BASIC TECHNIQUES IN BIOTECHNOLOGY

(Extraction of polysaccharides from diatoms)

AN INDUSTRY TRAINING REPORT

Submitted by

JAYALAKSHMI D (200401050)

JWALA G (200401053)

In partial fulfilment for the award of the degree of

BACHELOR OF TECHNOLOGY

IN

BIOTECHNOLOGY

RAJALAKSHMI ENGINEERING COLLEGE

UNIVERSITY OF MADRAS

CHENNAI 600005

JULY 2022
 UNIVERSITY OF MADRAS,
CHENNAI – 600005.

BONAFIDE CERTIFICATE

Certified that this training report titled “Basic Techniques in Biotechnology” is the

bona-fide work of JAYALAKSHMI D (200401050) and JWALA G (200401053)

who carried out work under my supervision. Certified further that to the best of my

knowledge the work reported herein does not form part of any other thesis or

dissertation on the basis of which a degree or award was conferred on an earlier

occasion on this or any other candidate.

SIGNATURE SIGNATURE

Dr. K. SATHYA Mrs. S. DIVYALAKSHMI

Professor and Head, Assistant Professor,

Department of Biotechnology, Department of Biotechnology,
Rajalakshmi Engineering College Rajalakshmi Engineering College,
Thandalam, Chennai-602 105 Thandalam, Chennai – 602 105

Submitted for the examination held on

INTERNAL EXAMINER EXTERNAL EXAMINER

 DECLARATION

We, Jayalakshmi D and Jwala G, hereby declare that the work entitled “Basic

Techniques in Biotechnology (Extraction of polysaccharides from Diatoms)”,

submitted by us to Rajalakshmi Engineering College, Thandalam in partial

fulfilment for the award of the degree of B. tech in Biotechnology department is a

record of bona-fide project work carried out by us under the guidance of

Dr. S. Elumalai, Registrar, University of Madras, Chepauk, Chennai-600 005, and

our supervisor Mrs. Divyalakshmi, Assistant Professor, Department of

Biotechnology, Rajalakshmi Engineering College, Thandalam, Chennai (affiliated

to Anna University, Chennai). We further declare that the work reported in this

report has not been submitted and will not be submitted, either in part or in full, for

the award of any other degree in this institute or any other university.

Jayalakshmi D (200401050)

Jwala G (200401053)

Date:

Place: Chennai
 ACKNOWLEDGEMENT

We wish to express our gratitude to the Chairperson DR. THANGAM

MEGANATHAN and the Principal DR. S. N. MURUGESAN, Rajalakshmi
Engineering College, who have enlightened our life quality and discipline along with
academic focus.
We express our earnest and deep sense of gratitude to Dr. JOHANNA
RAJKUMAR, Professor, Dean, Department of Biotechnology, and to Dr. K.
SATHYA, Associate Professor and Head, Department of Biotechnology, whohave
always stood as a moral support and guided us tirelessly with their valuable ideas
along with constant encouragement throughout the period of study.
We sincerely thank our Internal guide Mrs.S. DIVYALAKSHMI, Assistant
Professor, Department of Biotechnology, Rajalakshmi Engineering College, for her
guidance, encouragement, timely suggestions, keen interest and instructive
supervision throughout the work.
We owe our sincere regards to our guide, Dr. S. ELUMALAI, Registrar,
University of Madras, for his invaluable scientific advices, complete support,
guidance and encouragement throughout the process of carrying out this work.
We are grateful to Dr. Rajesh Kanna, Assistant Professor, Saveetha Dental
College and Hospitals, Chennai and Dr. Rajesh Damodharan, University of Madras,
Chennai, for guiding, keeping us on the correct track and allowed us to carry out the
project at his esteemed organization during the training. We are also thankful to all
the other staff in the laboratory for their valuable inputs, support andcare.

JAYALAKSHMI D
JWALA G
 ABSTRACT

Our training period was about basic and primary training in biotechnology, which

was part of the "Separation of polysaccharides from silicon" project. A

polysaccharide is an important biomacromolecule consisting of several

monosaccharides linked by glycosidic bonds. The biological activity of

polysaccharides depends on the physio-chemical and structural properties. Some

basic methods, starting with a diatomaceous earth-specific algae medium, were

prepared and sterilized in an autoclave. The culture was then inoculated into both

solid and liquid media. After incubation, the culture was observed under a

microscope to check if it was alive and motility. A DNA sample was prepared and

electrophoresed, and the gel plates were viewed under a UV transilluminator.

