline product. This brings up the pH changing bromothymol blue

indicator to the medium from green and deep purple blue following incubation citrate to

positive cultures are identified by growth on slant surface accompanying the blue

colorization.

REQUIREMENTS

• 24-48 hrs culture of E.coli and K. pneumonia

• Citrate agar and other lab instruments

PROCEDURE

• Simmons citrate agar was prepared and set into slants. Organisms were

streaked and incubated at 37°C for 18 -24 hrs and observed.

RESULT




Expt. No. 30

UREASE PRODUCTION TEST

AIM

To demonstrate the presence of Urease in the given bacterium

PRINCIPLE

Urea is di-amides of carbonic acid. Urease, the enzyme possessed by some bacterium

hydrolysis urea and releases ammonia acid and carbon dioxide. Ammonia reacts in

solution to form ammonium carbonate which is alkaline leading to increase in pH.

Phenol red which is incorporated to the medium changes its colour from yellow to

pinkish red in alkaline pH thus indicating the presence of Urease activity.

MATERIALS REQUIRED

• 24-48 hrs culture of E.coli and K. pneumonia

• Distilled water

• Inoculation loop

• Test tubes

• Christiansen`s agar

PROCEDURE

• Christiansen`s urea agar was prepared and set into slants

• The test organisms were streaked on the surface of slant and incubated at 37°C

for 18-24 hrs and observed

RESULT




Expt. No. 31

OXIDASE TEST

AIM

To demonstrate the presence of enzyme oxidizes in the given organisms

PRINCIPLE

Oxidase enzymes play an important role in the operation of the electron transport

system during aerobic respiration. Cytochrome oxidase uses O2 as an electron acceptor

during the oxidation of reduced cytochrome C to form water. The ability of bacteria to

produce cytochrome oxidase test reagent (Oxidase test disk) to calories that have gram

staining on a plate medium or using a wooden applicator stick. Bacterial sample can

either be rubbed on a dry side oxidase test reagent serves as an artificial substrate,

denoting electrons to cytochrome oxidase and in process becoming oxidized to a purple

and then dark purple colorization on the colonies. Indicator absence of oxidase and is a

negative test.

REQUIREMENTS

• Reagent impregnated disks

• P. aeruginosa

• Lab wares

PROCEDURE

• One of the reagent disks was carefully picked out using a sterile holder and

rubbed over one of the colonies in the cultured petriplate.

• The part of the disk in contact with the colony forming unit was withdrawn and

observed for change in blackish blue colour

RESULT




Expt. No. 32

ESTIMATION OF GLUCOSE BY GLUCOSE OXIDASE METHOD

AIM

Estimation of glucose by glucose oxidase method

PRINCIPLE

Glucose oxidase (GOD) oxidized to gluconic acid and hydrogen peroxide in the

presence of peroxide, release hydrogen peroxide is couple with phenol and 4-amino

antipyrine to form a colour phenolenine dye. Absorbance of colour dye was measured at

505 nm and directly proportional to glucose concentration in the sample.

REAGENT REQUIRED

• Glucose reagent

• Glucose standard

• Serum

PROCEDURE

• Take 3 test tube and mark it as A, B and C. Whereas A is blank, B is standard, C

is test.

• Blank is filled with glucose reagent of 1000 µL using Micropipette

• Standard is filled with glucose standard of 10 µl and glucose reagent of 1000 µL

• Test is filled with 10 µl of serum 1000 µL glucose reagent was mixed well and

incubated at 37°C for 10 min.

• Observe the result at 505 nm in calorimeter.

INCREASED CONCENTRATION

Hyperglycemia may occur in diabetics in patients reserving in told Venus fluid

containing glucose during severe stress and cerebro vascular accidents.




DECREASED CONCENTRATION

Hypoglycemia may be the result of an insulinoma, insulin administration of inborn errors

of carbohydrate metaboliser of fasting.

