Lab Manual 6th Sem- New

LABORATORY MANUAL

OF

B-Tech VI semester (OLD)

In

Biotechnology

DEPARTMENT OF BIOTECHNOLOGY

MOTILAL NEHRU NATIONAL INSTITUTE OF TECHNOLOGY ALLAHABAD

ALLAHABAD-211004 (U.P.)

Course name: BI601 (BIOINFORMATICS)

BT602 (ENZYMOLOGY)

BT604 (ANIMAL BIOTECHNOLOGY)

BT606 (MICROBIAL TECHNOLOGY)

SECTION 1: BT601 (BIOINFORMATICS)

S.NO EXPERIMENT

1 Retrieval of a protein/nucleotide sequence from NCBI GenBank

database.

2 Use of BLAST tool to identify it's homologs

3 Multiple sequence alignment of homologs

4 Identification of conserved regions in the MSA

5 Molecular Phylogeny using Mega 5

6 Homology Modelling

7 Molecular docking studies for screening drug candidates

SECTION 2: BT602 (ENZYMOLOGY)

S.NO EXPERIMENT

1 To produce amylase enzyme under solid state fermentation.

2 To extract the amylase enzyme produced and determining

enzymatic activity.

3 To extract the amylase enzyme produced and determining

enzymatic activity.

4 Study of enzyme (Xylanse) kinetics by the use of Michaelis Menton

equation.

5 To produce cellulase enzyme using solid state fermenatation.

6 Filter paper assay was estimated total cellulase activity in the crude

enzyme.

7 To recover proteins/enzymes from a solution by salting-out

(Ammonium sulphate precipitation of protein).

SECTION 3: (BT604 (ANIMAL BIOTECHNOLOGY)

S.NO EXPERIMENT

1 To study laboratory requirements for animal cell culture.

2 o determine the percentage of the viable cells in a suspension by

trypan blue exclusion test.

3 To study apoptosis by microscopic analysis.

4 To establish primary cell culture using fine dissection for

disaggregation of tissue.

5 To establish primary cell culture using enzymatic method for

disaggregation of tissue. (Warm trypsinization). 6 To establish primary cell culture using enzymatic method for

disaggregation of tissue. (Cold trypsinization).

7 To establish primary cell culture using enzymatic method for

disaggregation of tissue using collagenase. 8 To establish primary cell culture using mechanical method for

disaggregation of tissues.

9 To determine cell density after cell counting.

10 To perform MTT cell proliferation assay

SECTION 4: (MICROBIAL TECHNOLOGY)

S.NO EXPERIMENT

1 The production of Antibiotic (Neomycin) by Streptomyces fradiae in

Synthetic Media.

2 To determine the effectiveness of antibiotic using agar well dilution

bioassay test. 3 Fermentative production of amylase by Aspergillus Niger.

4 Demonstration of wine production by using grape juice.

5 To determine the minimum inhibitory concentration (MIC) of an

antibiotic.

SECTION 1: BT601 (BIOINFORMATICS)

Experiment No. 1

Aim: Retrieval of a protein/nucleotide sequence from NCBI GenBank database.

The Heat shock protein sequence from Homo sapiens will be retrieved from the GenBank

database available at ncbi.nlm.nih.gov.in

Select the type of sequence in the drop down box

Write the key words in search box

Click search

Right panel will show hits from top organisms.

Select the desired sequence

Use different key words to obtain the desired sequence.

Experiment No. 2

Aim: Use of BLAST tool to identify it's homologs

The Heat shock protein sequence from Homo sapiens retrieved from the GenBank database at

ncbi.nlm.nih.gov.in will be used as query in BLASTp search against the same database to

identify the homologs of the protein available in public domain.

Retrieval of Homolog sequences (As shown in Experiment No. 1)

Screenshot of BLAST webpage:

Select the type of BLAST from Basic BLAST list (Nucleotide blast, protein blast, blastx,

tblastn.

One can also Paste sequence in FASTA format in the query box.

Any GenBank Accession Nos can be used as a query sequence in BLAST search.

Choose the species genome to which you want to search the homology (for specific

results)

Enter your query sequence in query box

Can use algorithm according to your query.

Selection of homologs will be done on the basis of E value obtained from the BLAST

search for each protein/ nucleotide sequence. Ten best homologs from different organism

will be selected and saved.

Experiment No. 3

Aim: Multiple sequence alignment of homologs

Multiple sequence alignment option present in Bioedit will be used for MSA. Homologs

retrieved in previous step will be subjected to multiple sequence alignment by CLUSTALW

method, which is a heuristic method for fast MSA.

Load sequences as text file in FASTA format

Go to Menu> accessorry application> ClustalW MSA

Perform MSA

Result: Alignment file of all homologs was saved. The consensus region is observed.

Experiment No. 4

Aim: Identification of conserved regions in the MSA

The CD search tool available in BioEdit will be used to search conserved domains present in the

sequences. The identified conserved domains will be saved in a different file.

To search for conserved regions within an alignment (for example, to find possible targets for

PCR primers):

Select the sequences you want included in the analysis

Go to Main Menu> alignment> Find Conserved region

Choose Alignment->Find Conserved region.

Notes:

Dont allow gaps: No gaps in any sequence will be allowed for a reported region

Limit gaps in any segment to x: For a region to be reported as conserved, no sequence may have more than x gaps in that region.

Limit max contiguous gaps to x: For a region to be reported as conserved, no sequence may have more than x gaps in a row, regardless of how many total gaps are allowed.

Minimum length: This is the actual number of residues that must be present within the region in every sequence (not including gaps), regardless of the number of gaps allowed.

Experiment No. 5

Aim: Molecular Phylogeny Manual (by using MEGA 5.2.2)

1. Creating Multiple sequence Alignment File:

Open control panel>Open Align option>Edit/Build Alignment>Create New Alignment>Select

Protein/DNA> Paste your sequences in FASTA format>Select All sequences>Alignment>Align

by ClustalW>alignment parameters>OK> save file

Figure: Control Panel in MEGA 5.0

Figure: clustalW parameters

2. Building phylogenetic tree(By neighbour joining method):

Phylogeny>construct NJ tree>Open the MAS file saves in session 1>Analysis preferences> Save

tree

Figure: Options for building phylogenetic trees

Figure: Analysis preferences in creating phylogenetic trees

Figure: NJ Tree of plant CSPs

Arabidopsis thalianagi|330251603|gb|AEC06697.1| cold shock domain protein 3

Eutrema salsugineumgi|294470716|gb|ADE80750.1| cold shock domain protein 3

Medicago truncatulagi|357518027|ref|XP_003629302.1| Major cold-shock protein

Cucumis melo subsp. melogi|307136096|gb|ADN33944.1| cold-shock DNA-binding family protein

Ricinus communisgi|255545420|ref|XP_002513770.1| cold shock protein putative

Triticum aestivumgi|21322752|dbj|BAB78536.2| cold shock protein-1

Oryza sativa Japonica Groupgi|57900030|dbj|BAD88072.1| cold shock domain protein 2-like protein

0.1

Experiment No. 6

Aim: Homology Modelling

Select a protein sequence from GenBank with un known 3-D structure.

Search for homologs in Protein Data Bank.

Homolog with >40 % similarity selected as template.

Download and install Modeller, EasyModeller with compatiple version of Python.

Open EasyModeller Main Panel > Enter query >Browse Templates > select SALIGN > Perform Alignment>Generate Model and wait.

Draw Ramachandran Plot at Procheck. at http://www.ebi.ac.uk/thornton-srv/software/PROCHECK/

More than 90% residues in allowed regions.

Use model further.

Experiment No. 7

Aim: Molecular docking studies for screening drug candidates

Select and save the receptor and ligands coordinate files in PDB format.

Allow each ligand to bind with receptor by docking

Install Hex 6.0 on system.

Main Panel>File > Open> Receptor

Main Panel>File> Open> Ligand

Main Panel>Controls> Docking>Correlation type> Shape only> solutions 50 >Activate

Main Panel>Message> Note down the energies.

Select the ligand with lowest energy.

