9224 DETECTION OF COLIPHAGES*

9224 A. Introduction
coli C.10 Somatic coliphages, unlike the male-specific coliphages, are coliphages that do not require the presence of an F
pilus to infect host cells. They represent a broad assortment of
coliphage types and have often been included in environmental
studies. Also presented here is a procedure that uses an alternate
host bacterium, Salmonella typhimurium WG49. That host has
been used by many laboratories to detect male-specific RNA
coliphages and it previously has been used in one standard
method protocol.11 Although a double-agar-layer plaque assay
has been specified in these procedures, a single-agar-layer
method also is presented and can be used as an alternate plaque
assay. Such a single-agar-layer assay has been incorporated into
a method developed for the examination of ground waters.12 One
additional procedure, a membrane filter method for assaying
100-mL (and larger) sample volumes, is also presented here.
Other methods are available elsewhere. One, an enrichment
method, has particular usefulness as a presence-absence assay.13
Unless otherwise indicated in the procedures described here,
refer to Sections 9060A and B for guidance about sample collection, preservation, and storage.

1. General Discussion

Coliphages are bacterial viruses that infect and replicate in

Escherichia coli. They are shed in human and animal feces.
Although coliphages are not known to be hazardous to human
beings, they are potentially important microorganisms for monitoring the microbial quality of water and wastewaters.1
The detection of coliphages has been of increasing interest
since it has become clear that bacterial monitoring of waters and
wastewaters may not adequately indicate the presence of viruses
in those waters.2 The presence of pathogenic human viruses in
waters is a public health concern. Waterborne outbreaks of viral
illnesses such as gastroenteritis and hepatitis A occur in the
United States and elsewhere.3,4 Detection of human enteric viruses in water and wastewaters, however, is beyond the capabilities of most water laboratories. Such detection traditionally has
required the use of cell culture techniques.5 These techniques are
expensive, require skilled personnel, and have been both timeand labor-intensive. Coliphage assays, on the other hand, are
relatively inexpensive, are easier to perform with trained personnel, and yield overnight results. Coliphage assays have been
proposed as an alternative to human virus assays as an indicator
of the viral quality of waters. 6,7
Recent progress has been made in the development of specific
coliphage methods for evaluating waters and wastewaters. Much
of this work has focused on the detection of the group of
coliphages known as the male-specific RNA coliphages (also
referred to as the F-specific RNA coliphages or FRNA coliphages).8 These coliphages are 20 to 30 nm in size, contain a
single-stranded RNA genome, and have an isometric morphology. They exclusively infect bacterial cells that possess an F
pilus, an appendage used for bacterial conjugation. Their significance lies in the fact that these coliphages are structurally
similar to many human RNA viruses found in fecally contaminated waters. In particular, they resemble viruses of the picornavirus and calicivirus families, which include poliovirus; coxsackievirus; Norwalk and other noroviruses; hepatitis A virus;
and hepatitis E virus. The human viruses cannot replicate in the
environment. Similarly, the male-specific RNA coliphages have
only limited replication in the environment at temperatures below 30C.9 Male-specific RNA coliphages also resemble many
human enteric viruses in being relatively resistant to disinfection
treatment practices. Because of these characteristics, malespecific RNA coliphages are promising candidate indicators of
human viruses in environmental waters.
In the procedures presented here, methods have been included
for the detection of the male-specific RNA coliphages using host
E. coli Famp and for the detection of somatic coliphages using E.

2. References
1. ARMON, R. & Y. KOTT. 1996. Bacteriophages as indicators of
pollution. Crit. Rev. Environ. Sci. Technol. 26:299.
2. GOYAL, S.M. 1983. Indicators of viruses. In G. Berg, ed. Viral
Pollution of the Environment. CRC Press, Boca Raton, Fla.
3. CRAUN, G.F. 1986. Waterborne Diseases in the United States. CRC
Press, Boca Raton, Fla.
4. WILLIAMS, F.P., JR. & E.A. AKIN. 1986. Waterborne viral gastroenteritis. J. Amer. Water Works Assoc. 78:34.
5. BERG, G., R.S. SAFFERMAN, D.R. DAHLING, D. BERMAN & C.J. HURST.
1984. USEPA Manual of Methods for Virology. EPA-600/4-84-13,
Environmental Monitoring & Support Lab., Off. Research & Development, U.S. Environmental Protection Agency, Cincinnati,
Ohio.
6. KOTT, Y., R. NETTA, S. SHOSHANA & N. BETZER. 1974. Bacteriophages as viral pollution indicators. Water Res. 8:165.
7. GRABOW, W.O.K., P. COUBROUGH, E.M. NUPEN & B.W. BATEMAN.
1984. Evaluation of coliphages as indicators of the virological
quality of sewage-polluted water. Water S.A. 10:7.
8. HAVELAAR, A.H., W.M. HOGEBOOM & R. POT. 1984. F specific RNA
bacteriophages in sewage: methodology and occurrence. Water Sci.
Technol. 17:645.
9. WOODY, M.A. & D.O. CLIVER. 1995. Effects of Temperature and
Host Cell Growth Phase on Replication of F-Specific RNA Coliphage. Appl. Environ. Microbiol. 61:1520.
10. FOUT, G.S., F.W. SCHAEFER, III, J.W. MESSER, D.R. DAHLING & R.E.
STETLER. 1996. ICR Microbial Laboratory Manual. EPA-600/R-95/
178, National Exposure Research Lab., Off. Research & Development, U.S. Environmental Protection Agency, Cincinnati, Ohio.
11. TECHNICAL COMMITTEE ISO/TC 147, WATER QUALITY, SUBCOMMITTEE
SC 4, MICROBIOLOGICAL METHODS. 1995. Water Quality Detection and Enumeration of Bacteriophages. Part 1: Enumeration of
F-specific RNA Bacteriophages. ISO 1075-1:1995E, ISO, Geneva,
Switzerland.