CALCULATION

Absorbance of test- blank x 100

Absorbance of std- blank

OBSERVATION

Contents Blank Standard Test

Serum/ Plasma

Reagent 2

Reagent 1

OD @ 505 nm

RESULT




Expt. No. 33

TRANSFORMATION OF CELLS

AIM

To prepare competent cells and allow them to undergo transformation process

PRINCIPLE

The cells are made competent with an addition of magnesium chloride and calcium

chloride solutions. These competent cells are transformed by heat shock process. The

transformed cells are plated on a sob agar plate and are visualized.

MATERIALS REQUIRED

• E.coli

• DH5 culture

• Sterile chilled eppendorf tubes

• MgCl2 and 0.1 M CaCl2

• Sob agar plates

• Soc medium

• L.rod

PROCEDURE

Competent cell preparation

• Take sterile chilled eppendorf tubes add 1.5 mL of E.coli and DH5 culture to it

• Spin the tubes at 3000 rpm for 10 min at 4 °C to collect pellet of the cells

• Drain away the excess media by tapping to paper towels and wash it with PBS to

remove the left over media from the pellet

• Add 0.9 mL of MgCl2 and CaCl2 solution to the pellet

• Spin the tube to disaggregate the pellet formed and then centrifuge at 3000 rpm

at 4°C

• Pour the supernatant out drain away traces of MgCl2




• Dissolve the pellet in bowl of 0.1 M CaCl2

• Store in refrigerator (or) use directly transformation after in 0.1 M CaCl2 solutions

for 10 to 20 min.

TRANSFORMATION PROCESS

• 50 µl of competent cells are taken in sterile chilled eppendorf tube

• 2.5 µl of plasmid is added to the tubes

• The tubes are then immediately incubated in ice for 30 min

• Immediately transfer the tubes to a water bath maintained at 42°C. Incubated at

temperature for 90 sec (don’t shake)

• Immediately transfer the tubes to ice and incubate for 5 to 10 min

• Add 200 µl of soc medium and incubate the tubes in shaker for 45 min at room

temperature

• Plate the cells on the sob agar plates containing 100 mg/mL Ampicillin

• Spread the transformed cells on the plate using L-rod that was dipped in 10 %

ethanol heated and cool down

• Turn the plates over and incubate them for 20-24 hrs at room temperature

• Observe your plates for results and compare your plates with control

RESULT




Expt. No. 34

ISOLATION OF DNA FROM HUMAN BLOOD SAMPLE

AIM

To isolate of DNA from human blood sample

PRINCIPLE

Fresh blood from saturated suspended are lysed using the detergent trion x-100.

Proteins are removed by digestion with the non-specific protease, protinaese k, cell wall

debris; polysaccharide and remaining protein are pumped out by Triton x-100 in salt

high concentration retaining the nucleic acid in the solution. Then by using aqueous and

phase paultioning the DNA and the remaining complex are separated when DNA moves

to the top of aqueous phase are precipitated by addition of alcohol.

REAGENTS

Reagent A

• Tris HCl

• Glucose

• MgCl2

• Triton X-100

Preparation of Reagent A

• 1.211 gm of Tris HCl was added to 800 mL of distilled water and adjust to pH 8

• 109.5 gm of sucrose was added

• 1.107 gm of MgCl2 was added

• 10 mL of triton-x 100 was included and volume was made upto 1 litre with

distilled water

REAGENT B

• Tris HCl




• NaCl

• EDTA

• SDS

PREPERATION OF REAGENT B

• In 1 litre of water Tris HCl 48.44 gm was added

• NaCl will be 8.76 gm to be incorporated

• 17.53 gm of EDTA was involved

• 10 gm of SDS was added and 900 mL of water was poured and pH adjusted to 8

• 5M of sodium per chloride by dissolving 35.11.gm in 15 mL of water and make

the volume to 50 mL

PROCEDURE

• Collect 1.5 mL of blood in a 15 mL sterile falcon tube

• Then 6 mL of reagent A was added and mix in a shaker at room temperature and

centrifuged at 1500 rpm for 5 minutes at 4°C

• The supernatant was removed and the moisture was completely removed by

inventing the tube over a tissue paper

• Then resuspended the pellet in 1 mL of reagent B

• The tube was incubated at 65°C for 20 min in water bath and allows it to cool at

