United States
Environmental Protection
Agency
Office of Research
and Development
Washington DC 45260
EPA 600/4-84/013 (N16)
June 2001
&EPA
USEPA Manual of
Methods for Virology
Chapter 16
June 2001
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June 2001
Chapter 16
PROCEDURES FOR DETECTING COLIPHAGES1
1. Introduction
1.1 Scope
This chapter describes procedures
for the detection of coliphages in water
matrices. These procedures are based
on those presented in the Supplement to
the 20th Edition of Standard Methods
for the Examination of Water and
Wastewater and EPA Methods 1601 and
1602. Two quantitative procedures and
one qualitative, presence-absence proce-
dure are presented. The procedures can
be used, without supplementary
methods, to assay small volumes of wa-
ter (10 mL to 1L). For larger volumes
(> 100 L), large-scale concentration
methods such as described in Chapter
14 may be incorporated into the assay
scheme. However, as some concentra-
tion procedures may result in apprecia-
ble loss or inactivation of coliphage, it
is recommended that the suitability of
any large volume concentration method
be evaluated in measured recovery trials
before implementation.
1.2 Significance
Coliphages are bacterial viruses
that infect and replicate in Escherichia
coli. They are found in human and ani-
mal feces. Coliphages are potentially
important microorganisms for monitor-
ing the microbial quality of waters be-
cause: 1) traditional bacterial monitor-
ing does not accurately indicate the
presence of nonbacterial organisms
such as human pathogenic viruses, 2)
human virus detection is beyond the
capabilities of most water laboratories,
and 3) coliphage detection is relatively
inexpensive, easy to perform, and pro-
vides overnight results.
The procedures described below
provide for the detection of two groups
of coliphages commonly assayed for in
water, the male-specific coliphages and
the somatic coliphages. The male-
specific coliphages infect male bacterial
cells via the F-pilus, and the somatic
phages infect bacterial cells via the cell
surface. Male-specific coliphages have
only limited replication in the environ-
ment. That is similar to human viruses
which do not replicate outside the hu-
man body. The male-specific coliphages
that possess an RNA genome are also
similar to human enteroviruses in size,
structure and resistance characteristics.
Thus, male-specific RNA coliphages
are promising candidate indicators of
human viruses in waters. The somatic
coliphages, on the other hand, are com-
posed of a diverse group of bacterial
viruses that are highly varied in size
and structure and less resemble human
viruses. They can replicate in the envi-
ronment. While these factors make the
somatic coliphages somewhat less suit-
able as specific indicators of human
viruses in waters, they may be useful as
a general indicator of water quality.
Somatic coliphages are frequently found
in greater abundance than male-specific
RNA coliphages - an important consid-
eration for water analyses where pollu-
tion indices are expected to be low.
1.3 Safety
Although coliphages are not
pathogenic for humans, other microor-
ganisms that are human pathogens may
be present in waters impacted by un-
treated or inadequately treated domestic
wastes. Laboratories performing virus
analyses are responsible for establishing
an adequate safety plan and must rigor-
ously follow the guidelines on decon-
tamination and waste disposal given in
Chapter 2 (May 1991 Revision).
2. Apparatus, Materials, Media
and Reagents
2.1 Apparatus and Materials
2.1.1 Centrifuge, 0.5-1.0 mL sample
capacity, 5-10,000 x g performance
capability
2.1.2 Cryovials, 2 mL
2.1.3 Erlenmeyer flasks, 125 mL, 250
mL and2L
2.1.4 Glass bottles, capped, 100 ml, 1L
capacity
2.7.5 Graduated cylinders, 100 mL and
500 mL
2.1.6 Incubator, 36.5 ± 2°C
2.1.7 Inoculating loop
2.1.8 Laboratory balance
2.1.9 Pipets-Pipettors, 10 |iL, ImL,
5mL and 10 mL
2.1.10 Petri dishes, 100 x 15 mm, 150
x 15 mm
2.1.11 Filters, 0.22 |im, 0.45 urn —
Note: When passing material contain-
ing phage, always pass about 10 mL of
sterile 1.5% beef extract through the
filter just prior to use to minimize
phage adsorption to the filter.
