Biotechnology Perspectives

[BTY 150]

A Laboratory Manual

Approved for Autumn Term Session 2010-11 -1-

LABORATORY WORK:

S.No. Description

1 Principle and demonstration of paper chromatography.

2 Experimental analysis of Beer’s- Lambert law using spectroscope.

3 Principle and demonstration of colorimetry.

4 Calibration of pH meter and estimation of pH of different samples using pH meter.

5 Principle and demonstration of Electrophoresis.

6 Effect of different temperatures on bacterial growth.

7 Principle and demonstration of autoclave.

8 Principle and demonstration of Surface sterilization.

9 Effect of Various temperatures on nutrient content of media.

10 Introduction of various Biological Databases like PubMed and Rebase.

Approved for Autumn Term Session 2010-11 -2-

 Experiment No.1

Title: Principle and demonstration of paper chromatography

Equipments to be used: Whatman Paper no. 1 or 3, Chromatography Trough.

Learning Objectives:

Cellulose is an ideal support medium as paper sheets, which adsorbs water between
fibres and forms a stationary hydrophilic phase,

Separation of mixtures using the above stationary phase and organic solvent.

Procedure:

 The mixture is spotted onto the above mentioned paper, dried and the chromatogram
developed by allowing the solvent to flow along the sheet.

 The solvent front is marked and, after drying the paper the positions of the
compounds present in the mixture are visualized by a suitable staining reaction.

 The ratio of the distance moved by a compound to that moved by particular

compound, solvent system and paper under carefully controlled conditions of solute
concentration temperature and pH.

Precautions:

1. Sample spot should be as compact as possible.

2. Sample should not overrun the other end of chromatogram.

3. Solvent front should be marked before drying chromatogram.

Approved for Autumn Term Session 2010-11 -3-

 Experiment No. 2

Title: Experimental analysis of Beer’s-Lambert law using Spectroscope

Equipments/Chemicals required: Spectrophotometer, protein sample, and cuvette

Learning objectives:

Some colorimeters and spectrophotometers have two scales, a linear one of percent
transmittance and a logarithmic one of extinction. It is the latter scale that is related linearly
to the concentration and is the one used in the construction of the standard curve. With the aid
of such standard curve, the concentration of the unknown solution can be determined from its
extinction.

Procedure:

 Determine the extinctionof dye in turn against the range of the filters supplied with
the colorimeter.

 Carefully note the wavelength of the maximum transmission of each filter and plot the
graph of the absorbance recorded against each wavelength.

Scope of results:

Concentration of the sample can be determined.

Precautions

1. Solution should not be too concentrated.

2. Light used must be monochromatic

3. The wavelength of the light used should be at the absorption maximum of the
solution.

4. There must be no ionisation, association, dissociation or salvation of the solute with

concentration or time.

Approved for Autumn Term Session 2010-11 -4-

 Experiment No. 3

Title: Principle and demonstration of Colorimetry

Equipments to be used: Colorimeter, Cups for solutions

Learning Objectives:

1. The colour intensities of the unknown solution and the standard solution are
compared visually. The depgth of the unknown solution is varied until its colour
exactly matches that of the standard solution.

2. The depths of the standard and the unknown solutions are read on a scale and
the concentration of the unknown solution is determined.

Procedure:

 The cups and plungers are cleaned followed by turning on of the light source.
Both the cups are raised by the knobs provided their bottom touches the
plungers. Each scale at this time should read zero.

 A suitable volume of the standard solution is filled in each cup while one of
the cups (the left one) is adjusted to read 20.

 The other cup is moved until the colours in the two halves exactly match. Its
position is red on the scale (normally should be 20).

 The right cup is drained and filled with the unknown solution and replaced in
teh colorimeter. It is raised and lowered several times to rinse the plunger.

 It is drained and again filled with the unknown solution.

 The right cup is moved up and down until the colours in the two halves
exactly match.

 Its position is read on the scale. Three or four readings are taken and their
average is calculated.

 The cups and plungers are rinsed with distilled water and replaced in the
instrument after completion of the experiment.

Precautions:

1. Cuvettes should be handled with care.

2. After each reading cuvittes should be rinsed in distilled water.

Approved for Autumn Term Session 2010-11 -5-

 Experiment No. 4

Title: Calibration of pH meter and estimation of pH of different samples using pH meter

Equipments/Chemicals requirements:

pH meter calibration spreadsheet (hard copy), 50 ml glass beakers, 100 ml glass beaker, pH 4
buffered standard calibration solution, pH 7 buffered standard calibration solution, pH 10
buffered standard calibration solution, Kimwipes box, squeeze bottle filled with tap water,
stopwatch

