The Final Report for Field Training

Second Semester

Academic Year 2021-2022

Reported By

Student Name Unified Code

`fatma amr badway 19231084

Under the Supervision of

Names of Training Coordinators:
Dr. Mohamed Elawady

Report Submitted Date

Faculty of Agriculture – Cairo University

 Content Page No.#

1. Introduction………….. 3
2. Objectives of the Training Program…………….. 4
3. Identifying Training Entities/Locations of the Trainings….. 5
4. Training Activities that were Implemented…………… 6-23
5. Conclusion and Recommendations…………….. 24
 1-Report Introduction

This training learning us to gain experience in practical fields to qualify us

to work in public or private companies by mimicking the demanded tasks
needed to be achieved in each field and doing special experiments in each
laboratory to Enhance the general skills and gain the ability to solve
problems.
Training is divided to five main labs

1. Biotechnology (Bio,GMO)
2. Microbiology
3. Embryology
4. Chemistry
5. TEM
 2- Objectivities of the Training Program

 Acquire the knowledge and information about the practical

work of biotechnological techniques.
 Understand the topics related with the biotechnology like
bioinformatics, tissue culture, genome editing and protein
interaction.
 Gain the skills needed by the work market and work on
improving it, this skills like leadership, presentation skills,
report writing and stress management.
 Get the experience in working with my own hand and do
some analysis and tests like DNA extraction and preparation of
PCR reaction
.  Understand the criteria by which the devices in different labs
inside CURP work.
3- Identifying Entities/Location of the Training
CURP (Inside faculty of agriculture)
Duration: 2 weeks (practical)
First week:
1. Biotechnology (Bio) lab (1 day) (2/4)
2 .Biotechnology (GMO) lab (1 day) (3/4)
3. Microbiology lab (2 days) (4/4,5/4)
4. Embryology lab (1 days) (6/4)
Second week:
1. Embryology lab (1 days) (9/4)
2. Chemistry lab (2 day) (10/4,11/4)
3. TEM lab (2 days) (12/4,13/4)

4-Training Activities that was Implemented

Day 1 2/4/23023 Biotechnology (Bio)

Types of extraction
Manual and kit
CTAB DNA Extraction Protocol
1. Prepare CTAB Buffer (recipe attached) Prior to starting extraction add
polyvinylpyrrolidone and B-mercaptoethanol.
2. Transfer 50mg of frozen tissue in an eppendorf tube.
3. Grind tissue in liquid nitrogen with blue pestles, keeping tissue frozen the entire
time.
4. Add 500 ul of CTAB buffer and mix the tubes. Incubate at 55 for at 3 h, mixing
once after 30 minutes.
5. After incubating for 3 h add 1.5 ul RNase A. Incubate at 37°C for 15 minutes 6.
6. Remove samples from water bath and add 500 ul of chloroform and mix by
gently shaking tubes.
7. Centrifuge for 7 minutes at 16000 xg.
8. Transfer the aqueous phase into the new labeled tube. Be careful to avoid
transferring any chloroform.
9. Add 0.1 volumes cold 7.5 M ammonium acetate.
10. Add 0.6 volumes of cold isopropanolMix by inverting tubes 20-30 times.
11. Incubate on ice for 30-40 minutes.
12. Centrifuge for 3 minutes at 16000 xg.
13. Discard supernatant and add 700 ul. 70% EtOHinvert tubes 5-10 times.
14. Centrifuge for 1 minute at 16000 g.
15. Discard supernatant.
16. Hydrate pellets with 50 ul 11.
Vortex Centrifuge

Sheaker
Day 2 3/4/2023 Biotechnology (GMO)

Polymerase chain reaction (PCR)

Involves using short synthetic DNA fragments called primers to select a segment of
the genome to be amplified, and then multiple rounds of DNA synthesis to amplify
that segment.
PCR is based on three simple steps required for any DNA synthesis reaction:
1. Denaturation of the template into single strands
2. Annealing of primers to each original strand for new strand
3. Synthesis Extension of the new DNA strands from the primers.
A real-time polymerase chain reaction
(Real-time PCR or qPCR when used quantitatively) is a laboratory technique of
molecular biology based on the polymerase chain reaction (PCR). It monitors the
amplification of a targeted DNA molecule during the PCR (i.e., in real time), not at its
end, as in conventional PCR.
Microarray
Is a general laboratory approach that involves binding an array of thousands to
millions of known nucleic acid fragments to a solid surface, referred to as a “chip?”
The chip is then bathed with DNA or RNA isolated from a study sample (such as cells
or tissue).
Gel electrophoresis
Is a laboratory method used to separate mixtures of DNA, RNA, or proteins according
to molecular size? In gel electrophoresis, the molecules to be separated are pushed by
an electrical field through a gel that contains small pores.