Figure 1. The initial Hex scene showing the HyHel-5 antibody Fv domain as a

molecular skeleton.

Figure 2. A Hex scene showing the HyHel-5 antibody Fv domain and lysozyme in

van der Waals mode, and with the intermolecular axis drawn in white.

Figure 3. Illustration of the SPF steric density representations at various 3D

expansion orders for the complex between the HyHel-5 antibody Fv domain (left) and

quail lysozyme (right). From top left to bottom right: steric density isosurfaces shown

at expansion orders L=12, 16, 20, 24, and 26, with the subunits separated by 15 to

give a full view of each domain. The bottom right pair shows the van der Waals

surfaces from which the SPF expansions are derived.

Figure 4. Illustration of spherical harmonic surfaces to order L=12 for the

HyHel-5 antibody Fv domain (left) and lysozyme (right).

Figure 5. Illustration of the HyHel-5/Lysozyme complex shown as contoured

Gaussian density surfaces and coloured by chain colour, drawn using perspective

(keyboard P) and background (keyboard B) modes enabled.

Figure 6. Illustration of spherical polar docking with respect to the intermolecular

axis. An initial docking orientation may be defined by specifying which residues

should be located at the local coordinate origin for each molecule, and by defining

"interface residues" which will be located on the z-axis. The docking search may be

restricted by defining a range angle for the receptor and/or ligand orientations. If range angles are defined, then the interface residues will always be constrained to

appear within a spherical cone defined by the corresponding range angle. This

illustration shows two range angles, each of 45 degrees.

Figure 7. Screen shot of the Docking Control panel.

SECTION 2: BT602 (ENZYMOLOGY)

Experiment No. 1

Objective: To produce amylase enzyme under solid state fermentation.

Requirements: 500 ml conical flask, wheat bran, Czepkdox media, distilled water, test tubes,

autoclave, pipettes, weighing balance.

Procedure: (i) weigh 10 gm of wheat bran (solid substrate), using an electronic weigh balance

and transfer it to a clean and dry 500 ml conical flask.

(ii) Prepare 50 ml of Czepkdox media the compostion of which is as follows:

NaNO3 - 2.5 gm

KCl - 0.5 gm

KH2PO4 1.0 gm

MgSO4.7H2O - 0.5 gm

(iii) Pipette out 10 ml of the median and add o the conical flask containing wheat bran,uniformly

mixed the content.

(iv) Autoclave the conical flask (containing the substrate and media), test tubes and distilled

water.

(v) Prepare spore solution of the provided ATCC culture of Aspergillus oryzae using distilled

water under laminar hood.

(vi) Add around 5 ml of inoculums in to the 500 ml conical flask

(vii) Incubate at 30 degree centigrade for 4 to 5 days.

Observation:

Results:

Note: Clean all your glassware and the work bench after finishing your experiment.

Experiment No. 2

Objective: To extract the amylase enzyme produced and determining enzymatic activity.

Principle: Amylase assay is carried out which involves the following steps starch degrading

enzyme act on glycogen and related polysaccharides, -amylase causes endocleavage of substrate and hydrolysis -1,4 linkages in the random manner. The reducing sugar produced by the action of and/ or amylase react with dinitrosalicylic acid and reduce in to brown color product- 3, 5 nitrosalicylic acid.

Materials:

Sodium acetate buffer (0.1 M, pH 4.8)

1% starch solution - prepare a fresh solution by dissolving 1g starch in100ml

Dinitrosalicylic acid reagent

Maltose solution dissolve 50mg maltose in 50ml DW in standard flask and store it in a refrigerator

Centrifuge the extract (10000 rpm for 20 min). The supernatant is used as enzyme source.

Procedure:

1. Pipette out 0.5 ml of starch solution and 0.5ml of properly diluted enzyme in a test tube.

2. Incubate it at 50 C for 10 mints

3. Stop the reaction by 1ml of dinitrosalicylic acid reagent. 4. Heat the solution in a boiling water bath for 10 mints 5. Cool it in running tap water. 6. Make the volume to 10ml by addition of 8ml DW. 7. Read the absorbance at 560 nm wavelength. 8. Terminate the reaction at zero incubation time in the control test tube. 9. Prepare a standard graph with 0 100 g maltose.

Observation:

S. No. Test Tube Absorbance Net OD

1 Blank 0

2 Zero rxn (B1) E1- B1 =

3 Amylase (E1)

4 Zero rxn (B2) E2- B2 =

5 Amylase (E2)

Average optical density=

Calculation: One unit of an enzyme is amount of enzyme that releases one mol (1 U/ml) of

reducing sugar (glucose) is one min under assay condition.

Enzyme activity (U / ml) = OD x Dilution factor x Slope inverse

Incubation time x enzyme (ml)

Result:


Objective: To produce Xylase enzyme using solid state fermentation.

Materials: Erlenmeyer flasks (100 ml), wheat bran (substrate), Fungal strain Aspergillus niger

or Trichoderma reesei, Incubator, pH mater, Autoclave etc

Chemicals: (NH4)2HPO4, Na2HPO42H2O, KCl, MgSO47H2O, CaCl2.2H2O, FeSO4.7H2O, ZnSO4.7H2O.

Procedure: Xylanase production in Erlenmeyer flasks (100 ml) containing mineral salt solution

composition (grams per liter);

(NH4)2HPO4 - 2.5

Na2HPO42H2O - 10.0 KCl - 0.5

MgSO47H2O - 0.15 CaCl2.2H2O - 0.01

FeSO4.7H2O - 0.01;

ZnSO4.7H2O - 0.002

Birch wood xylan - 1.0

The fungi were cultured in Erlenmeyer flasks (250 ml) containing 10 g of wheat bran moistened

with 10 ml of mineral salts solution. The composition of the mineral salts solution was (g/l):

KCl, 0.5; MgSO4.7H2O, 0.5; (NH4)2HPO4, 2.5; NaH2PO4, 0.5; CaCl2.2H2O, 0.01; FeSO4.7H2O,

0.01; ZnSO4.7H2O, 0.002 and birch wood xylan, 1.0. The pH was adjusted to 5. The medium

was then autoclaved for 20min at 121C (15 lbs). After cooling, the flasks were inoculated with 1

ml of spore suspension containing 1x106 spores per ml. The spore suspension was obtained from

7 day-old pure cultures. After mixing, flasks were incubated at 30C under static conditions for 7

days.

After incubation, the enzyme was harvested in sodium citrate buffer (50 mM, pH 5.3). The

fermented slurry was filtered through cheese cloth and centrifuged at 10 000 x g for 20 min at

4C. The clear supernatant was used for enzyme assays.

Observation:

Results:


Experiment No. 3

Objective: To extract the amylase enzyme produced and determining enzymatic activity.

Principle: Xylanase catalyses the enzymatic hydrolysis of Xylan, (substrate) releasing Xylose.

The reaction is arrested by the addition of 3,5 dinitro salicylic acid (DNS). Also DNS forms a red colored complex with xylose. The amount of xylose released by the reaction is measured by

measuring its absorbance at 540 nanometers using a spectrophotometer.

Instruments used Spectrophotometer (calibrated to measure optical density at 540 nanometers) Water bath (maintained at 60 C) Water bath (maintained at 100 C) Vortex Mixer Micropipettes Enzyme sample (crude xylanase)

Sodium acetate acetic acid buffer (at a pH of 5.6) Xylose (substrate) 3,5 dinitro salicylic acid (DNS) solution Distilled water Procedure

1. The enzyme sample to be tested was diluted to a suitable level of dilution (the dilution factor lying between 50 and 500)

2. In a clean dry test tube, 1 ml of the diluted enzyme solution was taken. To it were added 1 ml of acetate buffer solution and 1 ml of substrate solution. The test tube was incubated

at 60 C in a water bath for 15 minutes.

3. Also a blank was similarly prepared, but with the 1 ml of substrate solution replaced with 1 ml of distilled water. The blank was also incubated along with the sample tube.

4. The reaction was arrested by adding 2 ml of DNS solution to each test tube. 5. 500 l of xylan solution was added to the blank test tube whereas an equal volume of

distilled water was added to the sample test tube.

