BIOTECHNOLOGY LAB MANUAL

Offered to
III YEAR B.TECH CHEMICAL ENGINEERING

DEPARTMENT OF BIOTECHNOLOGY
Experiment No:1
STERILIZATION TECHNIQUES

Sterilization is the process of destroying all forms of microbial life-vegetative and

sporulating. It is important that all equipment used in a microbiological experiment are
sterilized in order that a particular organism of interest is grown, without contamination by
the organisms present in the surrounding environment.
Several methods of this sterilization are employed in the process of sterilization to
sterilize the various equipments used in an experiment these methods are broadly classified
as PHYSICAL and CHEMICAL METHODS OF STERILIZATION.
I PHYSICALAGENTS:
The major physical agents used for the control of microorganisms are
TEMPERATURE, RADIATION AND FILTRATION.
1. TEMPERATURE: Microorganisms can grow over a range of temperatures, from
very low temperatures characteristic of Psychrophiles, to very high temperatures
characteristic of thermophiles. Temperatures above a maximum generally kill microbes.
Such high temperatures can be produced by DRYHEAT or MOISTHEAT.
DRY HEAT STERILIZATION: Can be used where it is either undesirable or unlikely
that steam under pressure will make direct and complete contact. This is true of glassware
such as Petriplates, erlynmeyer flasks, pipettes, test tubes. Such material can be sterilized
by placing in hot air, at a temperature of 1600C for 120 min.
Alternatively, equipment like forceps, inoculation needle etc can be sterilized by direct
heating on a flame till red hot. Thus it brings about destruction of unwanted organisms
without changing the nature (flavour) of the material. This process involves heating at that
temperature for 15 min and then cooking it quickly to 0- 50C.
PRINCIPLE INVOLVED IN DRY HEAT STERILIZATION: Dry heat oxidise
chemical components of organisms thus destroying them.
MOIST HEAT STERILIZATION: High temperatures combined with high moisture is
one of the most effective ways of sterilization.
PRINCIPLES INVOLVED IN MOIST HEAT STERILIZATION: Moist heat
coagulates microbial proteins, and is hence more rapid in killing microbes.
Moist heat can be applied in the following ways in order to bring about sterility.

2
STEM UNDER PRESSURE: Provides temperatures higher than those obtainable by any
other method. 2t has advantages of rapid heating, penetration and moisture which facilitates
coagulation of proteins. Autoclave is, a device used in the laboratory to sterilize media
solution and to kill discarded cultures. It is operated at 15lbs/ sq inch pressure, which yields
a temperature of 1210C effective in bringing about sterility in 15 min
FRACTIONAL STERILIZATION OR TYNDALLISATION: Some microbial
solutions cannot be heated over 1000C without being damaged. Such materials are
sterilized by Tyndallization, which involves heating at 1000C on three successive days with
incubation periods in between. Resistant spores germinate during the period of incubation
that is killed on heating on the subsequentday.
PASTEURISATION: Milk, cream and other alcohol beverages are subjected to controlled
heat treatment which kills microbes of a certain type alone.
2. RADIATION: When ionising radiation pass through cells, they create free
hydrogen radicals, hydroxyl radicals and peroxides that cause intracellular damage,
resulting in destruction of microbes. This method of sterilization is effective for
sterilization heat labile material / also called (OLDSTERILIZATION)

U.V.light is the most effective region of the electromagnetic spectrum, and is employed in
disinfecting of inoculation chamber and hospital operating rooms. UV light alters nucleic
acids and results in a pyrimidine diamer, thus inhibiting DNA replication.
3. FILTRATION: This technique is used when the material to be used is heat labile
and cannot be sterilized by heating for eg. Solutions of proteins, vitamins etc. filters of pore
size 0.02 -0.08 are used to filters off microbes, thus rendering the filtrate sterile. Pore
size, electric charge of the filter, charge carried by the organisms and nature of fluid being
filtered affect efficiency of filtration e.g. Acity filters, Berkefeld filter, Berkefeld filter,
membrane filter are all microbialfilters.
II CHEMICALAGENTS
Several groups of chemicals can be used as antimicrobial agents:

3
 S.No GROUP ACTION EXAMPLE
1. Alcohol Denature proteins and solubilize Ethyl
2. Aldehyde lipids Glutaraldehyde
3. Halogens Alkylate, reacts with – NH2, SH- Co I2,Cl2
4. Heavy oH
metals I2 inactivates proteins oxidise cells. Hg Cl2
5. Gases Precipitates and inactivates proteins Ethylene dioxide
6. Detergents (used for surface sterilization)
Alkylates organic compounds
Disrupt cellmembrane
7. Phenols Denature protein and disrupt cell
membrane

Ethyl alcohol (70%) finds indispensable use for disinfecting hands before and after
a microbiological experiments, and also to disinfect the inoculation chamber or area where
the experiment isconducted.

CONCLUSION: Sterilization is the first indispensable step of any microbiological

experiment. Clean and sterile equipment are pre-requisites for culture isolation and
characterisation of any microorganism in a laboratory. Several methods can hence be
employed to sterilize the various materials required for an experiment.