R.TpCR PCR

Incubator Nanodrop
Day 3-4 4/4/2023 Microbiology lab
5/4/2023

required skill gained from the training day

-Types and preparation of media.
-PCR component and how to work.
Training activities that were implemented in the field/Lab
1-Microbiology lab should have:
1- Preparation media room: In which we prepare the specific media for the
Specific microorganism that we detect.
-media that contain milk or high sugar level is not autoclaved but
sterilized in oven (180C \ 2h).
2- Sterilization room: In which we sterilize the prepared media.
3- Inoculation room: In which we inoculate the sample into the media.
4- Incubation room: In which we incubate the petri dishes containing the
inoculated media.

Food microbiology:
Food microbiology is the study of the microorganisms that inhibit, create, or
contaminate food. This includes the study of microorganisms causing food
spoilage; pathogens that may cause disease (especially if food is improperly
cooked or stored); microbes used to produce fermented foods such as
cheese, yogurt, bread, beer, and wine; and microbes with other useful roles,
such as producing probiotics.
2-Types of media:

1- Solid media.
2- Semi solid media.
3- Broth media.
Preparing the media in the lab or buying in bottels. Media used for
growing of bacteria and fungi to detect the effects of it.

3-PCR Component:
1-Water.
2-Nucleotides (dNTPs).
3-Primers (forward and reverse primers which are specific for the desired
gene) 4-Taq polymerase enzyme (which is resistant to the high temperature
used in the PCR process(.
5-Mgcl2 (which is essential cofactors for the polymerase enzyme to enhance
the amplification rate(.
6-Buffer (to control the optimum PH(.
7-The last thing we add to the PCR tube is the DNA sample to prevent its
damage or contamination.
-Instead of add each component one by one we can make master mix which is
a mixture contain all PCR components except the Primers and the DNA
samples.
 Now we are ready to run the PCR device.
The PCR process has three stages which are:
1-Denaturation:In this stage the temperature is about 94C which cause the
separation of the double strands of DNA and make it single strands.
2- Annealing :In this stage the temperature is 54-56, which allows the primers
to bind to its specific sites.
3- Extension :The temperature now is 74 which is optimum for the polymerase
enzyme to add the nucleotides one by one next to the primers.
Day 5-6 6/4/2023 Embryology lab
9/4/2023

The required skill gained from the training day

-Mendel’s Laws of Inheritance.
-Discovery of DNA .
-Stages of IVF and the media used in it.
-CBC ,Spectophotometer devices.
-ELISA
1-Mendel’s Laws of Inheritance:
1-Law of Dominance:This is also called Mendel’s first law of inheritance. According to
the law of dominance, hybrid offspring will only inherit the dominant trait in the
phenotype. The alleles that are suppressed are called the recessive traits while the
alleles that determine the trait are known as the dominant traits.
2-Law of Segregation:The law of segregation states that during the production of
gametes, two copies of each hereditary factor segregate so that offspring acquire one
factor from each parent. In other words, allele (alternative form of the gene) pairs
segregate during the formation of gamete and re-unite randomly during fertilization.
This is also known as Mendel’s third law of inheritance.
3-Law of Independent Assortment:Also known as Mendel’s second law of
inheritance, the law of independent assortment states that a pair of traits segregates
independently of another pair during gamete formation. As the individual heredity
factors assort independently, different traits get equal opportunity to occur together.

