United States
Environmental Protection
Agency
Office of Research
and Development
Washington DC 45260
EPA 600/4-84/013 (N14)
April 2001
&EPA
USEPA Manual of
Methods for Virology
Chapter 14
April 2001
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April 2001
Chapter 14
CONCENTRATION AND PROCESSING OF WATERBORNE VIRUSES BY POSITIVE CHARGE
1MDS CARTRIDGE FILTERS AND ORGANIC FLOCCULATION1
1. Introduction
1.1 Scope
This chapter describes the most
widely used virus adsorption-erution
(VIRADEL) method for recovering hu-
man enteric viruses from water matrices
(Fout et al., 1996). The method takes
advantage of positively charged car-
tridge filters to concentrate viruses from
water. The major advantage of methods
that use these filters over those de-
scribed in Chapters 5 and 6 that use
negatively charged filters is that addi-
tives are not normally needed to achieve
virus adsorption to filters.
1.2 Significance
Human pathogenic enteric viruses
can be present in surface or ground-
waters impacted by untreated or inade-
quately treated domestic wastes. The
presence of these viruses can cause
hepatitis, gastroenteritis and numerous
other diseases.
The surface water treatment rule
(40 CFR Part 141) established the maxi-
mum contamination level for enteric
virus in public water systems by requir-
ing that systems using surface water or
ground water under the influence of
surface water reduce the amount of vi-
rus in source water by 99.99%. The
rule requirements are currently met on
basis of treatment alone, and thus the
degree of actual protection against
waterborne viral disease depends upon
the source water quality. The actual
degree of protection can only be esti-
mated by monitoring source and fin-
ished waters over an extended period of
time.
The amount of environmental water
that must be sampled depends upon ex-
pected virus concentrations. In general,
it is recommended that one liter of high-
ly contaminated waters such as sewage
to 1,800 liters of drinking waters should
passed through a 1MDS cartridge filter.
1.3 Safety
The water samples to be monitored
may contain pathogenic human enteric
viruses. 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
Analytical Reagent or ACS grade
chemicals (unless specified otherwise)
and deionized or distilled reagent grade
water (dH2O) should be used to prepare
all media and reagents. The dH2O must
have a resistance of greater than 0.5
megohms-cm at 25 °C, but water with a
resistance of 18 megohms-cm is pre-
ferred.
Appendix 1 gives a list of potential
vendors for the apparatus, materials,
media and reagents used in this method.
Equivalent items from other vendors
may be substituted for those given in
the text.
2.1 Sampling Apparatus and Materials
Several configurations are given
below for the assembly of the filter
apparatus. The standard filter appara-
tus is used for all sampling, except
where a prefilter, dechlorination or pH
adjustment are required.
2.1.1 Standard filter apparatus (see Fig-
ure 14-1)
(a) Materials
Letters in bold print represent the
origin of the abbreviations used to iden-
tify parts in the figures.
(a. 1) One BR — Backflow Regulator
(Watts Regulator Product Series 8 — 3/4"
Hose Connection Vacuum Breaker).
(a. 2) Three SF — Swivel Female in-
sert with garden hose threads (United
States Plastic Product No. 63003).
(a. 3) Three sections of B T — Braided
Tubing, Yz" clear (Cole-Parmer Product
No. P-95636-02).
(a.4) Six HC1 — Hose Clamps (Cole-
Parmer Product No. P-06403-20).
(a. 5) One HF1 — Hose Fitting, nylon,
d" male NPT x !/2" tubing ID (United
States Plastic ProductNo. 61141).
(a.6) One PR — Pressure Regulator
(Watts Regulator Product No. d" 26A
(or 263A), Suffix B).
(a.7) One PN — PVC Nipple, d"
male NPT (Ryan Herco Product No.
3861-057; not required with the 263A
regulator).
(a. 8) One TE — PVC TEE with d"
female NPT ports (Ryan Herco Product
No. 3805-003; not required with the
263A regulator).
(a. 9) One RB1 — Reducing Bushing,
d" NPT(M) x i/4" NPT(F) (Cole-Par-
mer Product No. P-06349-32; not re-
quired with the 263 A regulator).
(a. 10) One PG — Pressure Gauge 0-60
pound per square inch (PSI; Cole-Par-
'Prepared by G. Shay Fout, D.R. Dahling and R.S. Safferman
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April 2001
WATER SOURCE
Insertion Pant for Additional Modules
(If required)
FQ1 CH MQ2 HF2
HC PR TE MQ1 RN1
BT
Figure 14-1 Standard Filter Apparatus
mer Product No. P-68004-03; place in
YA" gauge port if using the 263 A regula-
tor).
(a. 11) One RA — Reducing Adaptor,
!/2M female NPT x d" male NPT (Cin-
cinnati Valve and Fitting Product No.
SS-8-RA-6).
(a. 12) One MQ1 — Male Quick Con-
nect, !/2M male NPT (Cincinnati Valve
and Fitting Product No. SS-QF8-S-8PM
or Cole-Parmer Product No. P-31303-
36; appropriate hose fittings and braid-
ed tubing can be substituted for quick
connects).
(a. 13) Two FQ1 — Female Quick Con-
nects, !/2M female NPT (Cincinnati
Valve and Fitting Product No. SS-QF8
-B-8PF; Cole-Parmer Product No. P-
31303-01 may be used by substituting
the two reducing nipples (item a. 14) for
two reducing bushings (%" male NPT x
!/2M female NPT; Cole-Parmer Product
No. P-06349-35)).
(a. 14) Two RN1 — Reducing Nipples,
3/4" male NPT x !/2" male NPT (Cole-
Parmer Product No. P-06349-87).
