BT 5111 – M Tech Bioprocess Lab I

Experiment Manual

Dr. Smita Srivastava and Dr. Vignesh Muthuvijayan

July_ Nov 2022 (For 2022 Batch)

Department of Biotechnology
Indian Institute of Technology Madras
Chennai 600 036, INDIA
Table 1

S.N Experiment Day T.A Email.

o
1 Isolation of 04&05.08.22 Akhil
microorganisms from Thursday &
natural environment &Friday Aarsha
and determination of
antibiotic sensitivity
2 Estimation of reducing Akhil
sugars and Non- &
reducing sugars by Aarsha
DNS ,ethod
3 Estimation of Akhil
Ammonia, phosphate &
and total sugar Aarsha
4 Isolation of plasmid Manoj
DNA and agarose gel- &
electrophoresis Prashant
5 Transformation of Manoj
plasmid into E. coli &
Prashant
6 Study of the growth Manoj
kinetics of microbial &
cultures Prashan
7 Development of callus Rotation Rotatio Ritu
and plant cell basis. n basis: &
suspension cultures See table 2 Nisa

8 Agrobacterium Fenita
tumefaciens/rhizogene &
s mediated Prajna
transformation
9 Animal cell culture Demo
and fluorescent
imaging
Cell viability assay
using MTT/Alamar

Tentative Dates for Quiz, Practical exam:

Quiz : 15.09.22 Thursday

Viva : 22.09.22 Thursday
Practical Exam : 23.09.22 Friday
Table 2:

Experimen Experiment
t No
7 Development of callus G1 G2 G3 G4
and plant cell suspension
cultures
8 Agrobacterium G3 G4 G1 G2
tumefaciens/rhizogenes
mediated transformation

Staff TA: Aslam Basha Z (aslam@iitm.ac.in) 9677009588 (Whatsapp)

Laboratory Guidelines:

 Each student should maintain proper laboratory note book for recording the data
(no loose sheets please). After concluding the experiments, the lab records will be
signed by the respective TA’s.

 All students will submit a report (lab manual) on the experiment performed on the
previous week, after the experiment is concluded for evaluation. The completed report
should be submitted by 5.00 pm to the respective TA before the next class starts. Penalty
will be 5% of marks for delay. Report should be submitted individually.

 Follow the report format given below:

 Title Page (with name)

 Objective
 Introduction and Principle
 Materials
 Experimental Procedure
 Results and discussion (with pictures if need be)
 Inference
 Bibliography
 Appendix

 Only handwritten reports and plots on graph sheets (if needed) in pencil only will be
accepted.

 There will be a final laboratory exam along with a viva-voce where the student needs to
experiment alone, collect data, compile and analyze them to interpret the results.
 Wearing Laboratory coats and shoes is required for all laboratory classes.

 Report equipment problems or spillage immediately to Teaching Assistants or Mr.

Aslam.

 After the experiment is over, clean the equipment/surrounding before handing it over to
the respective TA.

 Report to the respective TA’s before you leave the lab.

 Marks distribution will be as follows:

Lab Reports 20%
Attendance/Conduct/ 20%
Attentiveness(Quizzes)
Mid sem viva 10%
Final exam (Experiment + viva) 30% + 20%

 Eating, gum chewing, browsing net, chatting, unnecessary fiddling with laboratory
equipment, and any kind of frivolous activity is prohibited during the laboratory class.

 Academic dishonesty and falsifying data is unacceptable. Students turning in laboratory

records with plagiarized or fudged data will be awarded a ‘U’ grade in the course.
Expt. No: 1. Isolation of microorganisms from natural environment and determination of
antibiotic sensitivity
A. Isolation of microorganisms from natural environment
Objective:To be able to isolate microbes from natural environment

Learning outcome:The student will be able to isolate microorganisms from the natural
environment and screen them to determine antibiotic sensitivity.

Materials: Soil, Water, sterile saline water, micropipettes, Nutrient agar plates, L-rod, test tubes

Equipment required: Laminar Air Flow unit, Incubator

Method:
SOIL
 Add 200 mg of soil sample to 1 ml of sterile saline water. Mix thoroughly for 5 min.
allow to solids to settle.
 Pipette out 100µl of appropriately diluted liquid onto corresponding agar plates in sterile
hood.
 Spread the plates using sterile L-rod.

WATER
 Collect 1 ml of tap water in a sterile test tube.
 Add 100 µl of tap water to the Nutrient agar plates in sterile hood.
 Spread the plates using sterile L-rod.

AIR
 Open the sterile Nutrient agar plates and expose to atmosphere for 10 min and close.
Keep one Nutrient agar plate without inoculation using for CONTROL
 Leave all the plates at 37 °C for 24 h. Observe and note colony morphology of the
colonies Wrap the plates with Para film and store at 4 °C for subsequent screening.

