AAM AND EBT LAB REPORT 2

LIST OF EXPERIMENTS

MIC determination for Cu and Zn

DNA extraction and quantification

Antibiotic Resistance Test

Cu and Zn metal estimation by Atomic Absorption Spectroscopy

 MIC determination of Cu and Zn
AIM: To Determine the Minimum Inhibitory Concentration of Copper and Zinc salts.

Chemicals Required: Zinc chloride, Copper Sulphate, LB Agar and Agarose.

Materials Required: Test-tube, Tips, Pipettes, and Petriplates

Principle:
Metals from human activity has the highest potential of toxicity and pollution and includes
wastewaters coming from metal plating industry, automobile, electrical and electronic materials,
home appliances, pipes, caps, guns, mechanics and dye industries. Microbes grown in these
environment show good tolerance to heavy metals using various mechanisms like adsorption,
complexation, precipitation and volatilization.
Minimum Inhibitory Concentration of the heavy metal resistant bacteria isolates was determined
by gradually increasing the concentration of the heavy metal and exposing the bacterial colonies.
This experiment was performed to determine the tolerance of the isolated bacteria to metals so
that it can be used later for bioremediation purposes.

For Copper
a) Using agar plates
1. LB agar plates were prepared.
2. Isolated colonies for white and yellow bacterial strains were picked up from master-plates
and inoculated in 10 ml LB media for primary inoculation.
3. It was incubated at 30°C overnight and O.D was estimated at 600nm.
4. When the O.D reached around 0.5, 300µl of the inoculum was taken and spread on the
agar plates.
5. Different concentrations of Cu were prepared in falcons (5mM, 6mM, 7mM, 8mM and
9mM respectively in duplicates) and one control was taken.
6. Discs were prepared and were dipped in different Cu concentrations.
7. They were then placed on the lawn and kept at 30°C overnight.

b) Using broth cultures

1. LB broth was prepared.
2. 100mM stock solution of CuSO4 was prepared.
3. Different concentrations of Cu were prepared ranging from 0.5mM to 20mM by adding
appropriate volumes of the stock solution into 10ml LB tubes.
 4. All the tubes were then inoculated with yellow and white colonies.
5. They were incubated at 30°C overnight.

For Zinc
1. Isolated colonies for white and yellow bacterial strains were picked up from master-plates
and inoculated in 10 ml LB media and incubated at 30°C overnight.
2. 100µl of the overnight culture was inoculated in LB broth having different
concentrations of Zn and incubated at 30°C.
3. OD at 600nm was obtained, and the MIC was estimated.

TABULATED RESULT FOR MIC DETERMINATION OF Cu AND Zn

Group Sample Used Salt Salt Zone of Inhibition/Zone Size
Concentration
A-Pond Yellow Cu 8 mM +(thin rim around the disc)
water
Cu 9 mM ++(a bit bigger)
Yellow Zn 10 mM
Zn 12 mM
Zn 14 mM
Zn 16 mM
Zn 18 mM
Zn 20 mM
White Cu No zone in any concentration
White Zn 10 mM +
Zn 12 mM ++
Zn 14 mM ++
Zn 16 mM ++
Zn 18 mM +++
Zn 20 mM ++

Group Sample Salt Salt Zone of Remarks

Used Concentration Inhibition/Zone
Size
B- Yellow Cu 5 mM +
Rhizospheric
soil
6 mM +
Zn 16 mM + Very Faint
18 mM + Diffused growth
20 mM +
 White Cu 5 mM Faint/thin rim
6 mM Faint/thin rim
7 mM Faint/thin rim
8 mM Faint/thin rim
9 mM Faint/thin rim
Zn 10 mM + Faint Rim
12 mM + Faint Rim
14 mM + Faint Rim
16 mM ++ Thin ZOI
18 mM ++ Faint Rim
20 mM +++ Faint Rim

Group Sample Salt Salt Concentration Zone of Inhibition/Zone Size

Used
C-Tailing Yellow Cu 6 mM +++
water
8 mM ++
10 mM ++
12 mM ++
Zn 10 mM +++
12 mM +++
13 mM +++
14 mM +++
White Cu 7 mM +
8 mM -
9 mM -
10 mM -
Zn 16 mM +++
18 mM ++
20 mM +
22 mM +

Group Sample Used Salt Salt Concentration Zone of Inhibition/Zone Size

D-Tailing Yellow Cu 5 mM +
soil
6 mM ++
7 mM +
8 mM ++
9 mM ++
Zn 10 mM +
12 mM ++
14 mM +
 16 mM ++
18 mM ++
20 mM ++
White Cu 5 mM -
6 mM +
8 mM +
9 mM +
Zn 10 mM +
12 mM +
14 mM +
16 mM +
18 mM +
20 mM +

