ENVR 133 – 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)

University Lake water (raw source for our water supply)-1000 ml

Secondary treated sewage effluent

Two 600-ml beakers

One 250 ml beaker

Bacteriophage MS-2 - about 1 x 107 PFU/ml

E. coli strain B - about 1 x 107 PFU/ml

Jar test flocculator 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 with 25mM MgCl2 and E. coli host.

MacConkey agar plates, for spot plate assay of E. coli B

Sterile screw cap tubes – polypropylene or glass, for dilution blanks; make 22 dilution blanks, 1.8 ml each

Diluent - 0.1% Peptone water

Two centrifuge tubes, 50-ml conical bottom, polypropylene, screw cap

Aspirator system: vacuum flask, vinyl tubing and large (blue) pipet tip with large opening

1, 5 and 10 ml pipets

Turbidimeter with sample cells and positive control samples

"Sand filter" – Cylindrical column containing washed filter sand.

Citrate buffer, 0.1 M, pH 5.5

NOTE: Plate 0.01 ml for each sample indicated below.

To plate for E. coli B, spot triplicate 0.01 ml 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 volumes of sample dilution onto a pre-marked TSA plate containing MgCl2 and 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

PROCEDURE

Place 500 ml of lake water or treated sewage effluent (chose one of these two samples for your group) 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: Record turbidity as NTU units:______

While stirring at about 100 rpm, add 1 ml each of E. coli B and MS-2 to each beaker, mix for about 1 minute, take a sample from each beaker, dilute 10- and 100-fold and plate the 10- and 100-fold dilutions. Mark as 0-time controls.

Add ____ ml of aluminum sulfate stock to one of the two beakers, mix at 100 rpm for another minute, and then reduce the mixing speed to about 20 rpm. Continue mixing for 20 minutes.

After 20 minutes, take a sample from each beaker, dilute 10- and 100-fold and plate the 10- and100-fold dilutions.

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.

After 20 minutes, take samples of the supernatants from just below the water level; dilute 10- and 100-fold and plate the undiluted sample and both 10- and 100-fold dilutions. Take an additional supernatant sample from each beaker and measure its turbidity:

With Al: ______NTU Without Al: ______NTU

Remove about 400 ml from each flocculation beaker, being careful not to disturb the settled floc ("sludge"). Place in another beaker.

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.

Take a sample from the second beaker of each filtrate, dilute 10- and 100-fold

and plate the undiluted sample and both the 10- and 100-fold dilutions.

Take samples from the same beakers and measure turbidity:

With Alum: ______NTU Without Alum: ______NTU

Return to the flocculation beakers and remove 35 ml samples of settled floc (sludge) from each as directed by the instructor. Place in 50 ml centrifuge tubes.

NOTE: For the no-alum beaker there may be no sludge, in which case just remove 5 ml of liquid from the bottom of the beaker

To each 35-ml floc sample add 5.0 ml of 0.1 M citrate buffer, pH 5.5, and mix vigorously (vortex) for several minutes. Take samples from each tube, dilute 10-, 100- and 1000-fold; plate for E. coli and MS-2

RESULTS AND QUESTIONS

Determine the concentrations of E. coli and of MS-2 as CFO 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.

Did the bacteria and virus become strongly associated with the alum floc? To what extent were the bacteria and virus detectable in the recovered alum floc after it was redissolved in citrate buffer?

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-flocculation- sedimentation and sand filtration from raw surface water and treated sewage effluent. Are the reductions similar or different?