(a. 15) One CH — Cartridge Housing
with wench (Cuno Product No. API IT
or 44857-05).
(a. 16) One FC — Filter Cartridge, posi-
tively charged 1MDS, ZetaPor Virosorb
(Cuno Product No. 45144-01-1MDS).
(a. 17) One MQ2 — Male Quick Con-
nect, !/2M female NPT (Cincinnati Valve
and Fitting Product No. SS-QF8-S-8PF;
Cole-Parmer Product No. P-31303-36
may be used by substituting the hose
fitting (item a. 18) for a 1A" female NPT
x ^"tubing ID hose fitting (Cole-Par-
mer Product No. P-06464-10)).
(a. 18) One HF2 — Hose Fitting, 1/2"
male NPT x !/2"tubing ID (United States
Plastic Product No. 62142).
(a. 19) One WM — Water Meter (Nep-
tune Equipment Product No. e" Tri-
dent 10).
The water meter should be used in
a horizontal position and protected
from freezing. The order should specify
that meters be rated in gallons (1 gal =
0.133 7ft3 or 3.7854 L). If not specified,
meters may be rated in cubic feet (1 ft3 =
7.481 gal or 28.316 L).
(a.20) One HF3 — Hose Fitting, nylon,
3/4" male NPT x !/2" tubing ID (United
States Plastic Product No. 61143).
(a.21) One FV — Flow Control Valve
(Plast-O-Matic Valves Product No.
FC075B-3-PVC).
(b) Apparatus assembly
The standard filter apparatus con-
sists of three modules: the regulator
module, the cartridge housing module
and the discharge module.
Teflon tape (Cole-Parmer Product
No. P-08782-27) must be used on all
threaded, non-compression fittings.
(b. 1) Regulator module — in order, as
shown in Figure 14-1, connect the
backflow regulator (BR) to a swivel fe-
male insert (SF). Clamp a piece of
braided tubing (BT) onto the tubing
connector of the swivel female insert
using a hose clamp (HC1). Clamp the
other end of the tubing to a d x y2"
hose fitting (HF1). Screw the fitting
into the inlet of the pressure regulator
(PR). Connect the outlet of the pressure
regulator to the PVC TEE (TE) via a
PVC nipple (PN). Connect the pressure
gauge (PG) to the top of the PVC TEE
using the reducing bushing (RB1). At-
tach a reducing adaptor (RA) to the re-
maining connection on the PVC TEE.
Add a male quick connect (MQ1) to the
reducing adaptor.
(b.2) Cartridge housing module —
Attach a female quick connect (FQ1) to
a reducing nipple (RN1). Connect the
reducing nipple to the inlet of the car-
tridge housing (CH). Attach another
reducing nipple to the outlet of the
housing. Attach a male quick connect
(MQ2) to the reducing adaptor.
(b.3) Discharge module — attach a
female quick connect (FQ1) to a hose
fitting (HF2). Connect a piece of braid-
ed tubing (BT) to the hose fitting (HF2)
with a hose clamp (HC1). Clamp the
other end of the braided tubing to a
swivel female insert (SF) with another
hose clamp (HC1). Attach the swivel
female insert (SF) to the inlet of the wa-
ter meter (WM). Attach another swivel
female insert (SF) to the outlet of the
water meter (WM) and connect a piece
of braided tubing (BT) with a hose
clamp (HC1). Clamp the other end of
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Prefilter Module
FQ1
MQ2
Double Injector Module
TE (1of3> /NflofJ)
Figure 14-2 Additional Modules for the Standard Filter Apparatus
the tubing to a hose fitting (HF3) with
another hose clamp(HCl). Screw the
hose fitting (HF3) into the inlet of the
flow control valve (FV). An additional
hose fitting (not shown) may be added
to the flow control valve for the attach-
ment of a sufficient length of tubing to
reach a drain. The discharge module
does not have to be sterilized.
(b.4) Connect the cartridge housing
module to the regulator module at the
quick connect. The combined regulator
and cartridge housing modules should
be sterilized with chlorine as described
in section 2.1. la. 10, 2.3.2. Presterilize
a 1MDS filter cartridge (FC) as de-
scribed in section 2.3.1 and place it into
the cartridge housing using aseptic tech-
nique. Replace the housing head of the
cartridge housing and tighten with a
cartridge housing wench. Check to en-
sure that the filter is adequately sealed
by shaking the housing. Adequately
sealed filters should not move. For con-
venience during shipping, the regulator
and cartridge housing modules may be
separated. Seal all openings into the
modules with sterile aluminum foil.
2.7.2 Prefilter module (see Figure 14-
2)
Use the prefilter module for waters
exceeding 75 nephelometric turbidity
units (NTU) or for other conditions that
prevent the minimum sampling volumes
from being obtained.
(a) Additional materials: One PC —
10 |im Polypropylene Prefilter Car-
tridge (Parker Hannifin Product No.
M19R10-A); in addition, a female quick
connect (FQ1), two reducing nipples
(RN1), a cartridge housing (CH) and a
male quick connect (MQ2) as described
for the standard apparatus are needed.
(b) Module assembly — in order, as
shown for the prefilter module in Fig-
ure 14-2, attach a female quick connect
(FQ1) to a reducing nipple (RN1).
Connect the reducing nipple to the inlet
of the cartridge housing (CH). Attach
another reducing nipple to the outlet of
the housing. Attach a male quick con-
nect (MQ2) to the reducing adaptor.
Sterilize the unit with chlorine as de-
scribed in section 2.1. la. 10, 2.3.2 and
add a presterilized polypropylene prefil-
ter cartridge using aseptic technique.
Cover the ends with sterile aluminum
foil.