DISCUSSION AND CONSLUSION

Discussion:
Microorganisms isolated from different locations in the mines were checked for resistance to the
metals zinc and copper. The microbes were exposed to increasing concentrations of zinc and
copper.
i. Pond Water Samples: The yellow coloured colonies showed a slight increase in resistance
as the copper concentration was increased, while the white colonies demonstrated high
resistance copper. Exposure of the yellow colonies to zinc did not affect their growth,
hence demonstrating good resistance. Whereas, the white colonies showed good tolerance
to zinc at a low concentration of 10mM and low resistance to zinc at higher
concentrations, such as 18mM.
ii. Rhizosphere soil: The yellow colonies isolated from rhizosphere soil showed resistance
to copper at low concentrations of 5mM and 6mM, and resistance to zinc at even high
concentrations of 20mM. Whereas, the white colonies showed excellent resistance to
copper even at high concentrations of 9mM. But these colonies showed increased
susceptibility to zinc as its concentration was increased.
iii. Tailing water: The yellow colonies of the tailing water sample showed slightly elevated
resistance as the concentration of copper was increased, whereas, its resistance to zinc
was reduced as the concentration of zinc was increased. The white colonies demonstrated
excellent resistance to copper even at high concentrations of 10mM and the zinc
resistance also increased with increasing concentrations of zinc.
iv. Tailing soil: The yellow colonies demonstrated growth inhibition at different
concentrations of copper and zinc at both high and low concentrations. The white
colonies showed good resistance to copper at low concentration and moderate resistance
to zinc even at elevated zinc concentrations.
Conclusion:
The following can be concluded about the metal resistance of the microbes:
1. The white colonies of the pond water sample are highly resistant to copper and have
some resistance to zinc at lower concentrations. The yellow colonies have excellent
resistance to zinc and slightly lower resistance to copper.
2. The yellow colonies isolated from the rhizosphere soil are resistant to both zinc and
copper, while the white colonies have excellent resistance to copper and resistance to low
concentrations of zinc.
3. The yellow colonies isolated from the tailing water samples have low resistance to copper
and zinc. The white colonies hold excellent resistance to copper and some resistance to
zinc at higher concentrations.
4. The yellow colonies isolated from the tailing soil hold poor copper and zinc resistance
and the white colonies have excellent copper resistance at low concentrations and decent
zinc resistance at higher concentrations.
The following values for Minimum Inhibitory Concentrations may be concluded:

Sample Colony Salt MIC

A – Pond water Yellow Copper 8mM
Zinc No inhibition
White Copper No inhibition
Zinc 10mM
B – Rhizosphere soil Yellow Copper 5mM
Zinc 16mM
White Copper No inhibition
Zinc 10mM
C – Tailing water Yellow Copper 6mM
Zinc 10mM
White Copper No inhibition
Zinc 16mM
D – Tailing soil Yellow Copper 5mM
Zinc 10mM
White Copper 6mM
Zinc 10mM
 DNA extraction and quantification
DNA EXTRACTION

AIM
To extract genomic DNA from environmental samples(soil and water)and quantify it using
nanodrop.

MATERIALS REQUIRED
DNeasyPowerSoil Kit, soil and water samples, sterile eppendorfs.

PRINCIPLE
Isolation of genomic DNA from environmental samples using the PowerSoil kit relies on a
proprietary method that removes inhibitory substances such as humic acid and gives highly pure
DNA. The kit uses a humic substance/brown colour removal procedure. This procedure is
effective at removing PCR inhibitors. Environmental samples are added to a bead beating tube
for rpid and thorough homogenization. Cell lysis occurs by mechanical and chemical methods.
Total genomic DNA is captured on a silica membrane in a spin column format. DNA is then
washed and eluted from the membrane.

