BTI619-Internship

• Biosafety levels
• Protocols of bacterial samples
• Genome and Genome sequence

Urooj Fatima

Student ID: BC180201166

SEMESTER Spring 2021

BTI619-Internship
 Biosafety Levels
• Biosafety levels (BSL) are used to identify the protective measures
needed in a laboratory setting to protect workers, the environment, and
the public. The levels are defined in Biosafety in Biomedical
Laboratories (the BMBL). Biosafety level designations in the BMBL
outline specific practices and safety and facility requirements. There are
many ways to combine equipment, practices, and laboratory design
features to achieve appropriate biosafety and bio containment. These are
determined through biological risk assessments specifically conducted
for each experimental protocol.
• The four biosafety levels are BSL-1, BSL-2, BSL-3, and BSL-4.
Biosafety Levels
Biosafety Level 1 (BSL-1)
BSL-1 labs are used to study infectious agents or toxins not known to
consistently cause disease in healthy adults. They follow basic safety
procedures, called Standard Microbiological Practices and require no
special equipment or design features. Standard engineering controls in
BSL-1 laboratories include easily cleaned surfaces that are able to
withstand the basic chemicals used in the laboratory.
Biosafety Levels
Biosafety Level 2 (BSL-2)
BSL-2 laboratories are used to study moderate-risk infectious agents or
toxins that pose a risk if accidentally inhaled, swallowed, or exposed to the
skin. Design requirements for BSL-2 laboratories include hand washing
sinks, eye washing stations in case of accidents, and doors that close
automatically and lock. BSL-2 labs must also have access to equipment
that can decontaminate laboratory waste, including an incinerator, an
autoclave, and/or another method, depending on the biological risk
assessment.
Biosafety Levels
Biosafety Level 3 (BSL-3)
BSL-3 will be used to work on Novel coronavirus
BSL-3 laboratories are used to study infectious agents or toxins that may be
transmitted through the air and cause potentially lethal infection through
inhalation exposure. Researchers perform all experiments in biosafety cabinets
that use carefully controlled air flow or sealed enclosures to prevent infection.
BSL-3 laboratories are designed to be easily decontaminated. These
laboratories must use controlled, or “directional,” air flow to ensure that air
flows from non-laboratory areas (such as the hallway) into laboratory areas as
an additional safety measure.
Other engineered safety features include the use of two self-closing,
or interlocked, doors, sealed windows and wall surfaces, and filtered
ventilation systems. BSL-3 labs must also have access to equipment that can
decontaminate laboratory waste, including an incinerator, an autoclave, and/or
another method, depending on the biological risk assessment.
Biosafety Levels
Biosafety Level 4 (BSL-4)

BSL-4 laboratories are used to study infectious agents or

toxins that pose a high risk of aerosol-transmitted
laboratory infections and life-threatening disease for
which no vaccine or therapy is available. The
laboratories incorporate all BSL 3 features and occupy
safe, isolated zones within a larger building or may be
housed in a separate, dedicated building. Access to BSL-
4 laboratories is carefully controlled and requires
significant training.
Test for Mycobacterium
Voges-Proskauer (VP) test

Principle:
The VP test shows if the bacterium has butanediol maturation and can part
glucose to acetoin by means of pyruvat and further to 2,3-butanediol as per: 2
pyruvate + NADH - > 2CO2 + 2,3-butanediol.
In the event that KOH (potassium hydroxide) is added, acetoin will be changed
over to diacetyl (= 2,3-butanedione), which responds with alpha-naphtol and
structures a pink complex.
Strategy
• Suspend one province from the unadulterated culture, which is to be
researched, in VP/MR medium.
•Incubate at 30-37ºC during 24-48 h.
•Add 0.2 ml of 40% KOH and afterward 0.6 ml of alpha-naphtol arrangement.
Voges-Proskauer (VP) test …

Result:
• Positive test outcome: shading change to pink.
• Negative test outcome: no shading change.
Use
• Klebsiella spp. what's more, Enterobacter spp. has the ability
to perform butanediole maturation rather than Escherichia
coli, Salmonella spp.
Indole test
Principle:

