30
Tecnología y Ciencias del Agua
, vol. VIII, núm. 3, mayo-junio de 2017, pp. 27-37
Aburto-Medina
et al
.
, Prevalence of
Enterobacteriaceae
and contaminants survey in sediments of the Atoyac River
•
ISSN 2007-2422
Determination of organic compounds
The organic compounds were extracted from
dried sediments by sonication Method 3550C,
(USEPA, 2007b) using 3 g of soil and 95 ml of
a mixture 1:1 n-hexane-ketone (Mallinckrodt,
HPLC grade). The extracts were concentrated
by roto-evaporation and changed to n-hexane
as a solvent. The recovery percentage of this
extraction method was validated using a
soil sample with known concentrations of
17 reference compounds. The percentage of
recovery of all compounds was higher than
90%. Regular controls with this soil were
performed to ensure the quality of the data.
The extracts were analyzed to identify organic
compounds by gas chromatography Method
8270D (USEPA, 2007c) coupled with a mass
spectrum detector (Agilent 6890N, MSD
5975B, USA) using a 5 MS column (Agilent,
USA). The temperatures of the detector and
injector were 250°C and 220°C, respectively.
The initial and final temperatures of the oven
were 70°C and 250°C at a rate of 7°C/min.
Helium was used as the carrier gas and the
mass scan range was from 50 to 450 z/m at 70
eV. Identification was made using the NIST05
Mass Spectral library.
Determination of total and fecal coliforms
The Most Probable Number (MPN) of total
coliforms, fecal coliforms, and
Escherichia
coli
was obtained for the three sampling points
(A1-A3) and followed the Mexican Norm NMX-
AA-42-1987, which agrees with the ISO norm:
ISO/DP 9308/2.
Biochemical Oxygen Demand (BOD)
The biochemical oxygen demand for five days
for the three sampling points (A1-A3) was
obtained with a Hach BOD Track II Respiro-
metric apparatus according to manufacturers´
instructions.
Determination of organic carbon
The organic carbon content was evaluated in
water samples for the sampling point A1 using
a TOC-LSCH analyzer (Shimadzu, Japan) which
principle of detection method is the combustion
catalytic oxidation at 680 ºC. Total and inorganic
carbon (TC and IC) were measured automati-
cally in triplicates by the equipment and organic
carbon was calculated by the difference between
those values. The detection limit of TC and IC
was 4 μg/L with a maximum coefficient of
variation of 1.5% between replicates.
DNA extraction and PCR
Microbial community DNA was extracted
directly from the slurries (A1) collected on
January 21, 2013, using the UltraClean Soil
DNA kit (MoBio Laboratories, Solana Beach
CA) and PCR amplification of 16S rDNA genes
was performed with the following primer pair:
63F (CAG GCC TAA CAC ATG CAA GTC)
and 1389R (ACG GGC GGT GTG TAC AAG)
(Marchesi
et al
., 1998) for bacteria as described
previously (Aburto
et al
., 2009). The cycling
conditions were as follows: 1 cycle at 94 °C for
2 min, 30 cycles of 1 min at 94 °C, 1 min at 55°C
and 2 min at 72 °C and a final elongation at 72
°C for 10 min.
PCR amplification of genes encoding Shiga
toxins 1 and 2 was performed on the A1 slurries
with the primer pair
stx
1 and
stx
2 as described
previously (Fagan
et al
., 1999). The cycling
conditions were as follows: an initial 95°C de-
naturation step for 3 min followed by 35 cycles
of 95°C for 20 s, 58°C for 40 s, and 72°C for 90 s.
The final cycle was followed by a 72°C incuba-
tion for 5 min.
Cloning and sequencing
PCR products were cloned with a CloneJet
cloning kit (Thermo) as described in the
manufacturer´s instructions using One Shot
TOP10 chemically competent
E. coli
cells. Re-
combinant colonies were recovered from LB