2.1.12 Test tubes 16 x 150 mm, screw
capped
2.1.13 Water bath set at 44.5 ± 1°C
2.2 Media and Reagents
'Prepared by P.P. Williams, R.E. StetlerandR.S. Safferman
16-1
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June 2001
The amount of media prepared
may be increased proportionally to the
number of samples to be analyzed.
2.2.1 Ampicillin solution — Dissolve
1.5 g of ampicillin in 100 mL of dH2O
and filter with the 0.22 |im filter (beef
extract pretreatment not necessary).
Store at 4°C. For use with male-spec-
ific coliphage assays that utilize E. coli
Famp.
2.2.2 Beef extract — prepare buffered
1.5% beef extract by dissolving 1.5 g of
beef extract powder and 0.375 g of
glycine (final glycine concentration =
0.05M)in90mLofdH2O. Adjust the
pH to 7.0 - 7.5, if necessary, and bring
the final volume to 100 mL with dH2O.
Autoclave at 121°C for 15 min and use
at room temperature. If beef extract
solutions are prepared for use at a later
date, store at 4°C.
2.2.3 Calcium chloride solution —
Add 0.22 g of CaCl2 to 50 mL of dH2O
and sterilize by autoclaving at 121°C
for 15 min. Use at room temperature.
Note: divalent cations such as Ca++ or
Mg++ are used to maintain phage stab-
ility and to facilitate efficient phage ad-
sorption to host.
2.2.4 Glycerol solution, 50% — Add
equal volumes of distilled water and
undiluted glycerol. Autoclave resulting
50% glycerol solution at 121°C for 15
min and use at room temperature.
2.2.5 Nalidixic acid solution — Dis-
solve 1.0 g of nalidixic acid sodium salt
in 100 mL of dH2O and filter with the
0.22 |im filter (beef extract pretreatment
not necessary). Store at 4°C. For use
with somatic coliphage assays that uti-
lize E. coli CN-13. Note: Nalidixic
acid is listed as a carcinogen by the
state of California. Review current Ma-
terial Safety Data Sheet for appropriate
handling of this chemical.
2.2.6 Ribonuclease (RNase) solution
— Dissolve 100 mg of RNase contain-
ing 50-100 Kunitz units/mg in 100 mL
of dH2O by heating to 100°C for 10
min. Store at -20°C in 0.5 mL aliquots.
For use with male-specific coliphage
assays.
2.2.7 Streptomycin solution — Dis-
solve 1.5 g of streptomycin sulfate in
100 mL of dH2O and filter with the 0.22
urn filter (beef extract pretreatment not
necessary). Store at 4°C. For use with
male-specific coliphage assays that uti-
lize E. coli Famp.
2.2.8 Tryptone agar slants — With
gentle mixing, add 1.0 g tryptone, 0.1 g
yeast extract, 0.1 g glucose, 0.8 g NaCl,
0.022 g CaCl2, and 1.2 g of Bacto-agar
to a total volume of 100 mL of dH2O in
a 250 mL flask. Further dissolve and
sterilize by autoclaving at 121°C for 20
min. Proceed accordingly.
For somatic coliphage (using host E.
coli C): After autoclaving, dispense 8
mL aliquots into 16 x 150 mm test
tubes. Prepare slants by allowing the
agar to solidify with the tubes at about a
20° angle. Slants may be stored at 4°C
for up to three months.
For somatic coliphage (using host E.
coli CN-13): After autoclaving, allow
the agar to equilibrate in water bath set
at 44.5 °C and then add 1.0 mL of fil-
tered nalidixic acid solution. Dispense
8 mL aliquots into 16 x 150 mm test
tubes. Prepare slants by allowing the
agar to solidify with the tubes at about a
20° angle. Slants may be stored at 4°C
for up to three months.
