Physical, Thermal, and Spectroscopic Characterization of Biofield Energy Treated Murashige and Skoog Plant Cell Culture Media

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Cell
Biology
2015; 3(4): 50-57
Published online December 22, 2015 (http://www.sciencepublishinggroup.com/j/cb)
doi: 10.11648/j.cb.20150304.11
ISSN: 2330-0175 (Print); ISSN: 2330-0183 (Online)
Physical, Thermal, and Spectroscopic Characterization of
Biofield Energy Treated Murashige and Skoog Plant Cell
Culture Media
Mahendra Kumar Trivedi
1
, Alice Branton
1
, Dahryn Trivedi
1
, Gopal Nayak
1
, Khemraj Bairwa
2
,
Snehasis Jana
2, *
1
Trivedi Global Inc., Henderson, USA
2
Trivedi Science Research Laboratory Pvt. Ltd., Bhopal, Madhya Pradesh, India
Email address:
publication@trivedisrl.com (S. Jana)
To cite this article:
Mahendra Kumar Trivedi, Alice Branton, Dahryn Trivedi, Gopal Nayak, Khemraj Bairwa, Snehasis Jana. Physical, Thermal, and
Spectroscopic Characterization of Biofield Energy Treated Murashige and Skoog Plant Cell Culture Media. Cell Biology.
Vol. 3, No. 4, 2015, pp. 50-57. doi: 10.11648/j.cb.20150304.11
Abstract:
The Murashige and Skoog medium (MS media) is a chemically defined and widely used as a growth medium for
plant tissue culture techniques. The present study was attempted to evaluate the impact of biofield energy treatment on the
physical, thermal, and spectral properties of MS media. The study was performed in two groups; one was kept as control while
another was subjected to Mr. Trivedi’s biofield energy treatment and coded as treated group. Afterward, both the control and
treated samples were analyzed using various analytical techniques. The X-ray diffraction (XRD) analysis showed 19.92%
decrease in the crystallite size of treated sample with respect to the control. The thermogravimetric analysis (TGA) showed the
increase in onset temperature of thermal degradation (T
onset
) by 9.41% and 10.69% in first and second steps of thermal
degradation, respectively after the biofield energy treatment as compared to the control. Likewise, T
max
(maximum thermal
degradation temperature) was increased by 17.43% and 28.61% correspondingly in the first and second step of thermal
degradation in the treated sample as compared to the control. The differential scanning calorimetry (DSC) analysis indicated
the 143.51% increase in the latent heat of fusion of the treated sample with respect to the control sample. The Fourier
transform infrared spectroscopy (FT-IR) spectrum of treated MS media showed the alteration in the frequency such as
3165→3130 cm
-1
(aromatic C-H stretching); 2813→2775 cm
-1
(aliphatic C-H stretching); 1145→1137 cm
-1
(C-N stretching),
995→1001 cm
-1
(S=O stretching), etc. in the treated sample with respect to the control. The UV spectra of control and treated
MS media showed the similar absorbance maxima (λ
max
) i.e. at 201 and 198 nm, respectively. The XRD, TGA-DTG, DSC, and
FT-IR results suggested that Mr. Trivedi’s biofield energy treatment has the impact on physical, thermal, and spectral
properties of the MS media. As a result, the treated MS media could be more stable than the control, and might be used as
better media in the plant tissue culture technique.
Keywords:
Biofield Energy Treatment, Murashige and Skoog Medium, X-ray Diffraction,
Fourier Transform Infrared Spectroscopy
1. Introduction
The optimum growth and development of plant tissues are
vary among the plants according to their nutritive
requirements [1]. The tissues from different plant parts may
also have different requirements for appropriate growth.
Furthermore, the plant species are sensitive to growth
regulators and high salts [2]. Therefore, the tissue culture
medium was selected based on the species to be cultured.