4
Experiment No:2

PREPARATION OF MEDIA
PURPOSE:
To prepare the nutrient agar medium for bacterial culture.
COMPOSITION OF NUTRIENT AGAR MEDIUM: [pH (7.2)]
Beef extract 20g 1L
Peptone 20g1L
Nacl 10 g1L
Agar 20g1L
Distilledwater 1L
COMPOSITION OF ROSE BENGAL AGAR MEDIUM pH: 5.6 ± 0.2 at 25 °C.
(g/litre)
Peptone5.0;
glucose10.0;
potassium dihydrogen phosphate 1.0;
dichloran 0.002;
magnesium sulfate 0.5;
Rose Bengal 0.025; agar-agar15.0.
Distilledwater 1L
PROCEDURE:
A clean erlynmeyer flask was taken with 200 ml of distilled water. The chemicals were
weighed accurately and dissolved one by one taking care to add the next chemical only
after the dissolution of the first one, expect agar and streptomycin in the case of rose bengal
medium agar was melted separately and added to other ingredients. The pH was adjustedto
7.2 for nutrient agar medium and 6.5 for rose bengal agar medium. The total volume of the
medium was made upto 1 litre. The medium was then distributed into 250 ml. Erlynmeyer
flasks of 100 ml each and were plugged tightly with paper (from) and tied with thread.
Then the flasks were sterilized at 1210C for 15 minutes with 15lbs/in2 in an autoclave.
Streptomycin was added only after the sterilization of the rose bengal agar medium.
PREPARATION OF PLATES, SLANTS ANDDEEPS:
The prepared media can be poured into plates, slants and deeps for cultures and pure-
cultures.
PLATES:
5
 The sterilized medium is cooled to 45 –500C and poured into sterile petriplates.
About 20 ml of the medium is poured into each of the sterile petriplates and allowed to
solidify. Incubate in an incubator overnight before use.
SLANTS:
The molten medium after adjusting the pH is poured into text tubes, 5 ml of each
and the test tubes are placed in a slanted position so that they with solidify with maximum
surface area.
DEEPS:
Deeps are prepared by pouring the molten medium in sterile glass text tubes and
then placed in a vertical position. The tubes are then cooled in cold water and are used to
maintain cultures for long periods of incubation.

RESULT:
Thus nutrient agar and rose bengal agar media were prepared and used to prepared
and used to prepare plates, slants and deeps for inoculation.

6
Experiment No:3

ISOLATION, ENUMERATION AND PURIFICATION OF MICROBES FROM A

GIVEN SAMPLE

PURPOSE:
To isolate, enumerate and purify various microbes from the given sample.

PRINCIPLE
The techniques commonly used for isolation of discrete colonies initially require that the
number of organisms in the inoculum be reduced. The resulting diminution of the
population size ensures that, following inoculation, individual cells will be sufficiently far
apart on the surface of the agar medium to effect a separation of the different species
present. The serial dilution is used to accomplish this. There are three techniques to do
isolation of pure cultures.

REQUIREMENTS: Sterile blanks (9ml), 12 sterile test tubes for slants, 1 sterile blank (10
ml), samples, 18-20 Petriplates(20 ml), 10 sterile pipettes (1 ml), bacterial growth medium-
NUTRIENT AGAR (5 X 100 Ml), inoculation loop and wire, burner, marker pen, sterile
chamber.
PROCEDURE:
1. SERIAL DILUTION: Exactly 1 ml of the given sample was added to 9ml of blank,.
This solution was labeled as 10-1. From this test tube, 1 ml of solution was taken and added
to a 9 ml blank, mixed evenly and labeled as dilution 10-2 for the same manner, the
procedure was separated during sterile pipettes for each transfer, until dilutions upto 10-5
are obtained. For the given soil sample, dilutions upto 10-3 and 10-4 were taken for fungal
isolation and dilutions 10-4 were taken for fungal isolation and dilutions 10-4 and 10-5 were
fungal isolation and dilutions 10-4 and 10-5were taken for bacterial isolation.

7
2. ISOLATION OF MICROBES:
POUR PLATE METHOD.
Pour plating is a technique useful for isolation and enumeration of microbes 1 ml of the
selected dilutions were pipetted into sterile petriplates in close proximity to the flame.
Molten agar (nutrient agar in case of bacterial culture and Rose bengal agar in case
offungal culture) was poured over the inoculum and the plates were swirled to evenly
distribute the inoculum. Plates of a particular dilution were prepared in duplicates. The agar
was allowed to solidify and the plates were incubated at a temperature of 350C in an
inverted fraction for a period of 24-72 hours.
SPREAD PLATE METHOD
The spread-plate technique requires that a previously diluted mixture of microorganisms
be used. During inoculation, the cells are spread over the surface of a solid agar medium
with a sterile, L-shaped bent rod. The step-by-step procedure for this technique is as
follows:
Place the bent glass rod into the beaker and add a sufficient amount of 95% ethyl alcohol
to cover the lower, bentportion.
a. With a sterile loop, place a loopful of culture in the center of the appropriately
labeled nutrient agar plate that has been placed on the turntable. Replace the
cover.
b. Remove the glass rod from the beaker and pass it through the Bunsen burner
flame, with the bent portion of the rod pointing downward to prevent the
burning alcohol from running down your arm. Allow the alcohol to burn off the