For male-specific coliphage (using
host E. coli Famp): After autoclaving,
allow the agar to equilibrate in water
bath set at 44.5 °C and then add 0.1 mL
of filtered ampicillin solution and 0.1
mL of filtered streptomycin solution.
Dispense 8 mL aliquots into 16 x 150
mm test tubes with. Prepare slants by
allowing the agar to solidify with the
tubes at about a 20° angle. Slants may
be stored at 4°C for up to three months.
2.2.9 Tryptone bottom agar — Prepare
one day prior to sample analysis using
the ingredients and concentrations listed
for tryptone agar slants (Section 2.2.8),
except use 1.5 g of Bacto-agar. Proceed
accordingly.
For somatic coliphage (using host E.
coli C): After autoclaving, pipet 15 mL
aliquots aseptically into sterile 100 x 15
mm petri dishes and allow the agar to
harden. Store the dishes inverted at
4°C overnight and warm to room tem-
perature for 1 h before use.
For somatic coliphage (using host E.
coli CN-13): After autoclaving, allow
the agar to equilibrate in water bath set
at 44.5 °C, and then add 1.0 mL of fil-
tered nalidixic acid solution to the 100
mL volume of warm agar. Pipet 15 mL
aliquots aseptically into sterile 100 x 15
mm petri dishes and allow the agar to
harden. Store the dishes inverted at
4°C overnight and warm to room tem-
perature for 1 h before use.
For male-specific coliphage (using
hostlt. coli Famp): After autoclaving,
allow the agar to equilibrate in water
bath set at 44.5 °C, and then add 0.1
mL of filtered ampicillin solution and
0.1 mL of filtered streptomycin solution
to the 100 mL volume of warm agar.
Pipet 15 mL aliquots aseptically into
sterile 100 x 15 mm petri dishes and
allow the agar to harden. Store the
dishes inverted at 4°C overnight and
warm to room temperature for 1 h be-
fore use.
2.2.10 Tryptone broth — Add 1.0 g
tryptone, 0.1 g yeast extract, 0.1 g glu-
cose, 0.8 g NaCl, and 0.022 g CaCl2 per
each 100 mL of dH2O. Sterilize by
autoclaving at 121°C for 20 min.
Proceed accordingly:
For somatic coliphage (using host E.
coli C): After autoclaving, cool and
store at 4°C.
For somatic coliphage (using host E.
coli CN-13): After autoclaving, cool
and add 1.0 mL of filtered nalidixic
acid solution per 100 mL of broth.
Store at 4°C.
For male-specific coliphage (using
hosts, coli Famp): After autoclaving,
cool and add 0.1 mL of filtered ampi-
cillin solution and 0.1 mL of streptomy-
cin solution per 100 mL of broth. Store
at 4°C.
2.2.77 Tryptone dilution tubes —
Aseptically, dispense 9 mL aliquots of
sterile tryptone broth into 16 x 150 mm
screw-capped test tubes that have been
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June 2001
sterilized by autoclaving at 121°C for
15 min.
2.2.12 Tryptone enrichment broth —
Add 10.0 g tryptone, 1.0 g yeast extract,
1.0 g glucose, 8.0 g NaCl, and 0.022 g
CaCl2 per each 100 mL of dH2O. Steril-
ize by autoclaving at 121°C for 20 min.
Proceed as described for tryptone broth
in 2.2.10
2.2.13 Tryptone SAL agar — Add 2.0
g tryptone, 0.2 g yeast extract, 0.2 g
glucose, 1.6 g NaCl, 0.022 g CaCl2, and
1.2 g of Bacto-agar per each 100 mL of
dH2O. Sterilize by autoclaving at
121°C for 20 min.