B. Determination of antibiotic sensitivity

Objective: To determine antibiotic sensitivity of bacterial strains

Learning outcome: The student will be able to screen the isolated microbes and determine their
antibiotic sensitivity

Materials: Soil, Water, sterile saline water, micropipettes, Nutrient agar plates, L-rod, test tubes

Equipment required: Laminar Air Flow unit, Incubator

Method:
1. Spread the test culture on nutrient agar plates to form lawn growth.
 2. Leave the plates for 5 minutes to air dry.
3. Using sterile forceps, place the discs impregnated with antibiotic on the surface of pre-
inoculated agar plates
4. Gently press the discs to make good contact with the agar.
5. Incubate the plates for 18 h at 37 °C
6. Measure the zone of clearance around the discs.

Expt. No: 2. Estimation of reducing, non-reducing sugars and polysaccharides

A. Estimation of Reducing Sugars by DNS Method

Objective: To estimate the amount of reducing sugars present in the given sample by DNS
method

Learning outcome: To be able to estimate reducing sugars present in different samples

Materials: Test tubes, water, glucose, DNS reagent – Add 1 g of NaOH and 20 g of sodium
potassium tartrate (Rochelle salt) to 70 ml of H2O. Then add 1 g of DNS and make up the
volume to 100 ml with H2O.
Standard – 1mg/ml of glucose
Working standard – 0.4 mg/ml of glucose

Equipment required: Water bath, UV Spectrophotometer

Procedure:

1. Take 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0 ml of working standard glucose solution
in clean test tubes and make up the volume to 1 ml with H2O.
2. Set a blank with 1 ml H2O.
3. Take 1 ml of test sample in another tube.
4. Add 1 ml DNS reagent to all the tubes.
5. Incubate the tubes for 10 min in boiling water bath.
6. Read the absorbance at 540 nm.
7. Prepare a standard graph by plotting amount of standard against absorbance at 540 nm.
8. Calculate the amount of reducing sugars in the test sample from the standard plot.
Standard table

Volume
Volume Volume Amount of DNS
of test
S. No. of stock of H2O glucose Reagent A540
sample
(ml) (ml) (mg) (ml)
(ml)
Blank - - 1 1
1 0.1 - 0.9 1
2 0.2 - 0.8 1 Boil for
3 0.3 - 0.7 1 10 min
4 0.4 - 0.6 1
5 0.5 - 0.5 1
6 0.6 - 0.4 1
7 0.7 - 0.3 1
8 0.8 - 0.2 1
9 0.9 - 0.1 1
10 1.0 - 0 1
Test - 1.0 0 1

B. Estimation of Non-Reducing Sugars

Objective: To estimate the amount of non-reducing sugars present in the given sample by
hydrolysis.

Learning outcome: To be able to estimate non-reducing sugars present in different samples

Materials: Test tubes, Water, Sucrose

DNS reagent – Add 1 g of NaOH and 20 g of sodium potassium tartrate to 70 ml of H 2O. Then
add 1 g of DNS and make up the volume to 100 ml with H2O.
Stock – 1mg/ml of sucrose

Equipment required: Water bath, UV Spectrophotometer

Procedure

1. Take 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0 ml of standard stock sucrose in clean test
tubes and make up the volume to 1 ml with H2O.
2. Set a blank with 1 ml H2O.
3. Take 1 ml of test sample in another tube.
4. Add 25 µl of 3 N HCl to all the tubes and keep in boiling water bath for 10 min.
5. Cool the tubes and add 1 ml DNS reagent.
6. Incubate the tubes for 10 min in boiling water bath.
7. Read the absorbance at 540 nm.
8. Prepare a standard graph by plotting amount of sucrose against absorbance.
9. Calculate the amount of reducing sugars in the test sample from the standard plot.
 Standard table

Volume Volume Volume Amount DNS

3 N HCl
S. No. of stock of test of H2O of sucrose reagent A540
(µl)
(ml) (ml) (ml) (mg) (ml)
Blank 0 - 1 25 1
1 0.1 - 0.9 25 1
2 0.2 - 0.8 25 1
3 0.3 - 0.7 25 Boil 1 Boil
4 0.4 - 0.6 25 for 1 for
5 0.5 - 0.5 25 10 1 10
min min
6 0.6 - 0.4 25 1
7 0.7 - 0.3 25 1
8 0.8 - 0.2 25 1
9 0.9 - 0.1 25 1
10 1.0 - 0 25 1
Test - 1 0 25 1

Expt. No: 3. Estimation of phosphates and ammonia

A. Estimation of Ammonia
Objective: To estimate the amount of ammonia in the given test sample

Learning outcome: To learn the process of ammonia and phosphate estimation to study the
residual nutrients (nitrogen and phosphorus) which are important for the growth of living
organisms.