8
 rod completely. Cool the rod for 10 to 15seconds.
c. Remove the Petri dish cover and spin theturntable.
d. While the turntable is spinning, lightly touch the sterile bent rod to the surface
of the agar and move it back and forth. This will spread the culture over the agar
surface.
e. When the turntable comes to a stop, replace the cover. Immerse the rod in
alcohol andreflame.
f. Keep the plate for incubation

3. ENUMERATION OF MICROBES OBSERVATIONS AND CALCULATIONS

BACTERIALCIULTURE

The number of bacteria in a given sample is usually too great to be counted directly.
However, if the sample is serially diluted and then plated out on an agar surface the
number of colonies can be used as a measure of the number of viable (living) cells in that
known dilution. However, keep in mind that if the organism normally forms multiple cell
arrangements, such as chains, the colony-forming unit may consist of a chain of bacteria
rather than a single bacterium. In addition, some of the bacteria may be clumped together.
Therefore, when doing the plate count technique, we generally say we are determining the
number of Colony-Forming Units (CFUs) in that known dilution. By extrapolation, this
number can in turn be used to calculate the number of CFUs in the originalsample.

Normally, the bacterial sample is diluted by factors of 10 and plated on agar. After
incubation, the number of colonies on a dilution plate showing between 30 and 300
colonies is determined. A plate having 30-300 colonies is chosen because this range is
considered statistically significant. If there are less than 30 colonies on the plate, small
errors in dilution technique or the presence of a few contaminants will have a drastic effect
on the final count. Likewise, if there are more than 300 colonies on the plate, there will be
poor isolation and colonies will have grown together.

Generally, one wants to determine the number of CFUs per milliliter (ml) of sample. To
find this, the number of colonies (on a plate having 30-300 colonies) is multiplied by the
number of times the original ml of bacteria was diluted (the dilution factor of the plate

9
counted). For example, if a plate containing a 1/1,000,000 dilution of the original ml of
sample shows 150 colonies, then 150 represents 1/1,000,000 the number of CFUs present
in the original ml. Therefore the number of CFUs per ml in the original sample is found by
multiplying 150 x 1,000,000 as shown in the formulabelow:

DILUTION P1 P2 AVG

10-4

10-5

No of bacterial C.F.U
No of colonies
Dilutions factor x amount ofsample added = C.F.U / ml ofsample.

4. STREAKPLATING:
The streak-plate method is a rapid qualitative isolation method. It is essentially a dilution
technique that involves spreading a loopful culture over the surface of an agar plate.
Although many types of procedures are performed, the four-way, or quadrant, streak is
described. Refer to figure, which schematically illustrates this procedure.
Streaking cultures for isolated colonies allows you to:
 separate mixedcultures
 purify a single type of bacterium
 propagate a clonal population ofbacteria
 help with the identification of abacterium

Streak plating is of two types:

Quadrant streak:
a.Place a loopful of culture on the agar surface in Area 1. Flame and cool the loop and drag
it rapidly several times across the surface of Area1.
b.Reflame and cool the loop and turn the petri dish 900. Then touch the loop to a corner of
the culture in Area 1 and drag it several times across the agar in Area
c. 2. The loop should never enter Area 1 again.
10
d.Reflame and cool the loop and again turn the dish 900.Streak Area 3 in the same manner
as Area 2.
e. Without reflaming the loop, again turn the dish 900 and then drag the culture from a
corner of Area 3 across Area 4, using a wider streak. Don't let the loop touch any of the
previously streaked areas. The flaming of the loop at the points indicated is to effect
dilution of the culture so that fewer organisms are streaked in each area, resulting in the
final desired separation.

SLANTS AND STABS: For long term storage of bacterial culture, suspended in a slow
state of growth, slants and states can be used. Desired bacterial colonies were picked up
with the sterile loop and streaked onto the surface of the slant (maximising the surface area)
sterile inoculation were dipped into bacterial culture maintained in nutrient broth, and were
stabbed into agar both slants and states were incubated.

OBSERVATIONS: Purified and stored bacterial cultures were deserved and various types
of colony morphology studied.
SIZE:
SHAPE/MARGIN:
ELEVATION:
PIGMENTATION:
TEXTURE:

RESULT: The given soil sample contained -------- C.F.U of bacteria per ml of sample.
Desired bacterial cultures more purified, their morphology studied and subcultured for

11
storage.

12
Experiment No:4
USE OF MICROSCOPE

A microscope is a scientific instrument with one or more lenses that allow you to observe
specimens so small it is not visible to the naked eye, e.g. microorganisms (bacteria) and
microsopic materials placed on the stage

General Procedures

1. Make sure all backpacks and junk are out of the aisles.
2. Plugyourmicroscopeintotheextensioncords.Eachrowofdesksusesthesamecord.
3. Always start and end with the Scanning Objective. Do not remove slides with the high
power objective into place - this will scratch the lens!
4. Always wrap electric cords and cover microscopes before returning them to the cabinet.
13
MicroscopesshouldbestoredwiththeScanningObjectiveclickedintoplace.
5. Always carry microscopes by the arm and set them flat on yourdesk.

