Experiment 1: Study of the effect of physical mutagen (UV) on bacterial cells

Theory
The most important part of the light spectrum is a narrow band called the visible light (380-750 nm).
Light wavelengths between 100 and 400 nm are considered ultraviolet (UV). Wavelengths below 100
nm are extremely hazardous (i.e.; ionizing radiation such as gamma rays and X-rays). Beyond 800
nm, the radiation is in the form of infrared (or heat rays), microwaves and radio waves. UV
irradiation is commonly used for surface sterilization of materials that would otherwise be damaged
by high heat (e.g., autoclaving) or hard to sterilize by other methods (e.g. operating rooms).
Although its rays have a very low power of penetration and are not as harmful as the rays of lower
wavelengths, they are still quite powerful mutagens. Their main effect on biological systems stems
from (1) production of a large number of free radicals that destabilize macromolecules and (2) the
fact that they are absorbed by the DNA molecules in the cells and cause alterations that are harmful
to cell survival. In humans, skin cancer is the most common effect of UV-induced mutations
observed. The interaction of the UV with skin cell DNA causes unrestricted growth of some skin cells
that ultimately produce a tumor.
The most common change in the DNA molecule brought about by UV irradiation and consequently
by the free radicals occur at locations on the molecule where two thymine (T) bases occur adjacent
to each other. UV irradiation causes the two T bases to fuse to each other on the same strand. Such
structures are called thymine dimers and cause a distortion in the shape of DNA. Thus, when it is
next time for DNA replication, a wrong base may be incorporated at the thymine dimer position on
the strand being synthesized. This would constitute a site of mutation and if it involves a protein that
plays a role in cell survival, it may be lethal.
Materials required

 E.coli strain K-12 or any other antibiotic sensitive strain

 MacConkey agar/ LB agar plates
 MacConkey agar/LB agar plates supplemented with amp (ampicillin) or rif (rifampicin)for
selection
 UV lamp (Philips UVC lamp, 15W) /UV chamber / laminar air flow cabinetfor irradiation
 UV Protective gloves and spectacles

Procedure
1. LB agar plates with distinct bacterial colonies are taken.
2. Individual colonies are picked and grown overnightin LB broth (20ml).
3. Next day,a series of dilutions of the culture are prepared; 10-1, 10-2, 10-3, 10-4, and 10-5. These
dilutions are then plated for the ultraviolet irradiation by spread-plating 0.1 ml of the diluted
cultures onto the LB agar plates .After plating, the plates should be kept in a black box (plastic
cooler box) before and after irradiation, to avoid any kind of photoreactivation
4. The dilutions of 10-2, 10-3, and 10-4 can be used as the ‘treatment’ set. For each dilution,one control
plate should be kept (untreated) and also 10-5 may be kept as control. . For eachtime of exposure,
there will be one set of plates i.e.,one plate from each dilution (10- 2, 10-3, and 10-4 dilutions). Plates
are randomly placed in the centre of the chamber and irradiated for the set time; the lids of the
‘treatment’ plates should be removed before placing the plates into the chamber to avoid shielding
by the lids. After irradiation, the lids are to be replaced and to be immediately placed into the black
box.
Exposure time should be maintained by using a digital timer and by manually adjusting the power
switch of the UV lamp.The UV lamp could be adjusted vertically to a desired height of up to 60 cm.
The experiments should be done in the dark to avoid photoreactivation. The only time a light
source can be present is during the transfer of the plates into the UV chamber.
5. Various time sets can be tested e.g., the set of exposures of 0, 10, 30 and 60 seconds and/or the
set of 0, 5, 10 and 15 seconds (or according to the teacher’s instruction). Before each irradiation,
the UV lamp should be warmed up for at least 30 minutes.
6. All the plates are then grown in the 37°C incubator for 24 to 36 hours before scoring the number
of colonies.
7. The experiment can be done with different bacterial species in order to compare the damages
induced by of UV radiation.
Screening of mutants
After a day of incubation, colonies from UV irradiated plates (UV survivors) are randomly picked and
transferd to a fresh LB agar plate to make a master plate of 100 colonies per plate. If the colonies in
the plates are countable then the plates can be directly used as master plate. The master plates are
then allowed to grow for 24 hours at 37°C before it is replicate-plated to the ‘screening plates’ such
as MacConkey agar/ LB agar supplemented with 100ug/ml of ampicillin. These ‘screening plates’ are
then incubated for 24 to 48 hrs.
Experiment 2: Preparation of Master and Replica plate for isolation of UV mutants

