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Transcript of BT I Sem 2013
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EXPERIMENT-1
AIM- To preparation acetic-Na acetate buffer system and validate the Henderson-
Hasselbach equation.
REQUIRMENT-
a) Chemicals - 0.1 M Sodium acetate.b) Reagents - 0.1 M acetic acid, 0.1 M HCl, 0.1 M HCl, distilled water.c) Equipments - test tubes, beakers, pipettes.d) Instruments - pH meter.
PRINCIPLE- A buffer solution is one that resists the pH change on the addition of acid
and alkali such solutions are used in many biochemical reaction where the pH needs to be
accurately controlled.
From the Henderson-Hasselbach equation, the pH of buffer solution depends on two
factors;
[1] The pK value.
[2] Ratio of salt to acid, this ratio is considered to be the same as the amount of
salt and acid mixture together over the PH range4-10. In the present case taking
the example of acetate buffer which consists of a mixture of acetic acid andsodium acetate:
Since acetic acid is only weakly dissociated, the concentration of acetic acid is almost the
same amount put in the mixture. Likewise the concentration of acetate ion can be
considered to be the same as the concentration of Na acetate placed in the mixture sincethe salt is completely dissociated.
HENDERSON-HASSELBACH EQUATION
Weak acids are only slightly ionized in solution and a true equilibrium is established
between the acid and the conjugate base.
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If HA represents a weak acid then
HA
According to the law of Mass action, Ka the acid dissociation constant is defined as
And, H+ is equal to Ka,
By definition, log 1/ [H+] = pH, and log 1/K
a = pK
a, so that by taking the log of the
equation above, we get the equation
In general terms,
PKa is equal to the negative logarithm of acid dissociation constant of a weak acid in
other words.
It is a pH at which the concentration of the acid its conjugated base are equal.
PROCEDURE-
[1] Put 4 ml of 0.1m acetic acid and 5 ml of Na acetate in a test tube and mix well.
[2] Measure or note the pH of solution.
[3] Now add 9 ml of distilled water and 1 ml of HCl and well. Note the pH value.
[4] 5 ml of Na acetate and 4 ml acetic acid and add 1 ml of HCl and measure the
pH value again.
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OBSERVATION TABLE-
SAMPLE pH
Buffer Solution 4.80
BUFFER + HCl 4.31
D/W = HCl 1.90
RESULT-By observation the percent change in pH with compare to D/W it is found that
the prepared buffer solution resist the change in pH on addition of HCl.
ASSESSMENT OF PRACTICAL BY INTERNAL (ON THE SPOT)
1. Participation/Involvement of candidate __________/102. Ability to perform himself/herself _________________/103. Results obtained_____________________________________/104. Accuracy_____________________________________________/105. On time submission of practical record____________/106. TOTAL________________________________________________/50
Internal Guide Student
(NAME AND SIGNATURE) (NAME AND SIGNATURE)
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EXPERIMENT-2
AIM:-To perform quantitative analysis of protein BSA and verification of beers law.
MATERIAL REQUIREMENTS -
a) Chemical: Alkaline solution carbonate solution, CuSO4, sodium potassium tartrate solution, Folin reagent, and Standard protein (Bovine Serum Albumin)
solution0.2 mg/ml, Unknown protein sample.
b) Equipments: mortar and pestle, volumetric flasks (50 ml), Pipettes,thermometer, test tubes, test tube stand.
c) Instruments: Spectrophotometer, weighing balance, centrifuge, water bath
PRINCIPLE:- Proteins are large biological molecules consisting of one or more chains
of amino acids. Proteins perform a vast array of functions within living organisms,
including catalyzing metabolic reactions, replicating DNA, responding to stimuli, and
transporting molecules from one location to another. Like other
biological macromolecules such as polysaccharides and nucleic acids, proteins are
essential parts of organisms and participate in virtually every process within cells. Many
proteins are enzymes that catalyze biochemical reactions and are vital to metabolism.
Proteins differ from one another primarily in their sequence of amino acids, which is
dictated by the nucleotide sequence of their genes, and which usually results in folding of
the protein into a specific three-dimensional structure that determines its activity.
The degree or extent of variation of proteins is also dependent upon its natural source, the
environment, physiological state etc. The isolation strategy for proteins would differ with
the type of source and the purpose of its extraction. The present experiment deals with the
extraction of proteins from pulses and its estimation by Folin Lowery method.
Proteins react with Folin Ciocalteau reagent to give a colored complex. The color so
formed is due to the reaction of the alkaline copper sulphate with the protein & the
reduction of phosphomolebdate by tyrosine & tryptophan in the protein. The intensity of
the color depends on the amount of these aromatic amino acids present & will thus varyfor different proteins.
max:- When measuring the absorbance of several samples that have different
concentration. It is important to identify an appropriate wavelength at which to make
measurements. This wavelength is the one at which the samples absorbs the most light
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i.e. it is the wavelength at which the maximum fraction of light is absorbed by the
samples is called as absorption maxima or max.
Beers Law:-Beer law states that for a parallel beam of monochromatic radiation passing
through homogenous solutions of equal path length the absorbance is proration to the
concentration. A C
Lamberts Law:- Lamberts law states that for a parallel beam of monochromatic
radiation passing through homogenous solution of equal concentration the absorbance is
proportional to the path length. A L
Lamberts law cannot be verified in lab as the path length of cuvette taken is constant.
PREPARATION:-
a) Reagent A: - Alkaline Sodium Carbonate - Alkaline Sodium Carbonate20gms/l in 0.1 mol/lt NaOH.
b) Reagent B: - Copper Sulphate - Potassium Tartrate solution CuSo4.5H2O in10gms/l of Na-k-tartrate 5gms/l
c) Alkaline Solution: - Alkaline solution is prepared on the day of use bymixing 50 ml of Reagent A and 1ml of Reagent B i.e., 50:1.
d) Folin Reagent: - Dilute commercial reagent with equal amount of water.
PROCEDURE:-
a) Finding the concentration of protein sample - Label the clean and dried test tubes from 1 to 13 and mark them as standard,
blank and test sample (In duplicates).
Take various aliquots of standard ranging from 0.1 ml to 1.0 ml with a gradationof 0.1 ml.
Make up the volume to 1 ml using distilled water. Take 1 ml of distilled water in the tube marked as blank to set zero. Take 0.5ml and 0.7 ml aliquots of test sample in duplicates and adjust the volume
to 1 ml by distilled water.
Add 5.0 ml of alkaline solution to all the test tubes and mix properly. Incubate the test tubes at 37C for 10 min in water bath. Add 0.5 ml of Folin reagent to all the tubes, mix well and incubater further for 30
min at 37C.
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Take the optical density (O.D) at 640 nm and calculate the concentration ofunknown from the standard graph or by calculation.
Verification of Beers law:-
i. Measure the absorbance of all the fine tubes using the optimal max.ii. Tabulate the reading
iii. The absorbance is directly proportional to the concentration of KMnO4.iv. A graph is plotted with absorbance of Y-axis and concentration of X-axis. v. A straight line will be obtained explaining the relation between absorbance
and concentration.
Also, beers law can also be written as:
ln IO/I C
ln IO/I = KC
Or 2.303 Log10Io/I = KC
Where:-
Io = intensity of indicant light
I = intensity of transmitted light
C = concentration of absorbing material
K = constant
Lamberts law:-
Ln I/ IO= e-Kb
or ln I/ IO= -Kb
lnIO/I= Kb or 2.303 log10 IO/I = Kb
Where:-
b = the observing thickness/ path length.
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RESULT:- The concentration of unknown protein is 212 mg/ml or 21.20 g%. A straight
line is obtained by plotting the graph between concentration and absorbance, verifying
the beers law.
ASSESSMENT OF PRACTICAL BY INTERNAL (ON THE SPOT)
1. Participation/Involvement of candidate __________/102. Ability to perform himself/herself _________________/103. Results obtained_____________________________________/104. Accuracy_____________________________________________/105. On time submission of practical record____________/106. TOTAL________________________________________________/50
Internal Guide Student
(NAME AND SIGNATURE) (NAME AND SIGNATURE)
0.00
0.20
0.40
0.60
0.80
1.00
1.20
10 20 30 40 50 60 70 80 90 100
O.Dat640n
m
Conc. (mg/ml)
Standard Protein (BSA as standard-0.2 mg/ml)
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EXPERIMENT NO.3
AIM - Separation of aliphatic aromatic and polar amino acids by Thin Layer
Chromatography.
REQUIREMENTS -
a) Chemicals - Silica gel, binder, solvent, standard amino acid,b) Equipments - Chromatography chamber, ninhydrin [sprayer], capillary tube,
micro-pipette.
PRINCIPLE-The separation of compounds on a thin layer is similar to that in paper
chromatography it has several advantages.
[1] Many different type of supporting media can be used because of which the
separation can be by adsorption, ion exchange or gel filtration upon the nature of
medium employed.
[2] The method is very rapid and many separations can be achieved in small time
duration.
[3] The compound after separation can be detection by corrosive sprays which is
not possible with paper as support.
[4] High temperature can also be used for detection with some thin layer, which in
no way is possible with paper chromatography. The detection of compound is onthe basis of their RF values.
The RF value depends upon the thickness of the layer below 200 um. Many adsorbents
are used, most common ones are silica gal, alumina etc. Even ready made thin layer
plates are available.
