SUMMER INTERNSHIP REPORT

FISH ENDOCRINOLOGY LAB OF PROF. NEETA SEHGAL

By- Bhor Srivastava, 2nd year graduate, Kalindi College, University Of Delhi.

Span of internship-

From 12th June'23 till 21st July'23

TABLE OF CONTENTS:

• Acknowledgement
• Work Performed
• Visit to Animal House
• Techniques Learned
• Chemical Preparation

ACKNOWLEDGEMENT-
First I wish to express my sincere gratitude to Prof. Neeta Sehgal for providing me an opportunity to do
my internship in her research lab based on the study of Fish Endocrinology located in the Zoology
Department of Delhi University. For me, it was a unique experience to study about various instruments
in the Laboratory & learning different techniques. This internship period was a great chance of learning
and professional development.

Next I express my deepest sense of gratitude to Dr. K. Vandana Rani, Assistant Professor, Zoology Dept. of
Kalindi College for providing us an opportunity to work in Fish Endocrinology Lab of Prof. Neeta Sehgal.

I also express my deepest thanks to the PHD scholars- Ila Ma’am, Rishikesh Sir, Ritu Ma’am, Uma ma’am,
& Nitika Ma’am, doing their research under the guidance of Prof. Neeta Ma’am, for giving necessary
advice and guidance. They arranged all facilities & made us familiar with all the machinery to make our
internship programme more meaningful. I thank them for their valuable time & all the teachings.

I sincerely thank Rishikesh Sir & Nitika Ma’am, for their careful and precious guidance which was
extremely valuable for my learning, both theoretically and practically, For teaching the techniques I
aspired to learn & showing us how to correctly use the equipments. I also wish to express my gratitude
to the staff member, Tekchand Sir, who rendered his help during my internship period.

Finally, I would like to extend my deep gratitude towards my family and my friends for their support in
carrying out this work successfully.
WORK PERFORMED-
In the beginning week, we were introduced with the main research topic of the fish endocrinology lab-
on Channa punctatus (Soli) & Heteropneustes fossilis (Catfish or Singhi). The machineries & equipments
present in the laboratory were made familiar with us & we’re assigned the task of learning their
principles & basic working. We were also taught the good laboratory practices which includes use of
different types of gloves for different work purposes, use of various kind of micropipettes & their
respective tips, tagging all the equipments (like Duran bottles, Eppendorf tube, etc.) used during an
experiment or chemical/ buffers synthesis & many similar, necessary practices.

The instruments we studied about were: Trans-illuminator, Weighing Machines, Northern Blotting &
Southern Blotting, Incubator/ Oven, Centrifuge, pH Meter, PCR, Spectrophotometer, Microbial Shaker,
CO2 Incubator, HPLC- Chromatography Machine, Microscopes of various power. We’re taught about the
use of different types of micropipettes for measuring what particular measures, & their respective tips;
spotting & recognizing various eppendorf & falcons.

CENTRIFUGE: Principle- A centrifuge stands on the grounds of sedimentation principle. The substances,
according to their density/ mass are separated, under the influence of gravitational force. When they are
spun at a high rpm, the particles with high density are settled at the bottom while the lighter particles
are at the top. We do phase separation & pallet aggregation by centrifugation only.

We learned about different kinds of centrifuge machines & rotors plus their fixing inside the centrifuge
machine. We were also taught how to set the centrifuge, for instance- setting the time, temperature,
soft start- soft stop and normal start-stop, etc.

NORTHERN- SOUTHERN BLOTTING: Principle- Northern and Southern blot analysis methods are based on
the movement of DNA, RNA fragments which are separated in gel according to their length & weight,
hereby allowing identification & detection of DNA or RNA.

Agarose Gel Electrophoresis was done in these blots. The DNA in the wells of the Agarose gel was
dispersed according to its weight & length from anode (-ve) to cathode (+ve).

UV TRANS-ILLUMINATOR: Principle- UV trans-illuminator works by emitting high levels of UV radiation

through the viewing surface.

We observed the Agarose gel under the low & high intensity to check the DNA & RNA bands and
compared them with the corresponding ladder.

pH METER: Principle- It measure the voltage between two electrodes, dipped in the solution and display
the result in the form of pH value.
Since the solution needed to be either on a specific pH to get dissolved completely or to maintain its
optimum pH for working effectively, for this, checking pH time to time was a vital step.

WEIGHING MACHINE: For accurate measurement of compounds for making different buffers &
chemicals, we used weighing machine.

We weighed NaOH pallets to make 1M NaOH solution, Agarose power for Agarose gel, NaCl, Tris-base,
EDTA, SDS, Soil Samples, etc.