Theory
Replica plating is the technique by which each colony is inoculated onto multiple plates according to
a numbered scheme. This method allows each colony to be tested by a variety of methods, while
retaining a master plate from which clones can be picked. As we need to screen in the order of tens
of colonies, so we will pick each colony individually and inoculate replicas using a toothpick. In cases
where hundreds or thousands of colonies must be replicated, it can be minimised by dividing the
plates into quadrant and then replicated ,one by one using a toothpick.
Procedure of Toothpicking
1. Take the plates grown in previous experiment obtained after UV irradiation
2. Using a sterile toothpick, touch a separate, distinct colony.
3. With the same toothpick, make a ¼-in. streak on the surface of LB agar plate.Make a second
streak with the toothpick on the corresponding place on an LB-Amp agar plate.
4. With a new sterile toothpick, touch a new colony.Repeat the process.
5. Incubate the plates at 37°C overnight.
6. Identify streaks that fail to grow on LB Amp agar plates. Find the corresponding streak on the LB
agar plate.
7. Count the number of mutant colonies from the streaks which can grow in LB Amp plates.

Result
Note down your observation in tabular form
Experiment 3: Study of survival curve of bacteria after exposure to UV light

Theory
A cell survival curve is a curve used in radiobiology. It depicts the relationship between the fraction
of cells survived or retaining their reproductive integrity and absorbed dose of radiation. Since a UV
monitor or lux monitor is not available, the actual amount of radiation (dose) exposed to the cells in
the direct plate irradiation remains unknown. Therefore various variables involving time of exposure
to UV and distance between the UV lamp and plates can be used to determine a UV Survival curve.
This kill curve or survival curve actually appears to be an ideal exponential decay curve.
Procedure
After a 24 hour (36ºC) incubation period (in dark), take out the Petri plates containing irradiated
microorganisms for evaluation. Count the colony forming units (CFU) in each plate. Calculate the
average number of CFU( from three plates) for each exposition time (as in equation 1) ti get survival
fraction (FS), where N represents the CFU after each irradiation and N0 corresponds to the CFU in
plates not exposed to radiation.

Result
Draw the survival curve by plotting survival fraction against exposure time with your experimental
set of data.
Experiment 4: Isolation of plasmid DNA from E.coli

Theory
The isolation of plasmid DNA from E. coli using an alkaline lysis is a well-established method. E. coli
with plasmid is cultured in media with antibiotics (selection media) to a high cell density, harvested,
and then lysed with a SDS/NaOH solution. Rapid acidification using concentrated potassium acetate
causes the precipitation of protein and chromosomal DNA. Plasmid DNA, which is supercoiled,
remains in solution. The plasmid DNA is washed with an propanol solution and then eluted in water
or TE buffer.
This method involves three steps:
1. growth of the bacterial culture,
2. harvesting and lysis of the bacteria,
3. purification of the plasmid DNA
Growth of the Bacterial Culture
Plasmids should be purified from bacterial cultures that have been inoculated with a single
transformed colony picked from an agar plate. At all times, the transformed bacteria should be
grown in selective conditions, i.e., in the presence of appropriate antibiotic.
Harvesting and Lysis of the Bacteria
Bacteria are recovered by centrifugation and lysed by a number of methods, including heat, alkali,
and detergents, and some other gentler means. The choice of methods is decided based on the
strain of E. coli, the size of the plasmid, and the techniques used to purify the plasmid. Smaller
plasmids (<15 kb) are fairly durable, and can withstand more severe methods of lysis such as those
listed above. Treatment of bacterial suspensions with detergent or heat disrupts the base pairing
and causes the linear stretches of sheared chromosomal DNA of the host to denature and fall away
from each other. The strands of the plasmid DNA, however, are unable to separate from each other
because they are topologically intertwined! Returning the conditions back to normal then allows the
two strands to fall perfectly back into alignment and renature into superhelical plasmid molecules.
Lysing the cells with base combined with the anionic detergent sodium dodecyl sulfate (SDS) has
been used for more than 20 years for plasmid isolation. Exposure of bacterial suspensions to the
strongly anionic detergent at high pH opens the cell wall, denatures chromosomal DNA and proteins,
and releases plasmid DNA into the supernatant. During lysis, the “cell debris” (broken cell walls,
proteins, and denatured chromosomal DNA) forms large complexes that are coated with SDS. These
complexes fall out of solution (precipitate) when sodium ions are replaced by potassium ions.
Purification of the Plasmid DNA
All methods of lysing bacteria will yield plasmid solutions contaminated with chromosomal DNA and
RNA. Centrifugation removes the vast majority of chromosomal DNA (it will form a pellet, while
plasmid DNA remains soluble), and treatment with RNase will eliminate contaminating RNA.