* Approved by Standard Methods Committee 2004.

Joint Task Group: Fred P. Williams, Jr. and Ronald E. Stetler (co-chairs), Samuel
R. Farrah, Pierre Payment, Mark D. Sobsey, William A. Yanko.

DETECTION OF COLIPHAGES (9224)/Somatic Coliphage Assay

12. U.S. ENVIRONMENTAL PROTECTION AGENCY. 2001. Method 1602:

Male-specific (F) and Somatic Coliphage in Water by Single
Agar Layer (SAL) Procedure. EPA 821-R-01-029, U.S. Environmental Protection Agency, Off. Water, Washington, D.C.

13. U.S. ENVIRONMENTAL PROTECTION AGENCY. 2001. Method 1601:

Male-specific (F) and Somatic Coliphage in Water by Two-step
Enrichment Procedure. EPA 821-R-01-030, U.S. Environmental
Protection Agency, Off. Water, Washington, D.C.

9224 B. Somatic Coliphage Assay

i. Test tubes, 16- 150-mm, screw-capped and with closures.
j. Water bath set at 44.5 1C.

1. General Discussion

This plaque assay procedure for detecting somatic coliphages

is a variant of the widely used double-agar-layer method;1 it is
based on the somatic coliphage assay procedure developed for
regulatory purposes.2,3 The procedure can be used, without supplementary methods, to assay small volumes of water and wastewater samples directly. In conjunction with large-scale concentration methods used to detect enteric viruses (Section 9510), the
procedure also can be used to assay samples of much larger
volume (100 L). However, the use of electronegative filters at
pH 3.5 is not recommended, because substantial loss of coliphage viability may occur. It is advisable to predetermine the
suitability of any large-volume method in measured coliphage
recovery trials. This plaque assay procedure uses E. coli C, one
of the most effective host bacteria for detecting somatic coliphages.4 Plaques produced with this host are readily distinguished, making plaque quantification relatively simple. The
major advantage in detecting somatic coliphages is that they
frequently are found in waters and wastewaters in greater abundance than the male-specific RNA coliphages. This can be an
important consideration, especially for the assay of waters where
pollution indices are expected to be low. The somatic coliphage
group, however, is composed of different phage types (including
both tailed and non-tailed phages) that exhibit widely varied
characteristics. Somatic coliphages can replicate in the environment if a suitable host is present. Because of these factors, the
somatic coliphage group may be less representative of human
enteroviruses than the more uniform RNA phages of the malespecific coliphage group. The somatic coliphage group thus may
not be the best group to serve as a specific indicator for human
viruses in waters and wastewaters. However, the detection of
somatic phages using E. coli C may be useful as a general
indicator of water quality.5
2.

3.

Media and Reagents

Adjust amount of media prepared proportionally to the number of samples. Use reagent-grade water (see Table 9020:II) in
preparing media and reagents.
a. Beef extract, 1.5%: Dissolve 1.5 g beef extract powder and
0.375 g glycine (final glycine concentration 0.05M) in 90 mL
water. Adjust pH to 7.0 to 7.5, if necessary, and bring final
volume to 100 mL with water. Autoclave at 121C for 15 min
and use at room temperature. Store at 4C.
b. Glycerol solution, 50%: Add equal volumes of water and
undiluted glycerol. Autoclave at 121C for 15 min. Store at 4C.
c. Tryptone agar slants:
Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.0
Yeast extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
0.1
Glucose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
0.1
NaCl . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
0.8
CaCl2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
0.022
Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.2
Reagent-grade water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100

g
g
g
g
g
g
mL

With gentle mixing, add ingredients to water in a 250-mL

flask. Dissolve and sterilize by autoclaving at 121C for 15 min
and dispense 8-mL portions into 16- 150-mm test tubes with
tube closures. Prepare slants by letting agar solidify with the
tubes held at about a 20 angle. Slants may be stored at 4C for
up to 3 months.
d. Tryptone bottom agar: Prepare before sample analysis,
using ingredients and concentrations listed for tryptone agar
slants, but use 1.5 g agar. After autoclaving at 121C for 15 min,
pipet 15-mL portions aseptically into sterile 100- 15-mm petri
dishes and let agar harden. Store dishes in sleeve bags at 4C for
up to 2 weeks and warm to room temperature for 1 h before use.
e. Tryptone broth: Prepare as for tryptone agar slants, excluding agar. Autoclave at 121C for 15 min, and allow to cool
before use. Broth may be stored at 4C for up to 3 months in
sterile, capped containers.
f. Tryptone dilution tubes: Aseptically, dispense 9-mL portions of sterile tryptone broth into 16- 150-mm screw-capped
test tubes presterilized by autoclaving at 121C for 15 min.
g. Tryptone top agar: Use ingredients and concentrations
listed for tryptone agar slants, but use 0.7 g agar. Autoclave at
121C for 15 min and place in the 44.5 1C water bath. Agar
may be stored at 4C for up to 3 months in sterile, capped
containers. Before using stored agar, melt solidified agar, then
place in 44.5 1C water bath.

Apparatus

a. Cryovials, 2-mL.
b. Erlenmeyer flasks, 125- and 250-mL, and 2-L.
c. Graduated cylinders, 100- and 500-mL.
d. Inoculating loop.
e. Laboratory balance.
f. Pipets, 1-, 5-, and 10-mL.
g. Petri dishes, 100- 15-mm.
h. Filters, 0.22-m. When passing material containing phage,
always pass about 10 mL 1.5% beef extract through filter just
before use to minimize phage adsorption to filter. Use presterilized filters or sterilize filters before use by autoclaving at
121C for 15 min. At the time of use, use sterile technique to
place filters into sterile sample filtration apparatus.
2

DETECTION OF COLIPHAGES (9224)/Somatic Coliphage Assay

4.