Focusing Specimens

1. Always start with the scanning objective. Odds are, you will be able to see something
on this setting. Use the Coarse Knob to focus, image may be small at thismagnification, but
you won't be able to find it on the higher powers without this first step. Do not use stage
clips, try moving the slide around until you findsomething.
2. Once you've focused on Scanning, switch to Low Power. Use the Coase Knob to
refocus. Again, if you haven't focused on this level, you will not be able to move to the
next level.
3. Now switch to High Power. (If you have a thick slide, or a slide without a cover, do
NOT use the high power objective). At this point, ONLY use the Fine Adjustment Knob to
focusspecimens.
4. If the specimen is too light or too dark, try adjusting thediaphragm.
5. If you see a line in your viewing field, try twisting the eyepiece, the line should move.
That's because its a pointer, and is useful for pointing out things to your lab partner or
teacher.
Drawing Specimens

1. Usepencil-youcaneraseandshadeareas
2. All drawings should include clear and proper labels (and be large enough to view
details).Drawingsshouldbelabeledwiththespecimennameandmagnification.
3. Labels should be written on the outside of the circle. The circle indicates the viewing
field as seen through the eyepiece, specimens should be drawn to scale - ie..if your
specimen takes up the whole viewing field, make sure your drawing reflectsthat.

Cleanup

1. Store microscopes with the scanning objective in place.

2. Wrap cords and cover microscopes.
3. Wash slides in the sinks and dry them, placing them back in the slide boxes to be used
later.
14
4. Throw coverslips away.
Experiment No:5
SIMPLE STAINING

PURPOSE
To perform the simple staining procedure to compare morphological shapes and
arrangements of bacterial cells.

PRINCIPLE
In simple staining, the bacterial smear is stained with a single reagent. Basic stains with a
positively charged chromogen are preferred, because bacterial nucleic acids and certain cell
wall components carry a negative charge that strongly attracts and binds to the cationic
chromogen. The purpose of simple staining is to elucidate the morphology and arrangement
of bacterial cells. The most commonly used basic stains are methylene blue, crystal violet,
and carbol fuchsin.

MATERIALS
Cultures
24-hour nutrient agar slant cultures of Escherichia coli and Bacillus cereus, and a 24-hour
nutrient broth culture of Staphylococcus aureus.

Reagents
Methylene blue, crystal violet, and carbol fuchsin.

Equipment
Bunsen burner, inoculating loop, staining tray, microscope, lens paper, bibulous paper, and
glass slides.

PROCEDURE
1. Prepareseparatebacterialsmearsoftheorganismsfollowingtheproceduredescribed.
Note: All smears must be heat fixed prior to staining.
2. Place a slide on the staining tray and flood the smear with one of the indicated stains,
using the appropriate exposure time for each: Carbol fuchsin, 15 to 30 seconds; methylene

15
blue, 1 to 2minutes.
3. Wash the smear with tap water to remove excess stain. During this step, hold the slide
parallel to the stream of water; in this way you can reduce the loss of organisms from the
preparation.
4. Using bibulous paper, blot dry but do not wipe theslide.
5. Repeat this procedure with the remaining two organisms, usinga
different stain for each.
6. Examine all stained slides under oilimmersion.
OBSERVATIONS AND RESULTS
In the space provided
1. Draw a representative field for eachorganism
2. Describe the morphology of the organisms with reference to their
shapes(bacilli,cocci,spirilli) andarrangements(chains,cluster,pairs).

16
Experiment No:6
GRAM STAINING

PURPOSE:
To become familiar with

17
1. The chemical and theoretical bases for differential stainingprocedures.
2. The chemical basis of the Gramstain.
3. Performance of the procedure for differentiating between the two principle groups of
bacteria: gram-positive andgram-negative.

PRINCIPLE
Differential staining requires the use of at least three chemical reagents that are applied
sequentially to a heat-fixed smear. The first reagent is called the primary stain. Its
function is to impart its color to all cells. In order to establish a color contrast, the second
reagent used is the decolorizing agent. Based on the chemical composition of cellular
components, the decolorizing agent may or may not remove the primary stain from the
entire cell or only from certain cell structures. The final reagent, the counterstain, has a
contrasting color to that of the primary stain. Following decolorization, if the primary stain
is not washed out, the counterstain cannot be absorbed and the cell or its components will
retain the color of the primary stain. If the primary stain is removed, the decolorized
cellular components will accept and assume the contrasting color of the counterstain. In
this way, cell types or their structures can be distinguished from each other on the basis of
the stain that isretained.
The most important differential stain used in bacteriology is the Gram stain, named
after Dr.Christian Gram. It divides bacterial cells into two major groups, gram-positive and
gram-negative, which makes it an essential tool for classification and differentiation of
microorganisms. The Gram stain uses four different reagents. Descriptions of these
reagents and their mechanisms of actionfollow.

