ENVR 421, 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 three assay methods: the single-agar layer (SAL) plaque assay, enrichment-spot
plate Most Probable Number (MPN) , and enrichment particle agglutination methods.
Each lab group will be doing the three methods to assay for coliphage: single agar layer
assay and two enrichment MPN assays (actually, one enrichment system but detection by two
confirmation assays: spot plate and immuno-agglutination).
Each lab group will perform each assay for a sample water (up- or down-stream) and a
wastewater (raw sewage or treated sewage effluent) and will assay for only one coliphage group,
somatic or F+ coliphages. Choose the one E. coli host and its corresponding media with
antibiotics ( E.coli F-amp = male specific coliphage with ampicillin and streptomycin agar or E.
coli CN13 = somatic coliphage with nalidixic acid agar). DO NOT MIX THESE UP or you will
get no results.
The lab will begin on Wednesday and end on Friday.
MATERIALS
For Both SAL and Enrichment 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 (or other) bottles (sterile) of peptone water: 99 ml of diluent each.
NOTE: Each pair of students will use only one of the two bacteria hosts and only one of the four
different samples to be analyzed; these will be assigned in the lab class.
Pipets
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Waterbath at 46oC
Incubator at 36oC
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 in waterbath or heat blocks (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
Analyze treated sewage effluent and water samples (morgan Creek) at undiluted and 10-fold
diluted.
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 Morgan Creek water and treated 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 plate 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 undiluted 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.
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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)
Save the plates for F+ coliphages (on host E. coli Famp) in the refrigerator. We will “pick”
plaques and save them for further analysis. Instructions to be provided later.
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.
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 sewage. 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
Spot Plate Confirmation of Overnight Enrichment Cultures
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 to harden
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:
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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 applied a drop
of clarified (centrifuged) enrichment broth as a spot in a marked square 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.
If you used E. coli host Famp to detect F+ coliphages, save the plates in the refrigerator for
subsequent lysis zone “picking” and F+ coliphage typing.
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.
Particle Immuno-agglutionation Confirmation of F+ Coliphage Enrichments
Obtain 2 agglutination cards and label/mark areas of the card near each circle on the card with
the enrichment culture volume you will apply to each circle: 3 circles for 30 ml enrichments, 3
circles for 3-ml enrichments and 3 circles for 0.3 ml enrichments. Label one card as F+ RNA
coliphages and another card as F+ DNA coliphages.
Then, using a sterile Pasteur or 1-ml serological pipet, apply one drop of the appropriate
enrichment culture to each spot on each card. Change pipets (separate pipet) for each
enrichment culture volume)!
Then, one-by-one to each sample drop in a circle on the card, add one drop of particle immunoagglutination reagent of the correct type (F+ RNA or F+ DNA to each sample drop on the card
and quickly mix by swirling briefly with a toothpick. Change toothpicks (freash tooth pick) for
each circle! Observe the circles in which the drops were mixed for the appearance of clumping of
particles within 1 minute. This is a positive reaction for F+ coliphage presence. (See Figure
below). If the particles do not clump and remain dispersed after one minute, this is a negative
reaction for F+ coliphage presence. Record the results for both cards (F+ DNA and F+ RNA) for
each enrichment volume. Bring these results to class for compilation.
+
Left: Positive reaction
Right: Negative reaction
Figure. Particle immuno-agglutination system and its positive and negative reactions.
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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/or positive by particle immuno-agglutination) and numbers of negative volumes (no lysis
zone on the plate and no particle agglutination) 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?
REFERENCES
Armon R. and Y. Kott A simple, rapid and sensitive presence/absence detection test for
bacteriophage in drinking water. Journal of Applied Bacteriology. 74(4). 1993. 490-496.
Dhillon, T.S. et al. (1970) Distribution of coliphages in Hong Kong sewage, Appl. Microbiol.
20:187-191.
Dhillon, E.K.S. and T.S. Dhillon (1974) Synthesis of indicator strains and density of ribonucleic
acid containing coliphages in sewage, Appl. Microbiol. 27:640-670.
Furuse, K. (1987) Distribution of coliphages in the environment: general considerations, pp.
87-123, In: Phage Ecology, S.M. Goyal, C.P. Gerba and G. Bitton (ed.), John Wiley and
Sons, New York
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Grabow, W.O.K. and P. Coubrough (1986) Practical direct plaque assay for coliphages in 100 ml
samples of drinking water. Appl. Environ. Microbiol., 52:430.
Havelaar, A.H. and W.M. Hogeboom (1984) A method for the enumeration of male-specific
bacteriophages in sewage, J. Appl. Bacteriol. 56:439-447.
Havelaar, A.H. and Th. J. Nieuwstad (1985) Bacteriophages and fecal bacteria as indicators of
chlorination efficiency of biologically treated wastewater, J. Water Pollut. Control Fed.
57:1084-1088.
Havelaar, A.H., Hogeboom, W.M. & R. Pot (1984) F-specific RNA bacteriophages in sewage:
methodology and occurrence, Wat. Sci. Tech. (Bilthoven) 17:645.
Hilton, M.C. and G. Stotzky (1974) Use of coliphages as indicators of water pollution, Can. J.
Microbiol. 19:747-751
Leclerc H, Edberg S, Pierzo V, and JM Delattre (2000) Bacteriophages as indicators of enteric
viruses and public health risk in groundwaters. J Appl Microbiol., 88(1):5-21. Review.
http://www.blackwell-synergy.com/doi/abs/10.1046/j.1365-2672.2000.00949.x
Love DC, Sobsey MD. (2007) Simple and rapid F+ coliphage culture, latex agglutination, and
typing assay to detect and source track fecal contamination. Appl Environ Microbiol.
73(13):4110-8. Epub 2007 May 4.
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
USEPA (2000b) Method 1602: Male-specific (F+) and Somatic Coliphage in Water by Single
Agar Layer (SAL) Procedure. Office of Water. EPA-821-R-00-010. United States
Environmental Protection Agency, Washington, D.C. 20460, April 2000.
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