The atmosphere of the separation chamber should be fully saturated with the solvent to
ensure accurate RF values. For this a small tank is used and the atmosphere of the tank is
fully saturated with solvent the walls of the chamber can also be rept lined with filter
paper soaked in the solvent to ensure complete saturation. The plate is developed usually
by the ascending chromatography technique.
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PRE-PRACTICAL LAB PREPARATION-
1. Silica gel plate preparation - Take silica gel [8 gm] dissolved in 10 ml distill wateradd little amount of binder, mix, and pour and spread on glass plate, allow to air
and incubate at 80C for 1 hour.
2. 0.2 gm ninhydrin dissolved in 100 ml acetone.3. Standard amino acid preparation - To prepare 10 gm/liter of solution add 10gm
[10% isopropanol10 ml in 90ml d/w]
4. Mobile phase preparation butanol, glacial acetic acid and distilled water in12:3:5 ratio.
PROCEDURE-
1. Prepare a thin layer of silica gel on the glass plate by spreading it evenly.2. Allow the plates to dry in air.3.
Keep the plates in hot air oven for 30 minutes.4. Make a line at a distance of 1.5 cm over the surface of plate using scale.
5. Label the amino acid alanine, tryptophan and glutamine on the left side at theback of glass plate.
6. Label the mixture of amino acid on the other side.7. Transfer 5 ml of 3 amino acid on the respective labeled side of plate using a
micropipette.
8. Similarly, put the drop of mixture on the 3 slides separately.9. Keep the slides in a chromatography chamber containing mobile phase
undisturbed for 30 minutes.
10.Measure the distance travelled by the solvent.11.Spray the ninhydrin reagent which indicates the separation of 3 amino acids
by purple color of the complex rhumanns purple.
12.Measure the distance of the colored spot of amino acid and mixture.13.Calculate the RF values of the amino acid separation.
Distance Of Solvent Distance Of Amino Acid Rf Value
6 cm 2.5 cm 0.41
6 cm 3.4 cm 0.56
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CALCULATION-
Rf1= 2.5/6 =0.41
Rf2= 3.4/6=0.56
RESULT- The Rf values of the amino acids are 0.41 and 0.56.
ASSESSMENT OF PRACTICAL BY INTERNAL (ON THE SPOT)
1. Participation/Involvement of candidate __________/102. Ability to perform himself/herself _________________/103. Results obtained_____________________________________/104. Accuracy_____________________________________________/105. On time submission of practical record____________/106. TOTAL________________________________________________/50
Internal Guide Student
(NAME AND SIGNATURE) (NAME AND SIGNATURE)
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EXPERIMENT-4
AIM : Quantitative estimation of reducing sugar by Nelson-Somogyi method.
REQUIREMENT:
a) Chemicals :-Alkaline copper tartrate, dissolve 2.5 gm anhydrous sodiumcarbonate, 2 gm sodium bicarbonate, 2.5gm potassium sodium tartarate,
20 gm anhydrous sodium sulfate in 80 ml & make up to 100 ml.
b) Equipment:-Test tube, test tube stand, beaker, conical flask, pipettes etc.c) Instrument:-Water bath, spectrophotometer, Balance etc.
THEORY: Sugar with reducing properties arising out of the presence of a potential
aldehyde or keto group are called reducing sugar like glucose, galactose, lactose,&
maltose .The nelson-somogye method is one of the classically &widely used method for
the quantitative & widely used method for the quantitative determination of reducingsugar. The reducing sugar when heated with alkaline Cupric tartrate sugar when heated
with cupric to cuprous state when the cuprous oxide is formed is treated with
arsenomolybdic acid. The reduction of molybdic acid to molybdenum free takes place.
The blue color developed is compared with set of standard in a colorimeter at 620 nm.
The concentration of unknown is estimated by plotting a standard of curve of O.D of
standard solution.
PREPARATION:
a)
Dissolve 15 gm CuSO4in a small volume of D/W. Add one drop of H2SO4&make up to 100 ml. mix 4 ml of solution b & 96 ml o solution a before using
it.
b) Arsenomolybdate reagent: Dissolve 2.5 gm ammonium molybdate in 45mlD/W add 2.5 ml H2SO4 & mix well. Then add 0.3 gm disodium hydrogen
arsenate then add 0.3 gm disodium hydrogen arsenate dissolve in 25 ml D/W
.Mix well & incubate at 37C for 24-48 h.
c) Standard glucose solution Stock = 100mg/100 ml D/Wd) Working standard =100 ml stock diluted to 100 to 100 ml with D/W.
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PROCEDURE:
1. Pipette out aliquots of 0.1 or 0.2 ml to separate test tubes.2. Pipette out 0.2, 0.4, 0.6, 0.8 and 1 ml of the working standard solution into a
series of test tubes.
3. Makeup the volume in both sample and standard tubes to 2 ml with distilledwater.
4. Pipette out 2 ml distilled water in a separate tube to set a blank.5. Add 1 ml of alkaline copper tartrate reagent to each tube.6. Place the tubes in boiling water for 10 min. s.7. Cool the tubes and add 1 ml of arsenomolybolic acid reagent to all the tubes.8. Make up the volume in each tube to 10 ml with water.9. For the Test Solution- U1 = 0.1 ml test solution + 1.9 ml D/W + 1ml alkaline
Copper tartrate and U2 = 0.2 ml test solution + 1.8 ml D/W +1ml alkaline
Copper tartrate.
10.Read the absorbance of blue colour at 620 nm after 10 min.11.From the graph drawn, calculate the amount of reducing sugars present in the
sample.
OBSERVATION TABLE:
Tubes Vol.
Std.
Vol.of
D/W
Conc.
Of Std.
Vol.of
Reagen
t A
Boilingwa
terbathfor10min
Vol.of
Reagent
B
Vol.
of
D/W
O.D.
at 540
nm
Blank - 2 ml - 2 ml 2 ml 4 ml 0
S1 0.4 ml 1.6 ml 40 g 2 ml 2 ml 4 ml 0.120
S2 0.8 ml 1.2 ml 80 g 2 ml 2 ml 4 ml 0.296
S3 1.2 ml 0.8 ml 120 g 2 ml 2 ml 4 ml 0.390
S4 1.6 ml 0.4 ml 160 g 2 ml 2 ml 4 ml 0.525
S5 2.0 ml - 200 g 2 ml 2 ml 4 ml 0.670
U1 0.8 ml 1.6 ml 2 ml 2 ml 4 ml 0.215
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CALCULATION:
For estimation the reading of standard is taken:
0.12 O.D is equivalent to 40 g. Therefore,
0.215 O.D is equivalent to: 71.66 g /0.8 ml
But for 1 ml the concentration would be : 89.576 g /ml.
RESULT:- The amount of reducing sugar estimated is 89.576 g /ml.
ASSESSMENT OF PRACTICAL BY INTERNAL (ON THE SPOT);
i. Participation/Involvement of candidate __________/10ii. Ability to perform himself/herself _________________/10
iii. Results obtained_____________________________________/10iv. Accuracy_____________________________________________/10v. On time submission of practical record____________/10
vi. TOTAL________________________________________________/50
Internal Guide Student
(NAME AND SIGNATURE) (NAME AND SIGNATURE)
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
40 g 80 g 120 g 160 g 200 g
O.D.at5
40nm
Concentration of the Carbohydrate (g/ml)
Graph for Standard Carbohydrate
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EXPERIMENT: 5
AIM: To study the effect of incubation time, temperature, pH and substrate concentration
on enzyme activity.
REQUIREMENT:
a) Chemical/cultures/inoculums/medium: Crude Enzyme extract, DNS,Starch (for amylase), Phosphate buffer.
b) Equipments: Test tube, Test tube stand, micropipette, Micro tips,Conical flak, beaker.
c) Instruments: Water bath, Incubator, Centrifuge, Hot air oven, Shaker,Spectrophotometer.
PRINCIPLE:
Enzymes are protein catalysts that, like all catalysts, speed up the rate of a chemical
reaction without being used up in the process. All enzymes are protein except ribozymes
(Which is the combination of both RNA). Enzymes are highly specific. Typically a
particular enzyme catalyzes only a single chemical reaction or a set of closely related
chemical reactions. As is true of any catalyst, enzymes do not alter the equilibrium point
of the reaction. This means that the enzyme accelerates the forward and reverse reaction
by precisely the same factor.
One reason for the efficiency and specificity of an enzyme is the way the enzyme
interacts with the reactant molecule, more commonly known as the substrate, in enzymecatalyzed reactions. The enzyme and substrate interact to form an enzyme-substrate
complex. The interactions between the substrate and active site are weak, non covalent
interactions (i.e. the substrate does not covalently bind to the active site but weakly
interacts with it through interactions like hydrogen-bonding, Vander Waals interactions,
etc).
Enzyme kinetics, the mechanism of enzyme catalyzed reactions is often studied by
making kinetic measurements on enzyme-substrate reaction systems. These studies
include measuring rates of the enzyme-catalyzed reactions at different substrate and
enzyme concentrations.
Every enzyme has some physical conditions required for their actions on their specific
substrate which comprises of incubation time, temperature, pH, substrate concentration
and enzyme concentration. The rate at which an enzyme works is influenced by several
factors, e.g. the concentration of substrate molecules (the more of them available, the
quicker the enzyme molecules collide and bind with them).
