ENVR 133, ENVIRONMENTAL HEALTH MICROBIOLOGY, SPRING
Quantification of Somatic and Male-specific Coliphages in Water and Wastewater
INTRODUCTION
Escherichia coli (E. coli) and other "coliform" bacteria are plentiful in feces and sewage.
Many different viruses capable of infecting E. coli, called coliphages, have become favorite
subjects and tools for virus research in the areas of biochemistry, molecular biology and genetics.
The natural history or ecology of these coliphages and their usefulness as indicators of fecal
contamination are becoming better understood. Various coliphages can be readily isolated from
raw, domestic sewage and sewage polluted water, foods and other environmental samples. They
are considered useful viral indicators of fecal contamination of water, foods and other
environmental media. They also have potential uses to track or identify sources of fecal
contamination as being either human or non-human in origin.
Coliphage detection can be done by (1) direct plating on their host bacteria using the
plaque technique, which is analogous to a "pour plate" technique for bacteria, (2) by enrichment
in broth culture followed by confirmation of positive broth culture by transferring some of it to a
host bacteria lawn in agar medium and incubating for development of a zone of lysis (US EPA,
2000a; 2000b) and by several alternative membrane filter methods.
In this lab exercise we will determine and compare the levels of somatic and malespecific coliphages in water and wastewater, and we will compare the levels of coliphages
detected by two assay methods: the single-agar layer (SAL) plaque assay and an enrichmentspot plate Most Probable Number (MPN) method.
Each lab group will be doing the two methods to assay for coliphage: single agar layer
assay and enrichment MPN assay.
Each group will perform each assay for a sample water (up- or down-stream) and a wastewater
(raw sewage or threared sewage effluent) and will assay for only one coliphage group, somatic or
F+ coliphages. Choose the one host and its corresponding agar with antibiotics ( E.coli F-amp
= male specific coliphage with ampicillin and streptomycin agar or E. coli CN13 = somatic
coliphage with nalidixic acid agar).
The lab will begin on Wednesday and end on Friday.
MATERIALS
For Both Methods:
Water or wastewater sample: as specified by the instructor
Somatic and male-specific E. coli hosts (CN-13 and Famp, respectively) - broth cultures in log
phase, about 108-109 cells per ml.
Sample diluent: tryptic soy broth with added MgCl2 (TSB-Mg) without antibiotics
Petri plates (for the SAL method and for the confirmation step of the enrichment method)
Milk dilution bottles of peptone water: 99 ml of diluent each.
NOTE: Each pair of students will use only one of the two bacteria hosts and only two of the four
different samples to be analyzed; these will be assigned in the lab class.
Pipets
Waterbath at 46oC
Incubator at 36oC
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100X antibiotics for media:
Ampicillin+Streptomycin for E. coli Famp (Male-specific host)
Nalidix acid for E. coli CN-13 (Somatic host)
For SAL Method:
Double strength, molten agar: tryptic soy agar, at 46oC (for the SAL method)
Culture tubes, glass or plastic, sterile
For Enrichment Method:
Bottles, 100-200 ml capacity, glass or plastic, sterile
Culture tubes, glass or plastic, sterile, 25, 10 and 3-5 ml capacity.
5X Tryptic Soy Broth with added MgCl2
1X Tryptic Soy Agar - molten at 46oC (to pour agar medium-host cell lawn plates for
confirmation of enrichment cultures)
Sterile Pasteur pipets
PROCEDURES
Dilute water and wastewater samples serially 10-fold by adding 11 ml of sample to 99 ml of
diluent in a milk dilution both.
Dilute water and sewage effluent samples 10-fold
Dilute raw sewage 10-, 100-, and 1000-fold
SAL method:
Dispense 10-ml volumes of molten 2X nutrient agar with appropriate antibiotic into large
test/culture tubes and keep in 46oC water bath.
Label 10-cm diameter Petri dishes with sample volume and host bacterium using one plates per
dilution
Dispense 10-ml volumes of the sample dilution into the test/culture tubes and place in the 46oC
water bath.
After 5 minutes, begin plating the samples one-at-a-time into Petri dishes as follows:
Add 0.1 ml of E. coli host to a pre-warmed water sample and place in the waterbath for 1
minute;
Add 10 ml of 2X TSA to the sample-host mixture, swirl briefly in your palms and pour
the contents into a sterile Petri dish
Repeat these steps with the remaining 10-ml sample volumes.
After the agar medium hardens, incubate the plates at 36oC, inverted.
After overnight incubation, remove plates from the incubator and count and record plaques.
