ESSENCE - International Journal for Environmental Rehabilitation and Conservation

Volume VII: No. 2 2016 [33 – 38] [ISSN 0975 - 6272]

[www.essence-journal.com]

Stress response of Escherichia coli on exposure to first transition series elements

in Liquid state

Bhandari, Prabhakar

Received: June 02, 2016  Accepted: August 23, 2016  Online: December 31, 2016

Abstract
Escherichia coli is non fastidious organism Introduction
that grows well in liquid state. It is known to Escherichia coli is known to grow profusely in
grow profusely in nutrient broth and hence this nutrient broth and hence this medium was
medium was considered for carrying out considered for carrying out studies in liquid
studies in liquid state. The test and reference state. The test and reference strain were first
strain were first inoculated in 100 ml sterile inoculated in 100 ml sterile nutrient broth and
nutrient broth and the inoculum size was the inoculum size was standardized by
standardized by MacFarland standard method. MacFarland standard.
15 ml of nutrient broth was dispersed in 6 Materials and Method
tubes for each compound and they were • Nutrient Broth - M001
supplemented with the salt concentrations and
Ingredients Gm/Lt
dilutions as described earlier. The tubes were
Beef Extract 3.00
sterilized and then subsequently inoculated
with 0.1 ml of standard inoculum. The growth Distilled water 1000
was recorded as Nephelometric Turbidity Peptone 5.00
Unit or NTU. The percent inhibition was pH 7.0 ± 0.2
calculated as follows. Sodium Chloride 5.00
Suspend all ingredients in distilled water stir to
Keywords: Metal salts | Broth media |
dissolve and boil the medium to dissolve agar-
Escherichia coli | Nephelometric Turbidity
agar completely and sterilization done by
Unit | Oligodynamic actions
autoclaving on 15 lb at 1200C for 15 min. (Hi-
For correspondence: Media Manual, 2003)
Department of Microbiology, Sevadal Mahila 15 ml of nutrient broth was dispersed in 6
Mahavidyalaya, Nagpur.
Email: prbhnadari02@gmail.com tubes for each compound and they were
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 Bhandari/Vol. VII [2] 2016/33 – 38

supplemented with the salt concentrations and
dilutions as described earlier. The tubes were
sterilized and then subsequently inoculated
with 0.1 ml of standard inoculum. Separate sets
were kept for EC-Test and EC-Ref ATCC
8739 strain, with every set an uninoculated
nutrient broth tube taken as control. The tubes
were incubated at 370C ± 20C for 24 hrs. After
24 hours, Turbidity was measured at 600 nm
using a nephelometer. The growth was
recorded as Nephelometric Turbidity Unit or
NTU. The percent inhibition was calculated as
follows.
• Development of Tolerance in liquid
state
The results are shown in Table 1.1 and Graph
1.1. One way analysis was done with Newman
Keuls Multiple comparison test to determine
the significance of difference among the metal
compound s for Oligodynamic actions. The
results are shown in Table 1.2 statistical and
relevant central tendencies are shown in Table
1.3 The results of EC-Ref ATCC 8739 are
shown in Table 1.4
Separate sets were kept for EC-Test and EC-
Ref ATCC 8739 strain, with every set an
uninoculated nutrient broth tube taken as
control. The tubes were incubated at 370C ±
20C for 24 hrs. After 24 hours, Turbidity was
measured at 600 nm using a nephelometer.
Observations and Result
The organisms which were viable at
0.2µgm/ml were taken out and grown in fresh
nutrient agar media for 6 days. 100 ml of
sterile nutrient broth was supplemented with
20 to 2000µgm/ml in different flask and they
were inoculated with 1 ml aliquot from 0.2
µgm/ml tube for each metal compounds. The
flasks were then incubated at 370C ± 20C for 6
days. After 6th day 5 ml culture was removed
aseptically and percent inhibition was
measured as described in 7.1. Optical Density
(OD) / NTU were measured at 6oo nm. The
results are shown in Table 1.5. After 6th day
1ml of culture was inoculated in to fresh sterile
nutrient agar plates by spread plate technique,
and plates were incubated at 370C ± 20C for 24
hrs. The tolerant organisms formed distinct
colonies suggesting there by the presence of
viable cells even after 6 days of exposure.
Statistical analysis was done by one way
ANOVA using Newman Keuls multiple
comparison test and the results are shown in
Table 1.6 and Table 1.7

Compound Vanadium Chromium Manganese Iron Cobalt Nickel Copper Zinc

0.2 µgm/ml 17 21 20 32 29 35 31 28
2 µgm/ml 18 20 25 35 32 33 32 30
20 µgm/ml 19 25 33 39 35 40 35 32
200 µgm/ml 40 43 35 48 45 48 42 41
2000µgm/ml 72 74 79 81 83 83 78 78
20000µgm/ml 89 90 92 94 95 94 93 93
N.B.: Values of percentage Table 1.1: Percent inhibition of growth due to first
were rounded off to the nearest transition series compounds against EC-Test
whole number as per standard
practice.