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1. Effect of substrate concentration on partially purified enzyme:The concentration of substrate is designated [S]and is expressed in units ofmolarity.
The optimum substrate concentration helps in determining the Km value. The
Michaelis constant (Km) is a means of characterizing an enzyme's affinity for a
substrate. TheKmin an enzymatic reaction is the substrate concentration at which the
reaction rate is half its maximum speed.
Significance of Km
The Km is equal to the substrate concentration at which the reaction rate is half its
maximum value. In other words, if an enzyme has a small value ofKm, it achieves its
maximum catalytic efficiency at low substrate concentrations. Hence, the smaller the
value of Km, the more efficient is the catalyst. Thus, a low Kmvalue means that the
enzyme has a high affinity for the substrate (as a little" substrate is enough to run the
reaction at half its max speed).
This is only true for reactions where substrate is limiting and the enzyme is NOT
allosteric. The value of Kmfor an enzyme depends on the particular substrate. It also
depends on the pH of the solution and the temperature at which the reaction is carried
out. For most enzymes Km lies between 10-1
and 10-7
M.
DeterminingKmand Vmaxexperimentally
To characterize an enzyme-catalyzed reaction Km and Vmax need to be determined.
The way this is done experimentally is to measure the rate of catalysis (reactionvelocity) for different substrate concentrations. In other words, determine V at
different values of [S]. Then plotting 1/V vs. 1/S we should obtain a straight line.
From the y-intercept and the slope, the values of Km and Vmax can be determined.
EXCEL can be used to plot the data which is then fitted to get a straight line, and
from the equation of the straight line the values of Kmand Vmaxare determined using
Lineweaver-Burkeplots for each experiment.
2. Effect of incubation time on activity of partially purified enzymeTime duration of assay is a very important step in characterization of enzyme as it
varies with source of the enzyme and its inherent properties. Even the incubation time
may vary for crude and purified enzyme for the similar assay procedure.
3. Effect of temperature on activity of partially purified enzyme
http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/M/Mole.htmlhttp://users.rcn.com/jkimball.ma.ultranet/BiologyPages/M/Mole.html -
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As the temperature rises, molecular motion and hence collisions between enzyme
and substratespeed up. But as enzymes are proteins, there is an upper limit beyond
which the enzyme becomes denatured and ineffective. The optimum temperature for
the activity of enzyme is determined during enzyme characterization.
4. Effect of pH on activity of partially purified enzymeThe conformation of a protein is influenced by pH and as enzyme activity is crucially
dependent on its conformation, its activity is likewise affected.
ASSAY METHOD:
There are many methods of measurement of enzyme activity. We can either do the
estimation of the substrate or determine the amount of product formed by a
spectrophotemetric method which we call as enzyme assay.
Spectrophotometricassays observe change in theabsorbanceof light between
products and reactants; radiometric assays involve the incorporation or release
ofradioactivityto measure the amount of product made over time.
Spectrophotometric assays are most convenient since they allow the rate of the
reaction to be measured continuously. Although radiometric assays require the
removal and counting of samples (i.e., they are discontinuous assays) they are usually
extremely sensitive and can measure very low levels of enzyme activity.
PREPARATIONS:-
Chemicals:-
Crude enzyme extract: Centrifuge the broth containing culture at 5,000 x g for 10
min. and collect the supernatant.
Starch: - 0.5% Starch was weighed for 50ml phosphate buffer (0.2M and pH 7.0).
a) ReagentsDNS as per the experiment no.1b) Buffers: Phosphate Buffer (0.1 N; 7.0 pH) - 17.5gm K2HPO4 in 100ml D/W was
dissolved in a conical flask, 13.6 gm KH2PO4in 100 ml D/W was dissolved and taken
in another conical flask., 61.5 ml from K2HPO4solution and 38.5 ml from KH2PO4
solution were taken and mixed to form 100 ml 0.1N phosphate buffer of pH 7.0.
c) 0.2M Phosphate buffer: - 0.56gm KOH (Sample A) was weighed for 50ml and 2.72gm KH2PO4(Sample B) was weighed for 100ml.
http://en.wikipedia.org/wiki/Ultraviolet-visible_spectroscopyhttp://en.wikipedia.org/wiki/Ultraviolet-visible_spectroscopyhttp://en.wikipedia.org/wiki/Absorbancehttp://en.wikipedia.org/wiki/Absorbancehttp://en.wikipedia.org/wiki/Absorbancehttp://en.wikipedia.org/wiki/Radioactivityhttp://en.wikipedia.org/wiki/Radioactivityhttp://en.wikipedia.org/wiki/Radioactivityhttp://en.wikipedia.org/wiki/Radioactivityhttp://en.wikipedia.org/wiki/Absorbancehttp://en.wikipedia.org/wiki/Ultraviolet-visible_spectroscopy -
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0.2 M Phosphate buffer preparation
with varying pH
pH SampleA (ml)
SampleB (ml)
6.4 1.3 8.7
6.6 1.8 8.2
6.8 2.4 7.6
7.0 3 7
7.2 3.5 6.5
7.4 4 6
7.6 4.3 5.7
PROCEDURE/METHODOLOGY:-
1. Optimization of incubation period:-a. 0.5 ml enzyme was taken in 5 test tubes 6 for test and 1 for control and 1.0 ml substrate
was added to start the reaction. In case of control immediately 3rd
step was followed to
stop the reaction. Blank was formed by transferring 1ml phosphate buffer in a test tube.
b. The mixture was incubated at 37 degree Celsius for different time: 5min, 10min, 15min,20min and 25min.
c. Immediately after incubation DNS was added to stop the reaction.d. The test tubes were kept in boiling water bath for 10mins.e. Reading was taken in spectrophotometer.
Sample(Crude
enzyme) (In ml)
Substrate (ml) Phosphate
Buffer
DNS (In ml)
Blank - 1ml 1ml
Control 0.5 0.5 - 1ml
Test (sample) 0.5 0.5 - 1ml
2. Optimization of temperature:-a. The optimization of temperature was done subsequent to the determination of optimum
incubation time which was found to be 15 min.
b. 0.5 ml enzyme was taken in 5 test tubes 4 for test and 1 for control and 0.5 ml substratewas added to start the reaction. In case of control immediately 3
rdstep was followed to
stop the reaction. Blank was formed by transferring 1ml phosphate buffer in a test tube.
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c. The mixture was incubated for 15mins at different temperature 20C,30C, 37C, 40Cand 50C
d. Immediately after incubation DNS was added to stop the reaction.e. The test tubes were kept in boiling water bath for 10min.f. Reading was taken in spectrophotometer.
Sample(Crude
enzyme) (In ml)
Substrate (ml) Phosphate
Buffer
DNS (In ml)
B - 1ml 1ml
C 0.5 0.5 - 1ml
T 0.5 0.5 - 1ml
3. Optimization of pH:-a. Substrate Starch was formed in 7 different of phosphate buffer of pH 6.4, 6.6, 6.8, 7.0,
7.2, 7.4 and 7.6.
b. 0.5 ml enzyme was taken in 7 test tubes for test and 0.5 ml substrate was added to startthe reaction. Blank was formed by transferring 1ml phosphate buffer in a test tube.
c. The mixture was incubated for 15mins at 30Ctemperature. Immediately after incubationDNS was added to stop the reaction.
d. The test tubes were kept in boiling water bath for 10 min.e. Reading was taken in spectrophotometer.
Sample(Crude
enzyme) (In ml)
Substrate
(ml)
Phosphate
Buffer
DNS (In
ml)
B - 1ml 1ml
C 0.5 0.5 - 1ml
T 0.5 0.5 - 1ml
4. Optimization of substrate concentration:-a. 0.1ml, 0.2ml, 0.3ml, 0.4ml, 0.5ml, 0.6ml, 0.7ml, 0.8ml, 0.9ml and 1.0ml substrate was
taken in 10 test tubes respectively.
b. 0.9 ml enzyme was added and volume was made up to 1ml by adding phosphate bufferof pH 7.4. Blank was formed by transferring 1ml phosphate buffer in a test tube.
c. The mixture was incubated for 20mins at 30Ctemperature. Immediately after incubationDNS was added to stop the reaction.
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d. The test tubes were kept in boiling water bath for 10mins. Reading was taken inspectrophotometer
Buffer
(in ml)
Substrate
(in ml)
Enzyme (in ml)
Incubationfor 15min
at 37C
DNS (In ml)
0.9 0.1 0.9 1
0.8 0.2 0.9 1
0.7 0.3 0.9 1
0.6 0.4 0.9 1
0.5 0.5 0.9 1
0.4 0.6 0.9 1
0.3 0.7 0.9 1
0.2 0.8 0.9 1
0.1 0.9 0.9 1
0 1 0.9 1
OBSERVATION
a. Effect of incubation time on activity of partially purified enzyme
No. Time(min)
O.D(580 nm)
U/ml
1 5 0.227 13.11
2 10 0.323 18.66
3 15 0.337 19.46
4 20 0.376 21.72
5 25 0.341 19.69
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No. pH O.D
(580 nm)U/ml
1 6.4 0.08 4.85
2 6.6 0.094 5.43
3 6.8 0.125 7.22
4 7.0 0.267 15.42
5 7.2 0.42 24.26
6 7.4 0.49 28.30
7 7.6 0.447 25.82
d. Effect of substrate concentration on partially purified enzyme
No.Substrate conc.
