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
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.
1. General Discussion
3.
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
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
4.
Procedure
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).
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.
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).
General Discussion
2.
4.
Procedure
Apparatus
See 9224B.2.
Adjust amount of media prepared proportionally to the number of samples. Use reagent-grade water (see Table 9020:II) in
making media and reagents.
where:
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.
2.
Apparatus
See 9224B.2.
3.
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
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
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
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:
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
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.
Procedure
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.
General Discussion
Apparatus
4.
Procedure
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.
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.