Development of culture medium was a consequence of
several systematic experimentation and trials [2, 3]. An ideal
plant tissue culture media should contain the constituents
such as micronutrients (<0.5 mM/L), macronutrients (>0.5
mM/L), amino acids, nitrogen supplements, vitamins, carbon
source, undefined organic supplements, plant growth
regulators and solidifying agents such as agar [4, 5]. The
Murashige and Skoog media (MS media) is a chemically
defined and most suitable classic plant cell culture medium,
consisting vitamins, carbohydrates, and inorganic salts [6].
51 Mahendra Kumar Trivedi et al.: Physical, Thermal, and Spectroscopic Characterization of Biofield
Energy Treated Murashige and Skoog Plant Cell Culture Media
Ammonium nitrate and potassium nitrate serves as the
nitrogen sources, while sucrose was used as the source of
carbohydrate. The MS medium was first invented by Toshio
Murashige and Folke Skoog to support the tobacco callus and
regeneration of shoots and plantlets from the explants [6, 7].
Autoclaving or heat treatment is the standard method of
culture media sterilization [8]. However, the extensive heat
treatment of complex culture media may result in nutrient
destruction via the direct thermal degradation or chemical
reactions between the components [9]. Therefore, it is
advantageous to find out an alternative approach that can
enhance the thermal stability of culture media.
Recently, the energy therapies have been reported for
several beneficial effects throughout the world. Biofield
energy treatment is the part of energy therapy that has been
reported to alter the numerous physicochemical and spectral
properties of organic compounds [10] and organic products
[11]. The National Institute of Health/National Center for
Complementary and Alternative Medicine (NIH/NCCAM)
considered the healing energy or the putative energy fields
treatment under the subcategory of energy therapies [12]. The
energy therapies such as magnet therapy, bio-electromagnetic
therapy, healing touch, etc. comprise low-level of energy
field interactions [13]. Numerous mechanism and
explanations are proposed in support of the biofield energy
therapies including the consciousness. It is described as
healers intent to heal, which may interact with the physical
realm [14]. Similarly, physical resonance is another concept
that includes subtle energies. According to this, the energy
might exchange between the energy fields of patient and
therapist [15]. Thus, the human can harness the energy from
Universe and transmit it to the objects (living or non-living).
The objects receive this energy and respond in a useful way.
This process is called as biofield energy treatment. Mr.
Trivedi is well known for his unique biofield energy
treatment (The Trivedi Effect
®
) that has been studied in the
field of biotechnology [16], agricultural science [17],
microbiology [18], etc.
Therefore, after considering the significant impact of
biofield energy treatment in different areas, this study was
attempted to evaluate its impact on the plant cell culture
media such as MS media. The biofield energy treated MS
media, and the respective control sample were analyzed
using several analytical techniques such as X-ray
diffractometry (XRD), thermogravimetric analysis-derivative
thermogravimetry (TGA-DTG), Differential scanning
calorimetry (DSC), Fourier transform infrared (FT-IR)
spectroscopy, and ultraviolet-visible (UV-vis) spectroscopy.
2. Materials and Methods
2.1. Study Design
The MS media was procured from HiMedia Laboratories,
India. It contained several inorganic salts such as ammonium
nitrate, EDTA, ferrous sulphate, magnesium sulphate, myo-
inositol, potassium nitrate, potassium phosphate, sucrose, etc.
(Table 1). The MS media was divided into two groups; one
was kept as control while another was handed over to Mr.
Trivedi to render the biofield energy treatment under standard
laboratory conditions. Mr. Trivedi provided the biofield
energy treatment to the treated group via his unique energy
harnessing process without touching the sample.
Subsequently, both the control and treated samples were
analyzed with respect to physicochemical and spectroscopic
properties using various analytical techniques such as XRD,
TGA-DTG, DSC, FT-IR, and UV-vis spectroscopy.
Table 1. Chemical composition of Murashige and Skoog media.