Materials Required
E.coli(or any other bacterial strain) harbouring plasmid
Solution I - Resuspension buffer (50 mM Glucose, 10 mM EDTA, 25mM Tris-Cl,100 mg/mL RNase A, pH 8)
Solution II- Lysis buffer (0.2 M NaOH, 1% SDS)
Solution III- Neutralization buffer (3M Potassium acetate, pH 5.6)
Isopropanol/Ethanol
Wash buffer (70% Ethanol)
Elution buffer (water or TE buffer- 10 mM Tris-Cl, 1 mM EDTA, pH 8)
Procedure
1. Inoculate 5 mL LB, supplemented with appropriate antibiotics, using a single colony. Grow over
night at 37˚C.
2. Transfer 1.5 ml of the overnight culture to a 1.5 ml microcentrifuge tube Centrifuge the tube for 5
minutes at 10,000 rpm in the microcentrifuge. If necessary, discard the supernatant and transfer
another 1.5 ml of overnight culture to the centrifuge tube, and centrifuge for 5 minutes (you
will pellet a total of 3 ml of culture).
3. Discard the supernatant and re-suspend the pelleted cells in 100l of ice cold solution 1,
(the cell resuspension solution) and mix by vortexing.
4. Add 20 µl 10 mg/ml lysozyme solution(perform this step if required)
5. Add 200 µl of freshly prepared Solution II.Mix by inverting and keep in ice for5 mins.
6. Add 150 µl of ice cold Solution III and mix gently by inverting, keep in ice for 5 minutes.
8. Centrifuge at highest speed (13,000rpm) for 5 minutes
10. Transfer the supernatant to a new tube.
11. Add equal volume of isopropanol (2 volumes of ethanol) at room temperature. Mix well and
stand at room temp for 15 minutes. Centrifuge 15 minutes at 13,000.00 rpm.
12. Pour off isopropanol(or ethanol).
13. Add 1 ml of 70 % ethanol to the pellet, invert the tubes several times and centrifuge at maximum
speed for 5-10 mins. Remove all the supernatant completely and keep the tubes open for sometimes
to make sure that ethanol has been removed completely.
13. Resuspend pellet in 50 µl of TE( may or may not containing 20 µl /ml RNase A) and store at 4oC.
Experiment 5: Study of different conformations of plasmid DNA by agarose gel
electrophoresis
Theory
Agarose gel electrophoresis is a method used to separate DNA and RNA molecules according
to their molecular size. This is achieved when negatively charged nucleic acids migrate
through an agarose gel matrix under the influence of an electric field (electrophoresis).
Shorter molecules with low molecular weight move faster and migrate farther than the
larger ones. This method is simple, rapid to perform, and capable of resolving fragments of
DNA that cannot be separated by other procedures such as density gradient centrifugation.
The position of DNA in the agarose gel is visualized under UV light by staining with low
concentration of fluorescent intercalating dyes, such as Ethidium bromide.
The following factors determine the rate of migration of DNA through agarose gels:
 Molecular size of DNA: Fragments of linear DNA migrate through agarose gel with a
mobility that is inversely proportional to the log10 of their molecular weight. Larger
molecules migrate more slowly because of greater frictional drag as they pass through the
pores of the gel less efficiently than the smaller molecules. Circular forms of DNA migrate in
agarose differently from linear DNA of the same mass. Undigested plasmids migrate more
rapidly than the same plasmid when linearized.
 Agarose Concentration: By using gels with different concentrations of agarose, DNA
fragments of different sizes can be resolved. Higher concentration of agarose facilitates
separation of small DNA, while lower agarose concentration allows resolution of larger DNA.
 Electrophoresis buffer: Several buffers have been recommended for electrophoresis of
DNA. The most commonly used are TAE (Tris-acetate-EDTA) and TBE (Tris-borate-EDTA).
DNA fragments will migrate at different rates in these two buffers due to difference in ionic
strength. Buffers not only establish a pH, but also provide ions required to support
conductivity.
 Effect of Ethidium bromide: Ethidium bromide is a fluorescent dye which fluoresces at
254–366nm. It intercalates between bases of nucleic acids and allows very convenient
detection of DNA fragments in gels on a UV transilluminator. Binding of ethidium bromide
to DNA alters its mass and rigidity, and therefore its mobility
 Voltage: As the voltage applied to a gel is increased, larger fragments migrate
proportionally faster than smaller fragments. The best resolution of fragments larger than
about 2 kb is attained byapplying voltage not more than 5 volts per cm to the gel (the cm
value is the distance between the two electrodes, and not the length of the gel).