2.3.14 Tryptone spot agar dishes —
Prepare one day prior to sample analy-
sis using the ingredients and concentra-
tions listed for tryptone agar slants
(Section 2.2.8), except use 0.75 g of
Bacto-agar for each 100 mL. After
autoclaving, place in water bath set at
44.5 °C and allow to equilibrate. Pro-
ceed accordingly.
For somatic coliphage (using host E.
coli C): With gentle mixing, add 2 mL
of a 4 h culture of host E. coli (Section
2.3.6). Pour the well mixed suspension
into five sterile 100 x 15 mm petri
dishes (approximately 20 mL per dish),
swirl gently, and allow the agar to
harden. Store the dishes inverted at
4°C overnight and warm to room tem-
perature for 1 h before use.
For somatic coliphage (using host E.
coli CN-13): Add 1.0 mL of filtered
nalidixic acid solution to the 100 mL
volume of warm agar. With gentle
mixing, add 2 mL of a 4 h culture of
host£. coli (Section 2.3.6). Pour the
well mixed suspension into five sterile
100 x 15 mm petri dishes (approxi-
mately 20 mL per dish), swirl gently,
and allow the agar to harden. Store the
dishes inverted at 4°C overnight and
warm to room temperature for 1 h be-
fore use.
For male-specific coliphage (using
host E. coli Famp): Add 0.1 mL of fil-
tered ampicillin solution and 0.1 mL of
filtered streptomycin solution to the 100
mL volume of warm agar. With gentle
mixing, add 2 mL of a 4 h culture of
host£. coli (Section 2.3.6). Pour the
well mixed suspension into five sterile
100 x 15 mm petri dishes (approxi-
mately 20 mL per dish), swirl gently,
and allow the agar to harden. Store the
dishes inverted at 4°C overnight and
warm to room temperature for 1 h be-
fore use.
2.2.75 Tryptone top agar — Prepare
the day of sample analysis using the
ingredients and concentrations listed for
tryptone agar slants (section 2.2.8), ex-
cept use 0.7 g of Bacto-agar. Autoclave
and place in the 44.5 ± 1°C water bath.
Note: nalidixic acid should be in the top
agar used for somatic coliphages when
host E. coli CN-13 is used; ampicillin
and streptomycin should be in the top
agar used for male-specific coliphages
with host E. coli Famp.
2.3 Host Bacteria and Coliphages
2.3.7 E. coli C, American Type Cul-
ture Collection (ATCC) #13706 —
Bacterial host for somatic coliphages.
E. coli CN-13 (ATCC #700609) can be
used as an alternate host for somatic
coliphages. CN-13 is a nalidixic acid
resistant variant of E. coli C that
appears to be comparable to E. coli C
for coliphage detection. Use of CN-13 is
indicated if indigenous bacteria in water
samples interfere with the coliphage
assay (using E. coli C). Use of CN-13
permits the addition of nalidixic acid to
the assay media to suppress the interfer-
ing bacteria.
2.3.2 E.coli Famp (ATCC #700891) —
Bacterial host for male-specific coli-
phages.
2.3.3 Coliphage 4>X174 (ATCC #
13706-B1)— Somatic coliphage to
serve as positive control.
2.3.4 Coliphage MS2 (ATCC # 15597-
Bl) — Male-specific coliphage to serve
as positive control.
2.3.5 Storage of host E. coli cultures:
For short term storage, inoculate host
culture onto tryptone agar slants with a
sterile inoculating loop by spreading the
inoculum evenly over entire slant sur-
face. Incubate the culture overnight at
36.5 ± 2°C. Store at 4°C for up to two
weeks. For long term storage inoculate
a 5-10 mL tube of tryptone broth with
the host culture. Incubate the broth cul-
ture overnight at 36.5 ± 2°C. Add l/5th
volume of 50% glycerol solution. Dis-
pense into 1 mL aliquots in 2-mL
cryovials and store at -70°C.