Materials: Test tubes, Water, Reagent bottles

Reagent A – Add 0.015 g of sodium nitroprusside and 4 ml of phenol to 50 ml of water and
finally make up the volume to 100 ml
Reagent B – Dissolve 2.5 g of KOH in 50 ml of water
Reagent C – Add 3.5 g of K2CO3 and 15 ml of sodium hypochlorite and make up the volume to
50 ml with distilled water
Standard
Stock – Dissolve 29.3 mg of (NH4)2SO4 in 10 ml of water
Working standard – Take 0.5 ml of stock and add 99.5 ml of water

Equipment required: Water bath, UV Spectrophotometer

Method:
1. Take 0.1, 0.2, 0.3, 0.4 ml of working standard into a series of test tubes. Make up the volume
to 0.4 ml in all the tubes.
2. Set a blank with 0.4 ml of water
3. Pipette out 0.4 ml of test sample into a test tube. Dilute the test sample if necessary
4. Add 0.4 ml of reagent A, 0.2 ml of reagent B and o.4 ml of reagent C to all the tubes
5. Incubate the tubes for 20 min at 37 °C
6. Measure the absorbance at 630 nm
7. Draw a standard graph by plotting absorbance vs concentration
8. From the standard plot calculate the amount of ammonia present in the test sample
Amount of ammonia = (Absorbance × dilution factor)/slope
Standard table

Volume
Volume Volume Reagent Reagent Reagent
Conc. of
S. No. of std of test A B C A630
(mg/l) water
(ml) (ml) (ml) (ml) (ml)
(ml)
1 Blank - - 0.4 0.4 0.2 0.4
2 1 0.1 - 0.3 0.4 0.2 0.4
3 2 0.2 - 0.2 0.4 0.2 0.4
4 3 0.3 - 0.1 0.4 0.2 0.4
5 4 0.4 - 0 0.4 0.2 0.4
6 Test - 0.4 - 0.4 0.2 0.4

B. Estimation of Phosphate
Objective: To estimate phosphate from the given sample (yeast fermentation broth) by
molybdate assay

Materials:
Ammonium molybdate, ascorbic acid, sulphuric acid and H2O*
Reagent A: Ammonium molybdate (1.25 g in 50 ml water)
Reagent B: Ascorbic acid (5 g in 50 ml water)
Reagent C: 6N sulphuric acid
Reagent D: water
Working reagent: 1:1:1:2 :: A:B:C:D
*Ultra-pure water will be used
Equipment required: Water bath, UV Spectrophotometer
Procedure
1. Make a stock of 2.8 mg KH2PO4 in 100 ml distilled water.
1g KH2PO4 = 0.698 g/L PO43-
2. Pipette 0.1, 0.2, 0.3, 0.4, 0.5 ml of stock solution in clean test tubes.
3. Take 1 ml of unknown sample in another tubes and 1 ml of water as blank and make
up the final volume to 0.5 ml
 4. Add 3 ml of working reagent to all tubes.
5. Incubate at 37 °C for 2 h.
6. Vortex well and measure absorbance at 820 nm.
7. Draw a graph taking amount of phosphate along x-axis and the OD along y-axis. The
amount of the phosphate present in the test sample can be calculated from the
standard graph.

Standard table

S. No. Volum Volume Working Amount of

e of of water reagent Phosphate A820
stock (ml) (ml) (mg)
(ml)
Blank 0 1 3 0
1 0.1 0.9 3 2
2 0.2 0.8 3 4 Incubate
3 0.3 0.7 3 6 at
4 0.4 0.6 3 8 37 °C
5 0.5 0.5 3 10 for
6 0.6 0.4 3 12 2h
7 0.7 0.3 3 14
8 0.8 0.2 3 16
9 0.9 0.1 3 18
10 1.0 -- 3 20

C. Estimation of Total Sugars by Phenol-Sulphuric Acid Method

Objective: To estimate the total sugars in the given sample by phenol-sulphuric acid method

Learning outcome: To be able to estimate total sugars present in different samples

Materials: Test tubes, water, glucose, phenol, sulphuric acid

 Phenol 5%: Redistilled (reagent grade) phenol (50 g) dissolved in water and make up to 1L.
 Sulphuric acid 96% reagent grade.
 Standard glucose: Stock—100 mg in 100 ml of H2O.
 Working standard—10 ml of stock diluted to 100 ml with H2O.

Equipment required: Water bath, UV Spectrophotometer

Procedure
1. Take 20, 40, 60, 80, 100, 120, 140, 160, 180 and 200 μl of standard stock glucose (1 mg/ml)
in clean tubes and make up the volume to 200 μl with H2O.
2. Set a blank with 200 μl of H2O.
3. Take 200 μl of the sample solution in a test tube.
4. Add 200 μl of phenol solution to each tube.
5. Add 1 ml of 96% sulphuric acid to each tube and shake well.
6. After 10 min shake the contents in the tubes and place in a water bath at 25–30 °C for 20
min
7. Read absorbance at 490 nm.
8. Plot a standard graph by taking amount of standard sugar on the x-axis and absorbance at
490 nm on the y-axis.
9. Calculate the amount of total carbohydrate present in the sample solution using the standard
graph.