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 Bhandari/Vol. VII [2] 2016/33 – 38

Parameter
Table Analyzed Data 1
ANOVA Table SS df MS
Are means significant different? (P <
0.05) No
Bartlett's statistic (corrected) 0.2921
Bartlett's test for equal variances
Do the variances differ signif. (P < 0.05) No
F 0.1595
Number of groups 8
One-Way analysis of variance
P-Value 0.9916
P-Value 0.9999
P-Value summary ns
P-Value summary ns
R squared 0.02715
Residual (within columns) 32380 40 809.5
Treatment (between columns) 903.6 7 129.1
Total 33290 47
Newman-Keuls Multiple Comparison Mean Diff. q Significant? Summary
Test P < 0.05?
Vanadium vs Nickel -13.00 1.119 No ns
Vanadium vs Iron -12.33 --- No ns
Vanadium vs Cobalt -10.67 --- No ns
Vanadium vs Copper -9.333 --- No ns
Vanadium vs Zinc -7.833 --- No ns
Vanadium vs Manganese -4.833 --- No ns
Vanadium vs Chromium -3.000 --- No ns
Chromium vs Nickel -10.00 --- No ns
Chromium vs Iron -9.333 --- No ns
Chromium vs Cobalt -7.667 --- No ns
Chromium vs Copper -6.333 --- No ns
Chromium vs Zinc -4.833 --- No ns
Chromium vs Manganese -1.833 --- No ns
Manganese vs Nickel -8.167 --- No ns
Manganese vs Iron -7.500 --- No ns
Manganese vs Cobalt -5.833 --- No ns
Manganese vs Copper -4.500 --- No ns
Manganese vs Zinc -3.000 --- No ns
Zinc vs Nickel -5.167 --- No ns
Zinc vs Iron -4.500 --- No ns
Zinc vs Cobalt -2.833 --- No ns
Zinc vs Copper -1.500 --- No ns
Copper vs Nickel -3.667 --- No ns
Copper vs Iron -3.000 --- No ns
Copper vs Cobalt -1.333 --- No ns
Cobalt vs Nickel -2.333 --- No ns
Cobalt vs Iron -1.667 --- No ns
Iron vs Nickel -0.6667 --- No Ns
Table 1.2: Tabular statistics of one way ANOVA tabular result
Percent inhibition of growth due to first transition
series compounds against EC-Test

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Vanadium Chromium Manganese Iron Cobalt Nickel Copper Zinc

Minimum 17.00 20.00 20.00 32.00 29.00 33.00 31.00 28.00
25% Percentile 17.75 20.75 23.75 34.25 31.25 34.50 31.75 29.50
Median 29.50 34.00 34.00 43.50 40.00 44.00 38.50 36.50
75% Percentile 76.25 78.00 82.25 84.25 86.00 85.75 81.75 81.75
Maximum 89.00 90.00 92.00 94.00 95.00 94.00 93.00 93.00
Mean 42.50 45.50 47.33 54.83 53.17 55.50 51.83 50.33
Std. Deviation 31.12 29.90 30.34 26.19 28.53 26.31 26.78 28.00
Std. Error 12.70 12.21 12.38 10.69 11.65 10.74 10.93 11.43
Lower 95% CI 9.844 14.12 15.50 27.34 23.23 27.89 23.73 20.94
Upper 95% CI 75.16 76.88 79.17 82.32 83.10 83.11 79.94 79.72
Number of values 6 6 6 6 6 6 6 6
Table 1.3: Column statistics of one way ANOVA: Percent inhibition of growth
due to first transition series compounds against EC-Test

100
% Inhibition

80
60 0.2 µgm/ml
40
2 µgm/ml
20
0 20 µgm/ml

Graph 1.1: Percent inhibition of growth due to first transition series compounds against EC-Test