(mg)OD U/ml 1/S 1/V
1 0.1 0.093 5.37 10.00 0.19
2 0.2 0.101 5.83 5.00 0.17
3 0.3 0.109 6.30 3.33 0.16
4 0.4 0.114 6.58 2.50 0.15
5 0.5 0.114 6.58 2.00 0.156 0.6 0.102 5.89 1.67 0.17
7 0.7 0.099 5.72 1.43 0.17
8 0.8 0.099 5.72 1.25 0.17
9 0.9 0.099 5.72 1.11 0.17
10 1.0 0.099 5.72 1.00 0.17
4.85 5.437.22
15.42
24.26
28.30
25.82
0.00
5.00
10.00
15.00
20.00
25.00
30.00
6.4 6.6 6.8 7.0 7.2 7.4 7.6
IU/ml
pH
Effect of pH on enzyme activity
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RESULTS AND DISCUSSION:
The optimum time for enzyme assay for amylase by DNS method was 15 min. while the
optimum temperature, pH and substrate concentration was 30C, 7.4 and 0.4mgl. TheKm
obtained was 0.47mg/ml. Thus, it indicates that a lower concentration of substrate is
needed to half saturate the enzyme to achieve the maximum velocity. The Vmax for the
enzyme is 20 moles/ml/min.
Precautions:
a) Handle the apparatus with care.b) Transfer the sample carefully to avoid the formation of bubble, and operate the pipette
such that solution removes completely from micro tips to enhance the chances of finding
correct optical density.
ASSESSMENT OF PRACTICAL BY INTERNAL (ON THE SPOT)
a. Participation/Involvement of candidate __________/10b. Ability to perform himself/herself _________________/10c. Results obtained_____________________________________/10d. Accuracy_____________________________________________/10e. On time submission of practical record____________/10f. TOTAL________________________________________________/50
Internal Guide Student
(NAME AND SIGNATURE) (NAME AND SIGNATURE)
y = 0.0232x + 0.05
-0.10
-0.05
0.00
0.05
0.10
0.15
0.20
0.25
0.30
-6.00 -4.00 -2.00 0.00 2.00 4.00 6.00 8.00 10.00 12.00
1/V
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EXPERIMENT-6
AIM-To isolate casein protein from milk
REQUIREMENTS-
a) Reagents/Chemicals - Milk 100ml, Sodium acetate buffer, Ethanol ethermixture (1:1), distilled water.
b) Instruments - pH meter, thermometer.c) Equipments - Glasswares [flask, beaker, pipettes, filter (muslin)],
Whatman filter paper, and funnel.
PRINCIPLE-
Casein, the protein found in milk, is present in cows milk to the extent 0.3-0.6%, it gets
precipitated by acidifying milk to a pH of 4.6-4.7. Casein is used to coat paper for
making books and magazines and also in the textile industry for fixing colors, loading,
sizing, refining, and water proofing, .Casein is a heterogeneous mixture of phosphorus
containing protein. Most proteins exhibit a minimum solubility in aqueous solution at
their isoelectric points, as they exist as uncharged zwitter ions at their isoelectric points.
Hence, casein may be isolated from milk by simply adjusting the ph of milk to the PI of
casein, at pH of 4.8 casein is also insoluble in ethanol ,which becomes handy to remove
unwanted fats during casein preparation by extracting fast using ethanol.
PROCEDURE-
1. Take 100 ml milk in a beaker and warm it to 40C gradually and add dilute aceticacid continuously with the dropper till the milk coagulates.
2. Cool to room temperature leave for 10 minutes and then filler the coagulatedprotein through a lean muslin cloth.
3. The precipitate formed is washed twice with distilled water and suspend it in 5 mlethanol.
4. Stir this solution and subsequently filter the suspension using a funnel.5. Wash twice with ethanolether mixture [1:1 ratio] and filter ether is inflammable
so handle with care.
6. Wash once more with pure ether, filter and spread out in a dish to allow it to dry.7. Weigh the casein isolated, calculate the percent yield.8. Calculate the percent recovery by comparing these two values and analyze the
amount of protein lost during the extraction process.
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OBSERVATION:-
Petri plate weight = 28.24 gm
Petri plate with casein protein = 30.25 gm
RESULT The amount of casein protein obtained from milk is 2.01 gm/100 ml i.e,
2.01%.
ASSESSMENT OF PRACTICAL BY INTERNAL (ON THE SPOT)
1. Participation/Involvement of candidate __________/102. Ability to perform himself/herself _________________/103. Results obtained_____________________________________/104. Accuracy_____________________________________________/105. On time submission of practical record____________/106. TOTAL________________________________________________/50
Internal Guide Student
(NAME AND SIGNATURE) (NAME AND SIGNATURE)
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EXPERIMENT NO.7
AIM:- To isolate starch from potato.
MATERIAL REQUIREMENT:-
Potato, weight, Petri plate, cold saline, grinder, chess cloth, ethanol etc.
PRINCIPLE:-amylase & amylopectin that hydrolysis, (leaves) starch a polysaccharides
(a molecule which consist of eight or more monosaccharide molecules) into maltose a
disaccharide (double sugar i.e. compound of 2 monosaccharide molecule &some
monosaccharide such as glucose these disaccharide &monosaccharide enter into the
cytoplasm of the bacterial cell through the semi permeable membrane & there by used by
the endo enzyme starch is a complex carbohydrate(polysaccharide composed of two
constituents amylase a straight chain polymers of 200-300 glucose units enzymehydrolysis of amylase with amylase hence yields maltose units mainly amylase may be
consider an anhydride of -D glucose units.
Amylopectin a linier branched polymer of phosphate groups the average chain length is
24 glucose units. Amylopectin upon incomplete hydrolysis yields the disaccharide
isomaltose. Starch is the most important reserve for material of the higher plant and is
found in equals legumes potato & other vegetables.
PROCEDURE:-
1. Weight 50 gm potato.2. Peel of them under cold water & put the weight pieces of potato in 100 ml cold
saline (0.9% NaCl 100 ml D\W.)
3. Grind the small pieces of potato by grinder.4. Filter the grieve thought cheese cloth.5. Keep the filtrate in the freezer to settle the starch particle.6. Decant the supernatant.7. Wash the residue of starch particle with ice cold saline water & finally with
ethanol.
8. Starch is insoluble in water & saline.9. Chilled saline is used to prevent the action of -amylase.10.Weigh the starch amount and express it in gm%.
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OBSERVATION:-
Potato weight=15 gm
Petri plate weight=29.83
Petri plate with starch=32.36
Dry weight=32.36-29.83
=2.53gm
RESULT:- 16.86 % of 15 gm potato is obtained in the form of starch.
ASSESSMENT OF PRACTICAL BY INTERNAL (ON THE SPOT)
1. Participation/Involvement of candidate __________/102. Ability to perform himself/herself _________________/103. Results obtained_____________________________________/104. Accuracy_____________________________________________/105. On time submission of practical record____________/106. TOTAL________________________________________________/50
Internal Guide Student
(NAME AND SIGNATURE) (NAME AND SIGNATURE)
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EXPERIMENT-8
AIM:- To understand fluorescence, Bright field and Phase contrast microscopy.
INTRODUCTION:- A fluorescence microscope is an optical microscope that uses
fluorescence and phosphorescence instead of, or in addition to, reflection and absorption
to study properties of organic or inorganic substances. The "fluorescence microscope"
refers to any microscope that uses fluorescence to generate an image, whether it is a more
simple set up like an epifluorescence microscope, or a more complicated design such as a
confocal microscope, which uses optical sectioning to get better resolution of the
fluorescent image.
PRINCIPLE:- The specimen is illuminated with light of a specific wavelength (or
wavelengths) which is absorbed by the fluorophores, causing them to emit light of longer
wavelengths (i.e., of a different color than the absorbed light). The illumination light is
separated from the much weaker emitted fluorescence through the use of a spectral
emission filter. Typical components of a fluorescence microscope are a light source
(xenon arc lamp or mercury-vapor lamp), the excitation filter, the dichroic mirror (or
dichroic beamsplitter), and the emission filter (see figure below). The filters and the
dichroic are chosen to match the spectral excitation and emission characteristics of the
fluorophore used to label the specimen. In this manner, the distribution of a single
fluorophore (color) is imaged at a time. Multi-color images of several types of
fluorophores must be composed by combining several single-color images.
Most fluorescence microscopes in use are epifluorescence microscopes (i.e., excitation
and observation of the fluorescence are from above (epi) the specimen). These
microscopes have become an important part in the field of biology, opening the doors for
more advanced microscope designs, such as the confocal microscope and the total
internal reflection fluorescence microscope (TIRF).
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Fluorescence microscopy requires intense, near-monochromatic, illumination which
some widespread light sources, like halogen lamps cannot provide. Three main types of
light source are used; xenon arc lamp or mercury-vapor lamps with an excitation filter,
lasers and high-power LEDs. Lasers are most widely used for more complex fluorescencemicroscopy techniques like confocal microscopy and total internal reflection fluorescence
microscopy while xenon and mercury lamps with an excitation filter or LEDs are
commonly used for widefield epifluorescence microscopes.