MICROBIAL SHAKER: Principle- It provides an artificial environment. We can manually set the
temperature (temperature range: 4°C to 60°C) & the circular shaking motion (max rotation of 400 RPM),
according to the experimental needs for the samples/ experiments.

We used microbial shaker to incubate our DNA samples at 37°C at 200rpm during DNA isolation
procedure.

PCR (POLYMERASE CHAIN REACTION) MACHINE: Principle- It synthesize multiple new strand copies of
DNA fragments from DNA complementary to the template strand.

We observed PCR technique, the chemicals used in the procedure & the precautions we need to take
while following the protocol.

CO2 INCUBATOR: Principle- A compressor and coolant are used to control the humidity inside the
incubator. CO2 is added into the incubator at a pre-set amount which helps maintain optimal conditions
for cell development.

It is primarily used in making cultures.

SPECTROPHOTOMETER: Principle- This method is used to measure how much light absorbance does a
solution have. The basic being- each & every chemical have a certain range of wavelength, & it absorbs
or transmits light according to its wavelength. In Spectrophotometer, we see how much is the intensity
of the sample (given chemical) by passing light through it.

VISIT TO ANIMAL HOUSE-

For learning Animal House Maintenance, we went to see the model organisms of this lab: Channa
punctatus (Soli) & Heteropneustes fossilis (Catfish).
In a room, Heteropneustes fossilis & Channa punctatus kept in different tanks. We were taught
siphoning- the technique by which the faecal contaminated water of the fishes was drained out (about
90%) using a rubber pipe & fresh, suitable water for the fishes was poured into the tanks, mixed with the
old water which was already there in the tank for not providing fishes with a whole new environment &
preventing stress to the fishes. There was also setup for the timely turning on & off the lights & air
conditioner for convenience of the experiments performed. The optimal temperature set in the room for
the fishes was 25°C.

We were also made familiar with the water tank which had motor setup to keep the water flowing in
order to make it de-chlorinated, the impurities to settle down & avoid insects from laying eggs. This
water was used to refill the tanks of fishes in room experimental room after siphoning. We also learned
that tanks covered with black material for the experiments which needs entirely dark environment.

There was a separate room for performing all the surgical related work.

TECHNIQUES LEARNED-

AGAROSE GEL ELECTROPHORESIS:

We were taught to make 0.8% & 1% Agarose Gel for viewing DNA bands; & 1.2 % Agarose Gel for RNA
bands; & Running Buffer.

The whole procedure in a nutshell for making 0.8% Agarose Gel:- Agarose power was weighed and
added in a Duran bottle along with 50× TAE + MQ water which was microwaved till the solution was
clear. When the Duran got to a touchable temperature, a very small quantity of Ethidium Bromide was
added to it with extreme care. Meanwhile, we already cleaned the gel plate & gel comb with 70%
alcohol and set it properly. The solution in the Duran was then emptied in the pre-set gel plate & left fir
30 minutes for the gel to get fixed. For the time being, we prepared Running Buffer so that the gel can be
submerged and for that- 50X TAE buffer measured in a measuring cylinder & the volume make up was
done by filling the rest of the cylinder with MQ till the 100ml reading, it was poured in the Northern/
Southern Blotting machine & rest of the MQ was also added to make it 700ml. The buffer was then set
on 60-70v for pre-run.

After the gel is set, the gel comb is removed & the gel plate is dipped in the Blotting machine till the level
that the gel is submerged, the wells are then loaded with ladder & samples with the help of
micropipettes. The samples are mixed with the tracking dye after which they were loaded- 1st well-
ladder, 2nd & 3rd wells with sample 1 & 2 respectively & the rest of the wells were blank. The voltage was
then set at 70v. The voltage supply was stopped when the DNA travelled 80% of the distance.

The gel was then taken out of the gel plate & viewed under U.V. Trans-illuminator.
DNA ISOLATION:

PHENOL CHLOROFORM ISOAMYL (PCI) DNA EXTRACTION METHOD, to isolate the DNA from the anal fin
tissue of Channa punctatus.

Firstly we prepared 10ml lysis buffer (since we only use it freshly prepared) by adding- 1M Tris-HCl +
0.5M EDTA + 0.5M NaCl + 10% SDS + rest MQ water to make up 10ml volume.