Compound Vanadium Chromium Manganese Iron Cobalt Nickel Copper Zinc

0.2µgm/ml 16 20 18 30 29 33 30 27
20 µgm/ml 17 26 32 39 33 41 33 32
200 µgm/ml 39 41 36 48 45 47 40 39
2000µgm/ml 71 73 76 82 84 81 74 76
20000µgm/ml 85 87 91 93 92 92 94 91
N.B.: Values of percentage were rounded off to the Table 1.4: Percent inhibition of growth due to
nearest whole number as per standard first transition series compounds
practice against
EC-Ref ATCC 8739

Compound Vanadium Chromium Manganese Iron Cobalt Nickel Copper Zinc

20 µgm/ml 4 7 12 5 22 24 8 7
200µgm/ml 11 16 18 11 24 24 10 12
2000µgm/ml 34 36 34 32 44 58 28 30
20000µgm/ml 78 82 89 91 90 92 78 81
N.B.: Values of percentage were rounded off to Table 1.5: Development of tolerance against first transition
the nearest whole number as per standard practice. series compounds by EC-Test organism

Parameter
Table Analyzed Data 1
One-way analysis of variance
ANOVA Table -18.50 1.087 No Ns
Are means significant different? (P < 0.05) No
Bartlett's statistic (corrected) 1253 7 179.0
Bartlett's test for equal variances SS df MS
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Do the variances differ signif. (P < 0.05) Mean Diff. q Significant? P < 0.05? Summary
F 0.1545
Number of groups 8
P-Value 0.9917
P-Value 27810 24 1159
P-Value summary ns
P-Value summary 29060 31
R squared 0.04312
Residual (within columns) -7.250 --- No ns
Treatment (between columns) -14.00 --- No ns
Total -4.250 --- No ns
Newman-Keuls Multiple Comparison Test -3.750 --- No ns
Copper vs Zinc -1.500 --- No ns
Copper vs Vanadium -0.7500 --- No ns
Vanadium vs Nickel -17.75 --- No ns
Vanadium vs Cobalt -13.25 --- No ns
Vanadium vs Manganese -6.500 --- No ns
Vanadium vs Chromium -3.500 --- No ns
Vanadium vs Iron -3.000 --- No ns
Vanadium vs Zinc -0.7500 --- No ns
Zinc vs Nickel -17.00 --- No ns
Zinc vs Cobalt -12.50 --- No ns
Zinc vs Manganese -5.750 --- No ns
Zinc vs Chromium -2.750 --- No ns
Zinc vs Iron -2.250 --- No ns
Iron vs Nickel -14.75 --- No ns
Iron vs Cobalt -10.25 --- No ns
Iron vs Manganese -3.500 --- No ns
Iron vs Chromium -0.5000 --- No ns
Chromium vs Nickel -14.25 --- No ns
Chromium vs Cobalt -9.750 --- No ns
Chromium vs Manganese -3.000 --- No ns
Manganese vs Nickel -11.25 --- No ns
Manganese vs Cobalt -6.750 --- No ns
Cobalt vs Nickel -4.500 --- No ns

Table 1.6: Tabular statistics of one way ANOVA

of tolerant EC-Test organism against
first transition series compounds

Compounds Vanadium Chromium Manganese Iron Cobalt Nickel Copper Zinc

Minimum 4.000 7.000 12.00 5.000 22.00 24.00 8.000 7.000
25% Percentile 5.750 9.250 13.50 6.500 22.50 24.00 8.500 8.250
Median 22.50 26.00 26.00 21.50 34.00 41.00 19.00 21.00
75% Percentile 67.00 70.50 75.25 76.25 78.50 83.50 65.50 68.25
Maximum 78.00 82.00 89.00 91.00 90.00 92.00 78.00 81.00
Mean 31.75 35.25 38.25 34.75 45.00 49.50 31.00 32.50
Std. Deviation 33.39 33.44 35.08 39.25 31.60 32.55 32.60 33.81
Std. Error 16.70 16.72 17.54 19.62 15.80 16.28 16.30 16.90
Lower 95% CI -21.38 -17.96 -17.58 -27.70 -5.286 -2.299 -20.87 -21.30
Upper 95% CI 84.88 88.46 94.08 97.20 95.29 101.3 82.87 86.30
Number of values 4 4 4 4 4 4 4 4
Table 1.7: Column statistics of one way ANOVA of
tolerant EC-Test organism against first
transition series compounds

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Discussion
As can be seen from the statistical analysis
those oligodynamic actions of all the metal
compounds are similar and there is no
significant difference between the different
metal compounds. However vanadium,
chromium and manganese, compounds showed
lesser inhibition than the other metal
compounds. It can be also observed that at
higher concentrations the percent inhibition is
extremely high. On prolong exposure the
organisms at lower concentrations showed high
degree of tolerance.
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