SAMPLE PREPARATION:-
A sample of herring sperm stained with SYBR green in a cuvette illuminated by blue
light in an epifluorescence microscope. The SYBR green in the sample binds to the
herring sperm DNA and, once bound, fluoresces giving off green light when illuminated
by blue light.
In order for a sample to be suitable for fluorescence microscopy it must be fluorescent.
There are several methods of creating a fluorescent sample; the main techniques are
labelling with fluorescent stains or, in the case of biological samples, expression of a
fluorescent protein. Alternatively the intrinsic fluorescence of a sample (i.e.,
autofluorescence) can be used. In the life sciences fluorescence microscopy is a powerful
tool which allows the specific and sensitive staining of a specimen in order to detect the
distribution of proteins or other molecules of interest. As a result there is a diverse range
of techniques for fluorescent staining of biological samples.
Epifluorescent imaging of the three components in a dividing human cancer cell.
(DNA is stained blue, a protein called INCENP is green, and the microtubules are red.
Each fluorophore is imaged separately using a different combination of excitation and
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emission filters, and the images are captured sequentially using a digital CCD camera,
then overlaid to give a complete image.)
LIMITATIONS:-
Fluorophores lose their ability to fluoresce as they are illuminated in a process calledphotobleaching. Photobleaching occurs as the fluorescent molecules accumulate chemical
damage from the electrons excited during fluorescence. Photobleaching can severely
limit the time over which a sample can be observed by fluorescent microscopy. Several
techniques exist to reduce photobleaching such as the use of more robust fluorophores, by
minimizing illumination, or by using photoprotective scavenger chemicals.
Fluorescence microscopy with fluorescent reporter proteins has enabled analysis of live
cells by fluorescence microscopy, however cells are susceptible to phototoxicity,
particularly with short wavelength light. Furthermore fluorescent molecules have a
tendency to generate reactive chemical species when under illumination which enhancesthe phototoxic effect.
Unlike transmitted and reflected light microscopy techniques fluorescence microscopy
only allows observation of the specific structures which have been fluorescently labeled.
For example observing a tissue sample prepared with a fluorescent DNA stain by
fluorescent microscopy only reveals the organisation of the DNA within the cells and
reveals nothing else about the cell morphologies.
LIGHT MICROSCOPY
The light microscope, so called because it employs visible light to detect small objects, is
probably the most well-known and well-used research tool in biology. Yet, many students
and teachers are unaware of the full range of features that are available in lightmicroscopes. Since the cost of an instrument increases with its quality and versatility, the
best instruments are, unfortunately, unavailable to most academic programs. However,
even the most inexpensive "student" microscopes can provide spectacular views of natureand can enable students to perform some reasonably sophisticated experiments.
A beginner tends to think that the challenge of viewing small objects lies in getting
enough magnification. In fact, when it comes to looking at living things the biggestchallenges are, in order,
Obtaining sufficient contrast Finding the focal plane Obtaining good resolution Rrecognizing the subject when one sees it
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The smallest objects that are considered to be living are the bacteria. The smallest
bacteria can be observed and cell shape recognized at a mere 100x magnification. They
are invisible in bright field microscopes, though. These pages will describe types ofoptics that are used to obtain contrast, suggestions for finding specimens and focusing on
them, and advice on using measurement devices with a light microscope.
TYPES OF LIGHT MICROSCOPES:-
The bright field microscope is best known to students and is most likely to be found in aclassroom. Better equipped classrooms and labs may have dark field and/or phase
contrast optics. Differential interference contrast, Nomarski, Hoffman modulation
contrast and variations produce considerable depth of resolution and a three dimensional
effect. Fluorescence and confocal microscopes are specialized instruments, used forresearch, clinical, and industrial applications.
Other than the compound microscope, a simpler instrument for low magnification use
may also be found in the laboratory. The stereo microscope or dissecting microscopeusually has a binocular eyepiece tube, a long working distance, and a range of
magnifications typically from 5x to 35 or 40x. Some instruments supply lenses for highermagnifications, but there is no improvement in resolution. Such "false magnification" is
rarely worth the expense.
BRIGHT FIELD MICROSCOPY:-
With a conventional bright field microscope, light from an incandescent source is aimed
toward a lens beneath the stage called the condenser, through the specimen, through anobjective lens, and to the eye through a second magnifying lens, the ocular or eyepiece.
We see objects in the light path because natural pigmentation or stains absorb lightdifferentially, or because they are thick enough to absorb a significant amount of light
despite being colorless. AParameciumshould show up fairly well in a bright field
microscope, although it will not be easy to see cilia or most organelles. Living bacteria
won't show up at all unless the viewer hits the focal plane by luck and distorts the imageby using maximum contrast.
A good quality microscope has a built-in illuminator, adjustable condenser with aperturediaphragm (contrast) control, mechanical stage, and binocular eyepiece tube. The
condenser is used to focus light on the specimen through an opening in the stage. After
passing through the specimen, the light is displayed to the eye with an apparent field that
is much larger than the area illuminated. The magnification of the image is simply theobjective lens magnification (usually stamped on the lens body) times the ocular
magnification.
Students are usually aware of the use of the coarse and fine focus knobs, used to sharpen
the image of the specimen. They are frequently unaware of adjustments to the condenser
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that can affect resolution and contrast. Some condensers are fixed in position; others are
focusable, so that the quality of light can be adjusted. Usually the best position for a
focusable condenser is as close to the stage as possible. The bright field condenserusually contains an aperture diaphragm, a device that controls the diameter of the light
beam coming up through the condenser, so that when the diaphragm is stopped down
(nearly closed) the light comes straight up through the center of the condenser lens andcontrast is high. When the diaphragm is wide open the image is brighter and contrast islow.
A disadvantage of having to rely solely on an aperture diaphragm for contrast is that
beyond an optimum point the more contrast you produce the more you distort the image.
With a small, unstained, unpigmented specimen, you are usually past optimum contrast
when you begin to see the image.
USING A BRIGHT FIELD MICROSCOPE:-
Mount the specimen on the stage:-
The cover slip must be up if there is one. High magnification objective lenses can't focus
through a thick glass slide; they must be brought close to the specimen, which is why
coverslips are so thin. The stage may be equipped with simple clips (less expensivemicroscopes), or with some type of slide holder. The slide may require manualpositioning, or there may be a mechanical stage (preferred) that allows precise
positioning without touching the slide.
Optimize the lighting:-
A light source should have a wide dynamic range, to provide high intensity illuminationat high magnifications, and lower intensities so that the user can view comfortably at low
magnifications. Better microscopes have a built-in illuminator, and the best microscopeshave controls over light intensity and shape of the light beam. If your microscope
requires an external light source, make sure that the light is aimed toward the middle of
the condenser. Adjust illumination so that the field is bright without hurting the eyes.
Adjust the condenser:-
To adjust and align the microscope, start by reading the manual. If no manual isavailable, try using these guidelines. If the condenser is focusable, position it with the
lens as close to the opening in the stage as you can get it. If the condenser has selectableoptions, set it to bright field. Start with the aperture diaphragm stopped down (highcontrast). You should see the light that comes up through the specimen change brightness
as you move the aperture diaphragm lever.
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Focus, locate, and center the specimen:-
Start with the lowest magnification objective lens, to home in on the specimen and/or thepart of the specimen you wish to examine. It is rather easy to find and focus on sections
of tissues, especially if they are fixed and stained, as with most prepared slides. However
it can be very difficult to locate living, minute specimens such as bacteria or unpigmentedprotists. A suspension of yeast cells makes a good practice specimen for finding difficult
objects.
Use dark field mode (if available) to find unstained specimens. If not, start with highcontrast (aperture diaphragm closed down).
Start with the specimen out of focus so that the stage and objective must be broughtcloser together. The first surface to come into focus as you bring stage and objectivetogether is the top of the cover slip. With smears, a cover slip is frequently not used, so
the first thing you see is the smear itself.
If you are having trouble, focus on the edge of the cover slip or an air bubble, orsomething that you can readily recognize. The top edge of the cover slip comes into focusfirst, then the bottom, which should be in the same plane as your specimen.
Once you have found the specimen, adjust contrast and intensity of illumination, andmove the slide around until you have a good area for viewing.
Adjust eyepiece separation, focus:-
With a single ocular, there is nothing to do with the eyepiece except to keep it clean.
With a binocular microscope (preferred) you need to adjust the eyepiece separation just
like you do a pair of binoculars. Binocular vision is much more sensitive to light anddetail than monocular vision, so if you have a binocular microscope, take advantage of it.
One or both of the eyepieces may be a telescoping eyepiece, that is, you can focus it.
Since very few people have eyes that are perfectly matched, most of us need to focus one
eyepiece to match the other image. Look with the appropriate eye into the fixed eyepiece
and focus with the microscope focus knob. Next, look into the adjustable eyepiece (withthe other eye of course), and adjust the eyepiece, not the microscope.
Select an objective lens for viewing:-
The lowest power lens is usually 3.5 or 4x, and is used primarily for initially findingspecimens. We sometimes call it the scanning lens for that reason. The most frequentlyused objective lens is the 10x lens, which gives a final magnification of 100x with a 10x
ocular lens. For very small protists and for details in prepared slides such as cell
organelles or mitotic figures, you will need a higher magnification. Typical highmagnification lenses are 40x and 97x or 100x. The latter two magnifications are used
exclusively with oil in order to improve resolution.