2.7.3 Injector modules (see Figure 14-
2)
Use injector modules for source or
finished water requiring pH reduction
and for finished waters requiring
dechlorination. Both single and double
injector modules should be assembled
so that they will be available on de-
mand.
(a) Materials:
(a. 1) Two FQ2 — Female Quick Con-
nects, !/2M male NPT (Cincinnati Valve
and Fitting Product No. SS-QF8-B-
8PM).
(a.2) Four ME — Male Elbows, d"
male NPT (Cincinnati Valve and Fitting
Product No. SS-6-ME).
(a.3) Two RN2 — Reducing Nipples,
d" male NPT * 1/2" male NPT (Cole-
Parmer Product No. P-06349-85).
(a.4) Two RB2 — Reducing Bushings,
d" female NPT * 1/2" male NPT (Cole-
Parmer Product No. P-06349-34).
(a.5) Three IN — In-line INjectors
(DEMA Engineering Product No. 203B
d" female NPT; a metering or peristal-
tic pump and appropriate connectors
may be substituted for an injector).
The use of injectors significantly
reduces the cost per apparatus. Injec-
tors can be used in the absence of an
electricity source, but are much more
difficult to control than pumps. If
pumps are selected, use a check valve
(e.g., Cole-Parmer Cat. No. P-98676-
00) on tubing between the pump and the
main body of the module.
(a.6) Three HC2 — Hose Clamps
(Cole-Parmer Product No. P-06403-10).
(a.7) In addition to the items specified
in sections 2.1.3a.l to 2.1.3a.6, four
reducing adaptors (RA), four PVC
TEEs (TE), two PVC nipples (PN), two
reducing bushings (RBI), two pressure
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April 2001
gauges (PG), two female quick connects
(FQ1), two male quick connects (MQ1)
and two male quick connects (MQ2) as
described for the standard apparatus are
needed. Two union ball joints, d" fe-
male NPT (not shown; Cincinnati Valve
and Fitting Product No. SS-6-UBJ) and
two PVC nipples may be used in place
of the two reducing nipples (RN2), male
quick connects (MQ2), female quick
connects (FQ1) and reducing bushings
(RB2) used with the double injector
module.
(b) Module assembly:
(b. 1) Single Injector Module — as-
semble the parts in order as shown for
the single injector module in Figure 14-
2. Attach a female quick connect (FQ2)
to a reducing adaptor (RA). Connect
the adaptor to the inlet of the injector
(IN). Connect the outlet of the injector
to a PVC TEE (TE) via a PVC nipple
(PN). Connect a pressure gauge (PG) to
the top of the PVC TEE using a reduc-
ing bushing (RB1). Attach a reducing
adaptor (RA) to the remaining connec-
tion on the PVC TEE. Add a male
quick connect (MQ1) to the reducing
adaptor.
(b.2) Double Injector Module — as-
assemble the parts as shown for the dou-
ble injector module in Figure 14-2.
Assemble the main portion by attaching
a female quick connect (FQ2) to a re-
ducing adaptor (RA). Connect the
adaptor to the top connector of a PVC
TEE (TE). Add a male elbow (ME) to
one of the connections on the PVC
TEE. Attach a reducing nipple (RN2)
to the other connection. If using a un-
ion ball joint in place of the quick con-
nects, attach a PVC nipple (not shown)
to the other connection. Add a male
quick connect (MQ2) to the reducing
nipple or add one portion of a union ball
joint (not shown) to the PVC nipple.
Connect the inlet side of an injector
(IN) to the male elbow. Attach another
male elbow to the outlet of the injector.
Connect the male elbow to another PVC
TEE. Connect a reducing nipple (RN2
or PVC nipple) to the other end of the
second PVC TEE. Add a male quick
connect (MQ2) to the reducing nipple
as above (or add one portion of the sec-
ond union ball joint to the PVC nipple).
Connect the top connector of the second
PVC TEE to a third PVC TEE via a
PVC nipple (PN). Connect a pressure
gauge (PG) to the top of the third PVC
TEE using a reducing bushing (RBI).
Attach a reducing adaptor (RA) to the
remaining connection on the third PVC
TEE. Add a male quick connect (MQ1)
to the reducing adaptor. Attach two
male elbows (ME) to the inlet and outlet
of a second injector (IN). Connect two
reducing bushings (RB2) or, if used, the
bottom portion or the two union ball
joints (not shown) to the male elbows.
Connect a female quick connect (FQ1)
to each reducing bushing. Orient the
second injector so that the direction of
flow is the same as the first injector (the
arrows on the injectors should both
point towards the pressure gauge side of
the assembly). Connect the two female
quick connects to the male quick con-
nects of the main portion to complete
the assembly or, if used, connect the two
portions of the union ball joints.
2.1.4 Other Sampling Materials
(a) Portable pH probe (Omega Product
No. PHH-1X)
(b) Portable temperature probe (Omega
Product No. HH110).
(c) Commercial ice packs (Cole-Par-
mer Product No. P-06346-76).
(d) One liter polypropylene wide-
mouth bottles (Nalgene Product No.
2104-0032).
(e) Insulated shipping box with carry-
ing strap (17" x 17" x 16"; Cole-Parmer
Product No. P-03742-00 and P-03742-
30).
(f) Miscellaneous — aluminum foil,
data card (see Appendix 2), hosecock
clamp, surgical gloves, screwdriver or
pliers for clamps, waterproof marker.
(g) Chemical resistant pump capable of
supplying 30 PSI at 3 gal/min and ap-
propriate connectors (for use where gar-
den hose-type pressurized taps for the
source or finished water to be monitored
are unavailable and for QC samples).
Follow the manufacturer's recommenda-
tions for pump priming.