2-Discovery of DNA:
-DNA was first recognized and identified by the Swiss biologist Johannes Friedrich
Miescher in 1869 during his research on white blood cells.
The double helix structure of a DNA molecule was later discovered through the
experimental data by James Watson and Francis Crick. Finally, it was proved that DNA
is responsible for storing genetic information in living organisms. -DNA is a group of
molecules that is responsible for carrying and transmitting the hereditary materials
or the genetic instructions from parents to offsprings.
There are three different DNA types:
 A-DNA: It is a right-handed double helix similar to the B-DNA form. Dehydrated
DNA takes an A form that protects the DNA during extreme conditions such as
desiccation. Protein binding also removes the solvent from DNA, and the DNA takes
an A form.
 B-DNA: This is the most common DNA conformation and is a right- handed helix.
The majority of DNA has a B type conformation under normal physiological
conditions.
 Z-DNA: Z-DNA is a left-handed DNA where the double helix winds to the left in a
zig-zag pattern. It was discovered by Andres Wang and Alexander Rich. It is found
ahead of the start site of a gene and hence, is believed to play some role in gene
regulation.
-DNA Structure

The following diagram explains the DNA structure representing the different
parts of the DNA. DNA comprises a sugar-phosphate backbone and the
nucleotide bases (guanine, cytosine, adenine and thymine).

1-Stages of IVF and the media used in it:

-IVF involves several steps : ovarian stimulation, egg retrieval, sperm retrieval,
fertilization and embryo transfer. One cycle of IVF can take about two to three
weeks. More than one cycle may be needed.

-There are two types of culture media available to support the growth of an
embryo: (1) single step media and (2) sequential, or two step, media. The
difference between the two types comes down to whether the culture media is
changed between fertilization check and embryo transfer,or cryopreservation.  
4)1-CBC Device :
A complete blood count (CBC), also known as a full blood count (FBC), is a
set of medical laboratory tests that provide information about the cells in a
person's blood. The CBC indicates the counts of white blood cells, red blood
cells and platelets, the concentration of hemoglobin, and the hematocrit (the
volume percentage of red blood cells). The red blood cell indices, which
indicate the average size and hemoglobin content of red blood cells, are also
reported, and a white blood cell differential, which counts the different types of
white blood cells, may be included.
 Faculty of Agriculture - Cairo University
The Final Report for Field Training
Second Semester
Academic Year 2021-2022

2-Spectophotometer Device:
The spectrophotometer is an instrument which measures the amount of
light that a sample absorbs. The spectrophotometer works by passing a
light beam through a sample to measure the light intensity of a sample.
These instruments are used in the process of measuring colour and
used for monitoring colour accuracy throughout production.

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 Faculty of Agriculture - Cairo University
The Final Report for Field Training
Second Semester
Academic Year 2021-2022

Day 7-8 10/4/2023 Chemistry lab

11/4/2023
The required skill gained from the training day:
-How to determine the contant of moisture in a sample?
-Chromatography and his function
-Difference between LC- MS and GC-MS
For moisture:
1- Weight dry cup “Ec”
2- Weight 1 gm. Of fresh sample in the dry cup “Sc”
3- put the cup in oven over night for 105 ℃
4- Take the cup and weight “Dc”
5-Calculate the Moisture with equation =(Sc-(Dc-Ec))×100/Wt. Of
Sample
-Chromatography:
It is a Technique which can fractionate the organic compounds which
have the same formula or have the same active formula by means of
stationary phase and Mobile phase.
-Function of Chromatography:
1) Detect Pesticides (in agriculture).
2) Detect organic and volatile substances.
3) Detect fatty acids in food and oils.

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 Faculty of Agriculture - Cairo University
The Final Report for Field Training
Second Semester
Academic Year 2021-2022
1-Liquid Chromatography (LC)
Definition :
-It is chromatography techniques which the stationary phase (Column)
and Mobile Phase is Liquid (Solvents or buffers).
Mechanism of LC MS/MS
the LC are worked throw there Phase:
1. Injection of sample.
2. Separation of sample.
3. Detection on MS/MS detector.
1- Injection of Sample
-Where the prepared sample was drawled with injection needle by High
pressure pump to next part to the column where the compounds in the
sample are separated by meant of mobile phase with definite flow
according to the method of injection
2- Separation of samples
-After injection the sample goes to the Stationary phase (Column)and is
separated by using the mobile phase (solvent or buffers) with specific
flow pumped to the column continuously .
-Every compound separated has a definite time for separation called
retention Time (RT).
-After injection the sample goes to the Stationary phase (Column)and is
separated by using the mobile phase (Helium gas) with specific flow
pumped to the column continuously with definite flow and pressure .
-Every compound separated has a definite time for separation called
retention Time (RT).
-The separated compounds goes to the Detector to define it .