Learning objectives: pH calculations of different samples

Procedure

 All pH meters must have probes that are thoroughly moistened 24 hours before
calibrations are done. To do this, soak the meters in tap water overnight before
beginning calibrations.
 Set the pH meters intended for calibration out on the lab bench. Rinse all meters with
tap water before beginning calibration. Gently shake off excess tab water. You may
use Kimwipes to gently dab-dry the excess water if needed, but do not wipe the
meters or the probes directly.
 Set the three beakers up in a line on the lab bench. Fill the first (farthest to the left) 50
ml beaker with pH 4 calibration solution. Make sure the level is just high enough so
that the meter probe will be completely submerged. Be conservative so not to waste
calibration solution.
 Fill the second 50 ml beaker with pH 7 stock solution. Make sure the level is just high
enough so that the meter probe will be completely submerged. Be conservative so not
to waste calibration solution.
 Fill the 100 ml beaker with pH 7 stock solution. Make sure the level is just high
enough so that the meter probe will be completely submerged. Be conservative so not
to waste calibration solution.
 Turn on and place pH meter being calibrated into the 100 ml beaker containing pH 7
calibration solution. Leave it submerged in the solution for three minutes (use the
stopwatch). Once this step is underway, go to Step 2.
 After three minutes has passed in Step 1, record the initial pH reading for the first pH
meter on the calibration spreadsheet in the appropriate space.
 Remove the pH meter and rinse the probe thoroughly with tap water. Dry by shaking
off the excess water and dabbing with Kimwipes. Do not wipe or try to dry the
meter’s probe completely. Immediately place it into the first beaker (farthest to the
left) containing pH 4 calibration solution.
 Press and hold the “CAL” button on the meter until the display numbers begin to
flash. Leave the probe submerged in the solution for three minutes.
 After three minutes passes, press the “HOLD” button to end calibration. Record the
after calibration reading on the calibration spreadsheet in the appropriate space. Note:
An initial reading is not recorded, just an after calibration reading.

Approved for Autumn Term Session 2010-11 -6-

  Remove the pH meter and rinse the probe thoroughly with tap water. Dry by shaking
off the excess water and dabbing with Kimwipes. Do not wipe or try to dry the
meter’s probe completely. Immediately place into the second (middle) beaker
containing pH 7 calibration solution.
 Press and hold the “CAL” button on the meter until the display numbers begin to
flash. Leave the probe submerged in the solution for three minutes.
 After three minutes passes, press the “HOLD” button to end calibration. Record the
after calibration reading on the calibration spreadsheet in the appropriate space. Note:
An initial reading is not recorded, just an after calibration reading.
 Remove the pH meter and rinse the probe thoroughly with tap water. Dry by shaking
off the excess water and dabbing with Kimwipes. Do not wipe or try to dry the
meter’s probe completely. Immediately place into the third (farthest to the right)
beaker containing pH 10 calibration solution.
 Press and hold the “CAL” button on the meter until the display numbers begin to
flash. Leave the probe submerged in solution for three minutes.
 After three minutes passes, press the “HOLD” button to end calibration. Record the
after calibration reading on the calibration spreadsheet in the appropriate space. Note:
An initial reading is not recorded, just an after calibration reading.
 Remove the pH meter and rinse thoroughly with tap water. Dry by shaking off the
excess water and dabbing with Kimwipes. Do not wipe or try to dry the meter’s probe
completely. Immediately place into the 100 ml beaker containing pH 7 calibration
solution. Leave the probe submerged in solution for three minutes.
 After three minutes passes, record the final calibration reading.
 Now you can directly determine the pH of your different samples one be one by
immersing the electrode in respective samples. Rinse electrode in distill water in
between each pH readings.

Scope of the result: Different buffers or samples will have different pH and so will be
manifested through the readings obtained through the pH-meter.

Cautions:

 You can safely pour all pH calibration solutions down the sink drain; they are not
hazardous.
 Return the pH calibration solutions and tap water squeeze bottle(s) to the appropriate
cabinet for storage.
 Wash all beakers and dry on the drying rack next to the sink; return to the appropriate
cabinet once dry for storage.
 Make sure all calibrated pH meters are turned off and put in the correct monitoring
box(es), or stored all together and clearly dated and labeled “calibrated.”
 Make sure you have filled out all the fields on the calibration spreadsheet and secure
it in the binder. Safe the electronic copy to the server accordingly.

Approved for Autumn Term Session 2010-11 -7-

 Experiment No. 5

Title: Principle and demonstration of Electrophoresis

Principle: Gel electrophoresis is a technique used to separate DNA samples based on their
size. DNA is loaded onto an agarose gel which is placed in a buffer solution. An electric
current will cause the DNA (which is positively charged) to migrate to the negative current
(down the gel). The small DNA molecules will move faster, and this results in small DNA
fragments moving farther down the gel.

Requirements: Agarose, TAE buffer, electrophoresis assembly, sample, loading dye.