2.2 Media and Reagents for Sampling
Apparatus
2.2.1 0.52% chlorine (HOC1; 5250
mg/L) — add 100 mL of household
bleach to 850 mL of dH2O and adjust
the pH of the solution to 6-7 with 1 M
HC1. Bring to 1 liter with dH2O. Store
for up to two weeks at room tempera-
ture.
2.2.2 2% sodium thiosulfate (Na2S2O3)
— dissolve 100 g of Na2S2O3 in a total
of 5000 mL dH2O to prepare a stock
solution. Autoclave for 30 min at
2.2.3 Hydrochloric acid (HC1) — Pre-
pare 0. 1, 1 and 5 M solutions by mixing
50, 100 or 50 mL of concentrated HC1
with 4950, 900 or 50 mL of dH2O, re-
spectively.
HCl solutions are self-sterilizing,
therefore, prepare those solutions to be
used for adjusting the pH of water sam-
ples at least 24 h before use.
2.3 Sterilization of Sampling Appara-
tus
2.3.7 1MDS filter cartridges and prefil-
ter cartridges — Presterilize 1MDS fil-
ter cartridges and prefilter cartridges by
wrapping the filters in Kraft paper and
autoclaving at 121°C for 30 min.
2.3.2 Chlorine Sterilization — Steri-
lize pumps, sampling apparatus mod-
ules, injector tubing and plastic bags for
transporting injector tubing by recircu-
lating or immersing the items in 0.52%
chlorine for 30 minutes. Drain the
chlorine from the objects being steril-
ized. Dechlorinate by recirculating or
immersing the items in a solution con-
taining 25 mL of 2% thiosulfate per
liter of sterile dH2O. Cover the appara-
tus module ends and the injector port(s)
with sterile aluminum foil. Place the
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April 2001
injector tubing in a sterile bag or wrap-
ping in such a way that the ends may be
removed without contaminating them.
Ensure that the solutions come in
full contact with all surfaces when per-
forming these procedures.
Used filter apparatus modules
should be decontaminated and then
cleaned according to the procedures in
Chapter 2 before final sterilization.
2.4 Apparatus and Materials for Elu-
tion and Organic Flocculation Steps
2.4.1 Positive pressure air or nitrogen
source equipped with a pressure gauge.
If the pressure source is a labora-
tory air line or pump, it must be
equipped with an oilfiller.
2.4.2 Dispensing pressure vessels — 5
or 20 liter capacity (Millipore Corp.
Product No. XX67 OOP 05 and XX67
OOP 20; see Figure 14-3).
Figure 14-3 Pressure Vessel
2.4.3 pH meter with combination-type
electrode and an accuracy of at least 0.1
pH unit.
2.4.4 Autoclavable inner-braided tub-
ing with screw clamps or quick con-
nects for connecting the vacuum source,
pressure vessel and cartridge housing.
2.4.5 Magnetic stirrer and stir bars.
2.4.6 Refrigerated centrifuge capable
of attaining 2,500 -10,000 xg and
screw-capped centrifuge bottles with
100 to 1000 mL capacity.
Each bottle must be rated for the
relative centrifugal force used.
2.4.7 Sterilizing filter — 0.22 |im
Acrodisc filter with prefilter (Gelman
Sciences Product No. 4525).
Use sterilizing filter stacks on sam-
ples that clog commercial filters. Pre-
pare sterilizing filter stacks using 0.22
fjmpore size membrane filters (Milli-
pore Corp. Product No. GSWP 47 00)
stacked with fiberglass prefilters (Milli-
pore Corp. APIS 47 00 andAP20 47
00). Stack the prefilters and 0.22 /j.m
membrane into a disc filter holder
(Millipore Corp. Product No. SXOO 47
00) with the AP20 prefilter on top and
0.22 [im membrane filter on bottom.
Disassemble the filter stack after each
use to check the integrity of the 0.22 jum
filter. Refilter any media filtered with a
damaged stack.
Always pass about 10-20 mL of
sterile beef extract, pH 7.0-7.5 (pre-
pared as above, without pH adjust-
ment), through the filter just before use.
This step will reduce virus adsorption
onto the filter membranes.
2.5 Media and Reagents for Elution
and Organic Flocculation Steps
2.5.7 Sodium hydroxide (NaOH) —
prepare 1 M and 5 M solutions by dis-
solving 4 g or 20 g of NaOH in a final
volume of 100 mL of dH2O, respec-
tively.
NaOH solutions are self-sterilizing
and may be stored for several months at
room temperature.
2.5.2 Iodine disinfectant, 0.5% — dis-
solve 5g of iodine in 1000 mL of 70%
ethanol.
2.5.3 Beef extract, desiccated powder
(Difco Product No. 0115-17-3) — pre-
pare buffered 1.5% beef extract by dis-
solving 30 g of beef extract powder and
7.5 g of glycine (final glycine concen-
tration = 0.05 M) in 1.9 L of dH2O.
Adjust the pH to 9.5 with 1 or 5 M
NaOH and bring the final volume to 2 L
with dH2O. Autoclave at 121°C for 15
min and use at room temperature.
Beef extract solutions may be
stored for one week at 4°C or for longer
periods at -20°C.
Screen each new lot of beef extract
before use to determine whether virus
recoveries are adequate. Perform the
screening by spiking one liter of beef
extract solution with 1 mL ofpoliovirus
containing a known virus concentration
ranging between 100 and 200 PFU/mL.
Process the spiked sample using the
organic flocculation concentration pro-
cedure given in section 4.2 and assay
the concentrate using the plaque assay
procedure given in Chapter 10 (Decem-
ber 1987 revision). The mean recovery
of poliovirus for three trials should be at
least 50%.