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 Faculty of Agriculture - Cairo University
The Final Report for Field Training
Second Semester
Academic Year 2021-2022
3- Detection by FID detector
-Compounds were entered to the flame Ionizer Detector (FID) to
analyze each compound
-Hydrogen gas and Air used for making of the flame
-The compounds were ionized by flame to be detect in the detector
-device parts:
1) Auto sampler:
It is the part where the rack is located, and it is where the samples are
placed and the syringe to withdraw the sample and inject it into the
device.
2) inlet:
It is the part in which the sample is converted from a liquid form to a
volatile form by raising the temperature in the presence of helium gas
(inert), which acts as a mobile phase.
3) oven and column:
The column represents the stationary phase that contains a substance
through which the sample is separated into the compounds of each
compound in its own retention time and is present inside the oven to
maintain the temperature to keep the sample in a volatile form.
4) MS unit:
In this part, an ionization process occurred for the compounds to
transform in the form of ions that express the compound to be separated
or to detect its presence according to the weight unit of these ions.
5) detector:
This part detects ions by converting ions in the form of a signal to collect
these signals and the peaks form expressing the presence of the

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 Faculty of Agriculture - Cairo University
The Final Report for Field Training
Second Semester
Academic Year 2021-2022
compounds to be detected in the sample and knowing their
concentration in the sample.
1-Gas chromatography-mass spectrometry-mass spectrometry (GC-
MS):
Sample: gas.
Mobile phase: gas such as H2, N2, Ar, or He.
Stationary phase must not react with the sample. It depends on
temperature.

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 Faculty of Agriculture - Cairo University
The Final Report for Field Training
Second Semester
Academic Year 2021-2022

2-Liquid chromatography-mass spectrometry-mass spectrometry(LC

MS):
Sample: liquid.
Mobile: liquid (solvent).
Stationary phase must not react with the sample. It depends on
pressure.

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 Faculty of Agriculture - Cairo University
The Final Report for Field Training
Second Semester
Academic Year 2021-2022
Day 9-10 12/4/2023 TEM Lab
13/4/2023
The required skill gained from the training day
-How to prepare the sample before examination?
-The component of electron microscope and the examination of
sample.
Training activities that were implemented in the field/Lab

 We have the block then we cut it using the Ultra Microtome and
diamond or glass knifes (100nm), Then we see the sample in the TEM.
Staining was made The fixation solution here is FAA, which preserve the
sample for months.
-We make the dehydration step using (Butanol + Ethanol).
-We use the paraffin wax to get rid of Butanol, and then it give us the
block, we cut it using manual microtome, we transfer the sample into
clean slide, stain it using erythrocerein, and crystal violet. And finally we
can see the sample under the light microscope.

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 Faculty of Agriculture - Cairo University
The Final Report for Field Training
Second Semester
Academic Year 2021-2022

-Transmission electron microscope: Consists of three units:

1- Gas unit.
2- Electric unit.
3- Water unit.
-The transmission electron microscope has three essential
systems:
1-An electron gun: produces the electron beam, and the condenser
system.
2-The image-producing system: consisting of the objective lens,
movable specimen stage, and intermediate and projector lenses, which
focus the electrons passing through the specimen to form a real, highly
magnified image.
3-The image-recording system: converts the electron image into some form
perceptible to the human eye. The image-recording system usually consists of a
fluorescent screen for viewing and focusing the image and a digital camera for
permanent records

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 Faculty of Agriculture - Cairo University
The Final Report for Field Training
Second Semester
Academic Year 2021-2022

5-Conclusion and Recommendations

 This field training was filled with lots of information and skills that will
help us as a senior student in our career either from the practical or the
theoretical parts of this training.
 Thanks a lot for all instructors in CURP, all are helpful and cooperative,
and we gain a lot in this training.
 It was a very useful training, as it gives us an experience in different
fields.
 The training inside our faculty at CURP was good and the instructors
were well experienced.

Recommendations
 Increase the duration of the field training to make sure that all devices
and all technique used in CURP is covered well for the next year
students.
 Increase the practical parts or the parts taught us how to work with our
hands to gain the experience.

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