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Move up in magnification by steps. Each time you go to a higher power objective, re-
focus and re-center the specimen. Higher magnification lenses must be physically closer
to the specimen itself, which poses the risk of jamming the objective into the specimen.Be very cautious when focusing. By the way, good quality sets of lenses are parfocal, that
is, when you switch magnifications the specimen remains in focus or close to focused.
Bigger is not always better. All specimens have three dimensions, and unless a specimen
is extremely thin you will be unable to focus with a high magnification objective. The
higher the magnification, the harder it is to "chase" a moving specimen.
Adjust illumination for the selected objective lens:-
The apparent field of an eyepiece is constant regardless of magnification used. So it
follows that when you raise magnification the area of illuminated specimen you see is
smaller. Since you are looking at a smaller area, less light reaches the eye, and the imagedarkens. With a low power objective you may have to cut down on illumination intensity.
With a high power you need all the light you can get, especially with less expensivemicroscopes.
WHEN TO USE BRIGHT FIELD MICROSCOPY:-
Bright field microscopy is best suited to viewing stained or naturally pigmented
specimens such as stained prepared slides of tissue sections or living photosynthetic
organisms. It is useless for living specimens of bacteria, and inferior for non-
photosynthetic protists or metazoans, or unstained cell suspensions or tissue sections.Here is a not-so-complete list of specimens that might be observed using bright-field
microscopy, and appropriate magnifications (preferred final magnifications are
emphasized).
Prepared slides, stained - bacteria (1000x), thick tissue sections (100x, 400x), thinsections with condensed chromosomes or specially stained organelles (1000x), large
protists or metazoans (100x).
Smears, stained - blood (400x, 1000x), negative stained bacteria (400x, 1000x). Living preparations (wet mounts, unstained) - pond water (40x, 100x, 400x), living
protists or metazoans (40x, 100x, 400x occasionally), algae and other microscopic plant
material (40x, 100x, 400x). Smaller specimens will be difficult to observe without
distortion, especially if they have no pigmentation.
PHASE CONTRAST MICROSCOPY
Most of the detail of living cells is undetectable in bright field microscopy
because there is too little contrast between structures with similar transparency and there
is insufficient natural pigmentation. However the various organelles show wide variationin refractive index, that is, the tendency of the materials to bend light, providing an
opportunity to distinguish them.
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A culture ofAmoeba proteusor a fresh suspension of Nagleria gruberimake good
practice specimens.
PRINCIPLE:-
Highly refractive structures bend light to a much greater angle than do structuresof low refractive index. The same properties that cause the light to bend also delay the
passage of light by a quarter of a wavelength or so. In a light microscope in bright field
mode, light from highly refractive structures bends farther away from the center of thelens than light from less refractive structures and arrives about a quarter of a wavelength
out of phase.
Light from most objects passes through the center of the lens as well as to the periphery.
Now if the light from an object to the edges of the objective lens is retarded a half
wavelength and the light to the center is not retarded at all, then the light rays are out ofphase by a half wavelength. They cancel each other when the objective lens brings the
image into focus. A reduction in brightness of the object is observed. The degree ofreduction in brightness depends on the refractive index of the object.
APPLICATIONS FOR PHASE CONTRAST MICROSCOPY:-
Phase contrast is preferable to bright field microscopy when high magnifications
(400x, 1000x) are needed and the specimen is colorless or the details so fine that color
does not show up well. Cilia and flagella, for example, are nearly invisible in bright field
but show up in sharp contrast in phase contrast. Amoebae look like vague outlines inbright field, but show a great deal of detail in phase. Most living microscopic organisms
are much more obvious in phase contrast.
http://www.ruf.rice.edu/~bioslabs/studies/invertebrates/naegleria.html#expthttp://www.ruf.rice.edu/~bioslabs/studies/invertebrates/naegleria.html#expthttp://www.ruf.rice.edu/~bioslabs/studies/invertebrates/naegleria.html#expt -
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Figure. (a) organelles are nearly invisible in bright field although they have different
refractive indexes; (b) light is bent and retarded more by objects with a high refractive
index; (c) in phase contrast a phase plate is placed in the light path. Barely refracted lightpasses through the center of the plate and is not retarded. Highly refracted light passes
through the plate farther from center and is held back another one quarter wavelength.;
(d) The microscope field shows a darker background (in this case the cell cytoplasm has ahigher refractive index than the contractile vacuole), with the organelles in sharp contrast.
USING PHASE CONTRAST:-
Phase contrast condensers and objective lenses add considerable cost to a microscope,
and so phase contrast is often not used in teaching labs except perhaps in classes in the
health professions and in some university undergraduate programs. This is unfortunatesince the images obtainable in phase contrast mode can be very dramatic.
To use phase contrast the light path must be aligned. An element in the condenser is
aligned with an element in a specialized phase contrast lens. This usually involves slidinga component into the light path or rotating a condenser turret. The elements are either
lined up in a fixed position or are adjusted by the observer until the phase effect isoptimized. Generally, more light is needed for phase contrast than for corresponding
bright field viewing, since the technique is based on a diminishment of brightness of most
objects.
ASSESSMENT OF PRACTICAL BY INTERNAL (ON THE SPOT)
1. Participation/Involvement of candidate __________/102. Ability to perform himself/herself _________________/103. Results obtained_____________________________________/104. Accuracy_____________________________________________/105. On time submission of practical record____________/106. TOTAL________________________________________________/50
Internal Guide Student
(NAME AND SIGNATURE) (NAME AND SIGNATURE)
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EXPERIMENT NO.9
AIM: To understand and perform Microtomy.
MATERIALREQUIREMENTs:
a) Equipments: 10 ml pipette, watch glass, beaker, slide, cover slip etc.b) Chemical: 70% alcohol, 90% alcohol, paraffin wax, safranine, glycerol, water
etc.
c) Instruments: microscope, microwave oven.d) Material: Stems
PRINCIPLE:
A microtome (from the Greek micros, meaning "small", and temnein, meaning "to
cut") is a tool used to cut extremely thin slices of material, known as sections. Important
in science, microtome is used in microscopy, allowing for the preparation of samples for
observation under transmitted light or electron radiation. Microtome use steel, glass, or
diamond blades depending upon the specimen being sliced and the desired thickness of
the sections being cut. Steel blades are used to prepare sections of animal or plant tissues
for light microscopy histology. Glass knives are used to slice sections for light
microscopy and to slice very thin sections for electron microscopy. Industrial grade
diamond knives are used to slice hard materials such as bone, teeth and plant matter for
both light microscopy and for electron microscopy. Gem quality diamond knives are usedfor slicing thin sections for electron microscopy.
Microtomy is a method for the preparation of thin sections for materials such as
bones, minerals and teeth, and an alternative to electro polishing and ion milling.
Microtome sections can be made thin enough to section a human hair across its breadth,
with section thickness between 50 nm and 100 m.
PREPARATION:
Preparing 30% alcohol: -
a) Take 5.8 ml water in beaker by the help of pipette.
b) Take 4.2 ml of 70% alcohol beaker.c) Mix both of them.
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PROCEDURE:
1) Cut the stem from plant.2) Then cut it into two pieces.3) Take one of them to dehydrated & remain another as undehydrated.4) Take 10-10 ml 70 % & 90% ethanol in beaker by the help of pipette.5) Then stored in alcohol.
In 30% ethanol: Dip the stem to be dehydrated in 30% ethanol for 20minutes.
In 70% ethanol: Then after 20 minutes, dipped the stem in 70%ethanol.
In 90% ethanol: Then after 20 minutes, dipped the stem in 90%ethanol.
6) Embedding tissues in paraffin blocks - Dehydrated stem processed intoparaffin are melted by placing the entire cassette in microwave oven for 15minutes. Hot paraffin is added to the mold and from the paraffin pot. Use
heated forceps to orient the stems in the mold. When the stems are in the
desired orientation remember the position of dehydrated & undehydrated
stems. Be sure there is enough paraffin to cover the face of the plastic
cassette. When the wax is completely cooled and hardened (~20 min.) the
paraffin block can be popped out of the mold. If the wax cracks or the stems
are not aligned well, simply melt them again and start over.
7) Sectioning stems:Turn on the water bath and check that the temp is 35-37C.Use fresh de ionized water. Put the block in a horizontal way, then sectioningthe both stems cut them in the form of thin chips. Place the dehydrated &
undehydrated stems chips in different watch glasses. Then placed these watch
glasses on water bath, so that if wax is present on stems chips, it will melt.
8) Staining: Take two slides one for dehydrated chips & another forundehydrated chips. Then placed one-one chips on both slides, add 1-1 drops
of safranin, then after 2 minute add glycerol then put cover slip & then
observe this slide under microscope.
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RESULT:- Rose stem cells where observed by microtomy process under light
microscope with great resolution on dehydrated slide.