6. The tubes were then boiled at 100 C in a water bath for 10 minutes to allow the color to develop.

The test tubes were then cooled in an ice bath.

The absorbance of the liquid in each tube was measured at 540 nm using a spectrophotometer.

The OD value of the blank corresponds to the amount of xylose present in the enzyme sample

and the substrate added. This is the amount of xylose that was already present and therefore was

not released due to the reaction.

Therefore the amount of xylose released due to the reaction is proportional to the difference between the OD values of the sample and blank.

A standard graph is plotted between the OD of the solution and the weight of xylose. The equation of the standard graph was found experimentally.

From this value the enzyme activity was calculated as follows:

Enzyme activity (U / ml) = OD x Dilution factor x Slope inverse

Incubation time x enzyme (ml)


Objective: Purifying the crude enzyme (xylanase) by ammonium sulfate precipitation method.

Enzyme Assay: Xylanase assay was conducted by dinitrosalicylic acid (DNS) method as a

modification of the method by Gawande and Kamat with xylose as the standard. One gram of

birchwood xylan was dissolved with stirring in 100 mL of 50 mM sodium acetate buffer, pH 5.5

at 60oC, boiled for several minutes and continued stirring for 3 h at room temperature. To 1.8

mL of this xylan solution, 200 L of enzyme solution was added and incubated at 35oC for 10 min. 3 mL DNS solution (a mixing of 16 g NaOH, 10 g DNS, 300 g sodium potassium tartrate

and 8 g sodium metabisulfite in 1 L water) was added to stop the reaction, and the solution was

boiled for exactly 5 min and then cooled down rapidly using ice bath until room temperature.

Xylose produced was followed by measuring the absorbance at 540 nm. Since xylan or xylose

added as inducers during incubation was present in the enzyme solution to be assayed that will

obviously affect the measurement, a blank for each sample in which no reaction take place (by

immediately stopping the reaction after adding enzyme solution) was carefully conducted in

order to obtain accurate data. One unit of xylanase activity was defined as the amount of enzyme

that produces 1 mol xylose per minute under the assay condition. Cellulase activity was measured using DNS method with glucose as the standard. The condition

was similar to that of xylanase assay except that 1 % carboxymethyl cellulose (Nacalai, Osaka,

Japan) replaced xylan as the substrate. One unit of cellulase activity was defined as the amount

of enzyme that produces 1 mol glucose per minute under the assay condition. The total extracellular protein concentration was measured by the Bradford method [5] with bovine serum

albumin (BSA) as standard.

Purification by Ammonium Sulfate Precipitation

Solid ammonium sulfate was added to the culture supernatant to 80% saturation. The resulting

precipitate was collected by centrifugation at 16,000 rpm for 30 min at 4oC. The precipitate was

redissolved in a 50 mM sodium acetate buffer (pH 5.5).

Purification by aceton precipitation

Prechilled aceton was added to the culture supernatant at one to one ratio or other (v/v) in an ice-

salt batch with stirring. The resulting precipitate was collected by centrifugation at 16,000 rpm

for 30 min at 4oC. The precipitate was dissolved in a 50 mM sodium acetate buffer (pH 5.5) and

dialyzed against the same buffer prior to enzyme assay.

Purification by ethanol precipitation

Prechilled ethanol was added to the culture supernatant at one to one ratio (v/v) or other in an

ice-salt batch with stirring. The resulting precipitate was collected by centrifugation at 16,000

rpm for 30 min at 4oC. The precipitate was dissolved in a 50 mM sodium acetate buffer (pH 5.5)

and dialyzed against the same buffer prior to enzyme assay.

Dialysis

Dialysis was performed using cellophane tubing having a cut-off molecular weight of 14,000 Da

(Wako size no. 8). The buffer was altered three times (after 2, 14 and 19 h) to ensure removal of

ammonium sulfate which may interfere separation during ion exchange chromatography.

Purification by Ion Exchange Chromatography

To find the best pH condition as well as type of column, purification by ion exchange

chromatography was first conducted batchwisely in 1.5 mL microtube. This microtube

functioned as a small chromatography column of DEAE-Toyopearl and Carboxymethyl (CM)-

Toyopearl. Larger volume of purification was conducted using a 2.8 _ 23 cm CM-Toyopearl

column preequilibrated with 10 mM sodium acetate buffer (pH 4.5) and eluted with a linear

gradient of 0 1 M NaCl (600 mL) in the same buffer.

Electrophoresis and zymogram

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed on a

15% acrylamide gels by the procedure developed by Laemmli [6]. The gels were run at a

constant current of 15 mA for approx. 2 h. Duplicate samples were run for simultaneous protein

(by Coomassie brilliant blue R-250) and activity staining (by 0.25% Congo red).


Experiment No. 4

Objective: Study of enzyme (Xylanse) kinetics by the use of Michaelis Menton equation.

Material required: Starch solution of different concentration (0.5 4%), amylase, acetate buffer, DNS reagent.

Principle: -amylase is a biological catalyst which catalyses the degradation of starch and other polysaccharides in to their mono-meric units by breaking - 1-4 glycosidase linkage. Michalies constant (Km) and maximum rate at which reaction can proceed (Vmax) are two important kinetic

properties of enzymatic reaction. The properties depend on nature of enzyme and on the

characteristics of the substrates. For homogeneous enzyme reaction, Michalies Menton relation

is V0 = Vmax. S / Km + S

1/ V0 = Km + S/ Vmax. S

= Km/Vmax. 1/S + 1/Vmax

Procedure:

1. Prepare the starch solution of different concentration ranging from 0.5 4% by mixing starch in acetate buffer.

2. In each of the test tubes add 0.5 ml of starch solution of different concentration. 3. Add 0.4 ml of acetate buffer and 0.1ml of amylase into each test tube for complete

reaction mixture.

4. Incubate the reaction mixture for 10 min. in water bath at 50C. 5. After incubation, immediate addition of 1ml of DNS for reaction termination. 6. Keep the test tubes in boiling water 15min. simultaneously. 7. Make up the volume in each test tube to 20ml by adding DW. 8. Blank and control should also be prepared according to above methods.

Observation: Stock solution = 50 ml of 4% starch

S.

No.

Volume of stock (ml) Starch concentration

(gm/ml)

Absorbance Velocity

1 0.05 4x10-3

2 0.10 8x10-3

3 0.15 1.2x10-2

4 0.20 1.6x10-2

5 0.25 2.0x10-2

6 0.30 2.4x10-2

7 0.35 2.8x10-2

8 0.40 3.2x10-2

Calculation: Vmax. =

Slope =

Km =


Experiment No. 5

Aim: To produce cellulase enzyme using solid state fermenatation.

Principles: This method tests for the presence of free carbonyl group (C=O), the so-called

reducing sugars. This involves the oxidation of the aldehyde functional group present in, for

example, glucose and the ketone functional group in fructose. Simultaneously, 3,5-

dinitrosalicylic acid (DNS) is reduced to 3-amino,5-nitrosalicylic acid under alkaline conditions:

oxidation

aldehyde group ----------> carboxyl group

reduction

3,5-dinitrosalicylic acid ----------> 3-amino,5-nitrosalicylic acid

Materials: Trichoderma ressei (fungus), wheat bran, DNS reagent, Carboxy methyl cellulose

(CMC), Whatman No 1 filter paper, spectrophotometer, 250-mlErlenmeyer flasks, centrifuge,

Incubator etc. The basal mineral salts solution used for the experiment had following

composition: KH2PO40.5%, NH4NO30.5%, MgSO47H2O0.1%, peptone0.1%, NaCl0.1%, and CaCl20.05% (trace elements: FeSO47H2O0.005%, MnSO47H2O0.001%, ZnSO47H2O0.001%, and CoCl20.0002%). The pH of the salt solution was adjusted with 1 N HCl or 1 N NaOH wherever required. It was then sterilized by autoclaving at

121C for 15 min at 15 lbs pressure.

Procedure: Erlenmeyer flasks containing 5 g of substrate moistened with the mineral salts

medium were inoculated with 1 ml of spore suspension containing the desired number of spores.