Procedure

premature solidifying of agar. Add 0.1 mL of host culture to each

of the 12 test tubes. Add 1 mL tryptone broth to test tube serving
as negative control. Add 1 mL X174 preparation (30 to 80
PFU/mL) to test tube serving as positive control. To each of the
remaining 10 tubes, add 1 mL sample. For each tube, mix and
immediately pour contents over bottom agar of a petri dish that
has been suitably labeled. Tilt and rotate dish to spread suspension evenly and place it on a level surface to let agar solidify.
Incubate at 36.5 2C overnight and examine for plaques the
following day. Count total number of plaques on the ten dishes
receiving the sample. Calculate the somatic coliphage concentration according to the formula:

a. Storage of E. coli C host culture: For short-term storage

inoculate an Escherichia coli C* host culture onto tryptone agar
slants with a sterile inoculating loop by spreading the inoculum
evenly over entire slant surface. Incubate the culture overnight at
36.5 2C. Store at 4C for up to 2 weeks. For long-term
storage inoculate a 5- to 10-mL tube of tryptone broth with host
culture. Incubate overnight at 36.5 2C. Add 1/5th volume of
50% glycerol solution. Dispense into 1-mL portions in 2-mL
cryovials and store at 70C, or lower.
b. Preparation of host: Inoculate 5 mL tryptone broth with E.
coli C from a slant with an inoculating loop and incubate
overnight (16 20 h) at 36.5 2C. Transfer 1.5 mL of the
overnight culture to 30 mL tryptone broth in a 125-mL flask and
incubate for 4 h at 36.5 2C with gentle shaking. The amount
of inoculum and broth used can be altered proportionally according to need.
c. Preparation of coliphage X174 positive control: Rehydrate a stock culture of somatic coliphage X174 according to
supplier directions and store at 4C. Prepare a 30-mL culture of
E. coli C as described above. Incubate for 2 h at 36.5 2C with
shaking. Add 1 mL rehydrated phage stock and incubate for an
additional 4 h at 36.5 2C. Filter through a beef-extract-treated
0.22-m filter (see 2h). Prepare 107, 108, and 109 dilutions
of the filtrate using tryptone dilution tubes. These dilutions
should be sufficient for most X174 stocks. Some stocks may
require higher or lower dilutions. Add 3 mL melted tryptone top
agar held in the 44.5 1C water bath to fifteen 16- 150-mm
test tubes. Keep these test tubes in heated water bath to avoid
premature solidifying of agar. Add 0.1 mL host culture to each
test tube. Add 1 mL 109 dilution to each of five test tubes.
Add 1 mL 108 dilution to five additional tubes and 1 mL
107 dilution to the remaining five tubes. Label tubes with
appropriate dilution. For each tube, mix and immediately pour
contents over bottom agar of a petri dish labeled with the dilution
assayed. Rotate dish to spread suspension evenly and place it on
a level surface to let agar solidify. Incubate at 36.5 2C
overnight and examine for plaques the following day. Count
number of plaques on each dish. To determine the titer of the
stock filtrate, use the dilution with dishes exhibiting 20 to 100
plaques. Average the plaque counts on these five dishes and
multiply result by the reciprocal of the dilution to obtain that
titer. For use as a positive control in the coliphage assay, dilute
stock filtrate to 30 to 80 PFU/mL in tryptone broth. Store original
filtrate and diluted positive control preparation at 4C. Before
using the positive control preparation for the first time, assay 10
mL by adding 1-mL volumes of the preparation to ten test tubes
containing agar and host culture, pouring their contents into ten
petri dishes and incubating overnight at 36.5 2C. Count
plaques on all dishes and divide by 10. If result is not 30 to 80,
adjust dilution of the positive control sample and re-assay.
d. Assay procedure: Add 3 mL melted tryptone top agar held
at 44.5 1C to each of ten 16- 150-mm test tubes for sample
assay and to each of two additional test tubes to serve as negative
and positive controls. Hold test tubes in water bath to avoid

C a (P 10) D

where:
Ca somatic coliphage concentration, PFU/mL,
P total number of plaques from the 10 dishes, and
D reciprocal of dilution made on the inoculum before plating
(D 1 for undiluted samples).

If the sample dishes are completely lysed or yield plaques that

are too numerous to count, dilute sample and assay again. In the
calculation for diluted samples, D will be greater than 1 (e.g., D
10, for a sample diluted 1:10 or D 100, for a sample diluted
1:100).
If the sample assayed is the product of a concentration procedure, such as an eluate from a large-volume filter sampling of
water or wastewater, calculate coliphage concentration of the
sampled water according to the formula:
Cb

Ca Va
Vb

where:
Cb somatic coliphage concentration of sampled water, PFU/L,
Ca coliphage concentration of concentrated material, PFU/mL,
Va volume of that material, mL, and
Vb volume of water processed in sampling procedure, L.

The resultant concentration generally is reported as PFU/L or

PFU/100 L. If the sample dishes are completely lysed or yield
plaques that are too numerous to count, dilute sample and assay
again. Count plaques on the positive control dish. Maintain a
record of the plaque count as a check on the virus sensitivity of
the E. coli C host. Assay any water eluate samples again where
the positive control counts are more than one log below their
normal average. Should plaques be detected on the negative
control dish, discard assay results and repeat assay.
5. References
1. ADAMS, M.H. 1959. Bacteriophages. Interscience Publishers, New
York, N.Y.
2. FOUT, G.S., F.W. SCHAEFER, III, J.W. MESSER, D.R. DAHLING & R.E.
STETLER. 1996. ICR Microbial Laboratory Manual. EPA-600/R-95/
178, National Exposure Research Lab., Off. Research & Development, U.S. Environmental Protection Agency, Cincinnati, Ohio.

* American Type Culture Collection Product No. 13706.

American Type Culture Collection Product No. 13706-B1.

DETECTION OF COLIPHAGES (9224)/Male-Specific Coliphage Assay Using Escherichia coli Famp

3. U.S. ENVIRONMENTAL PROTECTION AGENCY. 1996. Information collection requirements ruleNational primary drinking water regulations: Monitoring requirements for public drinking water supplies: Cryptosporidium, Giardia, viruses, disinfection byproducts,
water treatment plant data and other information requirements.
Federal Register 61(94):24354 (May 14, 1996).