Technique, spot indole test

•Place a few drops of Spot Indole Reagent on a piece of channel paper.
•Fill a plastic circle with microorganisms from a province developed for 24-48 hrs.
•Rub it onto the reagent soaked space of the channel paper.
Result:
Positive response: Appearance of a blue to blue-green shading change inside 10
seconds.
Negative response: Remain dreary or light pink.
 Coagulase test…
Principle:
A few microbes produce coagulase, which is a compound that changes fibrinogen
over to fibrin, which implies that it can coagulate plasma. The capacity to deliver
coagulase is thought to be related to the harmfulness of staphylococci. The test is
utilized to recognize coagulase positive and coagulase negative staphylococci.

Strategy
•Suspend one province from the associated unadulterated culture in 0.5 ml with
plasma from pony, hare or man.
•Incubate at 37ºC.
•Read the test after 4 h. In the event that the outcome is negative (see underneath),
proceed with the brooding.
•Perform the last perused after 24 h.
•Positive response if the plasma coagulates and the coagulate is steady. It should not
be disintegrated after blending.
•Negative response if the plasma doesn't coagulate or if the coagulate is disintegrated
again after blending.
•Coagulase trial of Stahylococcus spp. The upper cylinder shows positive outcome
(the plasma has coagulated) and the lower tube shows an adverse outcome.
Coagulase test
•Use
The coagulase test is utilized to recognize Staphylococcus aureus from
coagulase negative Staphylococcus spp A few strains of S. hyicus and S.
intermedius can br coagulase positive. S. pseudintermedius is coagulase
positive, however not until after 24 h.
Modified Oxidase Test …
Rule of Microdase (Modified Oxidase) Test
The microdase test is a fast test to separate Staphylococcus from
Micrococcus which are Gram positive cocci having catalase protein. For
the location of oxidase compound a channel paper round circles
impregnated with tetramethyl-p-phenylenediamine dihydrochloride
(oxidase reagent) in dimethyl sulfoxide (DMSO) are utilized. Within the
sight of climatic oxygen, the oxidase catalyst responds with the oxidase
reagent and cytochrome C to shape the hued compound, indophenol
demonstrated as blue or purplish blue tinge on the circle after the
presentation of bacterial state on the plate.
Materials:
Oxidase Disk
Channel paper circles impregnated with tetramethyl-p-phenylenediamine
dihydrochloride (oxidase reagent) in dimethyl sulfoxide (DMSO).
Modified Oxidase Test
Strategy of Microdase (Modified Oxidase) Test
•Using sterile forceps move a Microdase circle from the stock
container to a petri dish.
•Using a wooden utensil stick, rub a modest quantity of a few
settlements of a 18-24 hour unadulterated culture developed on blood
agar onto the highest point of microdase plate.
•Incubate at room calm for 2 minutes
•Observe for the shading advancement

Result:

Positive test: advancement of blue or purple blue on the plate inside 2

minutes
Negative test: no shading change
 Niacin Test

Principle
The niacin test strip is ordinarily made out of potassium thiocyanate, chloramine-T, citrus
extract, and 4-Aminosalicylic corrosive within the sight of citrus extract, chloramine-T and
potassium thiocyanate will respond to frame cyanogen chloride. This synthetic will fall to
pieces the pyridine ring of niacin to deliver y-carboxy glutaconic aldehyde and joins a sweet-
smelling amine to frame a yellow tone.
Materials required:
•Water , Culture plate , Test strip , Sterile cylinder , Niacin
Technique:
A niacin test strip is comparable in appearance to a pH test strip. It is little, slender,
rectangular, and white in shading. Water is put onto the way of life plate and contacted with a
test strip for 15–20 minutes inside a little, sterile cylinder.
Niacin Test
• Perception:
• Along with each clump of examples being tried, a positive control of M.
tuberculosis and a negative control with no life form will be
incorporated. In the event that the positive control tests negative, there
was presumably a blunder with the bunch, and similarly for a negative
test showing positive.
• Result:
• If abundance measures of niacin are distinguished, the fluid inside the
cylinder will become yellow, a positive test. On the off chance that the
fluid in the cylinder is clear, there are no overabundance measures of
niacin and the test is negative, A positive niacin test doesn't really
demonstrate the presence of M. tuberculosis in light of the fact that other
Mycobacterium species can test positive for overabundance niacin.
Isolation and Culturing of
bacterial samples
Solution:
Classification of bacterial culture media on the basis of consistency
1. Solid medium
Solid medium contains agar at an assembly of 1.5-2.0% or some other, generally
inactive solidifying subject matter expert. Solid medium has real development and
licenses microorganisms to fill in truly illuminating or accommodating habits (for
instance as states or in streaks). Solid medium is useful for confining microorganisms
or for choosing the area characteristics of the isolate.
2. Semisolid medium
Semisolid medium is set up with agar at assemblies of 0.5% or less. Semisolid
medium has a sensitive custard-like consistency and is important for the
improvement of micro-aerophilic minute life forms or for the confirmation of
bacterial motility.
3. Liquid (Broth) medium
These media contain unequivocal proportions of enhancements anyway don't have a
trace of gelling experts like gelatin or agar. Stock medium fills various necessities
like expansion of a colossal number of living creatures, maturing assessments, and
various tests. For instance sugar maturing tests, MR-VP stock.
Classification of bacterial Culture media…

1. Improved medium (added advancement factor)

Development of extra enhancements as blood, serum, egg yolk, etc, to
basal medium makes improved medium. Improved media are used to turn
out to be refreshingly requesting (fastidious) minuscule creatures. Blood
agar, chocolate agar, Loeffler's serum slant, etc a few examples of
improved media. Blood agar is set up by adding 5-10% (by volume) blood
to a blood agar base. Chocolate agar is generally called warmed blood agar
or lysed blood agar.
Classification of bacterial Culture media…
2. Explicit and improvement media
These media are planned to subdue unfortunate commensal or degrading
microorganisms and help to recover organisms from a blend of minuscule
creatures. While explicit media are agar-based, improvement media are liquid in
consistency. Both these media fill a comparable need. Any agar media can be made
explicit by the development of certain inhibitory experts that don't impact the
microorganism of interest. Various approaches to manage making a medium
specific join development of against contamination specialists, colors, engineered
materials, change of pH, or a mix of these.
Explicit and improvement media are proposed to control bothersome commensal or
spoiling minuscule living beings and help to recover microorganism from a mix of
organisms. While explicit media are agar based, improvement media are liquid in
consistency. Both these media serve the same explanation. Any agar media can be
made specific by extension of certain inhibitory experts that make an effort not to
impact the microorganism of interest. Various approaches to manage make a
medium specific fuse development of counter agents poisons, colors, manufactured
mixtures, adjustment of pH or a blend of these.
Classification of bacterial Culture media…
A) Selective medium
Principle: Differential advancement covering Specific medium is proposed to
cover the advancement of specific microorganisms while allowing the
advancement of others. Specific medium are agar based (solid) medium so
particular settlements may be separated.
Examples
Mannitol Salt Agar and Salt Milk Agar used to recover S.aureus contains 10%
NaCl
MacConkey's Agar used for Enterobacteriaceae people contains bile salt that
quells most gram positive organisms.
B) Enrichment culture medium
Headway medium is used to assemble the overall gathering of explicit
microorganisms in the lifestyle going before plating on solid specific medium.
Not in any way like specific media, upgrade culture is commonly used as a
stock medium. Improvement media are liquid media that moreover serves to
impede commensals in the clinical model. Selenite F stock, tetrathionate stock,
and solvent peptone water (APW) are used to recover microorganisms from
fecal models.
Classification of bacterial Culture media…
Differential medium: differential appearance
Certain media are arranged so different minute organic entities
can be seen dependent on their settlement tone. Various
systems join circuit of shadings, metabolic substrates, etc, so
those microorganisms that utilization them appear as
unmistakably concealed territories. Such media are called
differential media or marker media. Differential media license
the improvement of more than one microorganism of interest
anyway with morphologically discernable states.
Examples of differential media joined
Mannitol salts agar (mannitol maturing = yellow)
Mac Conkey agar (lactose fermenters, pink settlements (E.
coli) however non-lactose fermenter produces pale or dull
settlements.
Classification of bacterial Culture media…
3. Transport media
Clinical models ought to be delivered to the examination place
following collection to prevent wealth of soiling natural substances
or commensals. This can be refined by using transport media. Such
media thwart (drying) of a model, keep up the microorganism to
commensal extent, and ruin the abundance of unwanted minuscule
organic entities. A bit of these media (Stuart's and Amie's) are
semi-solid in consistency. Development of charcoal serves to
slaughter inhibitory parts.
Cary Blair transport medium and Venkatraman Ramakrishnan (VR)
medium are used to move feces from suspected cholera patients.
Sach's upheld glycerol saline is used to send crap from patients
suspected to encounter bacillary looseness of the bowels.
Pike's medium is used to move streptococci from throat models.
Isolation of DNA from human blood and bacteria
by Organic method
Principle
“The natural dissolvable based DNA extraction technique depends on the utilization
of natural substances like phenol and chloroform. Because of the unsafe idea of the
phenol and chloroform, the technique is limited”
MATERIALS
Reagents
•Chloroform
•Ethanol
•Ether (discretionary)
•Nucleic corrosive answer for be refined
•Phenol: Chloroform (1:1)
•Tris EDTA (pH 7.8) (discretionary)
•3 M sodium acetic acid derivation pH 5.2 or 5 M ammonium acetic acid derivation
•100% ethanol
Equipments
•Automatic pipette fitted with a dispensable tip
•Pipettes, huge bore (discretionary)
• Polypropylene tube
•Rotating wheel (discretionary)
Isolation of DNA from human blood and bacteria by Organic method