Primary stain
Crystal Violet This violet stain is used first and stains all cells purple.

Mordant
Gram's iodine This reagent serves as a mordant, a substance that forms an insoluble
complex by binding to the primary stain. The resultant crystal violet-iodine (CV-I) complex
serves to intensify the color of the stain, and all the cells will appear purple-black at this
point. In gram-positive cells only, this CV-I complex binds to the cell wall. The resultant
magnesium-ribonucleic acid-crystal violet-iodine (Mg-RNA-CV-I) complex is more
difficult to remove than the smaller CV-I complex.
18
Decolorizing Agent
Ethyl Alcohol, 95% This reagent serves a dual function as a lipid solvent and as a protein-
dehydrating agent. Its action is determined by the lipid concentration of the microbial cell
walls. In gram-positive cells, the low lipid concentration is important to retention of the
Mg-RNA-CV-I complex. Therefore, the small amount of lipid content is readily dissolved
by the action of the alcohol, causing formation of minute cell wall pores. These are then
closed by alcohol's dehydrating effect. As a consequence, the tightly bound primary stain is
difficult to remove, and the cells remain purple. In gram-negative cells,the high lipid
concentration found in outer layers of the cell wall is dissolved by the alcohol, creating
largeporesinthecellwallthatdonotcloseappreciablyondehydrationofcellwall

proteins. This facilitates release of the unbound CV-I complex, leaving these cells colorless
or unstained.
Counterstain
Safranin This is the final reagent, used to stain red those cells that have been previously
decolorized. Since only gram-negative cells undergo decolorization, they may now absorb
the counterstain. Gram-positive cells retain the purple color of the primary stain
The preparation of adequately stained smears requires that you bear in mind the
following precautions:

1. The most critical phase of the procedure is the decolorization step, which is based on
the ease with which the CV-I complex is released from the cell. Remember that over-
decolorization will result in loss of the primary stain, causing gram-positive organisms to
appear gram-negative. Under- decolorization, however, will not completely remove the
CV-I complex, causing gram-negative organisms to appear gram-positive. Strict adherence
to all instructions will help remedy part of the difficulty, but individual experience and
practice are the keys to correctdecolorization.
2. It is imperative that slides be thoroughly washed under running tap water between
applications of the reagents. This removes excess reagent and prepares the slide for
application of the subsequentreagent.
3. The best Gram stained preparations are made with fresh cultures , that is, not older than
24 hours. As cultures age, especially in the case of gram-positive cells, the organisms tend
to lose their ability to retain the primary stain and may appear to be gram-variable; that is,
some cells will appear purple, while others will appearred.
19
Materials
Cultures
24-hour nutrient agar slant cultures of Escherichia coli, Staphylococcus aureus, and
Bacilluscereus.

Reagents
Crystal violet, Gram's iodine, 95% ethyl alcohol, and safranin.

Equipment
Bunsen burner, inoculating loop or needle, staining tray, glass slides, bibulous paper, lens
paper, and microscope.

PROCEDURE
The steps are pictured in Figure
1. Obtain four clean glassslides.
2. Using sterile technique, prepare a smear of each of the three organisms and on the
remaining slide prepare a smear consisting of a mixture of S.aureus and E.coli. Do this by
placing a drop of water on the slide and then transferring each organism separately to the
drop of water on the slide with a sterile, cooled loop. Mix and spread both organisms by
means of a circular motion of the inoculatingloop.
3. Allow smears to air dry and then heat fix in the usualmanner.
4. Flood smears with crystal violet and let stand for 1minute.
5. Wash with tapwater.
6. Flood smears with the Gram's iodine mordant and let stand for 1minute.
7. Wash with tapwater.

8. Decolorize with 95% ethyl alcohol. Caution: Do not over-decolorize. Add reagent
drop by drop until crystal violet fails to wash fromsmear.
9. Wash with tapwater.
10. Counterstain with safranin for 45 seconds.
11. Wash with tapwater.
12. Blot dry with bibulous paper and examine under oilimmersion.

OBSERVATIONS AND RESULTS

20
Following your observation of all slides under oil immersion, record your results in the
chart.
1. Make a drawing of a representative microscopicfield.
2. Describe the cells according to their morphology andarrangement.
3. Describe the color of the stainedcells.
4. Classify the organism as to the gram reaction: Gram-positive orgram-negative.
Refer to photo numbers 2-4 in the color-plate insert for illustration of this staining
procedure.

21
Experiment No:7
PREPARATAION OF BUFFERS AND MEASUREMENT OF pH
PURPOSE:
To prepare the buffers of required pH

PRINCIPLE:
The pH meter measures the electrical potential developed by a pair of electrodes
dipping into a solution. For the measurement of pH of an electrode system sensitive to
changes in the hydrogen ion activity of the solution is chosen. This electrode system
consists of a sequence of electrodes whose potential varies with the pHof the solution.