S. No. Ingredients mg/L
1 Ammonium nitrate 1650.000
2 Boric acid 6.200
3 Cobalt chloride.6H
2
O 0.025
4 Copper sulphate.5H
2
O 0.025
5 EDTA disodium salt.2H
2
O 37.300
6 Ferrous sulphate.7H
2
O 27.800
7 Glycine (Free base) 2.000
8 Magnesium sulphate 180.690
9 Manganese sulphate.H
2
O 16.900
10 Molybdic acid (sodium salt).2H
2
O 0.250
11 myo-Inositol 100.000
12 Nicotinic acid (Free acid) 0.500
13 Potassium iodide 0.830
14 Potassium nitrate 1900.000
15 Potassium phosphate monobasic 170.000
16 Pyridoxine hydrochloride 0.500
17 Sucrose 30000.000
18 Thiamine hydrochloride 0.100
19 Zinc sulphate.7H
2
O 8.600
2.2. XRD Study
The XRD analysis of control and treated samples of MS
media was done on PW 1710 Phillips Holland X-ray
diffractometer with copper anode and nickel filter. The
wavelength of XRD system was set to 1.54056Å. The
percent change in crystallite size (G) was calculated using
following equation:
G = [(G
T
-G
C
)/G
C
] × 100
Here, G
T
and G
C
are the crystallite size of treated and
control samples, respectively.
2.3. TGA-DTG Analysis
The TGA-DTG analysis of control and treated samples
was carried out on Mettler Toledo simultaneous TGA/DTG
STAR
e
SW 8.10 thermal analyzer. The analytes were heated
up to 400°C from room temperature at the heating rate of
5°C /min under air atmosphere. The onset temperature of
thermal degradation (T
onset
) and the temperature at which
maximum weight loss occur (T
max
) were obtained from TGA-
DTG thermograms.
2.4. DSC Study
The melting temperature and latent heat of fusion of
control and treated MS media were determined using the
Pyris-6 Perkin Elmer differential scanning calorimeter. The
Cell Biology 2015; 3(4): 50-57 52
samples were heated up to 300°C at the heating rate of
10°C/min under air atmosphere with air flow rate of 5
mL/min.
2.5. FT-IR Spectroscopic Characterization
The FT-IR spectroscopic analysis of control and treated
samples of MS media was carried out on Shimadzu’s Fourier
transform infrared spectrometer (Japan) in the frequency
region of 500-4000 cm
-1
. The samples for FT-IR analysis
were prepared by mixing of MS media sample (1%) with
KBr powder and then pressed to a disc or pellets.
2.6. UV-Vis Spectroscopic Analysis
The UV-vis spectra of control and treated MS media were
recorded on Shimadzu’s UV spectrometer (2400 PC). The
instrument was equipped with quartz cell of 1 cm with a slit
width of 2.0 nm. The samples were analyzed at the UV
wavelength region of 200-400 nm.
3. Results and Discussion
3.1. XRD Analysis
Fig. 1. XRD diffractograms of control and treated Murashige and Skoog
media.
The XRD diffractograms of control and treated MS media
are shown in Fig. 1. The XRD diffractograms showed the
sharp and intense peaks in both the samples. This suggested
the crystalline nature of control and treated MS media. The
XRD diffractogram of control MS media showed the peaks at
Bragg’s angle (2θ) equal to 23.35º, 29.29º, 32.25º, 33.67º,
and 40.92º. Similarly, the XRD diffractogram of treated MS
media exhibited the XRD peaks at equal to 23.39º, 23.69º,
29.28º, 33.67º, and 46.64º. The result showed that positions
of XRD peaks in biofield energy treated sample were slightly
altered with respect to the control sample peaks. It is reported
that due to the presence of internal strain within the
molecules the positions and shape of X-ray diffraction peaks
can alter [19]. Based on this, it is hypothesized that the
biofield energy treatment possibly produced some internal
strain within the treated sample. Furthermore, the average
crystallite sizes of both the samples were determined using
Scherrer equation [20].
