Biology Reference
In-Depth Information
7.2.3 Analyses of Chemo-physical parameters
Temperature and EC were determined using a conductivity meter Cond340i (WTW GmbH,
Weilheim Germany) calibrated at 25
o
C and pH was determined using a pH meter pH340i
(WTW GmbH, Weilheim Germany). To analyze nitrate and chloride, as chemical indicators
of contamination, 250 ml of spring water was collected in sterile polypropylene bottles and
by means of a syringe 25 ml of sample was filtered through 0.45 µm cellulose acetate filter
into scintillation vials. Samples were then stored in a cool box and transported to the
Environmental Engineering and Public Health (EEPH) laboratory of the University of
Makerere and stored at -20
o
C until they were transported to the UNESCO-IHE laboratory in
Delft, The Netherlands, where nitrate and chloride were analyzed with an ICS-1000 AS 14A
Column (Dionex, Benelux BV).
7.2.4 Microbiological analyses and E. coli isolation
Total coliform (TC) in spring waters were analyzed by filter pressing 100 ml of spring water
and the filter paper was placed on a Chromocult agar (Merck, Whitehouse Station, NJ)
plate. Agar plates were then transported to the EEPH laboratory of Makerere University and
incubated at 37
o
C for 24 hours, respectively. The number of thermotolerant coliforms on
plates was then counted, and the purple colored colonies on the Chromocult agar plates
allowed for the detection and isolation of
E. coli
from other types of bacteria species growing
on the agar plates. To do so, a sterile toothpick was used to pick a single colony of
E. coli
from the agar plates and inoculated into 5 ml of Nutrient Broth followed by incubation at
37
o
C for 24 hours. Then, 1-2 ml of freshly grown
E. coli
cells were inoculated into sterile
vials (Microbank™-Dry, PRO-LAB DIAGNOSTICS, Toronto, Canada) containing porous
beads saturated with cryopreservative (cryovials), which serve as carriers to support
microorganisms. The vials were then stored at -20
o
C and transported to the UNESCO-IHE
laboratory in Delft, the Netherlands.
.
7.2.5 Bacteria growth and cell characterization
To conduct experiments, a sterile forceps was used to pick a bead from the cryovial, placed
into 25 ml of Nutrient Broth and then incubated for 24 hours at 37 °C while shaking at 150
rpm on an orbital shaker to obtain a cell concentration of ~10
9
cells/ml. Bacteria were washed
and centrifuged (4600 rpm) three times in Artificial Ground Water (AGW), which was
prepared by dissolving 526 mg/L CaCl
2
.2H
2
O and 184 mg/L MgSO
4
.7H
2
O, and buffering
with 8.5 mg/L KH
2
PO
4
, 21.75 mg/L K
2
HPO
4
and 17.7 mg/L Na
2
HPO
4
. The final pH-value of
the suspensions ranged from 6.6 to 6.8, while the electrical conductivity ranged from 980 to
1024 S/cm
To determine
motility
, a 2 mL fresh culture was centrifuged (14000 xg) and washed three
times in AGW, and by means of a sterile toothpick, cells were picked from the remaining
pellet in the test tube and inoculated at the centre of petri-dishes containing 0.35%
Chromocult agar. The plates were incubated at 37 °C for 24 hours after which growth and
diameter of migration was measured as motility (Ulett et al., 2006).
Hydrophobicity
was determined with the Microbial Adhesion To Hydrocarbons (MATH)
method (Pembrey et al., 1999; Walker et al., 2005), where percentage partitioning of cells
into dodecane was measured as cell hydrophobicity. Thereto, 4 mL of bacteria suspension of
known optical density and 1 mL of dodecane were vigorously mixed in a test tube for two
minutes and left to stand for 15 minutes to allow phase separation. Then, the optical density
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