The contents were mixed thoroughly and were incubated under controlled conditions of

temperature and humidity. Incubation was continued for the duration indicated in the

experimental designs and at the end of incubation period enzyme was recovered by extraction

with 0.1 N citrate buffer (pH 4.8). The extract was centrifuged to remove debris at 6,000 rpm for

10 min at 4 C and was used as the crude enzyme sample.

Enzyme Assay: The Carboxy methyl cellulose (substrate) was saturated with 0.5 ml of Nacitrate buffer (0.05 M, pH 4.8) and was equilibrated for 10 min at 50 C in a water bath. Half

milliliter of an appropriately diluted (in Nacitrate buffer0.05 M, pH 4.8) enzyme was added to the tube and incubated at 50 C for 60 min. Appropriate controls were also run along with the

test. At the end of the incubation period, each tube was removed from the water bath and the

reaction was stopped by addition of 3 ml of DNS reagent. The tubes were incubated for 5 min in

a boiling water bath for color development and were cooled rapidly. The reaction mixture was

diluted appropriately and was measured against a reagent blank at 540 nm in a UVVIS spectrophotometer.

Reaction 0.5 ml (substrate, CMC) + 450l buffer + 50l enzyme (broth) Control 0.5 ml (substrate, CMC) + 0.5ml buffer Calculation: Cellulase activity (U/ml) = OD x Dilution factor x Slope inverse

Incubation time x Enzyme (ml)

Result:


Experiment No. 6

Objective: Filter paper assay was estimated total cellulase activity in the crude enzyme.

Materials: Whatman no. 1 filter paper strip of dimension 1.0x 6.0 cm (50 mg), 0.05 M Na citrate

buffer, DNS reagent, Trichoderma ressei (fungus), wheat bran, DNS reagent, Spectrophotometer,

250-mlErlenmeyer flasks, centrifuge, Incubator etc. The basal mineral salts solution used for the

experiment had following composition: KH2PO40.5%, NH4NO30.5%, MgSO47H2O0.1%, peptone0.1%, NaCl0.1%, and CaCl20.05% (trace elements: FeSO47H2O0.005%, MnSO47H2O0.001%, ZnSO47H2O0.001%, and CoCl20.0002%). The pH of the salt solution was adjusted with 1 N HCl or 1 N NaOH wherever required. It was then

sterilized by autoclaving at 121C for 15 min at 15 lbs pressure

Procedure: Erlenmeyer flasks containing 5 g of substrate moistened with the mineral salts

medium were inoculated with 1 ml of spore suspension containing the desired number of spores.

The contents were mixed thoroughly and were incubated under controlled conditions of

temperature and humidity. Incubation was continued for the duration indicated in the

experimental designs and at the end of incubation period enzyme was recovered by extraction

with 0.1 N citrate buffer (pH 4.8). The extract was centrifuged to remove debris at 6,000 rpm for

10 min at 4 C and was used as the crude enzyme sample.

Analytical Method (Enzyme Assay): Filter paper assay was used to estimate total cellulase

activity in the crude enzyme preparation as given below. A rolled Whatman No 1 filter paper

strip of dimension 1.06 cm (50 mg) was placed into each assay tube. The filter paper strip was

saturated with 0.5 ml of Nacitrate buffer (0.05 M, pH 4.8) and was equilibrated for 10 min at 50 C in a water bath. Half milliliter of an appropriately diluted (in Nacitrate buffer0.05 M, pH 4.8) enzyme was added to the tube and incubated at 50 C for 60 min. Appropriate controls were

also run along with the test. At the end of the incubation period, each tube was removed from the

water bath and the reaction was stopped by addition of 3 ml of DNS reagent. The tubes were

incubated for 5 min in a boiling water bath for color development and were cooled rapidly. The

reaction mixture was diluted appropriately and was measured against a reagent blank at 540 nm

in a UVVIS spectrophotometer.

Calculation: FPU = 0.37/ Enzyme concentration

Result:


Experiment No. 7

Objective: To recover proteins/enzymes from a solution by salting-out (Ammonium sulphate

precipitation of protein).

The most common type of precipitation for proteins is salt introduced precipitation. Different

types of salts such as ammonium sulphate and sodium sulphate are widely used to precipitate out

proteins. Ammonium sulphate is the most widely used salt for the precipitation of proteins as it is

highly soluble, inexpensive, available in highest purity level, does not change the protein

solution to extreme pHs and in most of case it does not denature proteins.

Ammonium sulphate can be used for precipitation of total proteins at ~90% saturation or for

differential precipitation level of proteins using different saturation of salts. Up to 20%

saturation, ammonium sulphate precipitate particulate materials, and preaggregated and very

high molecular weight proteins and at 90% saturation it precipitates almost all proteins.

Principle

Proteins have polar amino acids such as glycine, serine etc. Usually in native proteins

hydrophilic amino acids are on the surface of proteins whereas hydrophobic amino acids are

buried. Attractive interactions between the nearby oppositely charged groups are ion pairs or salt

bridges. Analysis has revealed that in folded proteins, 4 attractive ion pairs and 1 repulsive ion

pair are present per100 amino acids. Water as powerful solvent, interacts with these surface

amino acids and keep them in solution.

Protein solubility depends on several factors. It is observed that at low concentration of the salt,

solubility of the proteins usually increases slightly. This is termed Salting in. But at high

concentrations of salt, the solubility of the proteins drops sharply. This is termed Salting out and

the proteins precipitate out. During ammonium sulphate precipitation the salt has to be added in

small amount under constant stirring to avoid accumulation of high concentration of salts. When

large amount of salt is added to an aqueous solution of proteins the salt requires more amount of

water for its dissolution. This leads to competition for water molecule on the proteins.

Completely ionized salts have more affinity for water molecules then protein hence addition of

salts takes up water molecule from the protein. Therefore the ionic interactions between water

molecules and protein are reduced and as result hydrophobic interactions dominate. The

hydrophobic amino acid patches present in all the proteins attract each other and forms

aggregates. These aggregates are nothing but the proteins in the form of precipitates.

In salt precipitation, the anions appear to be more significant. Temperature, pH and the most

important the protein concentration affect ammonium sulphate precipitation of proteins to large

extent. Higher ammonium sulphate is required for precipitating highly soluble proteins.

Procedure

1. Take sample in a beaker containing a stir bar and place on magnetic stirrer 2. While sample is stirring, slowly add ammonium sulfate of a desired saturation level (Can

refer ammonium sulphate precipitation chart)

3. Add ammonium sulfate very slowly to ensure that local concentration around the site of addition does not exceed the desired salt concentration.

4. Once total volume of ammonium sulfate is added, move beaker to 4C for 6 hours or overnight.

5. Collect the precipitate from the beaker and centrifuge the precipitate at 8000g for 10 minutes.

6. Carefully remove and discard supernatant. Invert the tube and drain well 7. Give two or three wash with distilled water 8. Dissolve the precipitate in phosphate buffer saline and dialyze protein solution at low

temperature overnight to get removed salt

9. Determine the concentration and store at -10C for long term storage.

The following table shows the weight (g) of ammonium sulfate to be added to one litre of

solution to produce a desired change in the concentration (% saturation) of ammonium sulfate.

SECTION 3: BT604 (ANIMAL

BIOTECHNOLOGY)

EXPERIMENT NO. 1

AIM: To study laboratory requirements for animal cell culture.

THEORY: During animal cell culture in laboratory following facilities are required

1. Sterile handling 2. Incubation 3. Preparation of glassware, media and tissue 4. Wash up 5. Sterilization 6. Storage

The clean area for handling should be available at one end of the room and wash-up and

sterilization at other end with preparation, incubation and storage in between.