Standard table

Volume Amount
Volume Volume
of test of Phenol H2SO4
S. No. of stock of H2O A490
sample glucose (µl) (ml)
(µl) (µl)
(µl) (mg)
Blank - - 200 200 1
1 20 - 180 200 1
2 40 - 160 200 1
3 60 - 140 200 1 Incubate at
4 80 - 120 200 1 25- 30 °C
5 100 - 100 200 1 for 20 min
6 120 - 80 200 1
7 140 - 60 200 1
8 160 - 40 200 1
9 180 - 20 200 1
10 200 - 0 200 1
Test - 200 - 200 1

Expt. No: 4. Isolation of plasmid DNA and agarose gel electrophoresis

Alkaline Lysis Method

Objective: To isolate plasmid DNA using the conventional alkaline lysis method

Learning objective: The student will be to isolate and quantify the DNA present in plasmids
and use it for genetic engineering techniques.

Materials: Micro-centrifuge tubes, micropipettes and tips, Agarose, Water, HCl, EDTA, Tris
base, RNase A, NaOH, SDS, KOH, Potassium acetate, Ethidium Bromide,
BUFFERS
Solution I: 50mM Tris pH 8.0 with HCl, 10mM EDTA, 100ug/ml RNase A
For 1 liter: Dissolve 6.06g Tris base, 3.72g EDTA 2H 20 in 800ml H20. Adjust the pH to 8.0 with
HCl. Adjust the volume to 1 liter with ddH20.
Solution II: 200mM NaOH, 1%SDS
For 1 liter: Dissolve 8.0g NaOH pellets in 950ml of ddH 2O, 50ml of 20% SDS solution. The
final volume comes out to 1 Liter.
Solution III: 3.0M Potassium Acetate, pH5.5
For 1 liter: Dissolve 294.5g potassium acetate in 500ml of H2O. Adjust the pH to 5.5 with glacial
acetic acid (~110ml). Adjust the volume to 1 liter with ddH2O.
For running the plasmid DNA in agarose gel electrophoresis
TE buffer 10mM Tris pH 8.0 with HCl, 1mM EDTA
For 1 liter: Dissolve 1.21g Tris base and 0.37g EDTA 2H 2O in 800ml of ddH2O. Adjust the pH
to 8.0 with HCl. Adjust the volume to 1 liter with ddH2O.

Equipment required: pH Meter, Gel doc, Centrifuge, Gel boat and electrophoresis unit

Procedure:
1) Inoculate a test tube containing 3-5ml of LB (which contains the antibiotic selective for the
bacterial culture) with a single isolated colony picked from an LB agar plate which contains the
selective antibiotic. Grow the culture overnight at 37⁰C with shaking.
2) Centrifuge 1.5ml of the overnight culture in a microfuge tube for 1 min at 14,000 rpm.
Discard the supernatant. Re-suspend the bacterial pellet in 200ul of Solution I. (Re-suspend the
pellet completely by pipetting up and down or by vortexing.)
3) Add 200ul of Solution II. Mix by inverting the tube gently.
4) Add 200ul of Solution III. Mix by inverting the tube gently. A white precipitate forms.
Centrifuge the tube for 10min at 14,000rpm. Transfer the supernatant to a fresh tube. Do not
transfer any of the white pellets.
5) Add 900ul of 100% ethanol to the supernatant. Mix well by inverting the tube several times.
Centrifuge at 14,000 rpm for 20 minutes. Remove and discard supernatant. To the DNA pellet
add 100ul of ice cold 75% ethanol. Centrifuge again for 30 seconds. Remove and discard
supernatant. Be sure that all of the ethanol has been removed from the pellet. If needed, air-dry
the pellet for10-30 minutes.
6) Re-suspend the pellet in 50ul of sterile ddH2O or TE buffer. Store at -20⁰C. A sample of 5ul
should be sufficient to see clear bands on an electrophoresis gel.

Expt. No: 5. Transformation of plasmid into E. coli

Objective: To perform Transformation of recombinant DNA into E. coli

Learning Objective: To learn the process of transformation of plasmids into bacterial cells
thereby, introducing new genes into a bacterial expression system.

Materials required: LB broth, LB-Amp plate, Micropipette and tips, Calcium chloride treated
competent cells, L-Rod
Equipment required: Incubator, Water bath
Procedure
1. To transform CaCl2 treated cells, transfer 200 µl of suspension of competent cells to a
sterile polypropylene tube using a chilled micropipette tip
2. Add 50 ng of DNA to competent cells. Concentration of DNA solution: 50 ng/ µl. Mix the
contents of the tube by gentle swirling.
3. Incubate the tube in ice for 20 minutes
4. Transfer the tube rack placed in a pre-heated 42◦C circulating water bath. Keep the tubes
in rack for 90 seconds
5. Incubate in ice for 2-3 minutes
6. Add 400 µl of LB broth to the tube
7. Incubate the culture for 45-60 minutes with shaking incubator
8. Centrifuge the culture at 4000 rpm for 5 minutes
9. Discard the supernatant and re-suspend the pellet in 100 µl of LB broth
10. Plate in agar plate with antibiotic and incubate at 37◦C for 16 hours
11. Calculate the number of colonies and hence the transformation efficiency
Transformation efficiency = No. of transformants x Final volume (ml)
___________________________________
µg of plasmid DNA x Volume plated (ml)