PROCEDURE

 0.25 g of soil sample (or 250 μl of water sample) was weighed and added to the PowerBead
tube provided in the kit. Gently vortex to mix.
 60 μl of Solution C1 was added to the soil and mixture and were invert several times
 The PowerBead Tubes were stuck horizontally to a Vortex Adapter tube holder and
vortexed at maximum speed for 10 min.
 Tubes were centrifuge at 10,000 x g for 30 s. 400-500ul of supernatant was transferred to
a clean 2 ml collection tube.
 250 μl of Solution C2 was added and vortex for 5 s. Incubated at 2–8°C for 5 min.
 The tubes were Centrifuged for 1 min at 10,000 rpm
 Avoiding the pellet, up to 600 μl of supernatant was transferred to a clean 2 ml collection
tube.
 Around 200 μl of Solution C3 was added and vortex briefly. Incubated at 2–8°C for 5 min.
 The tubes Centrifuged for 1 min at 10,000 rpm
 Avoiding the pellet, up to 750 μl of supernatant was transferred to a clean 2 ml collection
 tube.
 Solution C4 was thoroughly mixed by shaking and 1200 μl of it was added to the
supernatant. Vortexed for 5 s.
 675 μl of mixture was loaded onto an MB Spin Column and centrifuge at 10,000g for 1
min. Discarded the flow through.
 Above step was repeated twice, until all of the samples were processed.
 500 μl of Solution C5 was added and tubes were Centrifuged for 30 s at 10,000 rpm
 The flow through was discarded and centrifuged again for 1 min at 10,000 rpm
 The MB Spin Column was carefully placed into a clean 2 ml collection tube.
 100 μl of Solution C6 was added to the center of the white filter membrane.
 The tubes were Centrifuged at room temperature for 30 s at 10,000 rpm. The MB Spin
Column was discarded.
 Qualitative and quantitative assessment of the obtained DNA was done by agarose gel
electrophoresis and nanodrop.
 The DNA stored at -20°C for further applications.

QUANTIFICATION OF DNA

 Extracted DNA was quantified using nanodrop (UV/Vis spectrophotometer).

 Sample vol - 1μl; blank – nuclease free water.
 260/280 ratio (indicating purity of DNA) and concentration was measured.

Conc. Of DNA
GROUP SAMPLE 260/280 260/230
(μg/μl)
A Pond water 0.006 0.921 0.513
B Rhizospheric soil -0.005 0.823 0.650
C Tailing water 0.003 0.558 0.249
D Tailing soil -0.004 1 1.563

Inference:

 Presence of organic contaminants, DNA needs to be further purified.

 Very low concentration of nucleic acid in all the samples.
 low 260/230 ratio indicates – either residual phenol from extraction process or residual
guanidine (often due to column-based kits).

AGAROSE GEL ELECTROPHORESIS:

 0.8% agarose gel was prepared

 Samples were loaded with 6x loading dye; 1kb ladder was used to estimate size of DNA
  Gel was visualized in UV using GelDoc system.

Gel Image:

Inference:

 Bands were seen only in DNA samples of group B and D, as expected, since both were
soil samples (kit used was for extraction of DNA from soil)
 Size of the DNA was greater than 10kb

RESULT:
DNA was isolated from the collected environmental samples, quantified using nandrop UV/Vis
spectrophotometer and approximate size was estimated by gel electrophoresis.
 ANTIBIOTIC RESISTANCE TEST

Aim: Test the susceptibility of the metal resistant to various antibiotics.

Principle: The disk diffusion test, or agar diffusion test, is a test of the antibiotic
sensitivity of bacteria. It makes use of antibiotic discs to test the extent to which bacteria are
affected by those antibiotics. Agar plates with the bacterial colonies spread on them are used in
this test and discs containing antibiotics are placed and left to incubate. If an antibiotic stops the
bacteria from growing or kills the bacteria, there will be an area around the wafer where the
bacteria have not grown enough to be visible. This is called a zone of inhibition.
Once the zone diameter is measured it must be compared to a database of zone standards to
determine if the bacterium being studied is susceptible, moderately susceptible or resistant to the
antibiotic in question.
The Himedia (HX008) discs containing 6 different antibiotics were used namely Co-
Trimoxazole, Gentamycin, Tetracycline, Amoxyclave, Ofloxacin, Cefuroxime. Two different
discs were used for chloramphenicol and kanamycin.

Procedure:
1) Took 10 ml broth, inoculate with bacteria from the streak plates and incubated overnight
at 30̊ C (primary culture) for both yellow and white colonies and prepared agar plates.
2) Spread 100 ul of the culture broth on the plates and spread properly.
3) Placed the discs from Himedia (HX008) having 6 different antibiotics and 2 separate
discs for chloramphenicol and kanamycin.
4) Incubated the plates overnight at 30̊ C.
5) Observed the plates next day and measured the zone of inhibition.
Results and Discussion:
For White Colonies (Growth)

ANTIBIOTICS Group A Group B Group C Group D

Chloramphenicol -ve -ve -ve -ve
Kanamycin -ve -ve -ve -ve
Co- Trimoxazole -ve -ve -ve -ve
Gentamycin -ve -ve +ve -ve
Tetracycline -ve -ve -ve -ve
Amoxyclave +ve +ve +ve +ve
Ofloxacin -ve -ve -ve -ve
Cefuroxime -ve -ve -ve +ve