Tissue culture facilities required in any laboratory are:

MINIMUM REQUIREMENTS (essentials):

1. Sterile area: clean and quiet, no thorough traffic, separate from animal house and microbiology lab.

2. Preparation area: a. Storage area:

Liquid: ambient 4-20oc

Glass ware (shelving)

Plastics (shelving)

Small items (drawers)

Specialized equipment (slow turnover), cupboard

Chemicals- ambient 4-20oc (share with liquid but keep chemicals in sealed container over desiccant )

b. Desirable features (beneficial)

Filtered air(air conditioning)

Hot room with temperature recorder

Microscope room

Dark room

Service bench adjacent to culture media

Separate preparation room

Separate sterilizing room

Cylinder store

USEFUL ADDITIONS:

1. Piped CO2 and compressed air 2. Store room for bulk plastic 3. Containment room for biohazard work 4. Liquid N2 storage tank (500L)

The introduction of the laminar flow cabinet/ hoods to provide clean bench has greatly

facilitated the maintenance of the aseptic and sterile conditions, so that no separate room for

sterile handling is needed and the laminar flow cabinet can be kept in an unspecialized lab

space. Sometimes, however aseptic room is designed where filtered air is supplied from

ceiling so that the whole is regarded as the sterile working area.

Incubation is generally done in incubator at thermostatically controlled hot rooms. In hot

rooms, racks are designed for the placement of the cultures.

Media can be purchased, readymade or may be prepared in lab, the later being cheaper if

needed in large quantities.

The washing and sterilization facilities are often kept outside the tissue culture lab since they

would generate moisture (increase humidity and heat).

TISSUE CULTURE EQUIPMENT:

Essential equipment Desirable equipment Additional equipment

Oven CO2 incubator -70oC freezer

Incubator Cell counter High capacity centrifuge

Autoclave Osmo-meter Cell sizer

Microscope Magnetic stirrer Controlled rate cooler

Refrigerator Pipette drier FACS (florescence activated

cell sorter)

Pipette cylinder Pipette pluger Densitometer

Pipette washer Roller racks Glass wear washing machine

Centrifuge Automatic dispenser Time lapse cenimircographic

equipment

Water purification

system

Vacuum pump

pH meter Phase contrast microscope

Weighing machine Colony counter

Sink Trolley

Liquid N2 freezer

Laminar air hood

EXPERIMENT NO. 2

AIM: To determine the percentage of the viable cells in a suspension by trypan blue exclusion

test.

REQUIREMENTS: 0.4% trypan blue, cell suspension, microscope, haemocytometer,

micropipette and eppendroff.

PRINCIPLE: Dye exclusion test are made to determine the number of the viable cells present in

a cell suspension. It is based on the principle that live cells possess intact cell membrane which

excludes the dye, whereas the dead cells dont. These viable cells show a clear cytoplasm whereas dead cells shows blue cytoplasm with trypan blue

PROCEDURE 1. Mix 1 part of 0.4% trypan blue and 1 part of given cell suspension. 2. Incubate the mixture at room temperature for 3 minutes. Cells should be counted within 3-

5 minutes of mixing with trypan blue as longer incubation will lead to cell death.

3. Apply a drop of dye cell mixture to a haemocytometer and cover the grid area with the cover slip.

4. Count the stained and unstained cells separately under microscope.

OBSERVATIONS:

EXPERIMENT NO. 3

AIM: To study apoptosis by microscopic analysis.

REQUIREMENT:

0.1% trypan blue, cell suspension, microscope, Microfuge tubes.

PRINCIPLE: Trypan blue is the stain most commonly used to distinguish viable cells from non-

viable cells. Viable cells exclude the dye, while the non-viable ones absorb it and appear blue.

Before counting, cells should be placed in the buffer saline as a single cell suspension. Trypan

blue has a high affinity for serum protein that for cellular protein, so suspending cells in medium

containing serum will generate a dark background.

PROCEDURE: 1. 20l of trypan blue was taken in a vial/tube. 2. Add equal amount of cell suspension and mix gently. 3. Add 10l of cell-dye mixture in the depression of haemocytometer and place a cover slip

on the grid area.

4. Examine in a bright field microscope. 5. Count total of 300 cells 6. Record number of normal, apoptotic and dead cells (cell in apoptosis may appear white

due to exclusion of trypan blue with irregular shape and shrunken nucleus).

OBSERVATION:

Where, VN= Viable cells with normal nuclei (white cells)

VA = Viable cells with apoptotic nucleus (white cells with shrunken nucleus)

NVN = Non-viable cells with normal nuclei (blue cells with normal shaped and sized

nucleus)

NVA = Non-viable cells with apoptotic nuclei (blue cells with shrunken nuclei)

EXPERIMENT NO. 4

AIM: To establish primary cell culture using fine dissection for disaggregation of tissue.

REQUIREMENTS:

Growth medium (example 50:50 DMEM:F12 with 20% Fetal bovine serum), 100ml PBS

Petri-dishes, non-tissue culture grade. Forceps, scalpels, Pipettes, Centrifuge tubes, Culture flask

25cm2, tissue culture grade petri-dishes (the size of flask and the growth medium depend on the

amount of tissue, roughly five 25cm2/100mg of tissue).

THEORY: Cell culture is a complex process by which cells are grown under controlled

condition. Primary cell culture is the stage of cell culturing before sub-culturing.

A tissue/organ is required from which a particular cell line will be obtained. A prior permission

for isolation of tissue is required from ethical committee.

PROCEDURE:

1. Transfer tissue to fresh, sterile DBSS and rinse. 2. Transfer the tissue to a second dish, dissect of unwanted tissue such as fat or necrotic

material and transfer to a third disk.

3. Fine dissection is done. Tissue is chopped using crossed scalpels into pieces of 0.3-0.5 mm in sterile medium.

4. Transfer by pipette to a 15/50 ml sterile centrifuge tube. 5. Allow the pieces to settle. 6. Wash by resuspending the pieces in DBSS (washing solution or serum free media)

allowing the pieces to settle and removing the supernatant fluid. Repeat this step two or

more times.

7. Transfer the small pieces to a culture flask with about 20-30 pieces per 25 cm2 flask. (Culture flask should be percoated with fibronectin which enhances the attachment).

8. Remove most of fluid and add 1ml growth media per 25 cm2 growth surface. Tilt the flask gently to spread the pieces evenly over the growth surface.

9. Cap the flask and place it in an incubator at 37c for 18-24 hours. 10. If the pieces have adhered, then the medium volume may be made gradually over the next

3-5 days to 5ml per 25 cm2 and then changed weekly until a substantial outgrowth of

cells is observed. (Volume may be increased up to 25ml per 100 cm2).

11. Once an outgrowth has formed, the remaining explants may be picked off with a scalpel and transferred by prewetted pipette to a fresh culture and again it is cultured.

12. Replace the medium in first flask until the outgrowth has spread to cover at least 50% of growth surface, at which point cell may be subculture.

EXPERIMENT NO. 5

AIM: To establish primary cell culture using enzymatic method for disaggregation of tissue.

(Warm trypsinization).

REQUIREMENTS:

Tissue 1-5 gm, 50 ml DBSS. Trypsin(crude), 2.5% in D-PBSA or normal saline, D-PBSA(200

ml), Growth medium with serum(e.g. DMEM/F12 with 10% FBS), Culture flask, Petridish,

Preweighed vials, 250 ml centrifuge tubes, Trypsinization/stirrer flask(250 ml Erlenmeyer flask)

Magnetic follower, autoclaved in a test tube, Curved forceps, Pippets, Magnetic stirrer

Hemocytometer or cell counter

THEORY:

Cell-cell adhesion in tissue is mediated by variety of homotypic interacting glycopeptides, some

of which are calcium dependent (cadherins) and hence are sensitive to chelating agents such as

EDTA or EGTA. The easiest approach for disaggregation of tissues is to proceed from simple

disaggregation solution to a more complex solution with trypsin alone or trypsin/EDTA as a

starting point, adding protease to improve disaggregation and deleting unwanted trypsin if

necessary to increase viability.

The choice of which trypsin grade to use has always been difficult:

The purer the trypsin, the less toxic it becomes and the more predictable its action.

The cruder the trypsin, the more effective it may be because of other proteases. Crude trypsin is by far the most common enzyme used in tissue disaggregation, as

it is tolerated well by many cells and is effective for many tissues.

It is important to minimize the exposure of cells to active trypsin in order to

preserve maximum viability. Hence, when whole tissue is trypsinzed at 37 c dissociated cell should be collected every half hour and the trypsin should be

removed by centrifugation and neutralized with serum in medium.