4. HAVELAAR, A.H. & W.M. HOGEBOOM. 1983. Factors affecting the

enumeration of coliphages in sewage and sewage-polluted waters.
Antonie van Leeuwenhoek 49:387.
5. WENTSEL, R.S., P.E. ONEILL & J.R. KITCHENS. 1982. Evaluation of
coliphage detection as a rapid indicator of water quality. Appl.
Environ. Microbiol. 43:430.

9224 C. Male-Specific Coliphage Assay Using Escherichia coli Famp

1.

General Discussion

a. Ampicillin solution: Dissolve 0.15 g ampicillin in 100 mL

water and filter with the 0.22-m filter (beef extract pretreatment
not necessary). Store at 4C.
b. Beef extract, 1.5%: See 9224B.3a.
c. Glycerol solution, 50%: See 9224B.3b.
d. Ribonuclease (RNase) solution: Dissolve 100 mg RNase
containing 50 to 100 Kunitz units/mg in 100 mL water by
heating to 100C for 10 min. Store at 20C in 0.5-mL portions.
e. Streptomycin solution: Dissolve 0.15 g streptomycin sulfate
in 100 mL water and filter-sterilize with 0.22-m filter (beef
extract pretreatment not necessary). Store at 4C.
f. Tryptone agar slants: Mix ingredients, dissolve, and sterilize
as directed in 9224B.3c. Let autoclaved agar equilibrate in water
bath set at 44.5 1C, then add 1.0 mL filtered ampicillin
solution and 1.0 mL filtered streptomycin solution/100 mL volume of warm agar. Dispense portions, prepare, and store as
directed in 9224B.3c.
g. Tryptone bottom agar: See 9224B.3d. After autoclaving at
121C for 15 min, let agar equilibrate in water bath set at 44.5
1C, and then add 1.0 mL filtered ampicillin solution and 1.0 mL
filtered streptomycin solution/100-mL volume of warm agar.
Dispense portions and store as directed in 9224B.3d.
h. Tryptone broth: Use ingredients listed in 9224B.3c, excluding agar. Sterilize by autoclaving at 121C for 15 min. Cool and
add 1.0 mL filtered ampicillin solution and 1.0 mL filtered
streptomycin solution/100 mL broth. Store at 4C. Use for
growth medium. Store as directed in 9224B.3c.
i. Tryptone dilution tubes: See 9224B.3f.
j. Tryptone top agar: Use ingredients listed in 9224B.3c, but
use 0.7 g agar. After autoclaving at 121C for 15 min, let agar
equilibrate in a 44.5 1C water bath, then add 1.0 mL filtered
ampicillin solution and 1.0 mL filtered streptomycin solution
/100 mL volume of warm agar. Store as directed in 9224B.3g.

Like the somatic coliphage assay, this male-specific assay

is a variant of the double-agar-layer method1 and is based on
procedures developed for regulatory purposes.2 Similarly, this
male-specific assay can be used, without supplementary methods, to assay small volumes of water and wastewater samples
directly. In conjunction with large-scale concentration methods used to detect enteric viruses (Section 9510), the procedure also can be used to assay larger-volume samples (100
L). However, the use of electronegative filters at pH 3.5 is not
recommended because substantial loss of coliphage viability
may occur. Determine the suitability of any large-volume
method in measured coliphage recovery trials, before using it.
This procedure uses E. coli Famp as the host bacterium.3 This
strain is resistant to ampicillin and streptomycin antibiotics. It
is one of two bacterial strains that have been developed to
selectively detect male-specific coliphages. For a procedure
using the other bacterial strain, Salmonella typhimurium WG49,
see Section 9224D. It has not been universally established that one
strain performs better than the other. Individual laboratories may
evaluate both strains before selecting one for routine use. Malespecific plaques can be difficult to recognize in comparison to
somatic plaques. They are generally less well-defined than somatic
plaques. Use diligence when examining dishes for male-specific
plaques. As male-specific DNA coliphages and some somatic coliphages also can produce plaques on host E. coli Famp, preferably
confirm that plaques quantified by the assay are caused by RNA
phages. An RNase procedure that permits differential quantification
of the RNA coliphages also is presented below. The advantage in
detecting male-specific RNA coliphages is that these coliphages
more closely resemble human enteroviruses than do the varied
phage types of the somatic group. These male-specific RNA coliphages appear to be useful as a model for human viruses.4,5 As an
indicator of the presence of human viruses in waters and wastewaters, their role remains to be fully established.

2.

4.

Procedure

a. Storage of E. coli Famp host culture: Follow procedures of

9224B.4a, using Escherichia coli Famp.* For this strain, use
media containing ampicillin and streptomycin as described
above.
b. Preparation of host: Follow procedures of 9224B.4b. Place
on ice until used for assay to prevent loss of F pili (use within
3 h).

Apparatus

See 9224B.2.

3. Media and Reagents

Adjust amount of media prepared proportionally to the number of samples. Use reagent-grade water (see Table 9020:II) in
making media and reagents.

* American Type Culture Collection Product No. 700891.