Further hydrolysis was performed to obtain monosaccharide units from the algae.

Other techniques and processes such as size exclusion chromatography have also

been performed.
 TABLE OF CONTENTS

S.NO EXPERIMENTS PG.NO

1. Preparation of medium 1

2. Autoclaving and Inoculation 5

3. Slide preparation and observation 6

4. DNA Extraction 10

5. Gel Electrophoresis 12

6. Acid hydrolysis 14

7. Size Exclusion Chromatography 16

LIST OF FIGURES:

FIG.NO FIGURE PG.NO

1.1 &1.2 Preparation of media 1

1.3 BG 11 and PM media 2

3.1 Cyanobacteria 6

3.3 Diatom 8

3.2 & 3.4 Observation under microscope 6&8

6.1 Diatom after acid hydrolysis 14

7.1 Size exclusion Chromatography 16

1. PREPARATION OF MEDIUM
AIM:
To prepare the culture media

COMPOSITION:

INGREDIENTS gms/L

Potatoes, 200.0
infusion from

Dextrose 20.0

Agar 15.0

PROCEDURE:

• 100 ml of water was taken in a cleaned 250 ml conical flask.

• 3.9 g of Potato Dextrose Agar was weighed and added to the conical flask.
• The conical flask was shaken to dissolve the content.
• The conical flask was cotton plugged and wrapped.

1.1. ALGAL MEDIA PREPARATION:

Fig 1.1 Fig 1.2

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Two mediums were prepared:

1. BG-11 Medium: It is a universal media for the cultivation and maintenance of blue green
algae (cyanobacteria).
2. PM Medium: This media provides the necessary micro and macronutrients for the growth
of diatoms.
Fig 1.3

1.1.1 For BG Medium:

S.NO Ingredients Concentration(g/l)

BG-11 BG-11o
1. NaNO3 1.5 -
2. K2HPO4.3H2O 0.04 0.04
3. MgSO4.7H2O 0.075 0.075
4. CaCl2.2H2O 0.036 0.036
5. Citric acid 0.006 0.006
6. Ferric ammonium citrate 0.006 0.006
7. EDTA (disodium magnesium salt) 0.001 0.001
8. Na2CO3 0.02 0.02
9. Trace metal mix A5 + Co* 1 ml/l 1 ml/l
pH 7.4

2
 500 ml media

200 ml (500ml 100 ml (250 ml

200 ml ( 500 ml
conical flask) conical flask) conical flask) + 3g
agar.

Broth

3
1.1.2 For PM Medium:
Composition:

S.NO Nutrient Constituent Concentration

1 CaCl2.2H2O 0.37
2 K2HPO4 2.90
3 MgSO4.7H2O 3.70
4 Na2SiO3.9H2O 14.21
5 NaHCO3 3.15
6 NaNO3 56.70
7 Na2EDTA 2.180
8 FeCl3.6H2O 1.580
9 CuSO4.5H2O 0.005
10 MnCl2.4H2O 0.090
11 ZnSO4.7H2O 0.011
12 CoCl2.6H2O 0.005
13 Na2MoO4.2H2O 0.003
14 H3BO3 0.500

2 stock solutions each of 500 microliters were added to 350 ml and was made up to 500 ml. The
same procedure as mentioned in the above flowchart was repeated.

Autoclaving was carried out.

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 2. AUTOCLAVING AND INOCULATION:

AIM:
To sterilize the medium and inoculate

The cylinder was filled with sufficient water.

The materials to be sterilized were placed inside.

The lid was closed and the screws were tightened.

After the temperature reaches 100°C, the safety valve was closed.

Sterilization process was carried out at 121°C at 15 lbs for 15mins.

Inoculation was done by streaking technique and liquid medium was also inoculated.

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 3. SLIDE PREPARATION AND OBSERVATION:

AIM:

To observe the morphology of the given culture by preparing a slide.

MATERIALS REQUIRED:

• Clean glass slide

• Inoculation loop
• Microbial culture
• Tissue paper
• Immersion oil

PROCEDURE:

1. The slide was wiped using ethanol and dried.

2. A drop of the culture was placed on the centre of the slide.
3. Cover slip was placed and the slide was observed under the microscope.

OBSERVATION:

Cyanobacteria:

Fig 3.1 Fig 3.2

• Cyanobacteria exists in unicellular, colonial and filamentous forms.