The result exhibited the crystallite size of the control and
treated samples as 95.53 and 76.25 nm, respectively. The
result showed 19.92% decrease in the crystallite size of
treated sample as compared to the control (Fig. 2). It was
reported that increase in lattice strain may reduce the
crystallite size of the sample [21, 22]. Therefore, it presumed
that biofield energy had induced some lattice strain within
the treated molecules of MS media. As a result, the grains
were fractured into sub grains that could lead to decrease the
crystallite size of the treated sample as compared to the
control. This decrease in crystallite size of the treated sample
might enhance its solubility as compared to the
corresponding control during the preparation of culture
media for plant tissue culture [23].
Fig. 2. Crystallite size of control and treated Murashige and Skoog media.
3.2. TGA-DTG Analysis
The TGA/DTG thermograms of control and treated MS
media are shown in Fig. 3. The TGA thermogram of control
sample showed the three steps of thermal degradation
process. The first step was started at 85°C (T
onset
) and
terminated at 148°C (T
endset
) with 3.33% weight loss. During
this phase, the maximum thermal degradation (T
max
) was
observed at 120.5°C. The second step was started from
159°C and terminated at 202°C with a percent weight loss of
12.24% and T
max
of 180°C. Similarly, the third step was
initiated at 210°C and terminated at 258°C with T
max
of
233°C and 12.6% of sample loss
.
The results exhibited a
cumulative 28.17% weight loss during first, second and third
steps of thermal degradation. On the other hand, the treated
53 Mahendra Kumar Trivedi et al.: Physical, Thermal, and Spectroscopic Characterization of Biofield
Energy Treated Murashige and Skoog Plant Cell Culture Media
sample showed the two steps of thermal degradation. The
first step was initiated at 93°C, which was ended at 170°C
with 9.05% weight loss and T
max
of 141.5°C. The second step
was initiated at 176°C and terminated at 245°C with T
max
of
231.5°C, and 7.78% sample loss. This showed the cumulative
16.87% weight loss during the first and second steps of
thermal degradation.
Overall, the TGA/DTG study revealed the increase in T
onset
by 9.51% (in first step) and 10.69% (in second step) in the
biofield energy treated sample as compared to the control.
Furthermore, the T
max
was increased by 17.43% and 28.61%
in first and second steps of thermal degradation in treated
sample with respect to the control. The increase in T
onset
and
T
max
of both the steps in treated sample suggested the
increased thermal stability as compared to the control [24].
Further, the decreases in cumulative percent weight loss
during the thermal degradation process in treated sample also
suggested its enhanced thermal stability than the control [25,
26]. Based on this, it is assumed that the biofield treated MS
media is thermally more stable than the control sample.
Fig. 3. TGA-DTG thermograms of control and treated Murashige and Skoog media.
3.3. DSC Analysis
The DSC analysis was performed to determine the latent
heat of fusion (∆H) and the melting temperature of both the
control and treated MS media. A considerable amount of
interaction force present in the chemical bonds of any
substance holds them tightly on their positions. The energy
required to overcome the interaction force during the phase
change (i.e. solid into liquid) is termed as the ∆H. The DSC
thermograms (Fig. 4) of MS media showed the latent heat of
fusion as 28.68 J/g in the control and 69.84 J/g in the treated
sample (Table 2). This showed about 143.51% increase in the
latent heat of fusion of treated sample as compared to the
control sample. It might be due to increase in intermolecular
force after the biofield energy treatment of MS media than
the corresponding control. Consequently, the treated MS
Cell Biology 2015; 3(4): 50-57 54
media molecules absorbed more heat (∆H) to change the
phase from solid to liquid as compared to the control.
Previously, our group has reported the biofield energy
treatment induced alteration in ∆H in organic products such
as bile salt and proteose peptone [27]. Therefore, it is
supposed that biofield treatment might increase the
intermolecular interaction forces in treated MS media
molecules, which may lead to increase its latent heat of
fusion.
