Download Lab 10. Reduction of Microbes by Physical

ENVR 330 – Environmental Health Microbiology – Spring - Lab Exercise
Removal of E. coli and Bacteriophage MS-2 from Surface Water and Treated
Wastewater by Chemical Coagulation-Flocculation-Sedimentation and Rapid Sand
Filtration
INTRODUCTION
Besides disinfection, chemical coagulation with Al or Fe salts and filtration are the most
widely used conventional water treatment processes applied to source waters, especially
surface waters. Hence, the ability of these processes to remove and inactivate enteric
microbes is of importance and interest. The purpose of this experiment is to illustrate the
removal of a model virus, bacteriophage MS-2, and a model enteric bacterium, E. coli,
from raw surface water and treated sewage effluent by coagulation-flocculation and
sedimentation with aluminum salt, followed by rapid rate sand filtration.
MATERIALS (per group of students)
Secondary treated sewage effluent – ~1000 ml
6 600-ml beakers
1 250 ml beaker
MS-2 Coliphage - about 1 x 107 PFU/ml
Log phase E. coli strain B - about 1 x 107 PFU/ml
2 Jar test flocculators with variable speed control
Aluminum sulfate, Al2(SO4)3 – 18 H2O - 10 mg/ml
MS-2 spot plaque assay plates, consisting of tryptic soy agar, antibiotics, and E. coli
Famp host
MacConkey agar plates, for spot plate assay of E. coli B
Sterile polypropylene or glass tubes for dilution blanks; 22 dilution blanks per group, 1.8
ml each
Diluent - 0.1% Peptone water
1, 5 and 10 ml pipets
Turbidimeter with sample cells and positive control samples
"Sand filter" – Cylindrical column containing washed filter sand.
PROCEDURE
Notes on Spot Plating
To plate for E. coli B, spot triplicate 0.01 ml (10 µl) volumes of sample dilution onto a
pre-marked MacConkey agar plate. Allow sample spots to absorb into the agar medium
and then incubate the plate at 36oC overnight. Count pink E. coli colonies within spots
and record the number of colonies per 0.01 ml spot.
To plate for MS-2, spot triplicate 0.01 ml (10 µl) volumes of sample dilution onto a premarked TSA plate containing E. coli Famp host. Allow sample spots to absorb into the
agar medium and then incubate the plate at 36oC overnight. Count small plaques within
spots and record the number of plaques per 0.01 ml spot.
1.
Place 500 ml treated sewage effluent into each of two 600-ml beakers. Position
beakers under jar test flocculator and insert paddle blade to near the bottom of the
beaker. Take another sample of lake water and measure its turbidity:
_______NTU
2.
While stirring at about 100 rpm, add 1 ml each of E. coli B and MS-2 to each beaker.
Mix for 1 minute. Take a sample from each beaker and dilute 10- and 100-fold (0.2
ml into 1.8 ml diluent) and spot plate 10 µl of the 10- and 100-fold dilutions in
triplicate onto MacConkey and spot plate agars. Mark as time = 0 controls.
3.
Add ____ ml of aluminum sulfate stock to ONE of the two beakers. Mix at 100
rpm for 1 minute, then reduce the mixing speed to about 20 rpm for 20 minutes.
4.
After 20 minutes, take a sample from each beaker and dilute 10- and 100-fold (0.2
ml into 1.8 ml diluent) and spot plate 10 µl of the 10- and 100-fold dilutions in
triplicate onto MacConkey and spot plate agars.
5.
Turn off the mixer, remove the paddles from the beakers, and allow the beakers to
stand undisturbed for 20 minutes so that any formed floc can settle.
6.
After 20 minutes, take samples of the each supernatant from just below the water
level. Dilute 10- and 100-fold (0.2 ml into 1.8 ml diluent) and spot plate 10 µl of the
10- and 100-fold dilutions in triplicate onto MacConkey and spot plate agars. Take
an additional supernatant sample from each beaker and measure turbidity:
With Al: ______NTU
Without Al: ______ NTU
7.
Remove about 400 ml from each beaker by carefully decanting and/or using a pipet,
being careful to not disturb the settled floc ("sludge"). Place in another beaker.
8.
Gently pour recovered supernatants through separate sand filters (one for alum-
coagulated samples and one for uncoagulated samples). Collect the first 300 ml of
filtrates in one beaker and the next 100 ml of filtrates in a separate beaker.
9.
Take a sample from the second (100 ml) beaker of each filtrate, dilute 10- and 100fold (0.2 ml into 1.8 ml diluent) and spot plate 10 µl of the undilute, 10-, and 100fold dilutions in triplicate onto MacConkey and spot plate agars. Take an additional
supernatant sample from each beaker and measure turbidity:
With Al: ______NTU
Without Al: ______ NTU
RESULTS AND QUESTIONS
Determine the concentrations of E. coli and of MS-2 as CFU and PFU per ml,
respectively, in the control and test (alum-treated) samples for each sampling time and
tabulate these data. Be sure to correct for any dilutions made during sampling or in
plating. Also express these data as percent of initial E. coli and MS-2 detectable at each
sampling time, taking the zero time titer of the non-alum treated water as 100%.
Compare E. coli and MS-2 reductions to turbidity reductions by coagulation-flocculation
and filtration.
Explain the observed changes in E. coli and MS-2 levels and % initial viruses and
bacteria detected with each operation or sampling time.
How much of the bacteria and virus were removed from each sample by filtration?
Express as both percent and as log10 reduction. Explain any observed differences in
bacteria and virus removals by filtration between the two samples (alum-treated and
untreated).
Compare the extent of virus and bacteria reduction by coagulation-flocculationsedimentation and sand filtration from raw surface water and treated sewage effluent.
Are the reductions similar or different?