• The culture observed under the microscope was filamentous form.
• Filamentous forms exhibit functional cell differentiation such as heterocyst (for nitrogen
fixation), Akinetes (resting stage cells), and hormogonia (reproductive, motile
filaments).
• Hormogonia are motile filaments produced by many filamentous cyanobacteria that
function in dispersal, phototaxis and establishment of nitrogen fixing symbioses.

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• Nostocean cyanobacteria typically produce gliding filaments termed hormogonia at a low
frequency as part of their life cycle.
• The production of hormogonia inducing factor makes cyanobacteria to differentiate and
convert them to hormogonia filaments. Hormogonia travels by gliding motility.
• Heterocyst function as the sites for N2 fixation under aerobic conditions. They are
formed in response to lack of fixed N2. Cell enlarges, granular inclusions decrease,
photosynthetic lammel reorients and wall is triple layered.
• Akinetes is ana enveloped, thick walled, non-motile dormant cell formed by filamentous
heterocyst forming cyanobacteria. Akinetes are large compared to vegetative cells

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3.2 Diatoms:

Fig 3.3 Fig 3.4

• It is a unicellular organism, occur either as solitary cells or in colonies.

• Its cell wall is made up of silica (hydrated silicon dioxide), called a frustule.
• It has two distinct types namely centric and pennate diatoms based on shape of the
frustule. Diatoms typically range in size from 2 to 200 micrometers, while some species
are bigger.
• Their four-cell membraned, yellowish-brown chloroplasts, which are the site of
photosynthesis, are typical of heterokonts and contain pigments like the carotenoid
fucoxanthin.
• Flagella are typically absent from individuals, but they are present in the male diatom
gametes and have the typical heterokont structure, including the hairs (mastigonemes)
found in other families.
• By observing under microscope, sliding movements of diatoms can be seen caused by a
structure called striae and raphe.
• Striae are line of pores and raphe is a slit-like structure that extrudes jelly and confers
motility to many pennate diatoms.

Types:

1. Pennate diatom:

Pennate diatoms have bilateral symmetry. Each of their valves has slit apertures along the raphes,
and their shells are normally extended in a line parallel to the raphes. Through streams of
cytoplasm that constantly travel along solid surfaces known as raphes, they produce cell
movement.

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2. Centric diatom:

Centric diatoms are radially symmetric. They consist of upper and lower valves referred to as
hypotheca and epitheca. The enormous vacuole at the Centre of the centric diatom's cell is
surrounded by a hollow space created by the cytoplasm, which is found along the inner surface
of the shell. A substance known as "cell sap," which resembles seawater but varies depending on
the precise ion concentration, fills this substantial central vacuole. The centric diatom's nucleus is
at the Centre of one of the valves and starts to travel toward the Centre of the cytoplasmic layer
before division is finished. This happens before the diatom starts to expand. Centric diatoms
have a variety of shapes and sizes, depending on from which axis the shell extends.

Reproduction:

These organisms reproduce asexually through a process called binary fission, in which the
diatom splits into two pieces and creates two "new" diatoms with the same genetic makeup. Each
newly formed organism obtains one of the two frustules—one larger and the other smaller—that
the parent organism possessed. This newly formed epitheca is utilized to create a second, smaller
frustule, the hypotheca. The diatom that got the bigger frustule grows to be the same size as its
parent, whereas the one that got the smaller frustule stays smaller than its parent. As a result, this
diatom population's average cell size shrinks. But it has been noted that some taxa are able to
divide without shrinking their cell size. However, to restore cell size to a population of diatoms
for species undergoing size reduction, sexual reproduction and auxospore spore formation must
occur.

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 4 DNA EXTRACTION:

AIM:

To isolate DNA from Dolichospermum spiroides .

MATERIALS REQUIRED:

• Lysis buffer
• Proteinase – K
• DNA Extraction Buffer
• Chloroform and Isoamylalcohol (24:1)
• Sodium acetate
• Ethanol

PROCEDURE:

1. The culture was centrifuged and cells were obtained as pellet.

2. The supernatant was removed, TE buffer was added and centrifuged at 4000 rpm for 10
minutes twice.

3. Centrifuged DNA + TE buffer+ 0.1g silica powder was taken and grinded using mortar and
pestle.

4. Lysis buffer was added to the DNA sample ,grinded and the sample was taken in two
eppendorf tubes.

5. To one of the eppendorf tubes, proteinase was added, labelled as CPB and then both the
eppendorf tubes were placed in the hot water bath at 60°C for 30 minutes.

6. The tubes were cooled to room temperature and then 700 microliters of
chloroform:isoamylalcohol was added in the ratio 24:1.