NOTE; plaques appear as "clear circles, 1-10 mm diameter, in the cloudy, confluent lawn of host
bacteria. Sometimes plaques (especially the F+ coliphages) are very small and sometimes they
are "faint" (not entirely clear)
Record the number of plaques per plate and bring these data to the next class.
Observe the plaques for their different sizes (diameter) and morphologies (clear, cloudy,
bacterial colonies in center, "bull's-eye" appearance, etc.
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MPN Method
Enrichment:
Note: Do the steps for the enrichment procedure 1 sample volume and enrichment bottle at
a time. The bottle contents must be subdivided into aliquots of 3 x 30 ml, 3 x 30 ml and 3 x
0.3 ml within 5 minutes of adding the host bacteria to the sample.
For stream and sewage effluent samples, dispense a 100-ml volume of the undiluted and 10-fold
sample into a sterile bottle.
For the raw sewahe. Dispense a 100-ml volume of the 100-fold diluted and 1000-fold diluted
sewage.
Add 10 ml of 5X TSB to each sample bottle and mix briefly
Add 1-5 ml of host bacterium to a bottle (check with the instructor) and mix briefly.
Immediately remove sample volumes of 0.3 ml and dispense into three separate tubes.
immediately remove sample volumes of 3 ml and dispense into three separate tubes Then
immediately remove 30 ml volumes of sample and dispense into each of 3 tubes or bottles.
Incubate samples overnight at 36oC
Preparation of host lawn in agar medium:
Dispense 10 ml of molten 1X TSB into a sterile test/culture tube
Add 1 ml of host bacterium culture (either E. coli Famp OR CN-13)
Pour contents of the tube into a sterile petri dish and allow toharden
Store in refrigerator overnight
The next day:
Spot plating of enrichment cultures:
Remove plates with host lawn in agar medium from refrigerator and warm to room temperature
Using an indelible marking pen, draw a 1-cm square grid pattern on the bottom of the plate:
Mark each square with a number or other code to keep track of them
From each enrichment volume, transfer a 0.3 to 1.0 ml volume to a sterile microcentrifuge tube
and centrifuge at top speed for 10 minutes.
In each square, place 1 drop (~0.01 ml) of supernatant from a microfuge tube of broth
enrichment culture from one of the enrichment samples. The result will be to have applies a drop
of clarified (centrifuged) enrichment broth as a spot for each enrichment volume. KEEP
TRACK OF WHICH SPOT WAS INCULATED WITH WHICH ENRICHMENT VOLUME.
Allow the drops to dry at room temperature for ~0.25 to 0.5 hours
Then incubate the plate a 36oC overnight.
After overnight incubation, observe the plates for zones of lysis with each grid square that
received a drop.
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Record the results as positive (+) for a zone of lysis) or negative (-) for a zone of lysis (i.e.,
normal host cell lawn).
Bring these results to the next class meeting.
RESULTS CALCULATIONS
SAL:
From the numbers of plaques per plate, calculate the average numbers of somatic and malespecific coliphages per 100 ml and the 95% confidence intervals for these average values for
each sample. What were the ranges of plaque sizes and types of plaque morphologies on your
coliphage host?
Enrichment:
From the numbers of positive sample volumes (giving a positive spot or lysis zone on the plate)
and numbers of negative volumes (no lysis zone on the plate) out of three per dilution and per
sample, calculate (or look look up) the MPN and upper and lower 95% confidence intervals of
coliphages per 100 ml for each sample.
QUESTIONS
Are the coliphage titers for a given sample the same by both methods?
If not, what are possible reasons for this difference in detection?
For each group of coliphages (somatic and male-specific), compare the numbers of coliphages
detected in the different samples, as you have previously done for indicator bacteria.
Also, compare the coliphage levels in raw and treated sewage and calculate the coliphage
reductions (as percentages or log10) by the treatment plant.
How do the concentrations of somatic and male-specific coliphages obtained from this
experiment compare with the concentrations of the various indicator bacteria previously detected
in raw and treated sewage?
Do you think coliphages would be a reasonable adjunct or alternative indicators to coliform
bacteria or other enteric bacteria as indicators of fecal contamination? Why?
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http://www.blackwell-synergy.com/doi/abs/10.1046/j.1365-2672.2000.00949.x
USEPA (2000a) Method 1601: Male-specific (F+) and Somatic Coliphage in Water by Two-Step
Enrichment Procedure. Office of Water, EPA-821-R-00-009. Environmental Protection
Agency, Washington, D.C. 20460, April, 2000
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Environmental Protection Agency, Washington, D.C. 20460, April 2000.
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