DETECTION OF COLIPHAGES (9224)/Male-Specific Coliphage Assay Using Salmonella typhimurium WG49

Ca male-specific RNA coliphage concentration, PFU/mL,

c. Preparation of coliphage MS2 positive control: Rehydrate a

stock culture of male-specific RNA coliphage MS2 according
to supplier directions and store at 4C. Prepare a 30-mL culture
of E. coli Famp as described in 9224B.4b. Incubate and proceed
as directed in 9224B.4c, using the MS2 and E. coli Famp.
d. Assay procedure: Proceed as in 9224B.4d, using MS2 as
positive control. Calculate Ca as directed. If the sample dishes
are completely lysed or yield plaques that are too numerous to
count, dilute sample and assay again.
If the sample assayed is the product of a concentration procedure, calculate Cb as directed in 9224B.4d.
e. Assay procedure with additional confirmation: Proceed as
in 9224B.4d, using MS2 as positive control, but assay an additional 10 mL of sample. For the additional 10 mL of sample, add
RNase solution to the melted tryptone top agar to a concentration
of 40 g/mL. Before pouring tube contents to petri dishes, label
dishes so that the 10 dishes containing RNase are readily distinguished from the 10 dishes without RNase. Calculate the malespecific RNA coliphage concentration (Ca) in PFU/mL according to the formula:

P total number of plaques from the 10 dishes without RNase,

PRNase total number of plaques from the 10 dishes with RNase,
and
D reciprocal of the dilution made on the inoculum before
plating.

If the sample assayed is the product of a concentration procedure,

calculate coliphage concentration as previously described in d.
5. References
1. ADAMS, M.H. 1959. Bacteriophages. Interscience Publishers, New
York, N.Y.
2. FOUT, G.S., F.W. SCHAEFER, III, J.W. MESSER, D.R. DAHLING & R.E.
STETLER. 1996. ICR Microbial Laboratory Manual. EPA-600/R-95/
178, National Exposure Research Lab., Off. Research & Development, U.S. Environmental Protection Agency, Cincinnati, Ohio.
3. DEBARTOLOMEIS, J. & V.J. CABELLI. 1991. Evaluation of an Escherichia coli host strain for enumeration of F male-specific bacteriophages. Appl. Environ. Microbiol. 57:1301.
4. IAWPRC STUDY GROUP ON HEALTH RELATED WATER MICROBIOLOGY.
1991. Bacteriophages as model viruses in water quality control.
Water Res. 25:529.
5. SOBSEY, M.D., D.A. BATTIGELLI, T.R. HANDZEL & K.J. SCHWAB. 1995.
Male-specific coliphages as indicators of viral contamination of
drinking water. American Water Works Association Research Foundation, Denver, Colo.

C a [(P 10) (P RNase 10)] D

where:

American Type Culture Collection Product No. 15597-B1.

9224 D. Male-Specific Coliphage Assay Using Salmonella typhimurium WG49

1.

General Discussion

similarity to human enteroviruses not found with the somatic coliphage group. Assay of these male-specific phages can be useful in
evaluating water and wastewater treatment processes but as a specific indicator of the presence of human viruses, their role remains
to be clearly established.

This male-specific assay uses Salmonella typhimurium WG49 as

host bacterium. It is a variant of the double-agar-layer method1 and
has been adapted from several published procedures.2,3 The assay
can be used with large-scale concentration methods (see 9224C.1).
The use of S. typhimurium WG49 in this assay effectively eliminates plaque production by somatic coliphages. This is an advantage
over the E. coli Famp-based assay. However, somatic salmonella
phages do form plaques with S. typhimurium WG49 and these
somatic salmonella phages also may be present in waters under
investigation. Because it has not been established that either S.
typhimurium WG49 or E. coli Famp is universally superior as host
bacterium for the male-specific phages, individual laboratories may
find it useful to evaluate both assays before making a selection.
With either assay, take care in examining dishes for male-specific
plaques and confirm that the plaques detected are indeed caused by
RNA coliphages and not other phages. An RNase procedure for this
purpose is presented below. Also presented below is a treatment to
neutralize the somatic salmonella phage content of samples before
assay. This treatment may be necessary if assay interference by
somatic salmonella phages is encountered.4-6 Such interference may
be expected because somatic salmonella phages do outnumber
male-specific phages in some waters.7 The advantage of the S.
typhimurium WG49-based assay, as with the E. coli Famp-based
assay, is that the targeted male-specific RNA phages possess a

2.

Apparatus

See 9224B.2.
3.

Media and Reagents

Adjust amount of media prepared proportionally to the number of samples. Use reagent-grade water (see Table 9020:II) in
preparing media and reagents.
a. Beef extract: See 9224B.3a.
b. Glycerol solutions, 50%: See 9224B.3b.
c. Kanamycin solution: Dissolve 1 g kanamycin monosulfate
in 100 mL water and filter with the 0.22-m filter (beef extract
pretreatment not necessary). Store at 4C.
d. Lipopolysaccharide solution (LPS): Prepare stock solution
of phenol-extracted S. typhimurium LPS* by dissolving the

* Sigma Chemical Co., St. Louis, MO, item #L6511, or equivalent.

DETECTION OF COLIPHAGES (9224)/Single-Agar-Layer Method

lyophilized LPS in sterile phosphate-buffered saline to yield an

LPS concentration of 2 mg/mL. Store at 4C.
e. Nalidixic acid solution: Dissolve 1 g nalidixic acid in 100
mL water and filter with the 0.22-m filter (beef extract pretreatment not necessary). Store at 4C.
f. Phosphate-buffered saline: Dissolve 0.8 g NaCl, 20 mg KCl,
12 mg KH2PO4, and 91 mg Na2HPO4 in 100 mL water. Sterilize
by autoclaving at 121C for 15 min.
g. Ribonuclease (RNase) solution: See 9224C.3d.
h. Tryptone agar slants: Mix ingredients, dissolve and sterilize
as directed in 9224B.3c. Let autoclaved agar equilibrate in water
bath at 44.5 1C, then add 1 mL filtered nalidixic acid solution
and 0.2 mL filtered kanamycin solution/100 mL volume of warm
agar. Dispense portions and store as directed in 9224B.3c.
i. Tryptone bottom agar: See 9224B.3d. After autoclaving at
121C for 15 min, let agar equilibrate in water bath at 44.5
1C, then add 1 mL filtered nalidixic acid solution and 0.2 mL
filtered kanamycin solution/100 mL volume of warm agar. Dispense portions and store as directed in 9224B.3d.
j. Tryptone broth: Use ingredients listed in 9224B.3c, excluding agar. Sterilize by autoclaving at 121C for 15 min. Cool and
add 1 mL filtered nalidixic acid solution and 0.2 mL filtered
kanamycin solution/100 mL broth. Use for growth medium.
Store as directed in 9224B.3c.
k. Tryptone dilution tubes: See 9224B.3f.
l. Tryptone top agar: Use ingredients listed in 9224B.3c, but
use 0.7g agar. After autoclaving at 121C for 15 min, let agar
equilibrate in a 44.5 1C water bath, then add 1 mL filtered
nalidixic acid solution and 0.2 mL filtered kanamycin solution/
100 mL volume of warm agar. Store as directed in 9224B.3g.
4.

