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Molecular Microbiology Research (Online) 2013, Vol.3 No.2 9-20
ISSN 1027-5595
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17
plates were there incubated at 37 for 24 hrs.
℃
Repeated steps of 3 and 4 for each of the other two set
of tubes were also carried oil. The growth was
observed on the minimal agar plates.
3.10 Antibiotic marker studies using mutant strains
and treated strains of
S. typhimurium
Auxotrophic mutant strain of
S. typhimurium
MTCC
1251 and treated strains (UV and NTG) were taken up
for the study on bacterial genotics for antibiotic
markers. The procedure followed was from an
overnight grown culture, a loopful inoculum was
taken and inoculated into three sets of four tubes
containing LB broth and approximate antibiotics at
given concentrations. All the tubes were incubated at
37 for 1 hr, 2 hrs and 3
℃
hrs.
After one hour of incubation, the first set of tubes was
taken and a single streak was made on air dried LB
agar plates. The plates were then incubated at 37 for
℃
24 hrs. The steps 3 and 4 were repeated for each of the
other two set of tubes. The growth was observed in
LB agar plates.
3.11 Isolation of plasmids
Plasmids are extrachromosomal, covalently closed,
circular and automatically replicating DNA elements.
There are known to carry genes that specify resistance
like drug resistance, degenerative fertility, virulence,
tumour inducing and for the production of restriction
enzymes. Plasmids were isolated from strains by
alkaline lysis method.
3.12 Agarose gel electrophoresis
Before the electrophoretic run, agarose gel (0.7%) was
prepared along with ethidium bromide at a
concentration of 0.5 μg/mL. The boat (carrying the gel)
was placed in to the eletrophoresis tank carefully and
the tank was filled with 1 × TBE buffer which was
just enough to immerse the gel upto 1 mm top.
The plasmids and maker (10 μL) were loaded into
separate wells. The gel loading was carefully done
without touching the walls of the well but allowing the
samples to fall into the well white pipetting. The
eletrophoretic apparatus was than connected to the
power pack and electrophoresed at 50 mV using l ×
TBE as the running buffer. The migration of the
plasmid samples from the negatively charged to the
positive charged ends was carefully observed. The
electrophoretic run was stopped when the taking dye
had migrated and the distance judged as sufficient for
separation of the plasmid fragment (before 1 cm at
anodic end). The gel was taken carefully using a X-ray
film /OHP sheet/ scoop and transferred onto a
transilluminator and viewed for the occurrence of
bands and compared to the marker for molecular
weight determination.
4 Discussion
Ames et al.
(1990) estimated that about 99.9% of the
chemicals ingested by humans are natural. The
amount of pesticide residues in the plant food are
insignificant as compared to the amount of pesticides
produced by plants themselves against fungi, insects
and animal predators.
Ames et al. (1990) estimate state that the daily average
American exposure to burnt material in the diet is
about 2000 mg and to natural pesticide about 1500 mg.
Review findings of epidemiological literature
Steinmetz and Potter (1991) and Brock et al. (1992)
from nearly 200 studies show that the lack of adequate
consumption of fruits and vegetables were consistency
related to cancer.
Thun et al. (1992) observed the colon cancer rates
that are probably due to addition of diet, to
difference of physical activities which are inversely
related to colon cancer risks. Ames et al. (1993) also
suggested the role of antioxidants such as Vitamins
C, E and carotenoids in fruits and vegetables
account for a good part of their beneficial effects in
present epidemiological studies and related the role
of greater antioxidants consumption in human
cancer prevention.
In comparison, the total daily exposure to all synthetic
pesticides residues combined is about 0.09 mg based
on the sum of residues reported by the US Food and
Drug Administration (USFDA) (1993). Doll and Peto
(1981) were able to estimate that 35% of cancer was
due to dietary factors and the plausible contribution