Harshita sharma JNU , New Delhi Internship report SES.docx

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1.  INTERNSHIP
REPORT
 
Department of Biotechnology and Bioengineering 
School of Biosciences and Technology 
GALGOTIAS UNIVERSITY
Greater Noida, Uttar Pradesh 
Duration: 15th July 2025 – 19th August 2025 
Under the Guidance of 
Dr. Paulraj R. 
Professor 
Department of Environmental Science
Jawaharlal Nehru University
Submitted by 
Harshita Sharma
B. Tech Biotech (2
nd
year) 4
th
Sem. 
Enroll. No. 23071170041
Galgotias University 
RADIATION AND NANOTECHNOLOGY LABORATORY 
DEPARTMENT OF ENVIRONMENTAL SCIENCE 
JAWAHARLAL NEHRU UNIVERSITY
INDIA 
Dr. Paulraj R. Professor School of Environmental Science

2. CANDIDATE’S DECLARATION 
I hereby declare that the Summer Internship Report entitled: 
Cell Culture Techniques and Animal Handling
submitted to the Department of Biotechnology & Bioengineering, School of Biosciences and
Technology, Galgotias University, in partial fulfillment of the requirements for the award of the degree
of Bachelor of Technology (B. Tech) in Biotechnology, is an authentic record of my work carried out
during the period 15 July – 19 August, 2025 at Jawaharlal Nehru University. 
I further declare that this report has not been submitted by me or by anyone else for the award of any
other degree, diploma, or certificate. All information and data presented in this report are true to the best
of my knowledge, and due acknowledgement has been given wherever references have been made to
the work of others. 
Dated: Harshita Sharma
This is to certify that the above statement made by the candidate is correct to the best
of my knowledge. 
Date: 
(Signature) (Signature) 
Dr. Sacheen Kumar Dr. Gaurav Kumar 
Internship Coordinator Associate Professor and Head Department of Biotechnology and Bioengineering
Department of biotechnology and Bioengineering School of Biosciences and Technology School of
Biosciences and Technology Galgotias University, Greater Noida, U.P. Galgotias University, Greater
Noida, U.P. 
(Signature)  
Prof. (Dr.) Abhimanyu Kumar Jha 
Professor & Dean  
School of Biosciences and Technology  
Galgotias University, Greater Noida, U.P. 

3. Acknowledgement
I would like to express my sincere gratitude to Dr. Paulraj R. for providing me the opportunity to do this
internship and for their valuable guidance and support. I would also like to thanks Dr. Abhimanyu Kumar Jha
and Dr. Gaurav Kumar, Dean and HOD of school of Bioscience and Technology and school of Biological and
Biomedical Science department.
I also like to thanks all the PhD scholar of radiation and nanotechnology lab for their cooperation and special
thanks to Neha Jha and Pradeep Kumar (PhD scholar) as they helped us to learn techniques give exposure to
animal handling and cell culture.
I would also like to thank my co- partners Mr. Shashwat Sahu and Ms. Anandita Das also for cooperating
with me, supporting me.
Executive Summary
During the one-month internship, extensive exposure was gained to core laboratory practices, instrumentation, and
animal handling. The training included operation of key instruments such as the autoclave, laminar air flow, CO
₂
incubator, and spectrophotometer, along with proficiency in pH analysis and calibration. Practical experience was
obtained in animal house management, covering the handling of Wistar rats, their feeding systems, and grooming traits.
Technical skills acquired encompassed cell counting, gel electrophoresis (Native PAGE and SDS-PAGE), estimation of
protein concentration, lipid concentration, and lactate dehydrogenase activity, as well as cell culture techniques
including subculturing and cell splitting. Additional exposure was provided to laboratory fumigation procedures and
preparation of agarose plates, thereby enhancing both theoretical understanding and hands-on competence in modern
biological research methodologies.
Introduction
Biotechnology is a multidisciplinary field that integrates biology, chemistry, physics, engineering, and
computational sciences to develop products and processes for the benefit of society. It involves the use of living