supplier directions and store at 4C. Prepare a 30-mL culture of S.

typhimurium WG49 as in 9224B.4b. Incubate and proceed as directed in 9224B.4c, using the MS2 and S. typhimurium WG49.
d. Assay procedure: Proceed as in 9224B.4d, using MS2 as
positive control. Calculate Ca as directed. If the sample dishes
are completely lysed or yield plaques that are too numerous to
count, dilute sample and assay again.
If the sample assayed is the product of a concentration procedure, calculate Cb as directed in 9224B.4d.
e. Assay procedure with additional confirmation: See 9224C.4e.
f. Sample treatment procedure to reduce somatic salmonella
phages: Results of the confirmation procedure may reveal assay
interference by somatic salmonella phages present in some water
or wastewater samples. To neutralize these somatic salmonella
phages before assay, add enough LPS stock solution to the
sample to produce a final LPS concentration of 20 g/mL. Mix
thoroughly and hold at ambient temperature for 15 to 30 min.
Then assay as described above.

5. References
1. ADAMS, M.H. 1959. Bacteriophages. Interscience Publishers, New
York, N.Y.
2. HAVELAAR, A.H. & W.M. HOGEBOOM. 1984. A method for the
enumeration of male-specific bacteriophages in sewage. J. Appl.
Bacteriol. 56:439.
3. TECHNICAL COMMITTEE ISO/TC 147, WATER QUALITY SUBCOMMITTEE SC
4, MICROBIOLOGICAL METHODS. 1995. Water Quality Detection and
Enumeration of Bacteriophages. Part 1: Enumeration of F-specific
RNA Bacteriophages. ISO 1075-1:1995E, ISO, Geneva, Switzerland.
4. RHODES, M.W. & H.I. KATOR. 1991. Use of Salmonella typhimurium
WG49 to enumerate male-specific coliphages in an estuary and
watershed subject to nonpoint pollution. Water Res. 251315.
5. HANDZEL, T.R., R.M. GREEN, C. SANCHEZ, H. CHUNG & M.D. SOBSEY.
1993. Improved specificity in detecting F-specific coliphages in
environmental samples by suppression of somatic phages. Water
Sci. Technol. 27:123.
6. WILLIAMS, F.P. & R.E. STETLER. 1994. Detection of FRNA coliphages in groundwater: interference with the assay by somatic
salmonella bacteriophages. Lett. Appl. Microbiol. 19:79.
7. STETLER, R.E. & F.P. WILLIAMS. 1996. Pretreatment to reduce somatic salmonella phage interference with FRNA coliphage assays:
successful use in a one-year survey of vulnerable groundwaters.
Lett. Appl. Microbiol. 23:49.

Procedure

a. Storage of S. typhimurium WG49 host culture: Follow

procedures of 9224B.4a, using S. typhimurium WG49 host
culture. S. typhimurium WG49 is resistant to nalidixic acid and
kanamycin. Accordingly, use media containing nalidixic acid
and kanamycin as described above.
b. Preparation of host: Follow procedures of 9224B.4b. Place on
ice until used in assay to prevent loss of F pili (use within 3 h).
c. Preparation of coliphage MS2 positive control: Rehydrate a
stock culture of male-specific RNA coliphage MS2 according to
American Type Culture Collection Product No. 700730.
American Type Culture Collection Product No. 15597-B1.

9224 E. Single-Agar-Layer Method

1.

100-mL volumes of water and wastewater.1 Although originally

described for use with E. coli C, the procedure is suitable for use
with E. coli Famp and S. typhimurium WG49 host bacteria. The
general procedure is described below. For specific details relating to each of the three bacterial hosts, refer to Sections 9224B,
C, and D.

General Discussion

This method can be used as an alternative to the double-agarlayer procedures described above. It uses a single-agar-layer
format and larger petri dishes. The method enables more sample
material to be assayed per dish and can be used to directly assay

DETECTION OF COLIPHAGES (9224)/Single-Agar-Layer Method

2.

Apparatus

in the somatic coliphage assay. E. coli CN-13 is a nalidixic-acidresistant variant of E. coli C; using it permits the addition of
nalidixic acid to the assay media (see Section 9224D) to hinder
growth of indigenous bacteria. E. coli CN-13 appears equivalent
to E. coli C in somatic coliphage detection.2
e. Assay procedure: Place 100 mL sample in the 44.5 1C
water bath for 3 min. Add 5 mL CaCl2 solution and 5 mL
appropriate host bacterium preparation to the warmed sample.
Mix inoculated sample with 100 mL melted tryptone agar at 44.5
1C and distribute to eight 150- 15-mm petri dishes. For a
positive control, mix 1 mL of appropriate positive control preparation (30 to 80 PFU/mL) and 1 mL host bacterium with 12.5
mL warmed agar that has been diluted with an equal volume of
warm sterile water. Pour to a single 150- 15-mm petri dish.
Repeat for a negative control but omit the 1 mL of phage
preparation. Incubate the inoculated dishes at 36.5 2C overnight and examine for plaques the following day. Count total
number of plaques on the eight dishes that received the sample;
total is coliphage concentration/100 mL sample. However, when
assaying for male-specific RNA coliphages using E. coli Famp
and S. typhimurium WG49, this count could include some somatic and male-specific DNA coliphages or some somatic salmonella phages present in the sample. For appropriate procedures to address the presence of undesired phage, see s 4f and
g, below.
f. Assay procedure with additional confirmation (male-specific
assays only): Assay an additional 100-mL sample portion as
described above, but add RNase solution to the melted tryptone
agar. For the additional material, the melted tryptone agar should
contain RNase at a concentration of 60 g/mL (before sample is
added). Appropriately label dishes so that the eight dishes containing RNase are readily distinguished from the eight dishes
without RNase. Calculate the male-specific coliphage concentration according to the formula:

See 9224B.2. Use 150- 15-mm petri dishes for sample

assay.
3.