Materials Required
Plasmid DNA
Agarose
50XTAE buffer, control DNA
6X Gel Loading buffer
Ethidium bromide (10 mg/ml)
Procedure
1. Prepare gel tray by sealing the ends with adhesive tape. Place comb in the space provided on the
gel tray.
2. To prepare 50 ml of 0.8 % agarose solution, add 0.4 g agarose to 50 ml 1X TAE buffer in a glass
beaker or flask. Heat the mixture on a microwave/burner/hot plate by swirling the glass beaker/
flaskoccasionally, until agarose dissolves completely (Ensure that the lid of the flask is loose to avoid
buildup of pressure).
3. Allow solution to cool down to about 55-60 o C. Add 0.5 μl Ethidium bromide, mix well and pour
the gel solution into the gel tray. Allow the gel to solidify for about 30 minutes at room temperature.
4. To start the run, carefully remove the adhesive tape from both the ends of the gel tray, place the
tray in electrophoresis chamber, and fill the chamber (just until wells are submerged) with 1X TAE
electrophoresis buffer and gently remove the comb.
5. To prepare samples for electrophoresis, add 2 μl of 6X gel loading buffer for every 10 μl of DNA
sample. Mix well and load the sample into the well. Load 3 μl of 1 Kb DNA Ladder into one of the
well.
6. Connect the power cord to the electrophoretic power supply according to the conventions:
RedAnode and Black- Cathode. Electrophorese at 100-120 volts and 90 mA until dye markers have
migrated an appropriate distance, depending on the size of DNA to be visualized.
7. Electrophoresis apparatus should always be covered with the lid to avoid electric shocks. Avoid
use of very high voltage as it can cause trailing and smearing of DNA bands in the gel, particularly
with high molecular weight DNA. 8. Switch off the power supply once the tracking dye from the well
reaches
3/4th of the gel which takes approximately 45 minutes.

