2. Sampling and data
collection
2.1. Initial statement of the
experimental site
Before planting out the lettuce plants in the soil, the
samples of soil, compost, urine and greywater have been made to known the
initial concentration of the microbiological parameters.The parameters or
indicators which are analyzed in the different matrix are contained in the
table 1.And then, samples of soils are taken for each treatment per week to
analyze these parameters in table 1 below.Therefore, samplings were carried out
from 10 April to 26 May 2014.
Table 2: Different parameters
which are analyzed in the matrix
Matrix

Indicators/pathogens

Soil

E. coli/Faecal coliform, Salmonella, Helminthes eggs,
Faecal enterococci

Compost

Helminthes eggs, E.coli/Faecalcoliform, Salmonella

Urine

Faecal coliform, Faecal enterococci, Salmonella

Greywater

E. coli/ Faecal coliform, Salmonella, Faecal
enterococci

For all these parameters the microbiological analysis will be
used.
2.2. Microbiological analysis
of matrix (soil, compost, urine, and greywater)
2.2.1. Enumeration of bacteria in soil and compost
Compost or soil samples 25 g (w/v) were homogenized in 225 mL
of buffer phosphate water and a 10fold dilution series was performed in
maximum recovery diluents (ringer solution). Fecal coliforms and E.
coli and Enterococci were cultured following a method 9215 A in
Standard Methods for the Examination of Water and Wastewater (APHA, 1998).
Relevant dilutions were spread on plates in duplicate on the following
selective media; chromo cult coliform agar ES (Difco, France) incubated at
44,5°C and for 24 h for Fecal Coliforms,E. coli, and
Salmonella, Slanetz Bartley agar at 37°C for 48 h for
Enterococci. The bacteria load is expressed in (log_{10}
UFC/gDW soil or compost) through the equation 1:
(Equation 1)
Where:
N = Bacteria load in compost or soil (Log_{10}UFC/g
DW soil or compost);
n = Number of colonies in box of Petri;
P = Weight of compost or soil samples (25g);
V_{l} = Volume of Buffer phosphate used to
homogenization of compost or soil samples;
V = Volume of test (1 mL);
d = factor of dilution.
DW= Dry weight is expressed by this equation below:
(Equation 2)
Where:
M_{1}= 10g fresh weight + empty weight oftube,
M_{2}= 10gdry weigth+ empty weight of tube,
M_{0}= empty weight of tube.
2.2.2. Enumeration of bacteria in urine
The description of E.coli and Faecal Coliform(FC) or
Enterococci was done by the method of culture of spreading out in
depth.The samples were diluted with sterile ringer. After dilution, 1 mL of the
diluted sample was spread out over media (Chromocult Agar for E.
coli/Faecal coliform and Slanetz Bartley for Enterococci), contained in
box of Petri which were then carried to the drying oven for incubation with 44
°C during 24h for E. coli/Faecal coliform and with 37 °C
during 48 h for Faecal Enterococci. E coliwere identified by blue
colorantpurple and Faecal Enterococci by whitish. The colonies obtained were
counted thereafter and numbers obtained was allotted to the number of E coli or
enterococci present in the sample. This is why the concentration is expressed
in unit forming colony (UFC) reported to 100 mL of sample. Bacteria load is
expressed by equation (2):
(Equation 3)
Where:
N = Concentration of bacteria in urine (UFC/100
mL);
n = Number of colonies in box of Petri;
Vs = Reference volume (100 mL);
V = Volume of test (1 mL);
d = dilution factor.