PROCEDURE:

1. Transfer the tissue to fresh, sterile DBSS in petridish and rinse. 2. Transfer the tissue to second dish, dissect of the unwanted tissue, such as fat or necrotic

materials, and transfer to a third dish.

3. Chop with crossed scalpels. 4. Transfer the tissue with curved forceps to the preweighed vials or tubes. 5. Allow the pieces to settle. 6. Wash the tissue by resuspending the pieces in DBSS, allowing the pieces to settle, and

removing the supernatant fluid. Repeat this step two more times.

7. Drain the vial or tube and reweigh 8. Transfer all the pieces to the empty trypsinization flask, flushing the vials or tubes with

DBSS.

9. Remove most of the residual fluid and add 180ml of D-PBSA. 10. Add 20ml of 2.5% trypsin (other enzymes such as collagenase, hyauronidase or DNAse

may be added if required).

11. Add the magnetic follower to the flask 12. Cap the flask, and place it on magnetic stirrer in an incubator or hot room at 37c. 13. Stir at about 100 rpm for 30 min at 37c. 14. After 30 min , collect disaggregated cells as follows:

a) Allow the pieces to settle

b) Pour of the supernatant into a centrifuge tube and place it on ice. c) Add fresh trypsin to the pieces remaining in the flask, and continue to stir and

incubate further for 30 min.

d) Centrifuge the harvested cells from step 14(b) at approximately 500 rpm for 5 min. e) Resuspend the resulting pellet in 10ml of medium with serum and store the

suspension on ice.

15. Repeat step 11 until complete disaggregation occurs or until no further disaggregation is apparent.(usually 3-4 hours)

16. Collect and poll chilled cell suspension, count the cells by hemocytometer or electronic cell counter and check viability.

17. As the cells population will be very heterogenous, electronic cell counting will require confirmation with a hemocytometer, because calibration can be difficult.

18. Remove any large remaining aggregates by filtering through sterile muslin or a proprietary sieve.

19. Dilute the cell suspension to 1106 /ml in growth medium, and seed as many flasks as are required, with approximately 210

5 cells/cm

2. When the survival rate is unknown or

unpredictable, a cell count is of little value. In this case, setup a range of concentration

from about 5 to 25 mg of tissue per ml.

20. Change the medium at regular intervals (2-4 days as dictated by depression of pH) check the supernate for viable cells before discarding it, as some cells can be slow to attach or

may even prefer to proliferate in suspension.

EXPERIMENT NO. 6

AIM: To establish primary cell culture using enzymatic method for disaggregation of tissue.

(Cold trypsinization).

REQUIREMENTS:

Tissue 1-5 gm preweighed, Growth medium, DBSS, 0.25% crude trypsin in serum free RPMI

1640 or MEM/stirrer salt(S-MEM), Petridishes, non tissue culture grade, Forceps, straight and

curved, Scalpels, Erlenmeyer flask, Culture flask, Pipettes, Icebath

THEORY:

One of the disadvantage of using trypsin to disaggregate tissue is the damage that may result from

prolonged exposure of tissue to the trypsin at 37c, hence there is need to harvest cells after 30 minutes incubation in warm trypsin method rather than have them exposed for full time (3-4

hours) require to disaggregate the whole tissue.

A simple method of minimizing the damage to the cells during exposure is to soak the tissue in

trypsin at 4c for 6-18 hours to allow penetration of the enzyme with little tryptic activity. Following this procedure, the tissues will only require 20-30 minutes at 37c for disaggregation.

PROCEDURE:


materials, and transfer to a third dish.

3. Chop with crossed scalpels into about 3mm cubes, embryonic organs, if they exceed this size, are better left whole.

4. Transfer the tissue with curved forceps to a preweighed vial. 5. Allow the pieces to settle. 6. Wash the tissue by resuspending the pieces in DBSS, allowing the pieces to settle, and

removing the supernatant fluid. Repeat this step two or more times.

7. Carefully remove the residual fluid and reweigh the vial. 8. Add 100ml/gm of tissue of 0.25% trypsin in RPMI-1640 or S-MEM at 4c. 9. Place the mixture at 4c for 6-18 hours. 10. Remove and discard the trypsin carefully, leaving the tissue with only the residual

trypsin. (Other enzymes such as collagenase, hyaluronidase or DNAase, may be added in

1-2 ml amounts, if required.)

11. Place the tube at 37c for 20-30 minutes 12. Add warm medium, approximately 1ml for every 100 mg of original tissue and gently

pipette the mixture up and down until the tissue is completely dispersed.

13. If some tissue does not disperse, then the cell suspension may be filtered through sterile muslin or stainless steel sieve or a disposable plastic mesh strainer, or larger pieces may

simply be allowed to settle. When there is lot of tissue increasing the volume of

suspending medium to 20ml for each gram of tissue will facilitate settling and subsequent

collection of supernatant fluid, 2-3 minutes should be sufficient to get rid of most of the

larger pieces.

14. Determine the cell concentration in suspension by hemocytometer or electronic cell counter and check the viability.

15. The cell population will be very heterogeneous, electronic cell counter will initially require confirmation with hemocytometer, as calibration can be difficult.

16. Dilute the cell suspension to 1106 /ml in growth medium and seed as many flask as are required, with approximately 210

5 cells/cm

2. Where the survival rate is unknown or

unpredictable, a cell count is of little value. In this case, set up a range of concentration

from 5 to 25 mg of tissue per ml.

17. Change the medium at regular intervals (2-4 days as dictated by depression of pH) Check the supernatant for viable cells before discarding it, as some cells can be slow to

attach or may even prefer to proliferate in suspension.

EXPERIMENT NO. 7

AIM: To establish primary cell culture using enzymatic method for disaggregation of tissue

using collagenase.

REQUIRMENTS:

Collagenase(2000 units/ml), Culture medium, DBSS, Pipette, Petridishes, non tissue culture

grade , Culture flasks, Centrifuge tube, Scalpels, Centrifuge

THEORY:

Disaggregation in trypsin can be damaging or ineffective, so attempts have been made to utilize

other enzymes. Because the extracellular matrix often contains collagen, particularly in

connective tissues and muscles, collagenase has been the obvious choice.

This technique is very simple and effective for many tissues: embryonic, adult, normal and

malignant. It is of greatest benefit when the tissues are either too fibrous or too sensitive to allow

the successful use of trypsin.

Crude collagenase is often used and may depend, for some of its action on contamination with

other non specific protease. More highly purified grades are available if non specific proteolytic

activity is desirable, but they may not be as effective as crude collagenase,

PROCEDURE:


materials.

3. Transfer to a third dish and chop finely with crossed scalpels 4. Transfer the tissue by pipette to a 15-50 ml sterile centrifuge tubes. 5. Allow the pieces to settle 6. Wash the tissue by resuspending the pieces in DBSS, allowing the pieces to settle, and

removing the supernatant fluid. Repeat this step two or more times.

7. Transfer 20-30 pieces to a 25cm2 flask and 100-200 pieces to a second flask. 8. Drain off the DBSS, and add 4.5ml of growth medium with serum to each flask. 9. Add 0.5ml of crude collagenase, 2000 units/ml to give a final concentration of 200 units/ml

collagenase.

10. Incubate at 37c for 4-48 hours without agitation. Tumor tissue may be left up to 5 days or more if disaggregation is slow, although it may be necessary to centrifuge the tissue and

resuspend (i.e. < pH 6.5)

11. Check the effective disaggregation by gently moving the flask; the pieces of tissues will smear on the bottom of the flask and with gentle pipetting will break up into single cell and small cluster.

12. With some tissues (e.g. Lung, kidney, and colon or breast carcinoma) small cluster of epithelial cells can be seen to resist the collagenase and may be seprated from the rest by

allowing them to settle for about 2 minutes. If these cluster are further washed with DBSS by

resuspension and settling and the sediments is resuspended in medium and seeded, then they

will form islands of epithelial cells. Epithelial cells generally survive better if they are not

completely dissociated.