Expt. No: 6. Study of the growth kinetics of microbial cultures

Aim:

To determine the generation time of a bacterial culture

Principle:

Under optimum cultural conditions, a microbe reproduces rapidly and the dynamics of the
microbial growth can be charted by means of population growth curve. This facilitates
measurement of cell numbers and the rate of the growth of a particular organism under
standardized conditions. The stages of a typical growth curve are (1) Lag phase (2) logarithmic
or log phase (3) stationary phase and (4) Death or Decline phase.
Lag phase: The phase during which microorganisms adjust to the new environment. In this
phase, cellular metabolism is accelerated, cells increase in size, but no cell division occurs and
therefore no increase in cell mass. The length of the lag phase depends directly on the previous
growth condition of the organism. When a microorganism growing in a rich medium is
inoculated into nutritionally poor medium, the organism will take more time to adapt to the new
environment. Similarly when an organism from a nutritionally poor medium is transferred to a
nutritionally rich medium, the organism can easily adapt to the environment, and therefore will
have a very short lag phase.

Log/ exponential phase: During this phase, the microorganisms divide and grow rapidly. Their
metabolic activity increases and the number of cells increases logarithmically (exponentially)
i.e., single cell divides into two, two become four, eight, sixteen, thirty two and so on (That is 2 0,
21, 22, 23.........2n, n is the number of generations). The time taken by the bacteria to double in
number during a specified time period is known as the generation time. The generation time
tends to vary with different organisms. E.coli divides every 20 minutes; hence its generation time
is 20 minutes. Microorganisms are more sensitive to antimicrobials during this phase.
Stationary phase: As the bacterial population continues to grow, all the nutrients in the growth
medium get exhausted and accumulation of waste materials and toxic metabolites occur. This
shifts the conditions of the medium such as pH, thereby creating an unfavorable environment for
bacterial growth. The reproduction rate slows down; the rate of cell division equals that of cell
death.
Death phase: During this, the bacterium completely loses its ability to reproduce. Cells begin to
die due to the unfavorable conditions and death is rapid and at a uniform rate. The number of
dead cells exceeds the number of live cells. Some organisms which can resist this condition can
survive in the environment by producing endospores.

Materials required
LB broth
Culture- E.coli
Spectrophotometer
PBS (Phosphate-buffered saline)
250 ml conical flask, test tubes etc
Fig: Phases of a bacterial growth curve

Exponential growth can be described mathematically by the following equation

N t = N 0 × 2n
N0 = Initial bacterial number at the beginning of log phase
Nt = The bacterial number at time t
n = number of generations
Solving for ‘n’

n=
Generation time (Time per generation) = t/n

Procedure:
1. Using a sterile pipette, add 10 ml of E.coli culture into a 250ml conical flask containing 90
ml of sterilizedmedia. Add sterilized glucose solution equal to100 mg before inoculation.
2. Take a zero hour sample, as soon as the inoculum is added to the medium
3. Centrifuge the sample at 2000 rpm for 5 min.
4. The supernatant is saved and the precipitate is dissolved with PBS to the original volume
5. Measure the absorbance at 600 nm against PBS blank taken.
6. Keep the flask in a shaker set at 37 ˚C and 150 rpm.
7. After every 20 minutes take about 1ml sample from the flask under aseptic conditions and
repeat steps 3-6.
8. With GOD-POD reagent measure the concentration of glucose in the supernatant.
9. Draw a graph with the time on the x-axis and the A600 value on the Y-axis.

Observation and calculation:

Observe the pattern of cell growth. The concentration of cells in the log phase is taken to
calculate the generation time (GT) and find out the biomass yield from the amount of substrate
consumed.

Expt. No: 7. Development of callus and plant cell suspension cultures

A. Development of callus
Objective: To initiate callus cultures from carrots (Daucus carota)
Learning outcome: The student will be able to explain and carry out surface sterilization of ex-
plants and dedifferentiation in surface sterilized explants (i.e. callus induction).
Materials: Carrots, Anti-septic liquid (Dettol), forceps, scalpels, 70% v/v Ethanol, Sodium
Hypochlorite, sterile petridishes, Nutrient medium – Gamborg’s B5, supplemented with Plant
growth regulators – 2,4D and 0.8% agar
Equipment required: Laminar Air flow unit, autoclave, plant growth chamber
Method:

1. Fresh tap roots of carrot are washed thoroughly under running tap water to remove all
surface detritus.
2. The tap root is then dipped into an antiseptic liquid (Dettol) for 10 minutes and then the
root is washed.
3. The carrot root, sterilized forceps, scalpels, other instruments, autoclaved nutrient
medium on the petridishes are then transferred to laminar air flow.
4. The tap root is surface sterilized by immersing in 70% v/v ethanol for 60 seconds, fol-
lowed by 20-25 minutes in sodium hypochlorite. (as shown in Fig.2.)
5. The root is washed three times with sterile distilled water to remove the hypochlorite
completely.
6. The carrot is then transferred to a sterilized petridish containing a filter paper.
7. A series of transverse slice 1 mm in thickness is cut from the tap root using a sharp
scalpel.
[Each piece contains a whitish circular ring of cambium around the pith. An area of
4mm  across the cambium is cut from each piece so that each small piece contains part of the
2

phloem, cambium and xylem. Size and thickness of the explants should be uniform. The lid
of petridish should always be replaced after each manipulation.]
8. Each piece is transferred on to the surface of the nutrient medium in petri plates, using
sterile forceps. The culture plates are marked accordingly.
The nutrient medium is Gamborg’s B5 or MS medium supplemented with 0.5 mg/L 2, 4-
D and 0.8% clerigar.
9. After inoculation, the petri plates are taken to the culture room where they are placed in
the racks. Cultures are incubated in dark at 25°C.
 (Source http://www.biologydiscussion.com/plant-tissues/cell-suspension-culture/cell-suspension-culture-definition-principle-protocol-and-importance-plant-tissue/14601)

Fig.2. Callus initiation from carrots

Reference:
http://www.biologydiscussion.com/plant-tissues/callus-culture/callus-culture-history-principles-
and-significance-plant-tissue-culture/14597

B. Development of plant cell suspension cultures

Objective: To initiate plant cell suspension cultures

Learning outcome: To effectively establish plant cell suspension cultures which consist of only
single cells that are physiologically and biochemically uniform
Materials: 250ml conical flasks autoclaved with 50ml liquid medium, pre-established callus,
sterile spatula and cotton plugs, filter paper, Nutrient medium – Gamborg’s B5, centrifuge tubes
Equipment required: Laminar Air flow unit, autoclave, filtration apparatus, illuminated shaker,
centrifuge, plant growth chamber
Method:

1. A 250ml conical flask containing autoclaved 50ml medium is taken.

2. 3-4 pieces of pre-established callus tissue (approx. wt. 1 gm. each) from the culture tube using
the spoon headed spatula is transferred to the conical flasks. The flask mouth is flamed for a
moment and immediately covered with cotton plugs (as shown in Fig.3.)
3. The flasks are placed within the clamps of a rotary shaker moving at 120 rpm (revolutions per
minute)
4. After 7 days, the contents of each flask are poured through the sterilized sieve pore diameter -
60µ- 100µ and the filtrate is collected in a sterilized container. The filtrate contains only free
cells and small cell aggregates.
5. The filtrate is allowed to settle for 10-15 min. or centrifuged at 500 to 1,000 rpm. The
supernatant is discarded.
6. The residual cells are re-suspended in a requisite volume of fresh liquid medium and the cell
suspension is dispensed equally in several sterilized flasks (250 ml).
7. The flasks are placed on shaker and free cells are allowed to grow.

(Source http://www.biologydiscussion.com/plant-tissues/cell-suspension-culture/cell-suspension-culture-definition-principle-protocol-and-importance-plant-tissue/14601)

Fig.3. Plant cell suspension development

Reference: http://www.biologydiscussion.com/plant-tissues/cell-suspension-culture/cell-
suspension-culture-definition-principle-protocol-and-importance-plant-tissue/14601
 Expt. No: 8. Agrobacterium tumefaciens/rhizogenes mediated transformation

A. Agrobacterium tumefaciens mediated transformation

Objective: To transform calli of D. carota using Agrobacterium tumefaciens mediated
transformation
Learning Outcomes: At the end of this experiment the student will be able to
 Observe morphological changes in carrot roots due to transformation
 Differentiate between a wild type and transformed callus (by observing and comparing
growth in presence/absence of antibiotic and growth regulators)

Materials:
Biological materials – Carrots, Agrobacterium tumefaciensC58 (MTCC 609)
Sterile disposable petridishes (90mm), Erlenmeyer flasks (250ml) with 50ml of YEP (Yeast
Extract Peptone) medium, forceps, scalpels, inoculation loop, pipettes, sterile filter papers
(Whatman No.1), Parafilm, Cefotaxime, sterile needle, petridishes containing MS medium
(~20ml) with 0.8% agar, Petridishes containing MS medium (~20ml) with 0.8% agar and
500mg/l Cefotaxime
Equipment required: Spectrophotometer, LAF, autoclave, incubator, Plant growth chamber,
Shaker
Method:
Preparation of Bacterial culture
1. Streak the A. tumefaciens culture in YEP medium (with 1.5%) agar. Allow it to grow for
24-48 h, till the appearance of colonies.
2. Inoculate 1 loop of A. tumefaciens culture in 50ml of YEP medium. Place it on a gyratory
shaker at 150rpm at 28°C. Allow the culture to grow till it reaches an optical density of
0.6-0.8 at 600nm (OD600; Typically takes 24-32 h).