Procedure
• Transfer the nucleic corrosive example to a polypropylene cylinder and add an equivalent volume of phenol: chloroform.
(Subsequent to blending and centrifugation this will result in to two stages, lower natural stage and upper watery stage. The DNA is in the
watery stage and will be removed from it later. The nucleic corrosive will in general segment into the natural stage if the phenol has not been
enough equilibrated to a pH of 7.8-8.0.)
•Mix the substance of the cylinder until an emulsion structures.
• Centrifuge the combination at 80% of the greatest speed that the cylinders can endure for 1 moment at room temperature. On the off chance
that the natural and fluid stages are not all around isolated, axis again for a more extended time frame.
•Use a pipette to move the watery stage to a new cylinder.
•Repeat Steps 1-4 until no protein is apparent at the interface of the natural and watery stages.
•Add an equivalent volume of chloroform and rehash Steps 2-4.
• To recuperate DNA measure the volume of the watery stage.
•Add 1/10 volume of sodium acetic acid derivation, pH 5.2, (last centralization of 0.3M.
• Mix well and add 2 to 2.5 volumes of cold 100% ethanol (determined after salt option).
•Mix well.
• Place on ice or at - 20 degrees C for 20 minutes.
• Spin a greatest speed in a microfuge 10-15 min.
•Carefully empty supernatant.
•Add 1 ml 70% ethanol. Blend. Twist momentarily. Cautiously empty supernatant.
•Air dry or momentarily vacuum dry pellet.
•Resuspended pellet in the fitting volume of Tris EDTA cradle or twofold refined water.
• Proceed with measurement and proposed use after capacity
Isolation of DNA from human blood by inorganic method…
Principle
Addition of Proteinase K to nucleic corrosive arrangements quickly inactivates
nucleases that may in any case degrade the DNA or RNA during purification.
MATERIALS
Solutions/reagents
• Digestion Buffer (10mM NaCl, 10mM TRIS (pH 8.0), 10mM EDTA (pH 8.0),
0.5%
SDS)
•Proteinase K (20mg/ml)
• Sodium Acetate pH 5.2 (3M)
• Ice-cold 98% ethanol
• Ice-cold 70% ethanol
•1X TE
Equipment
•Incubator
•Centrifuge machine
•Sterile 1.5-ml micro-centrifuge tubes
Isolation of DNA from human blood by inorganic method…
Precipitation of Protein and cell Debris
•Add 0.1 volume Sodium Acetate pH 5.2 to sterile 1.5-ml micro--centrifuge tube.
•Close the tube tightly and gently mix the contents by inverting the tube.
• Incubate at -20°C for 15 minutes.
•Centrifuge at maximum speed for 20 minutes at 4°C and aspirate out the top layer.
•Transfer top aqueous layer into sterile 1.5-ml micro-centrifuge tube.
•Discard bottom layer.
•Be careful not to transfer any of the white solid (cell debris and SDS) into the fresh tube.
Precipitation of Nucleic Acid
•Add 2 volumes ice-cold 98% ethanol to sterile 1.5-ml micro-centrifuge tube.
•Close the tube tightly and gently mix the contents by inverting the tube.
•Incubate at -20°C for 15 minutes.
• Centrifuge at maximum speed for 20 minutes at 4°C, gently aspirate out the supernatant
and discard it.
• Add 1 ml of ice-cold 98% ethanol.
•Vortex the mixture for a few seconds
•Centrifuge at maximum speed for 5 minutes at 4°C, gently aspirate out the supernatant
and discard it.
•Add 1 ml of ice-cold 70% ethanol.
•Vortex the mixture for a few seconds
•Centrifuge at maximum speed for 5 minutes at 4°C, gently aspirate out the supernatant
DNA Extraction from bacteria culture