4. organisms, cells, and biological systems to produce useful goods, enhance crop yields, develop new medicines,
and improve environmental sustainability
The purpose of internship is to learn and gain experience from wet lab, improve our analytical power, hands on
experience on instrumentation.
Organization Profile
Jawaharlal Nehru University (JNU), established in 1969 by an Act of Parliament, is a premier public research
university located in Munirka, New Delhi. It is renowned for its interdisciplinary academic structure, with
schools and centers offering programs in social sciences, life sciences, environmental sciences, languages, and
applied sciences. JNU is recognized for advanced research facilities such as the University Library, Advanced
Instrumentation search Facility (AIRF), and University Science Instrumentation Centre (USIC).
The university’s vision is to be a global leader in higher education, research, and social responsibility. Its
mission is to provide high-quality education and innovative research, fostering values of national integration,
social justice, secularism, and scientific temper.
Internship Objectives
The objective of this internship was to gain practical exposure to fundamental laboratory techniques and
biological research methodologies. The training emphasized the development of technical proficiency through
pH measurement, observation and anatomical study of laboratory animals, understanding of sterilization
principles using autoclave, and quantitative estimation of protein concentration using the Bradford assay
with BSA standards. This internship aimed to strengthen analytical skills, enhance experimental accuracy, and
provide hands-on experience essential for future research and professional development in the field of
biotechnology.
Description of Work Done
The practical sessions included pH analysis and calibration of the pH meter, systematic observation of
laboratory animals and anatomical dissection of mice, study of the principles and operational procedure
of the autoclave for sterilization, and quantitative estimation of protein concentration using bovine serum
albumin (BSA) by the Bradford assay through standard curve analysis.

5. EXPERIMENTS PERFORMED
1.Protein Estimation by Bradford Assay
Methodology
Make a series of BSA standard solutions of known concentrations (e.g., 0, 2, 4, 6, 8, 10 µg/mL).
Add a fixed volume (usually 1 mL) of Bradford reagent to each standard.
Incubate at room temperature for ~5-10 minutes.
Measure absorbance at 595 nm.
Treat the unknown sample in the same way as standards.
Measure its absorbance at 595 nm
Plot a standard curve of absorbance vs. concentration.
Use the standard curve to find the concentration and absorbance of the sample.
Figure 1: Standard curve of Bovine Serum Albumin (BSA) using the Bradford assay. The graph shows a
direct proportionality between protein concentration and absorbance at 595 nm, with the regression
equation enabling determination of unknown protein concentrations.
2.Gel Electrophoresis (NATIVE PAGE):
(PAGE: Polyacrylamide Gel Electrophoresis)
Methodology
Gel Preparation (add gel composition table).
Cast the gel: Stacking gel (4% acrylamide) and a resolving/separating gel (12% acrylamide).

6. Allow it to polymerize. (time)
Mix protein samples with loading buffer.
Load 20ul of the sample in each well. (Volume adjusted according to total protein present).
Running of the Gel.
Fill tank with running buffer.
Run the gel at 80V (stacking) then increase to ~120V (resolving).
Staining:
After run completion, stain the gel with Coomassie Blue
Destain to remove background.
Analysis:
Compare bands to the molecular weight marker to estimate protein sizes.
3.Fumigation of Cell Culture Room
Using KMnO (potassium permanganate) and formalin is the classic method for fumigation of Cell culture
₄
room often used to sterilize biosafety cabinets, clean rooms, or incubators.
Materials:
Formalin (~37% formaldehyde)
KMnO crystals
₄
Heat-resistant container (ceramic or glass bowl)
Sealing tape.
Procedure:
Turn off equipment.
Seal the area or cabinet (plastic wrap/tape).
Place the KMnO in the container.
₄
Quickly add formalin to the KMnO .
₄
Immediately exit and seal the area—gas will begin to evolve rapidly.
Let sit overnight (usually ~6–12 hours).
Neutralize with Liquid ammonia.
Ventilate thoroughly before re-entering. Use exhaust fans or open windows if applicable.

7. Figure 2: Air quality test before and after fumigation. Plates on the right side (before fumigation) show
dense microbial growth, whereas plates on the left side (after fumigation) exhibit negligible colonies,
confirming the effectiveness of fumigation in reducing airborne contamination.
4.Revival of cells
To revive B16F1 (mouse melanoma) and HaCat cells (human skin keratinocyte) cells from cryostorage (liquid
nitrogen or -80°C)
Prepare Media and Equipment:
Warm complete growth media to 37°C.
Ensure biosafety cabinet is cleaned and ready.
Retrieve the cryovials from liquid nitrogen.
Thaw at 37
0
in water bath.
Sterilise the cryovial with 70% ethanol.
Transfer the cell suspension in 15ml falcon tube and add 10ml complete media to dilute the DMSO.
Centrifuge at 2500 rpm for 5minutes.
Carefully discard the supernatant (contains DMSO).
Resuspend and pellet in 5ml complete media.
Transfer to a flask (T25 or T75 depending on cell density needs).
Place in 37°C incubator with 5% CO .
₂