13. When complete disaggregation has occurred, or when the supernatant cells are collected after removing clusters by settling, centrifuge the cell suspension from the disaggregate at 50-100

g for 3 minutes.

14. Discard the supernatant DBSS or medium resuspend and combine the pellets in 5 ml of medium and seed in a 25cm

2 flask. If the ph fell during colllagenase treatment (to pH 6.5 or

less by 48 hours) then dilute the suspension 2 to 3 fold in medium after removing the

collagenase.

15. Replace the medium after 48 hours.

EXPERIMENT NO. 8

AIM: To establish primary cell culture using mechanical method for disaggregation of tissues.

REQUIRMENTS:

Growth medium (e.g. DMEM/F12 with 10% FBS), Forceps, Sieve or graded sieve of series from

100m down to 20m, Petridishes, Scalpels, Disposable plastic syringes, Culture flask

THEORY:

The outgrowth of cells from primary explants is relatively slow process and can be highly is

selective. Enzymatic digestion is rather more labor intensive, although potentially it gives a

culture that is more representative of the tissue. As there is a risk of proteolytic damages to cells

during enzymatic digestion, mechanical disaggregation is used as an alternative.

Example:

Collecting the cell that spill out when the tissue is carefully sliced

Pressing the dissected tissue through a series of sieve for which the mesh is gradually reduced in size (sieving)

Or alternatively forcing the tissue fragments through syringe (with or without wide gauge needle)

Or simply pipetting it repeatedly This process gives a cell suspension more quickly than enzymatic digestion but may cause

mechanical damage. Pipetting and syringing generate shear.

This method is moderately successful with soft tissues such as brain.

PROCEDURE:

1. Transfer the t issue to fresh, sterile DBSS and rinse.

2. Transfer the tissue to second dish, dissect of unwanted tissues such as fat or necrotic material and transfer to the third dish.

3. Chop the tissues into pieces to about 3-5 mm and place a few pieces at a time into a stainless steel or polypropylene sieve of 1mm mesh.

4. Force the tissues through the mesh into medium by applying gentle pressure with the piston of a disposable plastic syringe. Pipette more medium through the sieve to wash the

cells through it.

5. Pipette the partially disaggregated tissues from the petridish into a sieve of fine porosity, perhaps 100m mesh, and repeat step 4

6. The suspension may be diluted and cultured at this stage, or it may be sieved further through 20m mesh if it is important to produce a single cell suspension. In general. The

more highly dispersed the cell suspension the higher the shear stress required and the

lower the resulting viability.

7. Seed the culture flask at 2105, 1106 and 2106 cells/ml by diluting the cell suspension in medium.

EXPERIMENT NO. 9

AIM: To determine cell density after cell counting.

REQUIRMENTS:

Frozen animal cell line, Medium A , Medium B , Trypsin/EDTA, Trypan blue

CO2 incubator, Vortex mixer, Water bath, Autoclave, Laminar airflow, Hemocytometer

Micropipette, Tissue culture flask, Vented spinner

PRINCIPLE:

Cell culture is the process by which prokaryotic, eukaryotic or plant cell are grown under control

conditions. In practice the term cell culture refers to culturing of cells derived from multicellular eukaryotes especially animal cells.

There are two main types of animal cell culture:

1. Primary cultures

2. Continuous culture(cell lines)

Cells that are cultured directly from a subject are known as primary cell culture. With the

exception of some derived from tumors, most primary cell cultures have limited life span ,

continuous culture are comprised of single cell type that can be serially propagated in culture

either for a limited number of cell division or otherwise indefinitely. It helps us to learn about the

growth of cell under strict laboratory conditions of asepsis optimum temperature, gases and

pressure. It should be like in vivo condition, and then only cells are able to survive and

proliferate. In this practical, cell lines have to be grown with growth medium (minimum medium

with 10% FBS), continuous growth regulators and antibiotics. Incubate at optimum temperature

(37c) in a humidified CO2 incubator. Determine the cell count using haemocytometer. Steps to follow before starting the experiment:

1. Sterilize the laminar airflow working bench

2. Place al micropipette, tips, centrifuge tubes.

3. Thaw frozen vials of animal cell line in a 37c waterbath

4. Melt the medium A, B and trypsin/EDTA solution in water bath at 37c

5. Prepare CO2 incubator humidified with 10% CO2 at 37c

PROCEDURE:

1. Place the thawed vial of cells on a sterilized laminar flow working bench

2. Wipe the vial neck with 70% ethanol

3. Transfer 5ml of medium A into 25cm2 tissue culture flask aseptically

4. Transfer the dell suspension into medium a containing tissue culture flask aseptically.

5. Rotate the flask to evenly distribute the cell and incubate overnight in a humidified 10%CO2 incubator at 37c

6. Aspirate the medium carefully with sterile pipette without disturbing the bottom layer aseptically.

7. Add 0.5ml of trypsin/EDTA mixture and allow it for 30-40 seconds

8. Examine under inverted microscope for rounding of cells.

9. Once rounding of cells is noticed, completely decant the trypsin solution from the culture flask, to ensure that there is no more trypsin in culture flask, failing of which may cause cell

death.

10. Add 10ml of medium B mix by repeated pipetting and transfer the cell along with medium to a sterile 50ml centrifuge tube aseptically.

11. Centrifuge at 1800 rpm for 5 minutes at 4c.

12. Discard the supernatant aseptically

13. Suspend the pellet in 5 ml of medium B by repeated pipetting to disturb the clumps.

14. Take 1ml of cell suspension for counting.

15. Take 50l from the cell suspension and add 50 l of trypan blue solution.

16. Gently mix the cell suspension

17. Place the haemocytometer with cover slip over the microscope platform and focus the WBC chambers at 40 magnification.

18. Add 10l of cell suspension by placing the pipette tip in the corner of the cover slip and let the cell suspension get adsorbed between the coverslip and the haemocytometer

19. Wait for 10 minutes for the cell to settle

20. Count the live and dead cell in WBC chamber.

CALCULATION:

No. of live cells /ml = (total no. of live cells in a WBC chamber 104

dilution factor) / 4

Since equal amount of dye is added to the cell suspension, the dilution factor is 2.

Total number of dead cells = (total no. of dead cells in a WBC chamber 104

2) / 4

Cell viability (%) = total live cells (unstained) 100 % / total dead cells (stained) + total live

Cells (unstained)

21. Adjust the cell count to 3-4105 cells/ml by adding medium B.

22. Add 1 ml of counted cell (3-4105 cells/ml) into tissue culture flask containing 5ml of medium A

23. Incubate for 48 hours in a humidified 10% CO2 incubator at 37c

24. Aspirate the medium carefully with sterile pipette without disturbing the bottom layer aseptically.

25. Add 0.5 ml of trypsin//EDTA mixture and allow it for 30-40 seconds.

26. Follow step 8-20.

27. Count the cell using heamocytometer.

28. Cells are maintain and grown in medium B in vented spinner bottles at 37c without CO2.

29. Cells are diluted with fresh sterile medium B at 1-2 day interval to keep the cell density between 1.510

5 to 510

5 cells/ml.

EXPERIMENT NO. 10

AIM: To perform MTT cell proliferation assay

REQUIRMENTS:

MTT 3-(4,5-dimethylthizole-2-yl)-2,5-diphenyltetrazolium bromide, SDS sodium dodecyl

sulfate, Phosphate buffer saline(PBS), sterile HCL, Microplate, pipette

PRINCIPLE:

MTT cell proliferation assay provides simple method for determination of cell number using

standard microplate absorbance readers. Determination of cell growth rate is widely used in

testing of drug action, cytotoxic agents and screening other biologically active compounds.

Several methods can be used for such determination, but indirect approaches using florescent or

chromogenic indicators provide most rapid and large scale assays.

The MTT assay involve the conversion of the water soluble MTT 3-(4, 5-dimethylthizole-2-

yl)-2, 5-diphenyltetrazolium bromide to an insoluble formazan. The formazen is then solublized,

and the concentration determined by optical density at 570nm.