Visualization and Interpretation of Agarose Gel

The illumination of a stained gel under UV light (254–366 nm) allows DNA bands to be
visualized against a background of unbound dye. The gel image can be recorded by taking a
Polaroid™ photograph or using a gel documentation system.
Plasmid DNA can exist in three conformations: supercoiled, open-circular (oc), and
linear (supercoiled plasmid DNA is often referred to as covalently closed circular DNA, ccc). In
vivo, plasmid DNA is a tightly supercoiled circle to enable it to fit inside the cell. In the laboratory,
following a careful plasmid prep, most of the DNA will remain supercoiled, but a certain amount will
sustain single-strand nicks. Given the presence of a break in only one of the strands, the DNA will
remain circular, but the break will permit rotation around the phosphodiester backbone and the
supercoils will be released. A small, compact supercoiled knot of ccc-DNA sustains less friction
against the agarose matrix than does a large, floppy open circle of oc-DNA. Therefore, for the same
over-all size, supercoiled DNA runs faster than open-circular DNA. Linear DNA runs through a gel end
first and thus sustains less friction than open-circular DNA, but more than supercoiled. Thus, an
uncut plasmid produces two bands on a gel, representing the oc and ccc conformations. If the
plasmid is cut once with a restriction enzyme, however, the supercoiled and open-circular
conformations are all reduced to a linear conformation. Following isolation, spontaneous nicks
accumulate as a plasmid prep ages. This can clearly be seen on gels as the proportion of the two
conformations change over time: plasmids preps that have been thawed and refrozen many times,
show more oc DNA than fresh preps
Quantitation of DNA
Spectrophotometric analysis and agarose gel electrophoresis will reveal the concentration and the
purity of the genomic DNA. Use Elution Buffer to dilute samples and to calibrate the
spectrophotometer, measure the absorbance at 260 nm, 280 nm, and 320 nm using a quartz
microcuvette. Absorbance readings at 260 nm should fall between 0.1 and 1.0. The 320 nm
absorbance is used to correct background absorbance. Purity is determined by calculating the ratio
of absorbance at 260 nm to absorbance at 280 nm. The concentration of DNA is calculated by the
following formula: Concentration of DNA sample (μg/ml) = 50 x A260 x dilution factor
Experiment 6: Bacterial conjugation
Theory
Bacteria possess several methods of gene transfer for transmission of genes between individual
cells. These mechanisms not only generate new gene assortments, they also help to move genes
throughout populations and from species to species. The methods include transformation,
transduction and conjugation. These methods occur by lateral gene transfer which is a potent
evolutionary force that can create diversity within bacterial species. Conjugation is a recombination
process where two live bacteria come together, and the donor cell transfers genetic material to the
recipient cell. This process was first observed in 1946 by Joshua Lederberg and Edward Tatum in a
series of experiments with E. coli. Principle: Conjugation is the mode of gene transfer in many
species of bacteria. In 1950 William Hayes, Francis Jacob and Elie L. Wollman established that
conjugating bacteria are of two mating types. Certain “male” types (designated as F+ ) donate their
DNA and other “female types” (designated as F- ) receive the DNA as shown in Figure 1. Fcells
become F+ when they acquire a small amount of DNA. Hence the F factor is called as the Fertility
factor. In contemporary microbiology, the donor’s F factors are known to be plasmids which are the
extrachromosomal elements. The factors (plasmids) contain about 20-30 genes, most of which are
associated with conjugation. These genes encode enzymes that replicate DNA during conjugation
and structural proteins needed to synthesize special pili at the cell surface. Known as F pili or sex pili,
these hair like fibres contact the recipient bacteria, and then retract so that the surfaces of donor
and recipient are very close or touching one another. At the area of contact, a channel or
conjugation bridge is formed. Once contact via sex pili has been made, the F factor (plasmid) begins
replicating by the rolling circle mechanism. A single strand of the factor then passes over through the
channel to the recipient. When it arrives, enzymes synthesize a complementary strand, and a double
helix is formed. The double helix bends to a loop and reforms an F factor (plasmid), thereby
completing the conversion of recipient from F- cell to F+ cell. Meanwhile, back in the donor cell a
new strand of DNA forms, to complement the leftover strand of the F plasmid. The transfer of F
factors involves no activity of the bacterial chromosome; therefore the recipient does not acquire
new genes other than those on the F factor.On rare occasions an F-plasmid may become integrated
in the chromosome of its bacterial host, generating what is known as an Hfr (high frequency of
recombination) cell. Such a cell can also direct the synthesis of a sex pilus. As the chromosome of the
Hfr cell replicates it may begin to cross the pilus so that plasmid and chromosomal DNA transfers to
the recipient cell. Such DNA may recombine with that of its new host, introducing new gene variants.
Materials Required
Donor strain(tet +)
Recipient (strep +)
Tetracycline, Streptomycin, LB media
Procedure:
Day 1:
Pick up a loopful of Donor culture and streak onto LB plates with Tetracycline (30 μg/ml).
Pick up a loopful of Recipient culture and streak onto LB plates with Streptomycin (100 μg/ml).
Incubate overnight at 370 C.
Day 2:
Pick up a single colony from Donor and Recipient Strain grown overnight on LB plates and inoculate a
single colony in 6 ml of LB broth having respective antibiotics.
Incubate the test tubes overnight at 37o C.
Day 3:
Take 25 ml of LB broth and add 25 μl of tetracycline into it and inoculate 1 ml of overnight grown
culture into it. Incubate at 37o C in a shaker.
Take 25 ml of LB broth with streptomycin at a concentration of 100 μg/ml and inoculate 3 ml of
overnight grown culture in it. Incubate at 37o C in a shaker.
Grow the cultures till O.D of the donor culture reaches 0.8-0.9 at A600.
Add 0.2 ml of each donor and recipient cultures in a sterile test tube labeled as conjugated sample.
Mix by gentle pipetting and incubate at 37o C for 1-1.5 hours.
Take 2 sterile test tubes and label them as donor and recipient. Add 0.2 ml of respective cultures to
the test tubes and incubate at 37o C for 1-1.5 hours. NOTE: Do not place the tubes in shaker for
conjugation and further incubation.
Add 2 ml of LB broth into each tube after incubation. Incubate the tubes at 37o C for 1.5 hours.
Plate 0.1 ml of each culture on the antibiotic plates as indicated below.
Incubate the plates overnight at 37o C overnight.
Samples to be spread on respective plates as follows:
Donor Strain- 0.1 ml to each of a) LB + Streptomycin b) LB + Tetracycline c) LB + Streptomycin and
Tetracycline
Recipient Strain- 0.1 ml to each of a) LB + Streptomycin b) LB + Tetracycline c) LB + Streptomycin and
Tetracycline
Conjugated Sample 0.1 ml to each of a) LB + Streptomycin b) LB + Tetracycline c) LB + Streptomycin
and Tetracycline
Result
Note down the observations in the tabular form. Indicate bacterial growth with positive symbol and
absence of growth with negative symbol.
Interpretation
On observing colonies on different plates the following interpretation can be made:
1. Donor strains will grow only on tetracycline plates, similarly recipient strains will grow only on
streptomycin plates
2. Donor strain is sensitive to streptomycin and recipient strain is sensitive to tetracycline, hence no
growth will be seen in these plates.
3. The conjugated sample will grow on tetracycline and streptomycin plate. The reason being,
transfer of gene has occurred by means of conjugation.
4. The donor and recipient strain will not grow on tetracycline + streptomycin plate since each of the
strain is sensitive to one antibiotic in the plate.
Experiment 7: Demonstration of Ames test
Theory
A simple assay was developed by Bruce Ames to test the mutagenicity of various chemicals. The test
utilizes bacterial mutants containing specific transitions, transversions or frameshift mutations (small
insertions or deletions) in genes of the histidine operon. Each of these types of mutations may be
reversed by a specificcomplementary mutagenic event. The microbes are first grown in a rich broth
containing ample histidine. The cells are then washed twice and inoculated onto a minimal agar
plate. These cells, although auxotrophic for histidine, have accumulated enough of this amino acid to
be able to go through 3-4 cell divisions without requiring exogenous histidine. A disk or drop of the
test compound is placed in the center of the plate and the culture is incubated 48 hours at 37°C. A
ring of distinct colonies around the disk indicates the effectiveness of the chemical in inducing a
particular type of mutation. Ames has demonstrated the usefulness of this assay system for a quick
evaluation of the mutagenic properties of a chemical. It can be said that carcinogens are mutagens
but not all mutagens are carcinogens. Ames test has been applied in many surveys of chemicals to
define those substances that may present potential danger for humans who may be exposed to a
specific compound.The microbes are specific histidine-requiring mutants of Salmonella typhimurium
derived in Dr. Ames' laboratory. These mutants are extremely effective in detecting classes of
carcinogens that have little or no effect on the parent strains. The accuracy of the information
derived from this test may be concluded from an experiment where 85% of the carcinogens tested
(135/150) were detected as mutagens and less than 10% of 106 non-carcinogenic chemicals (based
on animal studies)were positive in the Ames test. Any standard mutagen (ethyl methane sulfonate)
can be tested to determine mutagenicity. This sample should be in liquid form and sterile.

Materials Required
Ames agar plates (or minimal media plates), spreader & forceps,Vial containing mutagen (ethyl
methane sulfonate or EMS), Sterile filter discs, Sterile Water,Culture of Salmonella (Ames strain;
NR115 or any mutants)

Procedure
1. Take 3 Ames plates and label them as EMS (mutagen), Unknown, and Control. Place 2-3 drops of
the bacterial culture on each plate and spread well with a sterile spreader. Allow the bacteria to dry
for a few minutes
2. Using sterile forceps transfer a sterile filter discto the center of each plate and tap gently to make
sure the filter is attached to the surface of the agar.
3. Using a sterile dropper, place a drop of sterile water on the filter disk which is on the Control
plate.
4. Repeat the above step with your unknown sample on the plate labeled Unknown.
5. Take the plate labeled EMS to the hood area. Use a sterile dropper to add one drop of EMS to the
filter disc
6. Place plates face up (do not invert) at 37°C for 48 hours. Seal the EMS plate with a strip of
parafilm. Your instructor will invert your plates later today, after making sure that the chemicals
have diffused sufficiently into the agar.
7. DO NOT OPEN THE EMS PLATE. Observe and record the growth and number of colonies on plates.
Draw what you observe and label properly.