2.3.6 Preparation of host for coliphage
assay: Inoculate 5 mL of tryptone broth
with host E. coli from a slant using a
sterile inoculating loop and incubate for
16 h at 36.5 ± 2°C. Transfer 1.5 mL of
the 16 h culture to 30 mL of tryptone
broth in a 125 mL flask and incubate
for 4 h at 36.5 ± 2°C with gentle shak-
ing. The amount of inoculum and broth
used can be proportionally altered ac-
cording to need.
2.3.7 Preparation of coliphage positive
controls: Rehydrate the ATCC coli-
phage stock and store at 4°C. Prepare a
30 mL culture of the appropriate host
(E. coli Famp for coliphage MS2; E.
coli C or CN-13 for coliphage 4>X174)
as described in Section 2.3.6. Incubate
culture for 2 h at 36.5 ± 2°C with shak-
ing. Add 1 mL of rehydrated phage
stock and incubate for an additional 4 h
at 36.5 ± 2°C. Filter the culture
through a beef extract-treated 0.22 |im
filter (see 2.1.11). Prepare 10'7, 10'8 and
10"9 dilutions of the filtrate using tryp-
tone dilution tubes. (These three dilu-
tions should be sufficient in most cases).
Add 3 mL of melted tryptone top agar
held in the 44.5 ± 1°C water bath to
fifteen 16 x 150 mm test tubes. These
test tubes should be kept in the heated
water bath to avoid premature solidify-
ing of the agar. Add 0.1 mL of the host
culture to each of the 15 test tubes. Add
1 mL of the 10"9 dilution into each of
five test tubes. Add 1 mL of the 10'8
dilution into five additional tubes and 1
mL of the 10"7 dilution into the remain-
ing five tubes. Be sure the tubes are
labeled with the appropriate dilution.
For each tube, mix and immediately
pour the contents over the bottom agar
of a petri dish labeled with the dilution
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June 2001
assayed. Rotate the dish to spread the
suspension evenly over the surface of
the bottom agar and place it onto a level
surface to allow the agar to solidify.
Invert and incubate the inoculated dish-
es at 36.5 ± 2°C overnight and examine
for plaques the following day. Count
the number of plaques on each of the 15
dishes. Five dishes from one of the as-
sayed dilutions should yield plaque
counts of 20 to 100 plaques. Average
the plaque counts on these five dishes
and multiply the result by the reciprocal
of the dilution to obtain the liter of the
undiluted stock. For use as a positive
control in the coliphage assay, dilute the
filtrate to 30 to 80 PFU/mL in tryptone
broth. Store the original filtrate and the
diluted positive control preparation at
4°C. 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, and pouring their
contents into ten petri dishes. Count
the plaques on all dishes and divide by
10. If the result is not 30 to 80, adjust
the dilution of the positive control sam-
ple and assay again.
3. Double Agar Layer (DAL)
assay
This is a quantitative method that
is based on the plaque assay. The pro-
cedure utilizes the traditional double
agar layer technique described by Ad-
ams. The technique has been in wide-
spread usage although the volumes as-
sayed are typically small (0.5-1.0 mL
per 100 mm assay dish). Refer to
Figure 16-1.