Preparation of carrot explants

1. Sterilize carrot roots as mentioned in Experiment 2 with sodium hypochlorite. The
sterilized carrots are incised into discs using sterile forceps and scalpel for subsequent
use.
 Transformation
1. Prepare the round sections of the carrot discs for transformation by pricking the exposed
area with needle.
2. Dip the cut carrot discs (4-5 nos) in A. tumefaciens C58 bacterial culture flask.
3. Place the bacterial culture flask with carrot discs in a gyratory shaker for 15-20 mins at
low speed (~75rpm).
4. Blot dry the discs on a sterile filter paper and place them onto petridish with no antibiotic
(co-cultivation medium; 4 discs per plate).
5. Wrap the petridish with Parafilm and incubate them in a growth chamber at 25°C under
dark conditions for 48 h.
6. After cocultivation period, transfer the discs to petridish containing MS medium with
antibiotic cefotaxime.
7. Observe the morphological changes in the carrot discs.
8. Transfer any tumerogenic calli formed to fresh MS plates with antibiotic maintained at
25℃ and 16h/8h light/dark photoperiod with subsequent transfer onto decreasing
concentrations of antibiotics until they are able to grow in an antibiotic and growth
regulator free medium.

Further reading: Gelvin SB (2003) Agrobacterium-mediated plant transformation: the biology

behind the “Gene-Jockeying” tool. Microbiology and Molecular Biology Reviews, 67(1):16-37
 Fig 4. Agrobacterium mediated transformation of carrot discs

B. Agrobacterium rhizogenes mediated transformation to induce hairy roots

Objective: To initiate hairy roots of D. carota

Learning Outcomes: The student will be able to induce hairy roots via Agrobacterium
rhizogenes mediated transformation of surface sterilized explants and establish liquid culture of
the hairy roots.

Materials: Biological materials – Carrots, Agrobacterium rhizogenes (MTCC 532)

Sterile disposable petridishes (90mm), Erlenmeyer flasks (250ml) with 50ml of YEP (Yeast
Extract Peptone) medium, Forceps, scalpels, inoculation loop, pipettes, sterile filter papers
(Whatman No.1), Parafilm, Cefotaxime, sterile needle, Petridishes containing MS medium
(~20ml) with 0.8% agar, Petridishes containing MS medium (~20ml) with 0.8% agar and
500mg/l Cefotaxime
Equipment used: Spectrophotometer, Laminar Air flow unit, autoclave, incubator, Plant growth
chamber, Shaker

Method:
Preparation of Bacterial culture
1. Streak the A. rhizogenes culture in YEP medium (with 1.5%) agar. Allow it to grow for
24 h, till the appearance of colonies.
2. Inoculate 1 loop of A. rhizogenes culture in 50ml of YEP medium. Place it on a gyratory
shaker at 150rpm at 28℃. Allow the culture to grow till it reaches an optical density of
0.6-0.8 at 600nm (OD600).
Preparation of carrot explants
1. The carrots are to be sterilized as mentioned in Experiment 2 with Sodium hypochlorite.
The sterilized carrots are incised into discs with the scalpel and used for transformation.
Transformation
1. Prepare the round sections of the carrot discs for transformation by pricking the
exposed area with needle.
2. When all the discs are prepared, transfer them to A. rhizogenes suspension. Make sure
the sections are fully submerged.
3. Place the flasks with immersed carrot discs in a gyratory shaker for 15-20 mins at low
speed (~75 rpm).
4. Blot the discs on a sterile filter paper and culture on petridish with no antibiotic (Co-
cultivation medium).
5. Wrap the petridish with Parafilm and incubate them in a growth chamber at 25℃
under dark conditions till 48h.
6. After cocultivation period, transfer the discs to petridish containing MS medium with
antibiotic.
7. Keep observing the plates for induction of hairy roots from the discs.
Maintenance
1. The hairy roots are incised from the discs after it has grown to ~2in length. It is
transferred to growth medium with antibiotic cefotaxime.
2. The concentration of antibiotic is reduced subsequently in further transfer until the
roots are free of bacterial contamination.
3. The hairy roots are maintained in antibiotic and growth regulator free medium at 25
℃ and 16h/8h light/dark photoperiod.
Expt. No: 9. Animal cell culture and fluorescent imaging & Cell viability using MTT/
Alamar blue

Objective: To image animal cells under a fluorescent microscope and to determine the cell
viability by Alamar assay

Learning outcome: To gain an understanding of basic assays involved in animal cell culture
studies which are crucial in carrying out in-vitro studies.

Materials:Animal cell lines (HEK-293), Tissue culture plates, Tissue culture dishes, Fluorescein
Diacetate(FDA), Propidium Iodide (PI), 4′,6-diamidino-2-phenylindole (DAPI), Fetal bovine
serum FBS, Phosphate buffer saline (PBS) (pH 7.4), sterile MilliQ® water, Cell culture media
(DMEM),