• DNA passes on in its sub-nuclear development the inherited information for

cell improvement and direct. In this manner, all living cells contain DNA.
DNA can moreover separate from cells of any plant, animal, or
microorganism. In this exploration office procedure, you will segregate DNA
from E. coli organisms.
• Step-1: Create your name or initials on a test tube containing chemical. Pour
the E. coli plan from a test tube into your holder of chemical. Cap the
chamber, switch it steadily a couple of times to mix the plan without
foaming, and recognize the chamber in a hot water shower at 55-60°C for 10
minutes. Notice changes in the clarity of the plan. Cleaning agent and
warming at 55-60°C separation the fats in the cell dividers of the organisms,
which frees the DNA.
• Step-2: (Optional) Remove the chamber from the bubbling water shower.
Add two level toothpicks overflowing with meat tenderizer to the chamber,
cap the chamber, mix carefully yet through and through, and return the
chamber to the 55-60°C water shower for 20 minutes. (Meat tenderizer
contains papain, a substance to isolate any proteins that may be associated
with the DNA.)
• Step-3: Take out the chamber from the bubbling water shower. Add cold
alcohol to the test chamber to make an alcohol layer on top of around 1 cm.
For best results, the alcohol should be just probably as cold as could truly be
The alcohol can be added to the course of action in
any occasion three unique ways.