Procedure for DNA isolation-

Add lysis buffer + lysozyme (1mg/ml) to the samples. Put under Microbial Shaker at 37°C, 200rpm for
30min. Add proteinase K to each tube. Incubate overnight in water bath at 55°C. Next day, vortex the
tubes for 15 seconds & centrifuge for 1 min at 4000xg & transfer the liquid to new eppendorf. Add PCI to
it. Vortex for 5 seconds then Centrifuge at 4°C for 10min at 12000xg. Aqueous phase was transferred to
new eppendorf. CI added, then centrifuged again at 4°C for 10min at 12000xg. Again the aqueous phase
was transferred to a new eppendorf & mix it with (1/10th volume) 5M NaCl & 2 volume of 100% ethanol.
Incubate overnight. The next day, Centrifuge at 20,000xg for 20 min at 4°C. Wash pallet with 70% ethanol
& dry it. (Decant the supernatant & suspend the pallet in 70% ethanol). Centrifuge at 20,000xg for 20
min (twice & Let the pallet dry out). Add RNase + 1X TE to each tube, dissolve the pallet & give the heat
shock treatment to them. Check DNA bands in Agarose Get Electrophoresis. DNA concentration was also
checked in Nano drop reader.

RESULTS-

RNA ISOLATION:

We took 2 eppendorf with the sample of Heteropneustes fossilis’ liver in one & ovary in another. Both
were poured with the fixative- TRI reagent & grinded with a homogenising stick & a homogenizer. TRI
Reagent was added again & stored in 4°C for a while & then shifted to ice bucket for 5 minutes after
which it was thawed & shook. The samples were then rotated in rotospin for 5 minutes after which
200μL chloroform was added to each eppendorf. The tubes were again rotated in rotospin for 5 minutes
& then centrifuged at 4°C for 30 minutes at 12,000rpm. The liquid phase was shifted to a new
eppendorf where isopropanol was added & the tubes were put in 0°C for 30 minutes. Then
centrifugation was again carried out at same settings as before & the samples were stored in -20°C
overnight & centrifuged the next day. RNA washing was done by 75% alcohol, thrice, with centrifugation
at 12,000rpm, 4°C at 5 minutes intervals. Then it was incubated at 50-60°C till the ethanol evaporates
completely. It was then dissolved in depC.

1.2%, 20ml Agarose Gel was prepared & electrophoresis was carried out in Northern- Southern Blotting
Machine & the gel was after viewed under UV trans-illuminator.

(The RESULTS cannot be shown as we ended the above experiment on 21st July’23.)

PRIMER DESIGNING:

Primers are needed while doing PCR as it requires template over which the single strand of DNA gets
attached and replicated further then.

We got the accession number and through a website, i.e., NCBI, we collected the sequence. After that
some other compatible sequences were found. They all were compared and their conserved regions
were identified. Then through hit and trial process, some regions of suitable characteristics were tested,
whether they can become primer- by checking the self complementary and other things. After that the
reverse and forward primers were checked as there should be no possibilities of primer dimers. The
protein sequencing of the desired primer was checked and then we are good to go.

RESULTS- We were given the accession number of Anabas and have to make a primer for Channa
punctatus, tho the self complementary of both the primers wasn’t as needful, but we got the whole
process precisely.

The pictures of results are in the PC of the lab only.

BACTERIAL STREAKING:

During the cell culture, it’s a major process in which the bacteria is taken from the culture by heating the
loop stick. It is then streaked over the culture medium in Petri dish. It’s sealed properly after that. This
whole process is conducted inside the Laminar Hood, with continuous aeration and all the equipment
before use are put under UV light.

RESULTS- We witnessed the whole procedure of streaking of bacterial culture on LB Agar medium and
learned some of the differences between the bacterial culture and cell culture.
FLUOROENZYMATIC ASSAY OF SOIL: Fluorometric analysis depends on the production of a
fluorescent compound as a result of enzyme activity between a substrate and enzyme. The rate
of production of the fluorescent compound is related to both the enzyme concentration and
substrate concentration.

We learned how to make suspension of soil; about the fluorescent molecule plus it’s overall role.
We saw the microplate reader and the protocol of it measuring the fluorescence of samples in
microplate by sending light through it. The readings were noted and used in further calculations
of the experiment.

WE ALSO WITNESSED FISH SURGERY & PCR.

CHEMICAL PREPARATION-
• Lysis Buffer
• Tris-HCl Buffer
• Running Buffer
• Ethylenediamine tetraacetic acid (EDTA)
• Sodium Dodecyl Sulphate (SDS)
• Sodium Chloride
• Tris EDTA (TE) Buffer
• Sodium Hydroxide
• Tris Acetate EDTA (TAE)- 50X Buffer
• Agarose Gel- 0.8%, 1%, 1.2%
• Sodium Acetate