8. Figure 3: Cell culture process showing observation of cultured cells using an inverted microscope (A) and
handling of Petri dishes containing culture medium under aseptic conditions (B).
5.Flow Cytometry.
Introduction
Flow cytometry is a laser-based technique used to analyze and quantify cells or particles as they flow in a fluid
stream through a beam of light. The technology measures multiple parameters simultaneously, including cell
size, granularity, and fluorescence intensity, which can be used to identify and characterize specific cell
populations.
The core components of a flow cytometer include:
1.Fluidics System: Suspends and transports cells in a stream to the laser intercept.
2.Optics System: Consists of lasers and detectors to illuminate cells and capture scattered and emitted
light.
3.Electronics System: Converts light signals into digital data for analysis.
4.Software: Analysis and visualizes the data, often in the form of scatterplots or histograms.
During the process, cells are typically labelled with fluorescent dyes or antibodies conjugated to fluorochromes,
which bind to specific cell surface markers or intracellular molecules. As cells pass through the laser, they
scatter light and emit fluorescence, which is detected and quantified.
A B

9. .
6.Detection of ROS by DCFDA of HaCaT cells through flow cytometry
Reagent preparation
DCFH-DA stock (10 mM): dissolve DCFH-DA in DMSO to 10 mM (e.g., for MW ≈ 439 g/mol;
calculate mass from vendor). Aliquot (10–20 µL) and store at −20°C, protected from light. Avoid >3
freeze–thaw cycles.
Working staining solution (10 µM): dilute stock in serum-free DMEM or PBS immediately before use.
For 1 mL final: add 1 µL of 10 mM stock to 999 µL medium (final DMSO 0.1%).
Positive control (H O )
₂ ₂
: prepare fresh; suggested starting concentration 200 µM in growth medium
(optimize for your cells).
Cell culture & treatments
Seed HaCaT cells so they reach ~60–80% confluence on day of experiment.
Apply experimental treatments (drugs, nanoparticles, UV, etc.) in culture plates according to your plan.
Include wells for positive control (treat a well with 200 µM H O for 30 min prior to processing) and
₂ ₂
controls above.
Harvesting cells
1. Remove media; gently wash once with PBS.
2.Add 0.05% trypsin-EDTA and incubate at 37°C until detached (monitor by microscope).
3. Neutralize with complete medium, pipette gently to single-cell suspension.

10. 4. Transfer cells to 15 mL tube; centrifuge 2500 rpm for 5 minutes at room temperature. Discard
supernatant.
Resuspension & staining
1. Resuspend pellet in 1 mL serum-free DMEM or PBS (per sample).
2. Prepare DCFH-DA working: make fresh 10 µM in serum-free medium or PBS.
3. Add the working DCFD-DA solution to each sample to achieve 10 µM final (for 1 mL suspension add 1 µL
of 10 mM stock). Mix gently. Protect from light.
4. Incubate 20–30 min at 37°C .
Post-staining washes
1. After incubation, centrifuge 2500rpm for 5 minutes, remove supernatant.
2. Wash cells twice with PBS.
3. Resuspend cells in 500 µL ice-cold PBS for acquisition,
4. Acquire the data.
Acquisition
1. Acquire immediately on flow cytometer using 488 nm laser and FITC channel (530/30 or 525/50).
2. Collect 20,000–50,000 singlet events per sample. Use FSC/SSC to gate cells, FSC-A vs FSC-H (or SSC) to
gate signal
Fluorescence microscopy image showing green fluorescent stained cells, indicating positive signal for ROS
generation.
7.Detection of mitochondria membrane potential of HaCaT cells by Rhodamine 123 using
flow cytometry:
Reagent preparation
Stock: dissolve Rh123 in DMSO to 1–10 mM. Aliquot, store −20°C protected from light.
Working: prepare fresh in serum-free medium; 5 µM final recommended starting point.
Staining:
1.Treat cells as required. CCCP control add FCCP (1–5 µM) to the CCCP well 10–30 min before
harvesting.
2.Detach cells using trypsin.

11. 3.Centrifuge at 2500rpm for 5 minutes, discard supernatant. Resuspend in 1 mL serum-free DMEM or
PBS.
4.Add Rh123 working solution to 2 µM final. Incubate 20–30 min at 37°C, protect from light.
5.Wash once with ice-cold PBS to remove extracellular dye.
6.Resuspend in 300–500 µL PBS and Acquire quickly (within 1 hr).
Acquisition
Excitation typically 488 nm; emission collected ~530 nm (FITC channel). CCCP sample should show
major loss of MMP.
Analysis
Use histogram or density plot to represent the data.
8.Cell subculturing
1. Preparation
Warm trypsin, PBS, and media to 37°C.
Sterilize workspace (biosafety cabinet) with 70% ethanol.
2. Examine Cells
Use a microscope to check:
oConfluency
oMorphology
3. Remove Spent Medium
Aspirate old media using a vacuum
4. Wash with PBS
Add PBS to wash away media.
5. Add trypsin and incubate for 15 minutes to detach the cells the.
6. Add complete media and centrifuge the cells and discard supernatant.
7. Resuspend the cells and count the cells using hemacytometer.
8. Seed 10
4
cells in each 60mm cell culture plate.