(A) Fill a pasteur pipette with alcohol, put it to bring down piece of the test
chamber, and conveyance the alcohol.
(B) Put around 1 cm of alcohol into the lower part of a test chamber and
add the game plan.
(C) Slowly pour the alcohol down inside the test tube with a pasteur pipette
or prescription dropper. DNA isn't dissolvable in alcohol. Right when
alcohol is added to the mix, all of the pieces of the mix, except for DNA,
stay in game plan while the DNA hurries out into the alcohol layer.
Step-4: Permit the response for sit for 2-3 minutes without disturbing it. It
is huge not to shake the test tube. You can watch the white DNA hurry out
into the alcohol layer. Right when incredible results are gained, there will
be adequate DNA to spool on to a glass bar, a pasteur pipette that has been
warmed at the tip to outline a catch, or relative contraption. DNA takes
after white natural liquid.
Kit-Method:
• Stage 1:
Step through an exam tube sterile it appropriately to stay away from defilement, presently
pour E. coli arrangement in the test tube which contains cleanser and reverse it appropriately
and gradually so all the arrangement can undoubtedly stir up. Rearrange it a few chance to
blend it appropriately, so there isn't development of shaping. Presently place the Test tube in
water shower at 55 degree celcius for 10 minutes. The cleansers and warming causes the
breakdown of fats present in the cell mass of microbes which assists with liberating DNA
from the microscopic organisms cell.
• Stage 2:
Presently eliminate the test tube from the shower and add meat tenderizer to the test cylinder
and blend it genetly which assists with dissolving of tenderizer in the arrangement. Presently
again place the test tube in water shower at 55 degree celcius for 20 minutes. In this
progression the tendenrizer contains a compound which is known as papain which causes
expulsion of protein from the DNA.
•Step 3:
Presently eliminate the test tube from the water shower and add cold liquor in the test
cylinder to accelerate the DNA from the arrangement, since DNA isn't solvent in liquor. The
liquor is included the test should be 1cm over the arrangement and it can pour in the test tube
with the assistance of Pasteur pippete.st tube with the assistance of Pasteur pippette.
• Stage 4:
Presently stay away from to shake to test cylinder and DNA is encouraged out from the
arrangement as bodily fluid.
Presently the DNA is spool out with the assistance of glass bar.
Gel Electrophoresis
Principle
Gel electrophoresis is a method used to isolate DNA sections as per their size. DNA tests
are stacked into wells (spaces) toward one side of a gel, and an electric flow is applied to
get them through the gel. DNA parts are adversely charged, so they move towards the
positive cathode.
Materials Required
•Glass flask
•Weighing balance
•Microwave oven
•UV Transiluminator
•Ladder
Chemicals required
•Agarose
•TAE 1x Buffer
•Ethedium bromide
Gel Electrophoresis Steps

The expansive advances associated with a typical DNA gel

electrophoresis convention:
1. Preparing the examples for running
The DNA is secluded and preprocessed (for example PCR,
enzymatic processing) and made up in arrangement with some
fundamental blue color to help envision the development of the
example through the gel.
2. Preparing an agarose TAE gel solution
TAE support gives a wellspring of particles to setting up the
electric field during electrophoresis. The weight-to-volume
centralization of agarose in TAE support is utilized to set up the
arrangement.
 Gel Electrophoresis Steps
3. Casting the gel
The agarose TAE arrangement is filled a projecting plate that, when the gel arrangement has
chilled off and cemented, makes a gel chunk with a line of wells at the top.
4. Setting up the electrophoresis chamber
The strong gel is set into a chamber loaded up with TAE support. The gel is situated so the
chamber wells are nearest to the negative terminal of the chamber.
5. Loading the gel: The gel chamber wells are stacked with the DNA tests and generally, a
DNA stepping stool is likewise stacked as reference for sizes.
6. Electrophoresis: The negative and positive leads are associated with the chamber and to a
force supply where the voltage is set. Turning on the force supply sets up the electric field
and the contrarily charged DNA tests will begin to move through the gel and away from the
negative cathode towards the positive.
7. Halting electrophoresis and envisioning the DNA
When the blue color in the DNA tests has relocated through the gel far enough, the force
supply is killed and the gel is eliminated and set into an ethidium bromide arrangement.
Ethidium bromide intercalates among DNA and is apparent in UV light. At times ethidium
bromide is added straightforwardly to the agarose gel arrangement in sync 2. The ethidium
bromide stained gel is then presented to UV light and an image is taken. DNA groups are
envisioned in from every path comparing to a chamber well. The DNA stepping stool that
was stacked is additionally imagined and the length of the DNA groups can be assessed
Genome and Genome sequence
• A genome is a complete set of genetic material. The human
genome, like any leftover cell living things, involves DNA
and consolidates both the nuclear and mitochondrial DNA. A
genome sequence is the finished rundown of the nucleotides
(A, C, G, and T for DNA genomes) that make up every one
of the chromosomes of an individual or an animal types.
• NCBI (https://www.ncbi.nlm.nih.gov/). Search “Novel
coronavirus”
• Then retrieve FASTA format of sequence. screenshot of
every step you performed are on the next slides …
Genome and Genome sequence
Genome and Genome sequence
THE END