3.1 Basic DAL Assay: Add 3 mL of
melted tryptone top agar held in the
44.5 ± 1°C water bath to each of ten 16
x 150 mm test tubes for sample assay
and to each of two additional test tubes
that are to serve as negative and
positive controls. The test tubes should
be kept in the water bath to avoid
premature solidifying of the agar. Add
0.1 mL of the host culture to each of the
12 test tubes. Add 1 mL of tryptone
broth to the test tube serving as negative
control. Add 1 mL of the appropriate
coliphage preparation (30-80 PFU/mL)
to the test tube serving as positive
control. To each of the remaining ten
test tubes, add ImL of sample. For each
tube, mix and immediately pour the
contents over the bottom agar layer of a
petri dish (see Section 2.2.9) that has
been suitably labeled with identification
information. Tilt and rotate the dish to
spread the suspension evenly over the
surface of the bottom agar and place it
onto a level surface to allow the agar to
solidify. Invert and incubate the
inoculated dishes at 36.5 ± 2°C
overnight and examine for plaques the
following day. Count the total number
of plaques on the ten dishes receiving
the sample. Calculate the coliphage
concentration (Ca) in PFU per mL
according to the formula:
(P -e- 10) x D PFU/mL
where P is the total number of plaques
from the ten dishes, D is the reciprocal
of the dilution made on the inoculum
before plating (D = 1 for undiluted
samples). 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,
then the coliphage concentration of the
sampled water (Q) can be calculated
according to the formula:
where Ca is the coliphage concentration
of the concentrated material in
PFU/mL, Va the volume of that material
in mL, and Vb is the volume of water
processed in the sampling procedure in
L. The resultant concentration is
generally reported as PFU/L or
PFU/100L. Count the plaques on the
positive control dish. Maintain a record
of the plaque count as a check on the
virus sensitivity of the host. Assay any
water 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 the assay.
Note: With host E. coli Famp, the
basic DAL assay will detect both DNA
andRNA male-specific coliphages as
well as some somatic coliphages. For a
procedure to selectively quantify the
RNA male-specific coliphages, see the
following section (3.2).
3.2 DAL Assay with RNase: In this
procedure an additional 10 mL of
sample is assayed. The procedure is the
same as that described above, except
that RNase solution is added to the
melted tryptone top agar in the assay of
the additional 10 mL of sample. For
the additional 10 tubes, the melted
tryptone agar should contain RNase at a
concentration of 40|ig/mL. Before
pouring tube contents to petri dishes,
the dishes should be appropriately
labeled so that the 10 dishes containing
RNase are readily distinguished from
the 10 dishes without RNase. Calculate
the male-specific RNA coliphage (Ca )
according to the formula:
[ (P + 10) -
10) ] * Z> PFU/mL
where P is the total number of plaques
from the 10 dishes without RNase, PR is
the total number of plaques from the 10
dishes with RNase, and D is the
reciprocal of the dilution made on the
inoculum before plating. 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, then
the coliphage concentration of the
sampled water (Cb) can be calculated as
previously described for section 3.1.
4. Single Agar Layer (SAL) assay
This is a quantitative method that
is also based on the plaque assay. This
procedure utilizes a single agar layer
technique adapted from Grabow and
Coubrough. It allows for the assay of
larger (100 mL) sample volumes in 150
mm assay dishes. Refer to Figure 16-2.
4.1 Basic SAL Assay: Place a 100 mL
volume of sample in the 44.5 ± 1°C
water bath for 3 minutes. Add 5 mL of
16-4
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June 2001
calcium chloride solution to the warmed
sample followed by 5 mL of the
appropriate host bacterium preparation.
Mix the host inoculated sample with
100 mL melted tryptone SAL agar also
held at 44.5 ± 1°C. Distribute the 210
mL of material to eight 150 x 15mm
petri dishes. For a positive control, add
to 12.5 mL sterile water, 0.6 mL of
calcium chloride solution. Then add 1
mL of the appropriate cj>X174
preparation or MS2 preparation (30-80
PFU/mL) and 0.6 mL of host bacterium.
Mix the host inoculated control with
12.5 mL of melted tryptone SAL agar.
Pour to a single 150 x 15mm petri dish.
Repeat for a negative control but add 1
mL of sterile tryptone broth in place of
the positive control phage preparation.
Invert and incubate the inoculated
dishes at 36.5 ± 2°C overnight and
examine for plaques the following day.
Count the total number of plaques on
the eight dishes receiving the sample.
That total is the coliphage concentration
per 100 mL of sample.
Note: With host E. coli Famp, the
basic SAL assay will detect both DNA
andRNA male-specific coliphages as
well as some somatic coliphages. For a
procedure to selectively quantify the
RNA male-specific coliphages, see the
following section (4.2).