2.5.4 Sodium phosphate, dibasic
(Na2HPO4 • 7H2O) — 0.15 M, pH 9.0 -
9.5 or 7.0-7.5.
Dissolve 40.2 g of sodium phos-
phate in a final volume of 1000 mL
dH2O. The pH of the solution should be
between 9.0-9.5. Adjust the pH to 9.0
to 9.5 with NaOH, if necessary, or to
7.0 to 7.5 with HCl. Autoclave at
121°C for 15 min.
3. Sample Collection
Operators must wear surgical
gloves and avoid conditions that can
contaminate a sample with virus.
Gloves should be changed after touch-
ing human skin or handling components
that may be contaminated (e.g., water
taps, other environmental surfaces).
3.1 Purge the water tap to be sampled
before connecting the filter apparatus
(see Figure 14-4). Continue the purg-
ing for 2-3 min or until any debris that
has settled in the line has cleared.
Figure 14-4 Purge of Tap
3.1.1 Remove the foil from the back-
flow regulator (BR; see Figure 14-1).
Loosen the swivel female insert (SF)
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April 2001
slightly to allow it to turn freely and
connect the backflow regulator to the
tap. Retighten the swivel female insert.
Disconnect the cartridge housing mod-
ule at the quick connect (MQ1/FQ1), if
connected, and cover the open end with
sterile foil.
3.1.2 Remove the foil from the ends of
the discharge module and connect it to
the regulator module. Place the control
flow valve or tubing connected to the
outlet of the flow control valve into a
one liter plastic bottle. Note that the
injector module, the prefilter module
and the cartridge housing module must
not be attached to the apparatus at this
stage of the procedure!
3.7.3 Slowly turn on the tap and adjust
the pressure regulator until the pressure
gauge on the regulator module reads 30
PSI (see Figure 14-5). If the tap is
incapable of 30 PSI, adjust the regulator
to achieve the maximum pressure.
Pressures less than 30 PSI will result in
a reduced flow rate and thus longer
sampling times. Flush the apparatus
assembly with at least 20 gal of the wa-
ter to be sampled. While the system is
being flushed, measure the pH, the
temperature and the turbidity on the
water collecting in and overflowing
from the one liter plastic bottle. Record
the values onto a sample data sheet (see
Appendix 2 for an example).
The pH meter should be calibrated
before each use for the pH range of the
water to be sampled.
Figure 14-5 Pressure Adjustment
3.1.4 If the sample has a pH above 8.0
or contains a disinfectant, turn off the
water at the tap and disconnect the dis-
charge module from the regulator mod-
ule. Remove the foil from the ends of a
single injector module (see Figure 14-2)
and connect the module to the male
quick connect of the regulator module.
Reattach the discharge module.
3.7.5 If the sample has a pH above 8.0
and contains a disinfectant, turn off the
water at the tap and disconnect the dis-
charge module from the regulator mod-
ule. Remove the foil from the ends of a
double injector module (see Figure 14-
2) and connect the module to the male
quick connect of the regulator module.
Reattach the discharge module.
3.7.6 If an inj ector module has been
added, remove the foil from the injector
port(s) and attach the injector tubing to
each port. Add a hosecock clamp to
each injector tubing and tighten com-
pletely to prevent flow into the
injector(s). Turn the fine metering ad-
justment screw on each injector (the
smaller screw) clockwise as far as it will
go to minimize the flow rate until the
injectors are adjusted (note that the
injectors were designed to have a mini-
mum flow rate of 20-30 mL/min; thus
completely closing the fine metering
adjustment screw does not stop the
flow). Place the other end of each tub-
ing into the appropriate sterile gradu-
ated container containing 0.1 M HC1 or
2% thiosulfate. Take care not to touch
or contaminate the surfaces of the injec-
tor tubing that will be placed in the
graduated containers. Slowly turn on
the tap again and readjust the pressure
regulator, if necessary.
3.7.7 If a single injector module has
been added, continue to flush the appa-
ratus and adjust the water bypass screw
on the injector (the larger adjustment
screw) until the pressure gauge on the
injector module is about 35% less than
the pressure gauge on the regulator
module (e.g., 19 PSI when the gauge on
the regulator module reads 30 PSI; a
minimum of a 35% pressure drop is
required to achieve suction). Loosen
the hosecock clamp and observe wheth-
er suction is occurring. If not, slowly
increase the pressure drop until suction
starts.
(a) If the pH value of the water sample
is greater than 8.0, ensure that the inject-
or tubing is placed into a graduated
container containing 0.1 M HC1. While
continuing to measure the pH in the one
liter plastic bottle, adjust the fine meter-
ing adjustment screw on the injector to
add sufficient HC1 to give a pH of 6.5 to
7.5. It may be necessary to use the
hosecock clamp to reduce the flow rate
to less than 20-30 mL/min or to use a
more dilute or concentrated HC1 solu-
tion with some water samples. When
the pH stabilizes at a pH of 6.5 to 7.5,
continue with step 3.1.9. Record the
adjusted pH onto a sample data sheet
(see Appendix 2).
(b) If the water to be sampled contains
a disinfectant, ensure that the injector
tubing is placed into a graduated con-
tainer containing 2% thiosulfate. Ad-
just the fine metering adjustment screw
on the injector to add thiosulfate at a
rate of 10 mL/gal (2.6 mL/L or 30
mL/min at a flow rate of 3 gal/min; note
that at this rate, approximately 3-4 L of
thiosulfate solution will be required per
sample). When the proper rate is
achieved, record the presence of the dis-
infectant and the addition of thiosulfate
on a sample data sheet (see Appendix 2)
and continue with step 3.1.9.