Learning objectives: Resolution of samples in a gel is a manifestation of its migration based

on size and is inversely proportional to the size.

Procedure:

1. A 1% agarose gel is poured and placed in the electrophoresis chamber, which is filled with
1 x TAE buffer.
2. The DNA products are mixed with a blue loading dye.
3. The DNA is then loaded into a “well” in the gel.
4. The voltage is applied until the dye has migrated down the gel.
5. The gel is soaked in an ethidium bromide solution to stain the DNA.
6. The DNA in the gel is visualized using UV light (which causes the ethidium
bromide to glow).

Scope of the results: Different samples will have different resolution fronts (Rf) which are
characteristics to their sizes. By calculating the Rf based on the distance travelled by the
sample vs tracking dye, the size of the sample (DNA) can be calculated through interpolation
of the same for a known standard.

Precautions:

1. Melted agarose should be poured immediately and its distribution should be even.

2. Combs should be placed in apparatus before pouring agarose.

3. Stop elctrophoresis before the sample reaches other end of the gel.

4. Ethidium bromide should be handled wearing gloves.

Approved for Autumn Term Session 2010-11 -8-

 Experiment: 6

Title: Effect of different temperatures on Bacterial Growth

Equipments/Materials Required:

 Nutrient broth cultures of Bacillus subtilis

 Czapek-Dox spore suspension of Aspergillus niger
 Nutrient agar medium (40 ml)
 Czapek-Dox agar medium (40ml)
 Hot water bath
 Centigrade thermometer
 Sterile petriplates (4)
 Inoculating loop/ needle
 Bunsen burner

Learning Objectives for students:

1: To study the lethal effects of temperature on microorganisms and understand its

relationship with time and temperature.

2: To determine the TDT of B. Subtilis and E.Niger required for a particular temperature.

Brief Procedure:

1. Divide the bottoms of each Petri plate into six sectors with the glass marking pencil.
2. Label four sets of plates with the time intervals as 0,3,6,9,12,18,21,24,27 and 30.
3. Pour melted and cooled nutrient agar medium into two sets and czapek-dox agar into
the other two sets and allow the media to solidify.
4. Label the bottom of nutrient agar plates with B. subtilis and Czapek-dox agar plate
with A. niger.
5. Inoculate “0” sector of NA plate with B. subtilis and of CDA plate with A. niger as
streak inoculations.
6. Suspend one each tube of B. subtilis and A. niger into the water bath set at a
particular temperature( as determined in the TDP experiment ) say 60◦C water bath for
4 minutes (one minute for the temperature of the broth and the water equalize and 3
minutes time of exposure).
7. Remove both of these tubes and cool these quickly under tap water.
8. Make streak inoculations on sector 3 on NA plate and CDA plate with the B. subtilis
and A. niger respectively.
9. Replace the tubes in the water bath and allow 1 minute for the broth and water to
become equalized before starting the next time interval, i.e. 6 minutes and repeat steps
7 and 8 quickly.
10. Incubate the B. subtilis inoculated plates at 30◦C (24-48 hours ) and A. niger
inoculated plates at 25◦C (3-5 days ) in an inverted position for the period-mention or
until growth occurs in the “0” time segments.

Approved for Autumn Term Session 2010-11 -9-

Scope of Results

Observe the inoculated plates for the presence of growth in various sectors and compare
with the “0” time sector and record the growth as + (growth present ) and –(no growth) and
determine the TDT of each organism i.e. the time to kill the test organism at the given
temperature.

Cautions

1. Water level of the water bath should be above the broth level.
2. Temperature of the water should be kept constant.
3. After a particular treatment the broth tubes should be immediately cooled.

Approved for Autumn Term Session 2010-11 - 10 -

 Experiment No. 7

Title: Principle and demonstration of Autoclave

Equipment required: Autoclave

Learning objective: Wet heat is the most dependable procedure for the destruction of all
forms of microbial life. Steam sterilization generally denotes heating in an autoclave
employing saturated steam under a pressure of approximately 15 psi to achieve a chamber
temperature of at least 121◦C. The critical factors in insuring the reliability of this
sterilization method is: 1) proper temperature and time; and 2) the complete replacement of
the air with steam (i.e. no entrapment of air). It was invented by Charles Chamberland in
1879, although a precursor known as the steam digester was created by Denis Papin in 1679.

Procedure:

1. Place object(s) to be autoclaved in an autoclave tray (for easy carrying when you take
them out!)

 If autoclaving liquid media, place aluminum foil loosely over the top of the container.

 If using a screw-on cap, loosen the cap before autoclaving.

2. Place the autoclave tray in the autoclave (be careful, the walls and inside panel of the
door is hot!)

* Minimum autoclaving time includes the time required for the volume to
reach sterilizing temperature (121 C) and 15 minutes at 121 C.