12. 9.MDA Estimation
MDA Estimation: Malondialdehyde (MDA) is a marker of lipid peroxidation and oxidative stress. It is
commonly measured in biological samples (e.g., tissues, blood, cultured cells) using the Thio barbituric Acid
Reactive Substances (TBARS) assay.
Principle of the TBARS Assay:
MDA reacts with Thio barbituric Acid (TBA) under high temperature and acidic conditions to form a
pink chromogen.
This MDA-TBA complex absorbs maximally at 532–535 nm, which can be measured using a
spectrophotometer or microplate reader.
Homogenize tissue in cold PBS or KCl buffer (e.g., 10% w/v).
Procedure:
Label Blank, Standards, Samples.
To each tube add:
Sample or standard: 200 µL
TCA (10%): 200 µL (precipitates protein)
Vortex, stand 10 min on ice; spin 10,000×g, 10 min; collect supernatant 200 µL.
Add to the supernatant:
TBA (0.4%): 200 µL
HCl (0.25 N): 400 µL (final mix 1.0 mL; acidic)
Cap tightly and heat 95–100 °C for 15 min (water bath).
Rapidly cool on ice 5 min.
Read at 532 nm.

13. 10.Animal Handling
Figure 4: Handling of laboratory mice demonstrating standard practices of careful lifting, gentle
restraint, and use of protective gloves to ensure animal welfare and safe experimental preparation.
Preparation Before Handling
PPE: Wear gloves, lab coat, and other necessary personal protective equipment.
Environment: Ensure the room is quiet and temperature-controlled (20–24°C).
Equipment Check: Prepare clean cages, handling tools (if needed), and recording sheets or software.
Approach: Calmly approach the cage to avoid startling the rats.
Picking Up: Use base of the tail and support under the body (not just by the tail).
Recording Observations
Use a daily log sheet or digital spreadsheet.
Include: Date, Time, Animal ID, Observer’s name, and all observation categories.
11.Vaginal Swab

14. A vaginal swab experiment is usually done in animal studies (like rats, mice) or in humans (clinical context)
Collect samples of vaginal secretions for microbiological, cytological, or molecular studies.
Monitor the estrous cycle in rodents.
Detect infections, pH changes, or microbiome composition.
General Experimental Steps:
1.Preparation
oSterile cotton swabs or micropipette tips are used.
oAnimals are gently restrained.
2.Collection
oA sterile swab is moistened with saline.
oInsert gently into the vaginal opening (2–3 mm in mice, deeper in rats).
oRotate carefully to collect epithelial cells and secretions.
3.Sample Processing
oThe swab is rolled on a clean glass slide.
oFixed and stained with crystal violet.
oObserved under a microscope.
4.Analysis
For estrous cycle determination: look for the proportion of epithelial cells, leukocytes, and cornified cells.
Observation:
Observed nucleated epithelium proestrus stage of estrous cycle in rats stained through crystal violet.

15. Figure: Microscopic image (20X) showing identified Proestrus stage with nucleated epithelial cells
stained with crystal violet.
Skills & Knowledge Gained
Lab Skills
Exposure to wet lab practices and experimental workflows
Handling and operation of laboratory instrumentation
Animal handling, care, and management practices
Sterilization techniques using autoclave
Working under laminar air flow for aseptic procedures
Operation of incubators for controlled culture conditions
Performance of standard laboratory techniques and assays
Challenges Faced & Solutions
To prevent or control contamination, cell culture laboratories are fumigated at regular intervals.
Contamination may also arise from improper pipette usage, such as reusing tips without replacement.
Additionally, careful preparation of gel plates with proper sealing is essential to avoid leakage and ensure
reliable results.

16. Conclusion
The internship provided comprehensive exposure to wet lab practices, instrumentation, and standard
experimental techniques. It enhanced understanding of laboratory workflows, safety precautions, and the
principles underlying essential equipment. Hands-on experience in assays and standard curve analysis improved
technical competence, while guidance from Ph.D. scholars offered academic direction and career insights.
Overall, the internship proved highly beneficial in strengthening practical skills, shaping research interests, and
preparing for future opportunities in the field of biotechnology.