Equipment required: Plate reader, Fluorescence microscope, Plate shaker

Method:
Viability assay: Alamar-blue assay
1. When cells are in log phase of growth, harvest them and determine the cell count. The
suggested optimal cell count is 1 x 104 cells/ml (cell density), which may vary between the cell
lines.
2. To appropriate wells of a tissue culture plate, add the test compound and vehicle controls.
Culture cells at 37°C in a cell culture incubator for desired test compound exposure period.
3. Remove the assay plates from 37°C incubator and mix by gently shaking it.
4. Aseptically add Alamar-Blue Reagent in an amount equal to 10% of the volume in the well. In
the blank sample well, add only Alamar-blue and media.
5. Incubate cultures with Alamar-Blue Reagent for the predetermined time in a cell culture
incubator at 37°C (the optimum incubation time may vary between the cell types).
6. After the incubation time ends, remove the plate and measure the fluorescence with excitation
wavelength at 570nm and 600nm wavelengths

Live/Dead staining: FDA-PI staining

1. Prepare the staining solution (Culture medium without FBS -5 ml, FDA (5 mg/ml) -8 µl PI
(2 mg/ml) -50 µl) and store in refrigerator.
2. Remove the cell culture medium.
3. Add staining solution. The volume is dependent on the geometries of the used culture dish.
4. Incubate cells at room temperature for 4 to 5 minutes in the dark.
5. Remove the staining solution.
6. Wash your sample with PBS.
7. Add PBS or medium without FBS to your sample.
8. Analyze sample with fluorescent microscopy.
Nucleus staining: DAPI staining
1. Dilute the DAPI stock solution 1:5000 in PBS (DAPI labelling solution).
2. Aspirate the cell medium from cells grown in tissue culture plates. Rinse the cells three
times with PBS.
Do not allow the cells to dry out at any time during the protocol.
3. Fix the cells for 10 min in 3.7% formaldehyde.
4. Aspirate the fixative. Rinse the cells three times, 5 minutes each, in PBS.
5. Permeabilize the cells by adding 0.2% Triton X-100.
6. Aspirate the Triton after 5 minutes. Rinse the cells three times, 5 min each, in PBS.
7. Incubate the cells for 1-5 min at room temperature in DAPI labelling solution.
8. Aspirate the labelling solution. Rinse the cells three times in PBS.
9. Mount the tissue culture plate on the fluorescent microscope.
10. Image the cells (excitation wavelength- 359 nm, emission wavelength- 461 nm when
DAPI is bound to DNA).

Format for a Laboratory Report

Reports are to be written in a manual and should contain the following sections:

Title
Objective
Principle
Materials
Experimental procedure
Results and discussion
Error analysis
Inference
Bibliography
Notation
Appendices

It should generally be written in the past tense and with a passive voice.

Title
Title should be brief and descriptive of the experiment under consideration. The title Page should
also include your name, date of submission of report and other relevant details.
Objective
This section should briefly describe the objectives of the current investigation; it should be
clearly stated.
Introduction
It should contain a short introductory remark concerning the experiment under study.
Principle
The theory underlying the phenomena and the computation method should be briefly outlined.
This section should only contain materials which are relevant to the experiments and which may
be used for the discussion of the results obtained.
Experimental Procedure
This section should clearly state what you did. Diagram of the experimental set-up and a brief
description of the apparatus used should be given. The operational procedure, preparation of
standard graph etc, should be given in fair detail. The scope and design of the experiment should
also be given.
Results and discussion
This is the most important section of the report. This section should summarize all the results
obtained in the form tables, graphs etc. You should include a sample calculation of one
experimental run. If possible an ‘error analysis’ should be carried out to determine the errors
associated with each calculated result. Main results should be tabulated and all subsequent
analysis of the main results should be clearly illustrated graphically or otherwise. All other data
and calculations should be given in the appendices.

Discussion section should include explanations of what the results means to you. The results and
observations reported should be discussed in reference to the theory given earlier in the report.
The experimental results should be compared with published results (standard texts and
handbooks), if possible. Any abnormal behavior noted should be pointed out and explained
suitably. For clarity of the report you may take one variable at a time to present the results and
discuss it immediately.

Error analysis
Student need to analyze the error involved in measurement and calculate % error in the measured
quantity

Inference
The conclusions that you can draw from the experimental results should be given here. This
section must also clearly indicate how far the objective stated at the beginning has been
achieved.

Bibliography
All references should be listed in formats as used in scientific journals and textbooks of science
and technology. An example is shown blow.

1. Biochemical Engineering Fundamentals, J.E. Bailey and D. Ollis, Second Edition, Mc-Graw
Hill (1986).
2. Benedetti, L., Bertucco, A. and Pallado, P. 1997. Production of Micronic Particles of
Biocompatible Polymer Using Supercritical Carbon Dioxide, Biotechnology and
Bioengineering, Vol. (53):232-237.

Notation
All symbols used should be defined when they first appear in the report, and should be listed
alphabetically in this section, together with their meaning. Follow SI units.

Appendices
Appendices should contain materials that are too bulky and detailed to include in the main body
of the report as inclusion of which may prevent the smooth flow of thought and presentation of
your results. These include unprocessed data, detailed calculations, intermediate results etc. All
materials in the Appendices should of course be referred to in the main body of the report.

Introduction - Why did you start?

Methods - What did you do?
Results - What did you find?
Discussion - What do the results mean?
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