White colonies of bacteria from different places show resistance to different antibiotics and its
evident from the table that all samples namely tailing soil, tailing water, pond water and
rhizosphere all are resistant to amoxyclave which is an amoxicillin that works by intereferes with
cell wall formation. Group A samples (pond water) and group B (rhizospheric soil) &D samples
(tailing soil) are resistant to only amoxyclave whereas group C samples (tailing water) are also
resistant to gentamycin in addition to amoxyclave.
For Yellow Colonies (Growth)
ANTIBIOTICS Group A Group B Group C Group D
Chloramphenicol -ve -ve -ve -ve
Kanamycin -ve -ve -ve -ve
Co- Trimoxazole +ve -ve -ve -ve
Gentamycin +ve -ve -ve -ve
Tetracycline -ve -ve -ve -ve
Amoxyclave +ve +ve +ve +ve
Ofloxacin -ve -ve -ve -ve
Cefuroxime -ve -ve +ve +ve

The yellow colony bacteria from group A are resistant to Co trimoxazole, gentamycin and
amoxyclave. For group B the resistance is present only for amoxyclave, group C & D samples
show resistance to cefuroxime in addition to amoxyclave.
None of the bacteria from either white or yellow colonies show resistance to chloramphenicol,
kanamycin or ofloxacin.
 Estimation of metal uptake using Atomic Absorption Spectroscopy
Aim: To Estimate the Metal content in sample using Atomic Absorption Spectroscopy
Principle:
The method relies on the principle of absorption method of spectroscopy. The liquid sample is
allowed to convert into free atoms (desolvated and atomized). These free atoms absorb the light
of a specific wavelength and remaining light is detected and recorded. The intensity of
absorption is directly proportional to the concentration of the sample.
Procedure
For the digestion of soil and water samples, 0.5 g of soil sample and 10 ml of water sample was
placed into a beaker and digested for 4 h at 90 °C with concentrated HCl:HNO3 (3:1) mixture (8
ml) and concentrated HClO4 (3 ml). The residue was filtered and diluted to 25 ml with deionized
water. The sample solutions were not clear. A blank digest was carried out in the same way.
Observation
Analysis of Cu in the samples (324.8 nm)
Sample Observed Conc. (ppm) Absorbance
STD 1 0.5148 0.0540
STD 2 0.9120 0.0954
STD 3 2.1390 0.2233
STD 4 2.9342 0.3062
Pond water 0.9916 0.1037
Rhizosphere soil 1.9631 0.2145
Tailing Water 1.0501 0.1098
Tailing soil 1.1321 0.1181
Analysis of Zn in the samples (213.9 nm)
Sample Observed Conc. (ppm) Absorbance
STD 1 0.4496 0.1154
STD 2 0.9823 0.3016
STD 3 2.1614 0.7137
STD 4 2.9067 0.9742
Pond water 0.5693 0.1637
Rhizosphere soil 1.2962 0.4113
Tailing Water 0.5297 0.1434
Tailing soil 0.5497 0.1504

Interpretation for Copper

 The OD and the ppm values complement each other. Higher OD indicates higher
concentration of metal and vice versa.
 Considering copper, the rhizospheric soil had a maximum copper content, 1.96 ppm,
owing to the fact that it could be due its presence near the roots in stable complex forms
rather than in free soil, where it could be dispersed.
 The tailing soil and water had relatively same levels of copper. These had lesser amounts
compared to rhizospheric soil sample as tailing are normally the by-products left over
from mining and extraction, and are most preferably contain less amount of all scrap
metals including copper.
 Pond water had least amount of copper as in water metals are mostly diffused and the
sample itself had only 0.001% of copper during sample collection.
Interpretation for Zinc
 The OD and the ppm values complement each other. Higher OD indicates higher
concentration of metal and vice versa.
 Samples from copper mine also have significant amounts of Zinc (less than copper) in
them due to the fact that many copper ores have zinc in them.
 Considering zinc, similar to copper, the rhizospheric soil had a maximum zinc content,
1.3 ppm, owing to the fact that it could be concentrated near the roots in stable complex
sforms rather than in free soil, where it could be dispersed. Microbial activity could also
aid in this process
 The tailing soil and water had relatively same levels of zinc (around 0.5ppm). These had
lesser amounts compared to rhizospheric soil sample, as tailings are normally the by-
products left over from mining and extraction, and are most preferably contain less
amount of all scrap metals including zinc.
 Pond water had least amount of Zinc because of the fact that in water the metal
concentration is highly diluted.