PROCEDURE:

1. Reagent preparation: prepare a 12mM MTT stock solution by adding 1 ml of sterile PBS to one 5 mg vial of MTT. Mix by vortexing or sonication until dissolved. Occasionally there

may be some particulate material that may not dissolve; this can be removed by filtration or

centrifugation. Once prepared , the MTT solution can be stored for four weeks at 4c protected from light

2. Add 10ml of 0.01 M HCL to one tube contain 1 gm of SDS. Mix the solution gently by inversion or sonication until SDS dissolves. Once prepared the solution should be used

promptly

3. Culture the cell in appropriate culture medium.

4. For adherent cells, remove the medium and replace it with 100 l of fresh culture medium. For non adherent cells, centrifuge the microplate, pellet the cells, carefully remove as much

medium as possible and replace it with 100l of fresh medium.

5. Add 10l of 12mM MTT stock solution prepared in step 1 to each well. Include a negative control of 10l of the MTT stock solution added to 100l of medium alone.

6. Incubate at 37c for 4 hours. At high densities (>100,000 cells per well) the incubation time can be shortened to 2 hours.

7. Add 100l of the SDS-HCL solution prepared in step 2 to each well and mix thoroughly using the pipette.

8. Incubate the microplate at 37c for 4-18 hours in humidified chamber.

9. Mix each sample again using pipette and read absorbance at 570nm.

SECTION 4: BT606 (MICROBIAL

TECHNOLOGY)

Experiment No.1

Aim: The production of Antibiotic (Neomycin) by Streptomyces fradiae in Synthetic Media.

Theory: The basic importance to of antibiotic production by microorganisms is a chemically

defined medium in which the effect of various nutrients, both organic and inorganic, can be

studied with a minimum of interference from ill-defined substances. Such a medium is reported

here for the production of neomycin by Streptomyces fradiae, together with studies on the

influence of various nitrogen and carbon compounds on this reaction.

Materials:

Basal medium gm/l

Glucose 5.0

K2HP04 2.0

MgSO4.7H2O 0.5

FeSO4.7H20 0.05

ZnSO4.7H20 0 .05

CaCO3 10

The neomycin-producing culture of S. fradiae 3535 (Waksman and Lechevalier, 1949) was used

throughout the study for neomycin biosynthesis. The culture was maintained on a potato-

dextrose agar slant at 28 C and was sub cultured at monthly intervals.

Procedure-

1-A well-sporulated slant culture (10 days old) was washed with 5 ml of sterile water,

2- 0.2 ml was used to inoculate each 100-ml Erlenmeyer flask containing 30 ml of medium.

3- After a night of stationary rest, the flasks were placed on a rotary shaker [250 rev/ min].

Incubation temperature was 28 C. Occasional checking of the flask to drop adhering cells into

the medium was necessary during the first 48 hr.

Result- checks the antibiotic sensitivity bacterial agar plate.

Experiment No. 2

Aim-To determine the effectiveness of antibiotic using agar well dilution bioassay test.

Theory-The effectiveness of antibiotics can be determined by agar well diffusion assay in which

sells are cut in agar media in a petriplate in which bacterial suspension have been inoculated

followed by addition of suitable dilution of antibiotics into well. Antibiotic well inhibits the

growth of microbes around well represent the inhibition zone.

Requirements- Autoclaved petridish, nutrient broth, bacterial culture, gel puncher.

Procedure

1- Prepare a bacterial medium and sterilized it.

2- Pour the media into the petriplate under aseptic condition.

3- Inoculate the agar plate with bacterial culture.

4- Allow the culture to grow for 24 hrs at 37C.

5- Put antibiotic of known dilution in well.

6- Let the antibiotic activity be completed for 24 hr and measure zone of inhibition.

Result- after 24 hr of incubation, a clear zone of inhibition was seen around wells to which

antibiotic was added indicating that antibiotic had inhibited the growth of bacteria around well

where it diffused.

Experiment No.3

Aim- Fermentative production of amylase by Aspergillus Niger.

Theory- Starch is broken into small units by the enzyme amylase prodused by

microorganisms.However starch react with iodine and develops blue colored comlex.Moreever,

intensity of the color developed is directly proportional to the concentration of starch present in

the sample.

Requirements- Iodine solution(0.01N),starch solution (0.1%),Distilled water (500ml),Growth

media-Yeast Extract(0.3gm),Malt extract(0.3gm),Peptone(0.5gm),soluble starch(1gm),Distilled

water(1lit),pH-6.

Procedure-

1- Prepare above mentioned media dispense in 200ml media in a flask and autoclave it.

2- Procure pure culture of Aspergillus Niger, and prepare its fresh culture in a tube.

3- Transfer 10ml inoculums in sterilized growth media and incubate at 30C for 24 hr,48 hr

and 72 hrs on a flask.

4- Filter the filtrate through sterile Wharman filter paper No.42, collect supernatant and

measure amylase activity of filtrate by starch iodine method.

5- Take different aliquots of starch solution ranging from 0-2ml and make initial volume to

16ml by adding distilled water, black will lack starch.

6- Add 4ml of 0.01N iodine solution, measure OD after 10min of incubation at 578nm.

7- Calculate the enzyme activity using the following formula:-

Volume activity = Eo-Et A 1000(V/l)

Eo T V

Where, Eo =OD2 OD1 Et =OD3-OD1

T=Incubation time(minutes)

A=12.35 constant

V= volume of starch

Result- Draw a graph between OD and starch, and calculate the enzyme activity.

Experiment No. 4

Aim- Demonstration of wine production by using grape juice.

Theory- Wine production is actually produced by fruit juice in which fermentation is carried out

by Saccharomyces cerevisiae. In most of the countries wine is produced from grape juice which

is called must. Three main character are required for wine production these are simple sugar,

yeast and anaerobic condition. This reaction is illustrated as below:-

C6 H12 O6 >2C 2H5OH + 2CO2

Requirements- Grape juice,Yeast culture, Flask, Balloon, lead acetate strip.

Procedure-

1- Collect fresh and healthy grapes and squeeze it to get juice.

2- Sterilize the grape juice passing through the Millipore filter paper.

3- Collect 100ml of juice in sterilized flask, incubate with 3ml of yeast culture and keep a

lead acetate test strip taped inside the neck of the flask.

4- Seal the mouth of the flask with rubber balloon before incubation.

5- Incubate it at 15-20C for 8-12 days.

6- Remove the balloon after incubation to check the odour of gas and examine the test strip

of lead acetate for change in color.

7- Record the pH before and after fermentation.

8- If H2S is produced, the paper will be dark due to formation of lead sulphide as hydrogen

sulphide react with lead acetate.

Result- Record the aroma.

Experiment No.5

Aim- To determine the minimum inhibitory concentration (MIC) of an antibiotic.

Theory-We need to establish the minimum inhibitory concentration (MIC) of an antibiotic

which may inhibit the growth of microorganism. Penicillin is effective against Gram-positive

bacteria while streptomycin kills Gram negative such as E.coli. Penicillin blocks the amino acid

synthesis .Osmotic pressure exerts on the wall and cell breaks and lyse.

Streptomycin is used against the Gram negative bacteria. It binds the protein of the30S subunit of

ribosome, blocking protein synthesis in the cell. The cells stop dividing due to check of new

protein synthesis and lose viability.

Requirements- Penicillin, streptomycin, Nutrient broth, Culture tube, Bacterial culture,

Spectrophotometer, Incubator, inoculation loop.

Procedure

1- Prepare the solution of measuring 4 units/ml of penicillin or 2mg/ml of streptomycin.

2- Mix 2ml of the antibiotic solution in 2ml of nutrient broth in test tube and shake well.

3- Transfer 2ml in tube 2 and subsequently transfer in rest of the tube containing 2ml of

nutrient broth except the last that does not contain any antibiotics solution. It is possible

to calculate the concentration of antibiotic in each tube.

4- Inoculate each tube with one drop of culture and incubate them at 37C for 48 hrs.

5- Measure the turbidity in term of optical Density (OD) by spectrophotometer and prepare

a table or plot a graph between antibiotic concentration and turbidity.

Result- Low concentration of antibiotic will show maximum optical density due to less

inhibitory effect; where as high concentration will reveal minimum optical density.

Lab Manual 6th Sem- New

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