3.7.8 If a double inj ector module is
being used (see Figure 14-6), continue
to flush the apparatus and turn the wa-
ter bypass screws on each injector clock-
wise as far as possible. Then turn the
water bypass screws on each regulator
one half turn counter clockwise. Con-
tinue turning the screws evenly one half
turn counter clockwise until the pres-
sure gauge on the double injector mod-
ule is 35% less than the pressure gauge
on the regulator module. Ensure that
the tubing from one injector is placed
into a graduated container containing
0.1 M HC1 and the other into a gradu-
ated container containing 2% sodium
thiosulfate. Loosen the hosecock
clamps. Since there may be slight
differences between the injectors and
since the pressure reading after the
injectors reflects an average pressure
drop from both injectors, some addi-
14-6
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April 2001
tional adjustment of the water bypass
screws may be required to obtain suc-
tion on each injector. After confirming
that each injector is drawing fluid, ad-
just the flow of HC1 and thiosulfate as in
step 3.4.8 (a)-3.4.8 (b) above. Record
the final pH, the presence of a disinfec-
tant and the addition of thiosulfate on a
sample data sheet (see Appendix 2) and
continue with step 3.1.9.
-
'$>'..
Figure 14-6 Double Injector
3.1.9 After adjusting the injectors, if
required, and flushing the system with
at least 20 gal, turn off the flow of water
at the sample tap and remove the dis-
charge module. If the water sample has
a turbidity greater than 75 NTU, remove
the foil from each end of the prefilter
module and connect the prefilter mod-
ule (see Figure 14-2) to the end of the
regulator module or to the end of one of
the injector modules, if used. Remove
the foil from the cartridge housing mod-
ule and connect it to the end of the reg-
ulator module, or to the end of the injec-
tor module or the prefilter module, if
used. Connect the discharge module to
the cartridge housing module.
3.1.10 Record the sample number,
location, date, time of day and initial
gallon (or cubic feet) reading from the
water meter onto a sample data sheet
(see Appendix 2).
Assign unique sample numbers to
each sample.
3.1.11 Slowly turn on the water
with the filter housing placed in an up-
right position, while pushing the red
vent button on top of the filter housing
to expel air. When the air is totally ex-
pelled from the housing, release the but-
ton, and open the sample tap com-
pletely. Readjust to 30 PSI, if neces-
sary. Check the thiosulfate usage rate
or the pH of the discharged water if an
injector(s) is being used and readjust, if
necessary.
Since the flow rate may change
during sampling due to filter clogging,
thiosulfate addition and the adjusted pH
of the sample must be monitored reg-
gularly during sampling.
Recommended sample volumes for
sewage are two to twenty liters. Recom-
mended minimum sample volumes for
surface or recreational wa-ters are 200
L and 1500 L for finished drinking wa-
ter or for untreated groundwater.
Recommended maximum sample vol-
umes for source and recreational waters
and finished waters/untreated
groundwaters are 300 L and 2000 L,
respectively. The total amount of sam-
ple that can be passed through a car-
tridge filter will depend upon water
quality, however, it should be possible
to obtain the minimum volume using the
procedures described above.
3.1.12 Turn off the flow of water at
the sample tap at the end of the sam-
pling period and record the date, time of
day, and final gallon (or cubic feet)
reading from the water meter onto a
sample data sheet (see Appendix 2).
Subtract the initial meter reading from
the final reading to obtain the total sam-
ple volume. Convert the total sample
volume to liters and record on a sample
data sheet (see Appendix 2 for appropri-
ate conversion factors).
Although the final water meter
reading may be affected by the addition
ofHCl and/or thiosulfate, the effect is
insignificant and may be ignored.
3.1.13 Loosen the swivel female
insert on the regulator module and dis-
connect the backflow regulator from the
tap. Disconnect the cartridge housing
module and the prefilter housing mod-
ule, if used from the other modules.
Turn the filter housing(s) upside down
and allow excess water to flow out as
waste water (see Figure 14-7). Turn
the housing(s) upright and cover the
quick connects on each end of the mod-
ules with sterile aluminum foil.
3.1.14 Pack the cartridge housing
module(s) into an insulated shipping
box. Add 6-8 small ice packs (prefroz-
en at -20°C) around the cartridge hous-
ings to keep the sample cool in transit
(the number of ice packs may have to be
adjusted based upon experience to en-
sure that the samples remain cold, but
not frozen). Drain and add the regula-
tor and injector modules used. Place the
sample data sheet (protected with a
closable plastic bag) in with the sample.
If the shipping box cannot be trans-
ported to the laboratory for virus analy-
sis by close of business on the day col-
lected, ship it to the laboratory by over-
night courier.
Figure 14-7 Remove Excess
Water
4. Elution and Concentration
4.1 Elution Procedure
The cartridge filters must arrive
from the utility in a refrigerated, but
not frozen, condition. The date of ar-
rival and the arrival condition should
be recorded on a sample data sheet (see
Appendix 2). Filters must be refriger-
ated immediately upon arrival. Ideally,
viruses should be eluted from filters
within 24 h of the start of the sample
collection, but all filters must be eluted
within 72 h of the start of the sample
collection.
Place a disinfectant-soaked sponge
over vents while releasing trapped air
or pressure throughout this procedure
to minimize dangers from aerosols.
14-7
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April 2001
4.1.1 Attach sections of braided tubing
(sterilized on inside and outside sur-
faces with chlorine and dechlorinated
with thiosulfate as described in section
2.1.1a.lO, 2.3.2) to the inlet and outlet
ports of a cartridge housing module
containing a 1MDS filter to be tested for
viruses. If a prefilter was used, keep the
prefilter and cartridge housing modules
connected and attach the tubing to the
inlet of the prefilter module and to the
outlet of the cartridge housing module.