room temperature

• 2 mL of ice cold chloroform was added and mix it for 20 min using shaker

• Then it was centrifuged at 1000 rpm for 5 min

• Then aqueous phase was transferred to centrifuge tube and twice the volume of

ice cold ethanol was added and invents the tube several times gently till DNA

appears as pale threads

• Again it was centrifuged at 10000 rpm for 10 min and air dry the pellet

• The pellet is resuspended in 150 µl of autoclaved water on TE buffer and

resolves it in 0.6% agarose gel

RESULT




Expt. No. 35

BLOOD GROUPING IN RH TYPING

AIM

To identify the blood group in RH type in given blood sample

PRINCIPLE

Blood grouping is based on the presence or absence of two antigens A and B on

surface of RBC. Based on their antigens four blood group is identified namely A,B, AB

and O groups.

• A group carries the antigen (A) in the surface of RBC

• B group carries the antigen (B) in the surface of RBC

• AB group carries the antigen (AB) in the surface of RBC

• group carries the antigen (O) in the surface of RBC

RH typing is based on the presence or absence of RH antigen or O antigen on the

surface of RBC two RH types were identified RH positive and RH negative

MATERIALS REQUIRED

• Glass slides

• Blood sample

• Types of Antiserum

PROCEDURE

• Take two microscopic slides and mark four circles and label them has A, B, AB

and O.

• Add one drop of freshly drowned anti-coagulated blood sample in all the four

circles.

• Add one drop of A antiserum in the circle marked as A like that respectively do

for all circles with different antiserum




• Mix the contents of all circles using applicator sticks separately and observe for

agglutination of RBC or clumbing of RBC with two sticks.

OBSERVATION

RESULT




Expt. No. 36

ANTI-STREPTOLYSIN O ACTIVITY

AIM

To determine Antistreptolysin O activity in serum by slide Agglutination method

PRINCIPLE

ASO latex test contain polystyrene latex coated with purified and stabilized streptolysin

O antigen which reacts with the corresponding Ag O antibody in the test sample

resulting in the agglutination of latex particles.

MATERIALS REQUIRED

• Patient’s serum

• Slide

• Applicators sticks

PROCEDURE

• Using disposable plastic dropper place one drop of test specimen in circle area of

the slides

• Add 1 drop of ASO latex antigen to the above drop and mix well with disposable

applicator sticks.

• Rock the slide gently to and fro for two minute and examine the agglutination

• For positive and negative controls follow the same procedure as mentioned

above by taking control serum from respective vials.

OBSERVATION

RESULTS




AUTHORS PROFILE

Dr. S. Mohana Roopan received his B.Sc. Chemistry at

V.H.N.S.N. College, Virudhunagar in 2004. He joined

immediate M.Sc. Organic chemistry at VIT University and

successfully completed his degree in the year of 2006. He

received M.BA. (HR) from Bharathiar University, Coimbatore

in 2009. Successfully completed his Ph.D. at VIT University

in the year of 2011. Currently working as Assistant Professor

(Senior) of Organic Chemistry Division at VIT University,

Vellore, Tamilnadu, India. He has taught academic courses in

organic chemistry, engineering chemistry and environmental

studies. He is having 3 funded projects (DST and DBT) and completed one project (Vilgro

funding). Prof. Mohana Roopan has received young scientist award from DST and DBT.

Continuously for the past 3 years (2010-2013) he has received “Research Award” from VIT

University, Vellore. He has authored or co-authored for more than >70 refereed journal

articles, professional proceedings papers, and technical reports. In addition, he has serving as

an editorial board member and reviewer in various reputed journal.

Ganesh Elango has received his M.Sc (Integrated) degree in

Biotechnology from VIT University, Vellore, Tamilnadu,

India in 2013. Now he is working as a research assistant in

the funded project by Department of Biotechnology (DBT).

His research focuses on the Nanoparticles synthesis and its

biological studies. He has presented his work in various

conferences. Recently he has published one review article in

Applied Microbiology and Biotechnology (I.F. 3.6) journal.

Also he has filled one Indian patent.

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