3. After sterilizing for the desired amount of time, turn the control handle to 'fast
exhaust' ('slow exhaust' for liquid media) and exhaust for ~10 minutes.

4. After exhausting, turn the control handle to 'dry' for ~10 minutes (if you are rushed,
this step may be skipped, but the contents may have condensation.)

5. Turn the control handle to 'off'.

6. Put on autoclave gloves, then open the door (turn counter-clockwise) and carefully
remove the contents.

 If the door won't open, just wait a few moments ñ the pressure will subside.

Approved for Autumn Term Session 2010-11 - 11 -

Cautions:

1. After completion of the operation, the lid should be opened with care because of the
hot material inside.

2. No inflammable liquid should be handled near autoclave.

Approved for Autumn Term Session 2010-11 - 12 -

 Experiment No. 8

Title: Principle and demonstration of Surface Sterilization

Equipments/Chemicals required: Rectified spirit, cotton, Sterile gloves etc.

Learning objectives: Raising pure cultures require making the place in use free
from contaminants. Various chemicals are employed for the purpose.

Procedure:

1. Wash hand with detergent and rinse with normal water.

2. Dry hands and rinse with rectified spirit.

3. Dry in air and apply rectified spirit onto the working surface.

4. Wipe the whole area carefully twice.

Scope of the result: Sterilization will ensure purity of the cultures raised

Caution: Sterilization of the surface should be maintained by avoiding sneezing and

coughing at the working place.

Approved for Autumn Term Session 2010-11 - 13 -

 Experiment No. 9

Title: Effect of various Temperatures on nutrient content (Glucose) of media

Equipments/Chemicals required: Test-tube, DNS reagent, NaOH, Sugar standards, Water

bath.

Learning Objective: Sugars can be assayed using their reducing properties. One compound
3,5-dinitrosalicylic acid (DNS) is reduced to 3-amino-5-nitrosalicylic acid in alkaline
solution. This principle is exploited in the assay of sugars in the present experiment.

Procedure:

 Prepare DNS reagent afresh when it has to be used.

 1mL of reagent is mixed with 3 mL of sugar solution in a test tube.

 Prepare a blank by adding 1mL of the reagent to 3 mL of distilled water.

 Cover each tube with a marble and place in a boiling water bath for 5 min
cool to room temperature and read the extinction at 540 nm against a
blank.

Scope of the result: Glucose content of the samples treated at temperature of boiling water
and normal temperature can be screened through DNS.

Caution:

1. DNS has to be prepared fresh i.e., just before the start of the expt.

2. Working sugar standards are also to be prepared afresh in order to yield optimum
results.

Approved for Autumn Term Session 2010-11 - 14 -

 Experiment No. 10

Title: Introduction of various Biological Databases like PubMed and Rebase.

Equipments Required :

 SYSTEM REQUIREMENTS
A computer with internet connection

 URL’s OF WEBSITES VISITED

NCBI: http://www.ncbi.nlm.nih.gov/pubmed.html

REBASE :http://rebase.neb.com/rebase/rebase.html

Learning objectives for the students:

To make students familiar with the concept of web browsing & introduction to a major
website of biotechnology like PUBMED & REBASE.

Brief Procedure:

A: To explore the PUBMED database

 Open the website of National Centre of Biotechnological Information NCBI via google or
through http/:www.ncbi.nlm.nih.gov/
 Select PUBMED from all databases on homepage
 Study about the organization & its structural layout
 Explore & observe the various types of search options with special reference to filtering of
data.
B: To explore the REBASE database

 Open the website of REBASE via google or through http://rebase.neb.com/rebase/rebase.html

Scope of Results to be reported:

Approved for Autumn Term Session 2010-11 - 15 -

OBSERVATIONS

 The website of National Centre of Biotechnological Information NCBI

is a huge reservoir of information. It is a composite database comprising of PUBMED
database for research articles.

RESULTS

The disease ___________________ was submitted to PUBMED server as query and

observed .

ANALYSIS

Approved for Autumn Term Session 2010-11 - 16 -

Approved for Autumn Term Session 2010-11 - 17 -
 CAUTIONS:

 Close all tabs before leaving.

 Shut down the computers.
Text Books:

1. Freifelder D, Physical Biochemistry: Applications to Biochemistry and

Molecular Biology, W.H. Freeman and Co., 1982.

Additional Reading:

2. Cooper, T.G. The Tools of Biochemistry, John Wiley and Sons, 1977.

3. Plummer D.T. An Introduction to Practical Biochemistry, T.M.H Publishing

Co., 1988.

Approved for Autumn Term Session 2010-11 - 18 -

Filename: LMBTY150.doc
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lab manuals for upload for BTY
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Title: Biotechnology Perspectives
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Author: Vachaspati
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Creation Date: 7/19/2010 11:17:00 AM
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Last Saved On: 7/21/2010 11:43:00 AM
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