7. The tubes were centrifuged at 12500 rpm at 25°C for 5 minutes.

8. The aqueous layer was separated, 500 microliters of ice-cold ethanol were added thrice and
centrifuged at 11500 rpm at 4°C for 5 minutes.

9. The pellet was obtained, ethanol added, hand vortexed.

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 10. Ethanol was removed and the pellet was air dried.

11. 100 microliters of TE buffer was added to the pellet. The DNA sample was run in gel
electrophoresis.

RESULT:

The quality of the DNA isolated was analyzed based on the absorption ratio between 260 nm and
280 nm. The ratio between 1.8 and 2.0 shows thegood quality of the DNA, the ratio below 1.8
shows protein impurities and the ratio above 2.0 shows the presence of RNA impurities.

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 5. GEL ELECTROPHORESIS:

AIM:

To separate DNA fragments by running agarose gel electrophoresis.

MATERIALS REQUIRED:

• Agarose powder
• TAE and TBE buffer
• Gel loading dye
• Ethidium bromide

PROCEDURE:

1. TAE Buffer Preparation:

Tris acetate EDTA buffer was prepared by diluting 50X stock solution to 0.5x working solution.

2. Agarose Gel Preparation:

500 mg of agarose is weighed and dissolved in 50 ml TAE buffer solution in a 250 ml conical
flask and boiled to form a transparent solution then 30 microlitre of EtBr was pipetted and added
to the agarose solution. (Ethidium Bromide shows fluorescence when exposed to UV radiation).

3. Casting of gel and making well using comb:

The transparent agarose solution was poured into the casting tray which has comb such that it
does not form any air bubbles. The casting tray was kept aside for 15 to 20 minutes to solidify.
The tank buffer solution (0.5X TBE buffer) was added and then the comb was removed without
disturbing the wells.

4. Preparation and loading of the sample:

8 microliters of gel loading dye were pipetted out in a flat polished surface to which 24
microliters of DNA sample was pipetted and added. About 10 μl of DNA was loaded into the
sample wells. DNA has negative charge so it was loaded at the wells near the cathode so that
once when voltage is applied migration of DNA is towards the anode.

5. Electrophoresis:

12
 Electrodes are attached to the power supply battery and 100V is applied for about 45 minutes
until it covers 3/4th of the casting tray. Sample starts migrating in the gel as bands.

6. UV illumination to view DNA band:

The gel slabs were exposed to UV light, sample bands start fluorescing.

RESULT:

The given sample DNA was separated by running agarose gel electrophoresis.

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 6 ACID HYDROLYSIS:

AIM:

To perform acid hydrolysis step for diatom culture.

MATERIALS REQUIRED:

• Tarson tubes
• Acetic acid and Sulphuric acid (9:1)
• Ethanol
• Distilled water
• Conventional microscope

Fig 6.1

• 10ml of diatom culture was taken into the tarson tube and centrifuged at 1000 rpm for 3
minutes.
• Obtain the pellet by discarding the supernatant. To this pellet acetic acid and sulphuric acid
was added in the ratio of 9:1 and kept in water bath for 30 minutes at 80-100C.
• Acid was removed from the pellet after centrifuging at 1000 rpm for 3 minutes.
• To remove the debris from the pellet 2 ml of ethanol was added and then centrifuged at 1000
rpm for 2 minutes.
• Then the pellet was dispersed in 2ml distilled water and centrifuged for 2 minutes at 1000
rpm.
• The water was drained and the pellet was observed under conventional microscope.

14
RESULT:

The given algal cell wall was broken down and the complex sugar from cell wall is converted into
simple sugars.

15
 7 SIZE EXCLUSION CHROMATOGRAPHY:

Fig 7.1
AIM:

To elute the protein pigments.

MATERIALS REQUIRED:

• Sephadex G-2 5 and buffer

• glass wool
• external pump
• potassium phosphate buffer
• sample to be eluted(protein)

PROCEDURE:
• In order to prepare potassium phosphate buffer, required amount of potassium phosphate
monobasic and dibasic were calculated, weighed and added to the measuring cylinder.
• Formula: (Required molarity x molecular weight x Volume needed)/1000
• It was stirred using magnetic stirrer.
• pH 7 was attained by the addition of 10 M NaOH and checked using pH meter.
• The buffer was sterilized using autoclave.
• The column was packed using glass wool and resin which was already allowed to soak in
the sephadex buffer.
• The column was washed several times using the potassium phosphate buffer.
• Then the sample was loaded and then buffer.

16
RESULT:
Different colours of pigments (proteins) were eluted and stored.

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