1. Acetic acid-sodium acetate buffer:

Reagents:
1. Acetic acid(0.2m)
1.55 ml of glacial acetic acid is made up to 100 ml with distilled water.
2. Sodium acetate solution(0.2m)
1.62 g of sodium acetate 0r 2.72 g of sodium acetate trihydrate is dissolved in 100
ml of water.

PROCEDURE:
Pipetted out exactly 35.2ml of sodium acetate solution into a 100ml standard flask.
To this added exactly 14.8ml of acetic acid. This is made up to 100ml with distilled water.
This gives 0.2m acetic acid-sodium acetate buffer whose pH could be measured a pH
meter.
The pH meter was first standardised using standard buffer. Washed the electrode
with distilled water. Now introduced 02.m acetate buffer the pH was found to be.5.

2. Carbonate-Bicarbonate Buffer.
Reagents:
i) Sodium Carbonate solution (0.2m) Dissolved 2.12 g of anhydrous sodium carbonate in
100 ml ofwater.

ii.Sodium Bicarbonate Solution (0.2M)

Dissolved 1.68 g of sodium bicarbonate in 100 ml of water.
22
PROCEDURE:
Pipetted out exactly 27.5ml of sodium carbonate solution (0.2M). To this added
22.5ml of sodium bicarbonate solution (0.2M). This gives 0.2M carbonate-bicarbonate
buffer, Whose pH is measured.
Standardized the pH measured the pH of the prepared buffer. The pH was found to
be 10.
3. BarbitoneBuffer:
REAGENTS:
1. Diethyl barbituricacid
2. Sodium diethylbarbiturate

PROCEDURE:
Dissolved 2.589g of diethyl barbituric acid and 14.2 g of sodium diethyl barbiturate
in distilled water and made up 1 liter. This gives borbitone buffer, Its pH was found to be
8.6

4. Citric acid Buffer:

Reagents:
Citric acid solution (0.1M)
Dissoloved 2.101 g citric acid in 100 ml of water.

PROCEDURE:
Mixed 46.5ml of citric acid (0.1M) and 3.5 ml of sodium citrate (0.1M) and made
upto 100 ml with water. This gives 0.1M citric acid buffer. Standardized the pH meter and
measure the prepared solution, this give citrate buffer of pH 3.

5. Phosphate Buffer:

Reagents:
1. Monobasic sodium phosphate solution (0.2M)
Dissloved 2.78 g at monobasic sodium phosphate in 100ml ofwater
2. Dibasic sodium phosphate solution(0.2M)
Dissloved 5.365g of dibasic sodium phosphate heptahydrate and 7.179 g of dibasic sodium
phasphate deco carbonate in 100 ml of water.
23
PROCEDURE:
Mixed 39ml of monobasic sodium phosphate solution with 61ml of dibasic sodium
phosphaste solution. This is made up to 200 ml with distilled water. This gives phosphate
buffer whose pH can be measured. The pH of this was found top be 7.

DIGITAL pH METER

RESULT:
The buffers of the required pH was prepared

24
 Experiment No:9

ESTIMATION OF PROTEIN BY LOWRY’SMETHOD

To estimate the amount of protein in the given sample by Lowry’ method.

PRINCIPLE:

The principle behind the Lowry method of determining protein concentrations lies in the
reactivity of the peptide nitrogen[s] with the copper[II] ions under alkaline conditions
and the subsequent reduction of the Folin-
Ciocalteayphosphomolybdicphosphotungsticacidtoheteropolymolybdenum blue by the
copper-catalyzed oxidation of aromatic acids [Dunn, 13]. The Lowry method is sensitive
to pH changes and therefore the pH of assay solution should be maintained at 10 -10.5.

The Lowry method is sensitive to low concentrations of protein. Dunn [1992] suggests
concentrations ranging from 0.10 - 2 mg of protein per ml while
Price[1996]suggestsconcentrationsof0.005-0.10mgofproteinperml.The
majordisadvantageoftheLowrymethodisthenarrowpHrangewithinwhich
itisaccurate.However,wewillbeusingverysmallvolumesofsample,which will have little or
no effect on pH of thereactionmixture.A variety of compounds will interfere with the
Lowry procedure. These include some amino acid derivatives, certain buffers, drugs,
lipids, sugars, salts, nucleic acids and sulphydryl reagents [Dunn, 1992]. Price [1996]
notes that ammonium ions, zwitter ionic buffers, nonionic buffers and thiol compounds
may also interfere with the Lowry reaction. These substancesshould be removed or
diluted before running Lowry assays.

Reagents

1. 2% Na2CO3 in 0.1 NNaOH

2. 1% NaK Tartrate inH2O

3. 0.5% CuSO4.5 H2O inH2O

4. Reagent I: 48 ml of A, 1 ml of B, 1 mlC

5. Reagent II- 1 part Folin-Phenol [2 N]: 1 partwater

6. BSA Standard - 1 mg/ ml

Procedure:
1. 0.2 ml of BSA working standard in 5 test tubes and make up to 1ml using
distilledwater.
2. Thetesttubewith1mldistilledwaterserveasblank.