4.1.2 Place the sterile end of the tubing
attached to the outlet of the cartridge
housing module into a sterile two liter
glass or polypropylene beaker.
4.1.3 Attach the free end of the tubing
from the inlet port of the prefilter or
cartridge housing modules to the outlet
port of a sterile pressure vessel and at-
tach the inlet port of the pressure vessel
to a positive air pressure source. Add
pressure to blow out any residual water
from the cartridge housing(s). Open the
vent/relief valve to release the pressure.
4.1.4 Remove the top of the pressure
vessel and pour 1000 mL of buffered
1.5% beef extract (pH 9.5, prewarmed
to room temperature) into the vessel.
Replace the top of the pressure vessel
and close its vent/relief valve.
Acceptable alternatives to the use
of a pressure vessel include 1) the use
of a peristaltic pump and sterile tubing
to push the beef extract through the fil-
ter and 2) the addition of beef extract
directly to the cartridge housing and
the use of positive pressure to push the
beef extract through the filter.
4.1.5 Open the vent/relief valve(s) on
the cartridge housing(s) and slowly ap-
ply sufficient pressure to purge trapped
air from them. Close the vent/relief
valve(s) as soon as the buffered beef
extract solution begins to flow from it.
Turn off the pressure and allow the so-
lution to contact the 1MDS filter for 1
min.
Wipe up spilled liquid with
disinfectant-soaked sponge. Carefully
observe alternative housings without
vents to ensure that all trapped air has
been purged.
4.1.6 Apply pressure to force the buf-
fered beef extract solution through the
filter(s) (see Figure 14-8).
The solution should pass through
the 1MDS filter slowly to maximize the
elution contact period. When air enters
the line from the pressure vessel, ele-
vate and invert the filter housing to per-
mit complete evacuation of the solution
from the filters.
4.1.7 Turn off the pressure at the
source and open the vent/relief valve on
the pressure vessel. Place the buffered
beef extract from the two liter beaker
back into the pressure vessel. Replace
the top of the pressure vessel and close
its vent/relief valve. Repeat steps 4.1.5
-4.1.6.
Figure 14-8 Elution of 1MDS Filter
(a) Turn off the pressure at the source
and open the vent/relief valve on the
pressure vessel. Thoroughly mix the
eluate. Record the date and time of elu-
tion on a sample data sheet.
(b) Proceed to the Organic Flocculation
Concentration Procedure (section 4.2)
immediately. If the Organic Floccula-
tion Concentration Procedure cannot be
undertaken immediately, adjust the pH of
the eluate to 7.0 to 7.5 with 1 M HC1
and store the eluate at 4°C for up to 24
h or for longer periods at -70°C.
4.2 ORGANIC FLOCCULATION
CONCENTRATION PROCEDURE
Minimize foaming (which may inac-
tivate viruses) throughout the procedure
by not stirring or mixing faster than
necessary to develop a vortex.
4.2.1 Place a sterile stir bar into the
beaker containing the buffered beef ex-
tract eluate from the cartridge filter(s).
Place the beaker onto a magnetic stirrer,
and stir at a speed sufficient to develop
a vortex.
4.2.2 Insert a combination-type pH
electrode into the beef extract eluate.
Add 1 M HC1 to the eluate slowly while
moving the tip of the pipette in a circu-
lar motion away from the vortex to fa-
cilitate mixing. Continue adding 1 M
HC1 until the pH reaches 3.5 ± 0.1 and
then stir slowly for 30 min at room tem-
perature (see Figure 14-9).
The pH meter must be standardized
at pH 4 and 7.
Sterilize electrodes before each use
by immersing the tip of the electrode in
0.1% chlorine for at least 1 min.
Dechlorinate using a solution contain-
ing 2.5 mL of 2% sterile sodium thiosul-
fate per liter of sterile dH2O. Sterilize
electrodes after each use by rinsing to
remove particulates followed by immer-
sion of the tip of the electrode in 0.1%
chlorine again for at least 1 min.
Dechlorinate again using a solution
containing 2.5 mL of 2% sterile sodium
thiosulfate per liter of sterile dH2O.
A precipitate will form. If pH falls
below 3.4, add 1 MNaOHto bring it
back to 3.5 ± 0.1. Exposure to a pH
below 3.4 may result in some virus
inactivation.
Figure 14-9 Floe Formation
4.2.3 Remove the electrode from the
beaker, and pour the contents of the
beaker into a centrifuge bottle. Cap the
bottle and centrifuge the precipitated
beef extract suspension at 2,500 *g for
15 min at 4°C (see Figure 14-10). Re-
move and discard the supernatant.
14-8
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April 2001
To prevent the transfer of the stir
bar into a centrifuge bottle, hold an-
other stir bar or magnet against the
bottom of the beaker while decanting
the contents. The beef extract suspen-
sion will usually have to be divided into
several centrifuge bottles.
Figure 14-10 Centrifuge Floe
4.2.4 Place a stir bar into the centri-
fuge bottle that contains the precipitate.
Add 30 mL of 0.15 M sodium phos-
phate, pH 9.0 - 9.5. Place the bottle
onto a magnetic stirrer, and stir slowly
until the precipitate has dissolved com-
pletely (see Figure 14-11).
Since the precipitate may be diffi-
cult to dissolve, it can be partially dis-
persed with a spatula before or during
the stirring procedure. It may also be
dissolved by repeated pipetting or by
shaking at 160 rpmfor 20 min on an
orbital shaker in place of stirring.