ASSESSMENT OF PRACTICAL BY INTERNAL (ON THE SPOT)
1. Participation/Involvement of candidate __________/102. Ability to perform himself/herself _________________/103. Results obtained_____________________________________/104. Accuracy_____________________________________________/105. On time submission of practical record____________/106. TOTAL________________________________________________/50
Internal Guide Student
(NAME AND SIGNATURE) (NAME AND SIGNATURE)
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2. Clarify cell and tissues structure and morphology.3. Demarcate functional boundaries.
Limitations
1. Can not to be used for real time in vivo analysis of any tissue ( requires the removal andkilling of the tissue).
2. Uses in humans limited to biopsied tissues3. For looking at changes in tissue over time, each point in time requires a new tissue
sample from a new animal.
4. Tissue preparation a histochemical analysis may alter specimen morphology or chemistrydepending on the methods and material.
PROCEDURE-
1. Stomata Sataining-
a) Take a section of aloevera leaf then peel a thin layer from the surface of leaf.
b) Cut a thin layer into two parts. c)
Place these thin layers on two slides A & B.
d) On slide A, add safranin& then add iodine.
e) On slide B add safraninonly.
f) Add drop of glycerol on both slides & then put cover slips.
g) Observe these slides under microscope.
2. Nucleus Staininga) Take an onion then peel the thin layer from the surface of the onion
b) Cut a thin layer in a small piece.
c) Place this thin layer in the watch glass.d) In the watch glass, add few drops of safranine.
e) Then put the watch glass on a water bath for 10 minutes to heat.
f) Then after 10 min. pick out a thin layer from watch glass & put it on slide.
g) Add drop of glycerol & then put cover slip.
h) Observe this slide under microscope.
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3. Cell Staininga) Take a slide, put few drops of methylene blue.b) Take a tooth pick & scrape it on chicks surface (on surface of jaw).c) Then dip this tooth pick in methylene blue.d) Add glycerol & then put a cover slip.e) Observe this slide under microscope.
RESULT
For Stomata Staining -
1. Inslide A, pinkish stomata & bluish starch are observed.2. In slide B, at the central red stomata are observed.
For Nucleus Staining - Prominent nucleus is observed after staining.
For Cell Staining -Squamous cells are observed after staining.
ASSESSMENT OF PRACTICAL BY INTERNAL (ON THE SPOT)
1. Participation/Involvement of candidate __________/102. Ability to perform himself/herself _________________/103. Results obtained_____________________________________/104. Accuracy_____________________________________________/105. On time submission of practical record____________/106. TOTAL________________________________________________/50
Internal Guide Student
(NAME AND SIGNATURE) (NAME AND SIGNATURE
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EXPERIMENT:11
AIM:- Tostudy various stages of mitosis.
REQUIREMENTS:-
a) Chemicals/Reagents1 N hydrochloric acid, 1% acetocarmine stain, 45% aceticacid.
b) EquipmentsMicroscope slide, cover glasses, watch glasses,dissection needles,scalpel or razor blade, forceps, filter paper.
c) Instruments - Microscope, onion bulbs growing cell in beaker.
PRE-LAB PREPARATION:-
a) Prepared acetocarmine stain (dissolve 1.0 g carmine in pre-boiled 100 ml of 45%glacial acetic acid, in pre boil it for 2 min, cool and filter using whatman no. 1
filter paper.
b) Well growing onion in water.
PRINCIPLE: -
The increase in the number of body or somatic cells that accomplished by a process
called mitosis. This important life process is responsible for the increase in the number of
individuals in population of unicellular organism and replaced cells. The most prominent
feature of this process are changes in the nucleus, which passes through a series of easily
stages. Istthe chromosome appear as thin threads which gradually thicken, separate and
migrate as daughter chromosomes to the two opposite poles of the cell, where they
rebuild the two daughter At nucleus . At this stage the division of the nucleus is complete.
The process of mitosis is basically the same in all organisms, both plants and animals.
PROCEDURE:-
1. Take the terminal 12 cm of the onion root tips can be cut with the helpof needles or razor blade.2. Place the tip in 1 N HCl in water glass.3. Incubate for 5 min. 60C.4. Discard the HCl.5. In the same glass wash glass add few drops of 1% acetocarmine.6. Warm to gently & incubate for 15-20 min.
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7. Take a single root on microscopic slide add 1-2 drop of 45% acetic acid.8. Cut 1mm of root tip using a sharp blade.9. Apply a clean cover glass to the slide & remove the excess stain by filter
paper.
10.Tap the slide with match stick.11.Observe under microscope 10X than 40X.
OBSERVATION AND RESULT:- All the four dividing stages of mitosis cell division
. Prophase, Metaphase, Anaphase, & Telophase can be observed.
FigOnion root tip showing anaphase stage
PRECAUTION:- Excess heating during staining of root tips would damage the
chromosomes staining time is crucial.
ASSESSMENT OF PRACTICAL BY INTERNAL (ON THE SPOT)
1) Participation/Involvement of candidate __________/102) Ability to perform himself/herself _________________/103) Results obtained_____________________________________/104) Accuracy_____________________________________________/105) On time submission of practical record____________/106) TOTAL________________________________________________/50
Internal Guide Student
(NAME AND SIGNATURE) (NAME AND SIGNATURE)
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EXPERIMENT-12
AIM:- Preparation of media for growth of various microorganisms.
REQUIREMENTS:-
a) Cultures Algae, E.coli, proteaus, Pseudomonas, S.aureus Micrococci cultures,Actinomycetes, Rhizobium, Azotobacter, Aspergillus, Trichoderma, Yeast culture.
Mediums - N. agar, N. broth, Bennet agar, MacConkey agar/broth, E.M.B Agar.,
Muller Hinton agar, Blood agar, TSI slant, S.C. slant, Urea broth, Gelatin medium,
peptone 1%, 2%, PDA, MRBA, Czapek agar, GYE, Ashbys mannitol agar, YEM,
Thioglycolate medium.
b) Equipments - Wire loop, burner.
THEORY:- To understand, study, isolate, identify, purify, and maintain an organism it is
essential to grow them. To provide them various nutrients required for growth. It is
essential to provide them the favorable conditions like suitable temperature, pH, aeration,
moisture, etc. The environment from which an organism gets all these is called as a
culture medium. Depending upon the type of microorganism & nutritional specification
there are different kind of media prepared. If the purpose is isolation, identification &
maintained then preferably solid media (containing agar) are used and when growth &
study of physiological state metabolic reactions and production is it be dealt then the
liquid medium is used (which is called as broth).
The medium consists of macronutrients & micronutrients suspended in water (D/W).The
macronutrients comprises of C/N/H requirements while the macronutrients can be S/P /K
/Na/ Ca/ Mg/ Mn/ Fe/Cu/Zn/coenzyme etc. which are required small quantities as a
cofactor or coenzyme for various enzymatic reactions. Depending on the energy
requirement, electron source & carbon requirement of the microorganism organic or
inorganic source are used. Generally Peptone Meat Extract, Urea are used as nitrogen
source. Some grow on atmospheric Nitrogen, others thrive on inorganic nitrogen
compounds such as nitrates or ammonium salts and still other derives nitrogen from
organic compounds such as amino acids.
All organisms require carbon in some form to use synthesizing cell component. All
organisms require at least small amount of CO2 . Some use CO2 sole source of carbon
such organisms are called as autotrophs whereas others require organic compounds. All
organisms require oxygen, sulphur & phosphorus for cell compounds. Oxygen is
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provided in various forms such as water component atoms of various nutrients or
molecular oxygen.
Sulphur is needed for synthesis of certain amino acid (cysteine, cystine & metheonine).
Some bacteria require organic sulphur compounds, some use inorganic & some even use
elemental sulpher. Phosphorus is usually supplied in from of phosphate. It is an essentialcomponent of nucleotides, nucleic acids, phospholipids, teichoic acid & other
compounds.
All living organisms require metal ions such as K+, Ca
2+, Mg
2+, Fe
2+, Zn
2+,
Cu2+
,Mo6+
,Ni2+
,B3+
,Co2+
in small quantities as trace metals (few known to act as co-
factors) Water is required by all living organisms to disperse nutrients for its entry in the
cell. Provides environment for metabolic reaction, its high specific heat provides
resistance to sudden transient temperature changes in the environment. H2O is also a
chemical reactant required for many hydrolytic reactions carried out in a cell. Besides the
basic media required to promote the growth of a particular micro organism some mediaare of significance to identify biochemical character & finally the identification of a
micro organism. They also help in distinguish various groups of micro organisms as
Grampositive & Gram
negative such media are differential & selective. Nutrient agar and
nutrient broth is the medium used for general purposes for growth of all kinds of bacteria.
Bennets agar is the medium used for cultivation of Actinomycetes, PDA, Czapeks agar
MRBA, GYE are used for fungi (yeast & molds) Muller Hinton is used for screening of
micro organisms antibiotic susceptibility (Ashbys) Mannitol agar & Yeast extract
mannitol agar medium are used for Azaotobacter andRhizobium respectively E.coli &
other Gram
negative microorganisms are identified on MacConkey agar/broth and EosinMethylene Blue agar medium.
The biochemical media used for identification of Grampositive microorganisms are
Blood agar, Milk agar and nutrient (for catalase and coagulase) similarly the biochemical
media used for identification of Gramnegative microorganisms are TSI, SC, Urea,
Gelatin,Glucose phosphate broth 1%, 2% peptone. Blood agar differentiates between ,
, hemolytic streptococci (greenish zone, clear zone, & no heamolysis respectively),
Milk agar gives good pigmentation. MacConkey medium contains Bile salts that inhibits
growth of Grampositive organism while netural red helps in differentiating lactose
fermenting & non fermenting microorganism by pink colored colonies.