3. Add4.5mlofReagentIandincubatefor10minutes.

4. Afterincubationadd0.5mlofreagentIIandincubatefor30minutes

5. Measuretheabsorbanceat660nmandplotthestandardgraph.

6. Estimate the amount of protein present in the given sample from the
standardgraph.

Tabulation:

Vol. Vol. of Vol. of Vol. of

Conc OD
of Distilled reagent Reagent
of BSA
S.No BSA water I II At
Incubation for 30 min
Incubation for 10 min

(mg/ml)
(ml) (ml) (ml) (ml) 660nm

Calculations:
Result:
The amount of protein present in the given sample was found to be …………
 AQUEOUS TWO PHASEEXTRACTION

AIM:To isolate the given protein by aqueous two phase extraction and to find the
partition co-efficient.

PRINCIPLE:

Downstream processing is an integral part of any product development, and the

final cost of the product depends largely on the cost incurred during extraction
and purification techniques. The conventional techniques used for product
recovery, for example precipitation and column chromatography, are not only
expensive but also result in lower yields. Furthermore since solid– liquid
separation by centrifugation or filtration results in some technical
difficulties,forexamplefilterfoulingandviscousslurries,therefore,thereisan ongoing
need for new, fast, cost-effective, eco-friendly simple separation techniques.
Thus, for separation of biomolecules, aqueous two phase systems (ATPS) offer an
attractive alternative that meets the above-mentioned requirements as well as the
criteria for industrially compatible procedures. Hence, it is increasingly gaining
importance in biotechnological industries. The advantage of using this technique
is that it substantially reduces the number of initial downstream steps and
clarification, concentration, and partial purification can be integrated in one unit.
Furthermore, scale-up processes based on aqueous two phase systems are simple,
and a continuous steady state ispossible.

An aqueous two-phase system is an aqueous, liquid–liquid, biphasic system

which is obtained either by mixture of aqueous solution of two polymers, or a
polymer and a salt. Generally, the former is comprised of PEG and polymers like
dextran, starch, polyvinyl alcohol, etc. In contrast, the latter is composed of PEG
and phosphate or sulphate salts. This polymer-salt system results in higher
selectivity in protein partitioning, leading to an enriched product with high yields
in the first extraction step.
Partitioning of the two phases is a complex phenomenon, taking intoaccount
theinteractionbetweenthepartitionedsubstanceandthecomponentofeach
phase.Anumberofdifferentchemicalandphysicalinteractionsareinvolved, for
example hydrogen bond, charge interaction, van der Waals’ forces, hydrophobic
interaction and steric effects. Moreover the distribution of molecules between the
two phases depends upon the molecular weight and chemical properties of the
polymers and the partitioned molecules14 of both thephases.
Thus, the distribution of molecules between the two phases is characterized by the
partition coefficient, Kpart, defined as the ratio of the concentrate in the top (Ctop) and
bottom (Cbottom) phase,respectively.
Kpart = Ctop/Cbottom

MATERIALS REQUIRED:

PEG 4000,Sodium carbonate, Sodium phosphate, Sodium sulphate, Distilled

water, Centrifuge tube (15 ml), Alkaline copper sulphate solution,Folin’sreagent.
PROCEDURE:

 Prepare 40% (w/w) of PEG 6000 and 25% (w/w) of Sodiumsalt solutions.

 Take 5 ml of salt solution in three different centrifuge tube and add

preweighed 30 mg of BSA and mix well.

 Add 5 ml of PEG solution to all the tubes.

 Centrifuge the tube at 5000 rpm for 10 mins.

 Carefully transfer the top phase solution to the test tube.

 Estimate the amount of protein present in the top and bottom phase by
Lowry’s method.

 Calculate the partition coefficient

Calculations:
Result:
Partition coefficients of BSA in following systems are
PEG 4000 – Carbonate system =
PEG 4000 – Phosphate system =
PEG 4000 – Sulphate system =
PROTEINPRECIPITATION.(AmmoniumSulfatePrecipitation)

AIM:
To precipitate the given protein sample by using Ammonium sulphate and to
determine the protein recovery.

PRINCIPLE

Proteins are usually soluble in water solutions because they have

hydrophobic amino acids on their surfaces that attract water molecules and interact
with them. This solubility is a function of the ionic strength and pH of the solution.
Proteins have iso electric points at which the charges of theiramino acid side
groups balance each other. If the ionic strength of a solution is either very high or
very low proteins will tend to precipitate at their iso electric point. The solubility is
also a function of ionic strength and as you
increasetheionicstrengthbyaddingsalt,proteinswillprecipitate.Ammonium sulfate is
most common salt used for this purpose because it is unusually soluble in cold
buffers (we have to keep proteins cold!) and is economically viable. Ammonium
sulfate fractionation is commonly used in research laboratories as the first step in
protein purification because it provides some crude purification of proteins away
from non- proteins and also separates some proteins. Ammonium sulfate also
yields the precipitated proteins slurry
thatisusuallyverystable,sothepurificationcanbestoppedforafewhours.