When the centrifugation is performed in
more than one bottle, dissolve the pre-
cipitates in a total of 30 mL and com-
bine into one bottle. The precipitate
should be completely dissolved before
proceeding or significant virus loss may
occur in step 4.2.5. Because virus loss
may also occur by prolonged exposure
to pH 9.0-9.5, laboratories that find it
difficult to resuspend the precipitate
may dissolve it initially in 0.15 M so-
dium phosphate, pH 7.0 - 7.5. If this
variation is used, the pH should be re-
adjusted to 9.0-9.5 with 1 MNaOH af-
ter the precipitate is completely dis-
solved and mixed for 10 min at room
temperature before proceeding to step
4.2.5.
4.2.5 Check the pH using an electrode
sterilized as in step 4.2.2 and readjust to
9.0-9.5 with 1 M NaOH, as necessary.
Remove the stir bar and centrifuge the
dissolved precipitate at 4,000 -10,000
xg for 10 min at 4°C. Remove the
supernatant and discard the pellet. Ad-
just the pH of the supernatant to 7.0-7.5
with 1 M HC1.
4.2.6 Load the supernatant into a 50
mL syringe and force it through a beef
extract-treated sterilizing filter. This
step is to remove bacteria, fungi and
other contaminating agents.
If the sterilizing filter begins to
clog, empty the loaded syringe into the
bottle containing the unfiltered
supernatant, fill the syringe with air,
and inject air into filter to force any
residual sample from it. Continue the
filtration procedure with another filter.
4.2.7 Measure the amount of filtered,
concentrated supernatant and record the
amount as the Final Concentrated
Sample Volume (FCSV) onto a sample
data sheet. Also record the date and
time of concentration on a sample data
sheet. Divide the filtered supernatant
into subsamples, if appropriate, and la-
bel the containers with appropriate sam-
pling information for identification.
Figure 14-11
Precipitate
Dissolving
4.2.8 Hold any portion of the sample
that can be assayed within 24 h at 4°C
and freeze all other portions at -70°C.
Assay the sample for viruses using the
plaque assay described in Chapter 10
(December 1987 revision) or the total
culturable assay described in Chapter
15.
5. References
Fout, G.S., F.W. Schaefer III, J.W. Mes-
ser, D.R. Dahling and R.E. Stetler.
1996. ICR Microbial Laboratory
Manual. U.S. Environmental Pro-
tection Agency Publication No.
EPA/600/R-95/178. Office of Re-
search and Development, Washing-
ton, D.C.
14-9
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April 2001
Appendix 1. VENDORS
The vendors listed below represent one
possible source for required products.
Equivalent items from other vendors
may substituted for items given in the
text.
Cincinnati Valve and Fitting Co.
11633DeerfieldRd.
Cincinnati, OH 45242
(513)469-1212
Cole-Parmer Instrument Co.
7425 N. Oak Park Ave.
Niles, IL60714
(800) 323-4340
Cuno, Inc.
400 Research Parkway
Meriden, CT 06450
(800) 243-6894 (Ask for a local
distributor)
DEMA Engineering Co.
10014 Big Bend Blvd.
Kirkwood,MO63122
(800) 325-3362
Difco Laboratories
P.O. Box 331058
Detroit, MI 48232
(800) 521-0851 (Ask for a local dis-
tributor)
Gelman Sciences
600 S. Wagner Rd.
Ann Arbor, MI 48103
(800) 521-1520
Millipore Corp.
397 Williams St.
Marlboro, MA 01752
(800) 645-5476
Nalgene Nunc International
P.O. Box 20365
Rochester, NY 14602
(716) 586-8800 (Ask for a local
distributor)
Neptune Equipment Co.
520 W. Sharon Rd.
Forest Park, OH 45240
(800) 624-6975
OMEGA Engineering, Inc.
P.O. Box 4047
Stamford, CT 06907
(800) 826-6342
Plast-o-matic Valves, Inc.
1384Pompton Ave
Cedar Grove, NJ 07009
(973) 256-3000 (Ask for a local distribu-
tor)
Parker Hannifin Corp.
Commercial Filters Div.
1515 W. South St., Lebanon, IN 46052
(765) 482-3900
Ryan Herco
2509 N. Naomi St.
Burbank, CA91504
(800)848-1141
United States Plastic Corp.
1390NeubrechtRd.
Lima, OH 45801
(800) 537-9724
Watts Regulator
Box 628
Lawrence, MA 01845
(978)688-1811
14-10
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April 2001
Appendix 2. SAMPLE DATA SHEET
SAMPLE DATA SHEET
SAMPLE NUMBER:
SITE OR UTILITY NAME:
SITE OR UTILITY ADDRESS:
STREET:
CITY:
STATE:
ZIP:
SAMPLER'S NAME:
WATER TEMPERATURE:
TURBIDITY:
MTU
WATER pH:
ADJUSTED WATER pH:
WATER DISINFECTED:
(CHECK) _ YES _ NO
THIOSULFATE ADDKI):
(CHECK) _ YES _ NO
nvrr. METER READING:
Date:
CHECK UNITS:
Time:
gallons
ft3
FINAL METER READING:
Date:
CHECK UNITS:
Time:
allons
.ft3
TOTAL SAMPLE VOLUME:
(Final-Initial meter readings * 3.7854 (for readings in gallons)
or x 28.316 (for readings in ft3))
SHIPMENT DATE:
ARRIVAL DATE:
CONDITION ON ARRIVAL:
ANALYST/TECHNICIAN NAME:
DATE ELUTED:
TIME:
DATE CONCENTRATED:
TIME:
FINAL CONCENTRATED SAMPLE VOLUME (FCSV):
ml
Comments:
14-11
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