4.2 SAL Assay with RNase: In this
procedure an additional 100 mL of
sample are assayed. The procedure is
the same as that described above, except
that RNase solution is added to the
melted tryptone agar in the assay of the
additional 100 mL of sample. For the
additional material, the melted tryptone
agar should contain RNase at a
concentration of 80|ig/mL (before
sample is added). Appropriately label
dishes so that the dish containing
RNase are readily distinguished from
the dish without RNase. Calculate the
male-specific coliphage concentration
(Ca) according to the formula:
C=P-
PK PPU/lOQml
where P is the total number of plaques
from the dish without RNase, PR is the
total number of plaques from the dish
with RNase.
5. Enrichment Spot assay
This is a qualitative, presence-
absence method. It employs an initial
enrichment step to amplify coliphage
numbers in samples, prior to assay with
a spot technique. The procedure is
adapted from EPA Method 1601 and
allows for the assay of 100 mL or 1 L
sample volumes. The procedure is
described for 100 mL samples, for 1L
samples scale up the volumes described
in Section 5.1 by a factor of 10. Spot
assay volumes in Section 5.2 remain the
same. Refer to Figure 16-3.
5.1 Enrichment: Place 100 mL of
sample into sterile bottle. Add 1.25 mL
of calcium chloride solution. Add 0.5
mL of a 4 h culture of host E. coli
(Section 2.3.6). Then add 5 mL of
tryptone enrichment broth. Repeat the
procedure for the positive control,
replacing the 100 mL of sample with
100 mL of sterile distilled water
inoculated with 0.5 mL of coliphage
positive control preparation (Section
2.3.7). Again repeat procedure for the
negative control, replacing the 100 mL
of sample with 100 mL of sterile
distilled water and no coliphage
inoculum. Proceed as follows:
For somatic coliphage (using host E.
coli C): Mix well and incubate at 36.5 ±
2°C overnight.
For somatic coliphage (using host E.
coli CN-13): Add 1.0 mL of filtered
nalidixic acid solution and mix well.
Incubate at 36.5 ± 2°C overnight.
For male-specific coliphage (using
host£. coli Famp): Add 0.1 mL of
filtered ampicillin solution and 0.1 mL
of filtered streptomycin solution and
mix well. Incubate at 36.5 ± 2°C
overnight. Note: As used in this
presence-absence assay, both DNA and
RNA male-specific coliphages as well
as some somatic coliphages will be
detected with host E. coli Famp.
5.2 Spot assay: Remove enrichment
cultures from incubator and again mix
well. Remove 10 |iL and carefully
deposit the volume to a preselected area
on a tryptone spot agar dish (Section
2.2.14). Repeat for positive and
negative controls using the same spot
agar dish and being careful to keep the
deposited volumes, or spots, separate.
Spot dish should be adequately labeled
prior to spotting and it is advisable to
mark a grid pattern on the spot dishes
as a guide when multiple samples are
assayed. Allow deposited volumes to
absorb into agar for up to 60 min.
Invert inoculated spot dish and incubate
overnight at 36.5 ± 2°C. After
overnight incubation, examine dish for
lysis (clearing) at the spot where the
enrichment inoculum was applied. Use
the positive and negative control spots
for comparison. A positive result is
indicated where lysis is observed at the
spot of application. The entire spot
need not be lysed. In some cases, lysis
will be observed as one or more small
plaques within the spot of application.
5.3 Interference: Interfering bacterial
growth may occasionally be observed at
the application spot. This growth is
noticeably different from host bacterial
growth and can prevent accurate
assessment of the spot assay. Such
interference may be addressed by
including filtration or centrifugation
treatment at the end of the enrichment
step. Proceed with either filtration or
centrifugation treatments as follows:
Filtration: Take 0.5 to 1.0 mL of
the incubated enrichment medium and
place through a beef extract-treated
0.45 umpore size sterile filter. Spot
assay the filtered material as in 5.2.