MATERIALS REQUIRED

Ammoniumsulphate,BSA,Alkalinecoppersulphatesolution,Folin’sreagent,
Magneticstirrer,Centrifugetube,Centrifuge,Testtube,Spectrophotometer.

PROCEDURE

 Dissolve5mgofBSAin10mlofdistilledwaterin50mlbeaker.

 Add the corresponding saturation amount (50-100 %) of ammonium sulphate to the

beaker with stirring.

 After adding ammonium sulphate completely to the beaker , leave it undisturbed for
30 mins.
 Transfer the solution the 15 ml centrifuge tube abd centrifuge at 10000 rpm for 5
mins.

 Discard the supernatant and dissolve the pellet in distilled water and centrifuge again.

 Discard the supernatant and dissolve the pellet in 1 ml of distilled wate

 Take 200µl of sample and estimate the amount of protein present by Lowry’s
method.

 Calculate the percentage of recovery by using the formula

Recovery percentage =(Final protein concentration / Initial protein concentration) *100

Tabulation:

Percentage of
Ammonium Protein Percentage
Sulphate concentration of recovery
50
60
70
80
90
100

RESULT
MaximumpercentagerecoveryofBSAis----------------------------at----------- % of

Ammonium sulphate saturation.

Experiment No:
SEPARATION OF AMINO ACIDS BY THIN LAYER CHROMATOGRAPHY
PURPOSE:
To separate amino acids by thin layer chromatography.
PRINCIPLE:
Chromatography is a method by which a mixture of substances in smaller quantities can be
separated both qualitatively and quantitatively. In chromatography there are twophases-the
stationary phase and other mobile phase. When the mobile phase moves along stationary
phase, separation of substances takes place. In thin layer chromatography, the thin layer of
gel functions as an inert supporting material. When the mobile phase moves along the gel
solvent, as the partition coefficient differ for different sugars, the rate of flow differs and
therefore separation occurs.
Materials Required:
1. Silica gel G
2. Microscopic slides
3. 3.n-butanol
4. Acetic acid
5. Spraying reagent (0.3% solution of Ninhydrin in butanol containing 3 ml aceticacid)

6. Aminoacids
PROCEDURE:
A slurry of silicagel G is prepared in 0.02M sodium acetate buffer. Taken the microscopic
slides and keeping them flat, pipetted out about 1-2 ml of the slurry into them. By tilting
the slides spread the slurry evenly on the surface. Lining the edges with Vaseline will be of
help. Allowed the slides to dry completely leaving them flat. 5 l samples of amino acid (or
mixture) are spotted and the slide is then dipped in a trough containing n- butanol-acetic
acid-water in the ration 8:2:2. The slide must be handled with care not to break the surface.
After development, that is, when the solvent has reached the top, the slide is dried and
sprayed with the developing reagent. The slide is then heated in an oven at 1100 C for 10
minutes and Rf values of the spots aremeasured.

Rf = Distance moved bysolute

---------------------------------
Distance moved by solvent
RESULT:
Experiment No:

SEPARATION OF SUGARS BY PAPER CHROMATOGRAPHY

PURPOSE:
To separate the sugars by paper chromatography.

PRINCIPLE:
Chromatography is a method by which a mixture of substances in smaller quantities can be
separated both qualitatively and quantitatively. In chromatography there are two pahses the
stationary phase and other mobile phase. When the mobile phase moves alongstationary
phase, separation of substances takes place. In paper chromatography the paper functions
as an inert supporting material. When the mobile phase moves along the paper solvent, As
the partition coefficient differ for different sugars the rate of flow differs and therfore
separation occurs.

MATERIALS REQUIRED:
1.Whatmann No :1 filter paper
2.N-butanol
3.Glacial acetic acid
4.Sugars
5.Spraying reagent-Alkaline permanganate spraying agent 0.1% KmnO4 in 2% sodium
carbonate

PROCEDURE:
3 strips of whatman No 1 filter paper of size 12 2 cm are cut. A line is drawn at a distance
of 2.5 cm from the base and a pencil mark made at the mid point. Sugar solutions of
glucose and fructose at a concentration of 100 mg/10ml is prepared. The chromatography
paper is placed on a box having a slit with lighting arrangment underneath the slit. Spotting
is done on the paper using a micropipette. Care is taken to see that the spot does not spread
beyond a particular diameter. 10ml each glucose and fructose are spotted on a paper A and
B. To the strips a mixture is applied. The three strips are placed in three different boiling
tubes each containing 5ml of n-butanol acetic acid, water in the ration 4:2:1. The boiling
tubes are plugged with cotton, The paper are kept in a a pre-saturated chromatographic
chamber and the solvent is allowed to rise. When the solvent front has reached three fours
of the paper the strips are removed and air dried. It is then sprayed with the spraying agent
and dried in hot air oven at1000C.

The sugars appeared as yellow spots in a purple background. The distances travelled by
the solvent are measured. The distance from the base line to the centre of each spot are
measured, Rf values is then calculated asfollows.

Rf = Distance moved bySugar

---------------------------------
Distance moved by solvent

RESULT: The Rf values of sugar is found tobe