Media and Reagents

The amount of media prepared may be increased proportionally to the number of samples to be analyzed. Use reagent-grade
water (see Table 9020:II) in preparing media and reagents. For
all host cultures:
a. Beef extract: See 9224B.3a.
b. Calcium chloride solution: Add 22 g CaCl2 to 50 mL water
and sterilize by autoclaving at 121C for 15 min. Use at room
temperature.
c. Glycerol solution, 50%: See 9224B.3b.
d. Tryptone agar slants: See 9224B.3c.
e. Tryptone agar:
Tryptone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.0
Yeast extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
0.2
Glucose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
0.2
NaCl . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.6
CaCl2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
0.022
Agar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.2
Reagent-grade water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100

g
g
g
g
g
g
mL

Sterilize by autoclaving at 121C for 15 min. Place in the 44.5

1C water bath.
f. Tryptone broth: See 9224B.3d, excluding the agar.
g. Tryptone dilution tubes: See 9224B.3f.
For E. coli Famp host only:
h. Ampicillin solution: See 9224C.3a.
i. Streptomycin solution: See 9224C.3e.
For S. typhimurium WG49 host only:
j. Kanamycin solution: See 9224D.3c.
k. Lipopolysaccharide solution (LPS): See 9224D.3d.
l. Nalidixic acid solution: See 9224D.3e.

C a P P RNase

where:
Ca male-specific coliphage concentration, PFU/100 mL,
P total number of plaques from dishes without RNase, and
PRNase total number of plaques from dishes with RNase.

For E. coli Famp and S. typhimurium WG49 hosts:

m. Ribonuclease (RNase) solution: See 9224C.3d.
4.

g. Sample treatment procedure to reduce somatic salmonella

phages (WG49 host assay only): To neutralize these somatic
salmonella phages before assay, add enough LPS stock solution
to sample to produce a final LPS concentration of 20 g/mL.
(NOTE: Add LPS after adding CaCl2 to sample and before adding
WG49 host.) Mix thoroughly and hold at ambient temperature
for 15 to 30 min. Then add WG49 host and assay as described
above.

Procedure

a. Storage of host cultures: Follow procedures of 9224B.4a,

using appropriate host culture.
b. Preparation of host: Follow procedures of 9224B.4b.
c. Preparation of coliphage positive controls: Rehydrate a
stock culture of appropriate positive control according to supplier directions and proceed as directed in 9224B.4c.
d. Sample filtration (somatic coliphage assay, only): The bacteria present in samples for analysis can interfere with the plaque
assay. To eliminate such interference, filter sample before assay.
Use sterile 0.22-m filter unit. To minimize phage adsorption to
the filter, pass about 10 mL 1.5% beef extract through filter
before passing sample through filter. NOTE: As an alternative to
sample filtration, use E. coli strain CN-13* in place of E. coli C

5. References
1. GRABOW, W.O.K. 1986. Practical direct plaque assay for coliphages
in 100-mL samples of drinking water. Appl. Environ. Microbiol.
52:430.
2. SOBSEY, M.D., A. AMANTI & T.R. HANDZEL. 1996. Detection and
occurrence of coliphage indicator viruses in water. In Proc. Amer.
Water Works Assoc. Water Quality Technol. Conf., New Orleans,
La., Nov. 1216, 1995. American Water Works Assoc., Denver,
Colo.

* American Type Culture Collection Product No. 700609.

DETECTION OF COLIPHAGES (9224)/Membrane Filter Method

9224 F. Membrane Filter Method

1.

General Discussion

b. Magnesium chloride solution, 2M: Dissolve 19.04 g MgCl2

in 100 mL water and sterilize by autoclaving at 121C for 15
min. Use at room temperature.
c. Glycerol solution, 50%: See 9224B.3b.
d. Phosphate-buffered saline: Dissolve 0.8 g NaCl, 20 mg
KCl, 12 mg KH2PO4, and 91 mg Na2HPO4 in 100 mL water.
Sterilize by autoclaving at 121C for 15 min.
e. Tetrazolium violet solution: Add 3 g tetrazolium violet to
100 mL water and filter-sterilize with 0.45-m filter. Use 1 mL
tetrazolium violet solution/100 mL agar.
f. Tryptone agar slants: See 9224B.3c.
g. Tryptone agar: Use ingredients and concentrations listed for
tryptone broth, and add 0.9 g agar and 15 mg MgSO4/100 mL of
broth. Sterilize by autoclaving at 121C for 15 min. Place autoclaved agar in the 44.5 1C water bath. After agar has cooled,
add 1 mL filtered polysorbate solution and 1 mL filtered tetrazolium violet solution/100 mL volume of warm agar. Then add
2 mL of appropriate host bacterium preparation per 100 mL agar.
Dispense agar by pipet in 5-mL portions to sterile 60- 15-mm
petri dishes and let agar harden. If plates are to be used the
following day, store at 4C overnight and warm to room temperature for 1 h before use.
h. Tryptone broth: See 9224B.3e.
i. Tryptone dilution tubes: See 9224B.3f.
j. Polysorbate solution: Add 30 g polysorbate* to 100 mL
water and filter with 0.22-m filter. Use 1 mL/100 mL agar.