Moreover, the DSC thermograms of MS media exhibited
the melting temperature at 132.21°C in control and 132.07°C
in treated sample (Table 2). The result showed no significant
change in the melting temperature of the treated sample with
respect to the control.
Table 2. Thermal analysis of control and treated samples of Murashige and
Skoog media.
Parameter Control Treated
Onset temperature (°C)
85.00 93.00
159.00 176.00
210.00
T
max
(°C)
120.50 141.50
180.00 231.50
233.00
Latent heat of fusion (J/g) 28.68 69.84
Melting point (°C) 132.21 132.07
T
max
: temperature at maximum weight loss occurs.
Fig. 4. DSC thermograms of control and treated Murashige and Skoog
media.
3.4. FT-IR Spectroscopic Analysis
FT-IR spectra of the control and treated MS media are
shown in Fig. 5. The contents like inorganic salt, plant growth
regulators, and carbohydrates comprised in MS media may
contain functional groups such as O-H, N-H, C-H, C=C, N=O,
C-O, etc. Therefore, the FT-IR spectra of MS media may be
expected with the vibrational frequencies associated with these
groups. The vibration peak at 3546 cm
-1
in control and 3543
cm
-1
in treated sample were assigned to O-H stretching. The
vibration peak at 3165 cm
-1
in control sample was assigned to
aromatic C-H stretching that was shifted to lower frequency
region i.e. at 3130 cm
-1
after the biofield energy treatment. The
position of IR peak in the FT-IR spectrum depends on the
force constant and the dipole moment of respective bond. The
increase in dipole moment or force constant in the same atomic
bond cause to increase in the frequency (upstream shift) and
vice versa [28, 29]. Based on this, it is hypothesized that the
force constant or dipole moment of aromatic C-H might
decrease; therefore, the IR peak of aromatic C-H was shifted to
lower frequency region.
The IR peak at 2895 cm
-1
in control sample was assigned to
aliphatic C-H
3
stretching. This peak was shifted to 2902 cm
-1
in
the treated sample. Furthermore, the peak at 2813 cm
-1
in
control sample was assigned to aliphatic C-H
2
, which was
shifted to lower frequency region i.e. at 2775 cm
-1
in the
treated sample. This might be due to alteration in force
constant or dipole moment of CH
3
and CH
2
group in treated
sample with respect to the control. The IR peak at 1762 cm
-1
in
control and treated sample were assigned to N=O stretching
due to nitrate salt (ammonium nitrate or potassium nitrate)
present in the sample.
In addition, the peaks at 1398 cm
-1
in control and 1402 cm
-1
in treated samples were assigned to asymmetric NO
3
vibration.
Likely, the IR peak at 825 cm
-1
in both the control and treated
sample was attributed to symmetric vibration of NO
3
group,
possibly due to nitrate salts [30]. The IR peak at 1626 cm
-1
in
control was assigned to C=C stretching, which was slightly
sifted to 1620 cm
-1
in the treated sample. This showed the
decrease in force constant of C=C bond of treated sample as
compared to the control. The peak at 1145 cm
-1
in the control
sample was assigned to C-N stretching (might be due to
EDTA) that was shifted to 1137 cm
-1
in the treated sample. It
suggested the reduction in force constant or dipole moment of
C-N bond after the biofield energy treatment as compared to
the control. The IR peak at 995 cm
-1
in control was attributed
to S=O stretching possibly due to presence of sulfates salts
such as magnesium sulphate and manganese sulphate
comprised in MS media [31]. This S=O stretching peak was
sifted to 1001 cm
-1
in the treated sample. The IR peaks at 603-
663 cm
-1
in control and 603-657 cm
-1
in the treated sample
were assigned to C-S stretching, possibly due to thiamine
hydrochloride, present in the MS media [32]. Overall, the FT-
IR data suggested that biofield energy treatment acted at the
molecular level of treated MS media and altered the dipole
moment or force constant with respect to the corresponding
control sample.
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