Eosin methylene blue helps differentiating between E.coli & Klebsiella. E.coli gives
greenish metabolic shining colonies because of acid production on fermenting lactose
while Klebsiella gives purplish pink & mucoidal colonies. Glucose phosphate broth is
used to detect Voges Proskeaur (VP) & methyl red (MR) test. Gelatin helps in
identification of liquefication of & production of Gelatinase enzyme. Simmon citrate agar
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contains Bromothymol blue indicator color which indicates citrate utilization &
production of Ammonia by turning blue (in alkaline condition).
Triple suger iron medium (TSI) help in identification of H2S production and acid
production. The H2S produced reacts with Iron present in media forming black precipitate
while acid reacts with neutral red indicator giving yellow colour which turns red onalkaline conditions.
1% peptone is used to check IAA production by using Kovacs reagent. A positive
reaction is indicated by pink ring formation. Urea utilization is seen by formation of
ammonium carbonate which is alkaline & indicated by light pink colour turning to purple
pink detected by colour change of phenol red indicator.
PROCEDURE:-
1. Inoculate the given culture in various media.
2. S.aureus & Micrococciin Blood agar, Milk agar, N. agar, N. broth.
3. Clostridium (soil sample) in thioglycollate broth.
4.Aspergillus, Yeast, Trichoderma,in PDA, MRBA, Czapek, GYE.
5. E.coli in Mac. Conkey, EMB, TSI, S.C., Urea, Gelatin, Peptone, Proteus,
Psuedomonas.
6.Rhizobiumon YEM & Azotobacter on Ashbys mannitol.
7.Actinomyceteson Bannets agar.
8. Algae (Nostoc)in M medium.
9. Incubate the cultures of bacteria at 37c for 24 hour & fungi at 26C for 4 days, Algae
in sunlight at room temp.10. Observe the pattern of growth in various media.
OBSERVATIONS: -
FigE.colion MacConkey agar, S.aureuson Blood agar andAspergilluson PDA
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RESULT:- Different patterns of growth are observed in different medias.
ASSESSMENT OF PRACTICAL BY INTERNAL (ON THE SPOT)
1. Participation/Involvement of candidate __________/102. Ability to perform himself/herself _________________/103. Results obtained_____________________________________/104. Accuracy_____________________________________________/105. On time submission of practical record____________/106. TOTAL________________________________________________/50
Internal Guide Student
(NAME AND SIGNATURE) (NAME AND SIGNATURE)
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EXPERIMENT-13
AIM:- To sterilization methods that are used in biotechnology.
REQUIREMENTS:-
a) Autoclave or pressure cooker ( or wet sterilization)b) Laminar flow sterile cabinet (fitted with UV lamp)c) Oven ( for dry sterilization)d) Ethyl alcohol sodium hypochlorite (5%) ( for chemical sterilization)e) Filter sterilization unit vacuum pump and 0.22 m ultra filters (for ultra
filtration
f) Distilled water (glass double distilled water)g) Sprit seeds of any plant material.h) Glassware, plastic ware, cotton plugs, sprit lamp, absorbent cotton.i)
Forceps, surgical blades and holder.
PRINCIPLE:-
The maintenance of sterile environment (devoid of micro-organisms) is very important
during the transfer and culture of microbes. Plant and animal cells as most of the nutrient
culture media can also support the growth of bacterial, fungal contaminants which spoil
the culture the inoculation of microbial plant and animal cultures is usually done in a
laminar flow cabinet which is designed to maintain sterile working area and fitted with
germicidal lamp.
Another important thing is the sterilization of tools (e.g. surgical instruments) Culturevessels. Nutrient media and plant material (e.g. leaves) used in culture, there are several
method by which these materials can be sterilized the common methods employed for
this purpose are given below.
Dry heat sterilization:- This procedure is mainly used for the sterilization of glassware
and metal instruments that are not damaged by elevated temperature, generally laboratory
dying ovens are used.
Wet heat sterilization:- This method is employed for the sterilization of glassware,
paper products, tissue culture tool (e.g. surgical blades and scalpels) and liquid (e.g.
tissue culture medium) an autoclave ( which function like a home pressure cooker) is
used for this purpose.
Sterilization by ultra filtration:-This method is mainly used for chemical which our
heat-sensitive (heat labile) or unstable at high temperature e.g. enzymes, gibberellin acid
(ga3) and antibiotics, membrane filters with varying pore size (0.22, 0.45 and 0.50 mm)
which are commercially available (e.g. nalgene, millipore and whatman membrane filter
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are makeup of cellulose acetate or cellulose nitrate are available in pre-sterilized plastic.
disposable unit or as individual autoclavable units. liquids in small quantity will be
sterilized by passing through a membrane filters which is fitted to a membrane filter unit
these unit are equipped for vacuum facility.
Chemical sterilization:- This procedure is mainly employed for the surface sterilizationof plant materials such as leaves, shoot tips, seeds etc. the plant materials to the sterilized
are usually incubated in an aqueous solution of optimum concentration of any
disinfectant (chlorax, mercuric chlorite and sodium hypochlorite (commercial bleach)
foran appreite time after washing with tap after then sterile double distilled water the
most commonly used surface sterilant are chlorax or solution contain agents and this
should be prepared in 0.05% [v/v] detergent like twen 80 or teepal or 0.1 0.2%
mercuric chloride (this compound is poisonous and mutagenic, hence it should be
handed carefully).
PRE-LAB PREPARATION:-
Preparation of 70% ethyl alcohol and 5% sodium hypochlorite. Autoclaving the filtration unit. Post-grow seeding of any plant (15 days old) Making of cotton plugs.
PROCEDURE:-
Sterilization of glassware & tools-
Complete sterilization can be achieved by keeping glassware & various tools like forceps,
surgical blade, holder, needs any other metal instruments (wrapped in heavy duty
aluminum foil) in an oven for about 2 hour at 160oC.
Sterilization of nutrient media-
Liquid (e.g. nutrient media) or other materials (e.g. forceps, glassware etc.) can be
sterilized by keeping them in an autoclave with a 0.22 m pore size for sterilizing such
chemicals they should be passed through the ultra filters using vacuum pump.
OBSERVATION:- Sterilization of liquid media by autoclave, if note done properly can
lead to contamination of the media by microbes the excess autoclaving can lead to
browning of the media.
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Surface sterilization for longer durations can affect the viability of seeds & the culture
ability of plant part (e.g. leaves)
ASSESSMENT OF PRACTICAL BY INTERNAL (ON THE SPOT)
1. Participation/Involvement of candidate __________/102. Ability to perform himself/herself _________________/103. Results obtained_____________________________________/104. Accuracy_____________________________________________/105. On time submission of practical record____________/106. TOTAL________________________________________________/50
Internal Guide Student
(NAME AND SIGNATURE) (NAME AND SIGNATURE)
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EXPERIMENT NO.13
AIM:Identification of bacteria by Bergeys manual.
REQUIREMENT:
a) Medium - Nutrient agar,1% peptone broth,2%peptone broth, milk agar, Mc.Conkeyagar, urea broth, gelatin broth, blood agar, EMB agar, Simon citrate slant, TSI slant.
b) Reagents - Methyl red indicator, H2O2, blood plasma.c) EquipmentsTest tubes, Petriplates, Slides, and Pipettes.d) InstrumentsIncubator, Hot air oven.
THEORY:
Bergeys manual was first published in 1923 byDavid Hendricks Bergey, it is used to
classify bacteria based on their structural and functional attributes by arranging them
into specific familial orders. Bergey's Manual of Determinative Bacteriology was
published first as a guide for identifying unknown bacteria and is still published. In
order to have no discrepancy and to include the "relationships between organisms" and
to have "overall expanded scope" The Bergeys Manual of Systematic
Bacteriologywas introduced and is the main resource for determining the identity
of bacteria species, utilizing every characterizing aspect.
This new style (Systematic Bacteriology)was picked up for a four volume set that first
began publishing in 1984. The information in the volume were separated as follows.
Volume 1 includes information on all type of Gram negative bacteria that wereconsidered to have medical and industrial importance.
Volume 2 includes information on all type of Gram positive bacteria.
Volume 3 deals with all of the remaining slightly different Gram negative bacteria alongwith theArchea.
Volume 4 has information on filamentous actinomycetes and other similar bacteria.As we know huge battery of tests are done to identify an unknown organism which
would result in a lot of media and time being wasted dealing with irrelevant tests. Thus
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we would like to proceed in stages performing only those tests which are applicable to
what ever basic knowledge we have about unknown. There is no media that can possibly
support the growth of all of the different species of bacteria. As an example formulation
exists for media to detect glucose fermentation based on nutrient requirement of various
group of bacteria. To utilize glucose of medium as a routine test for glucose catabolism
is unwise, as it was designed to differentiate Gram negative bacteria while Gram
positive bacteria happen to grow poorly in the medium. To assist in explaining the
general principles of pH-based differential media, glucose of medium can be valuable in
a demonstration of the relative effect of amino acid deamination, glucose respiration and
glucose fer