Centrifugation: Take 0.5 to 1.0 mL
of the incubated enrichment medium
and centrifuge at 5,000 to 10,000 x g
for 10 min. Recover the supernatant
and spot assay as in 5.2.
6. References
Adams, M.H. 1959. Bacteriophages.
Interscience Publishers, New York.
APHA. 2001. Standard Methods for the
Examination of Water and Waste-
water (L. S. Clesceri, A. E.
16-5
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June 2001
Greenberg, and A. D. Eaton ed),
20th Edition Supplement. Amer-
ican Public Health Association,
Washington, D.C.
Grabow, W.O.K. and P. Coubrough.
1986. Practical direct plaque assay
for coliphages in 100-mL samples
of drinking water. Appl. Environ.
Microbiol. 52:430-433.
U.S. EPA. 2001. Method 1601: Male-
specific (F+) and somatic coliphage
in water by two-step enrichment
procedure. EPA 821-R-01-030.
U.S. EPA. 2001. Method 1602: Male-
specific (F+) and somatic coliphage
in water by single agar layer (SAL)
procedure. EPA 821-R-01-029.
Disclaimer: Mention of trade names or
commercial products does not constitute
endorsement or recommendation for use.
16-6
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Figure 16-1 basic double agar layer (DAL) procedure
June 2001
1
Add 3.0 ml melted tryptone top agar to 12 test tubes and place in 44.5 ± 1 C water bath
44.5 C
10 tubes for samples
2 tubes for controls
Label 12 pre-poured bottom agar plates and keep at room temperature
Samples
Controls
For each top agar tube in water bath:
A. Add Q.1ml of a 4hr culture of host bacterium
*\
C. Gently mix tube in palm
B. Add 1 .OmL of sample OR:
For negative control add
1.0 mL of tryptone broth
For positive control add
1.0mL of r.oliphage prep
("30-80 PFU/mL)
D. Pour tube into pre-poured
bottom agar plale
Invert and place in incubator at 36.5 + 2 C for 16-24 hr
Samples Controls
Examine for plaques and record results
Samples
Negative
Control
Positive
Control
16-7
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June 2001
Figure 16-2 basic single agar layer (SAL) procedure
1
Place flasks with 100 mL of sample and two 12.5 mL volumes of sterile water
into a 44.5 + 1 C water bath
100 mL sample
12.5 ml aerile water
44.5 C
A. After 3 min, add calcium chloride solution
5 ml CaCI jSOln 0.6 ml C«C1 fOn
Sample
44.5 C
o To negative control add
1.0 mL of tryptone broth
For positive control add 1.0 ml of
coliphage prep (30-80 PFU/mL)
A. Add 4hr cuNure of host bacteria, followed by tryptone SAL agar
5 mL host, then 100 mL agar 0.6 mL hosl. then 12.5 mL agar
44.5 C
Sample
NBJJIJJ.
B. Gently swirl to mix
^ Pour sample evenly to 8 large petri dishes;
'' pour 1 dish for each control
Invert and place in incubator at 36.5 ± 2 C for 16-24 hr
Samples Controls
Examine for plaques and record results
Samples
Negative Positive
Control Control
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June 2001
Figure 16-3 basic enrichment spot procedure
1
Place 100 ml of sample and two 100 ml volumes
of sterile water into sterile bottles
100 ml sample
100 mL sterile waler
Sample
Negative Positive
control contrcJ
Add calcium chloride solution
1.25mLCaCI2san
"'c<; live
contra
Remove 10/JL from each bottle to labeled grid on spoti dish
Sample
Sta dish
f Allow to absorb for up to 60 min
Samp,,
Add 4hr culture of host bacterium
0.5 mL host culture
'
8
Invert and incubate 16-24 hr at 36.5 ± 2 C
Eta ash
36.5 C
Add tryptone enrichment broth
5 ml tryptone enrichment bran
Mix each bottle well and incubate 16-24 hr at 36.5 ± 2 C
Examine spots for zones of lysis and record results
Sample
Spot dish
16-9
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