This method uses a filter-adsorption technique to recover

coliphages from water samples. With this method, 100-mL and
larger volume samples can be assayed effectively by a simple
procedure in which the sample is first passed through a membrane filter. Coliphages present in the sample are adsorbed onto
the filter and the phage-adsorbed filter is then directly plaque
assayed.1,2 The advantage of this method is that, unlike Methods
B through E, it does not require multiple assay dishes for each
sample. A single assay dish is utilized for each phage-adsorbed
filter. This significantly reduces the time and materials required.
The disadvantage is that extraneous material, which can interfere
with subsequent plaque assay, can accumulate on filters as water
is passed through them. The amount of accumulation depends on
the quality of the water being sampled and increases in proportion to the sample volume. Keep sample volumes as small as
possible to prevent observable accumulation of discoloring material on the filter. However, with poor-quality waters, rapid
accumulation may be observed with even small sample volumes.
With such waters, use larger-diameter filters to increase sample
volume. In the procedure below, the use of 47-mm membrane
filters is described, but 90-mm membrane filters may be used if
sample volumes prove inadequate with the smaller filters. Although originally described for use with S. typhimurium WG49,
the procedure is suitable for use with E. coli C, and E. coli Famp
host bacteria. The general procedure is described below. For
specific details relating to each of the three bacterial hosts, refer
to the preceding sections (Sections 9224B, 9224C, and 9224D).
Although a membrane-filter method is presented here, other
approaches also may prove effective for the assay of 100-mL and
larger volume samples. Methods such as the most probable
number method could be adapted to larger volumes,3 and such
non-filter-based methods probably would be less susceptible to
poor water quality. Consider their use if water conditions are
encountered that make the membrane filter method impractical.
2.

For E. coli Famp host only:

k. Ampicillin solution: See 9224C.3a.
l. Streptomycin solution: See 9224C.3e.
For S. typhimurium host only:
m. Kanamycin solution: See 9224D.3c.
n. Lipopolysaccharide solution (LPS): See 9224D.3d.
o. Nalidixic acid solution: See 9224D.3e.
For E. coli Famp and S. typhimurium WG49 hosts:
p. Ribonuclease (RNase) solution: See 9224C.3d.

Apparatus

4.

See 9224B.2a through f and h through j, and in addition:

a. Petri dishes, 60 15 mm.
b. Sample vacuum filtration apparatus, including a 47-mm
membrane filter holder and filtrate receiving vessel. Sterilize
apparatus before use by autoclaving at 121C for 15 min.
c. Sample membrane filters, 0.45-m pore size with a 47-mm
diam and a cellulose nitrate and acetate composition. Sterilize
filters before use by autoclaving at 121C for 15 min (or use
pre-sterilized filters). At the time of use, use sterile technique to
place sterile filter into sterile sample filtration apparatus.
3.

Procedure

a. Storage of host cultures: Follow procedures of 9224B.4a,

using appropriate host culture.
b. Preparation of host: Follow procedures of 9224B.4b.
c. Preparation of coliphage positive controls: Rehydrate a
stock culture of appropriate positive control according to supplier directions and proceed as directed in 9224B.4c.
d. Assay procedure: For each 100 mL sample, use a single
petri dish assay. After dishes have been prepared, add 2.5 mL
MgCl2 solution to sample to yield a final concentration of 0.05M.
Pass magnesium-chloride-supplemented sample through sterile
sample filter at a rate sufficient to clear entire 102.5 mL within
1 to 3 min. Using sterile technique, remove filter from sample
filter apparatus. Taking care to prevent formation of bubbles,
apply filter, face down, onto surface of one of the pre-poured
agar petri dishes. Incubate at 36.5 2C overnight and examine

Media and Reagents

The amount of media prepared may be increased proportionally to the number of samples to be analyzed. Use reagent-grade
water (see Table 9020:II) in preparing media and reagents.
For all host cultures:
a. Beef extract: See 9224B.3a.

* Tween 80 or equivalent.

DETECTION OF COLIPHAGES (9224)/Membrane Filter Method

for plaques the following day. Count total number of plaques.

That total is the coliphage concentration/100 mL sample. However, in assays for the male-specific RNA coliphages using E.
coli Famp and S. typhimurium WG49, this count could include
some somatic and male-specific DNA coliphages or some somatic salmonella phages present in the sample. For appropriate
procedures to address the presence of undesired phage see 4e and
4f, below.
e. Assay procedure with additional confirmation (male specific coliphage assay only): Assay an additional 100-mL sample
portion. Proceed as described above, but add RNase solution to
the melted tryptone agar to give a concentration of RNase of 40
g/mL. Label assay dish containing RNase so that it is readily
distinguished from the corresponding assay dish without RNase.
After overnight incubation at 36.5 2C, determine malespecific RNA coliphage concentration/100 mL sample by subtracting total number of plaques counted on the RNase-containing dish from total number counted on the dish without RNase.
f. Sample treatment procedure to reduce somatic salmonella
phages (WG49 host assay only): To neutralize somatic salmo-

nella phages before assay, treat sample filters with an LPS

solution made in PBS that contains 50 mM CaCl2 and has an LPS
concentration of 20 g/mL. After passing 100 mL sample
through filter, pass 5 mL of LPS solution through filter. Handle
LPS-treated filter as described in the above assay procedure with
untreated filters.

5. References
1. SOBSEY, M.D., K.J. SCHWAB & T.R. HANDZEL. 1990. A simple membrane filter method to concentrate and enumerate male-specific RNA
coliphages. J. Amer. Water Works Assoc. 82(9):52.
2. SOBSEY, M.D., D.A. BATTIGELLI, T.R. HANDZEL & K.J. SCHWAB. 1995.
Male-specific coliphages as indicators of viral contamination of
drinking water. American Water Works Association Research Foundation, Denver, Colo.
3. KOTT, Y. 1966. Estimation of low numbers of Escherichia coli
bacteriophage by use of the most probable number method. Appl.
Microbiol. 14:141.