United States
Environmental Protection
Agency
Environmental Monitoring and
Support Laboratory
Cincinnati OH 45268
EPA/600/4-84/013(R7)
September 1989
Revision
Research and Development
&EPA
USEPA Manual of
Methods for Virology
Chapter?
Revised September 1989
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September 1989
Chapter 7
Method for Recovering Viruses from Sludges, Soils, Sediments and Other Solids
1. Introduction
This chapter describes a procedure
that may be used to monitor for the
presence of solids-associated
viruses. Its design requires that the
virus be eluted directly from solids.
Samples which may have a portion of
the virus content suspended
unattached in a liquid matrix, such as
in sludges and sediments, must first
be treated to bind the free viruses to
the solids. Thus, initial steps in this
procedure will differ based on
whether or not the solids are
suspended in liquid. Because of the
naturally high association of viruses
with solids, the virus content of the
liquid matrix is likely to represent
only a small fraction of the viruses
present in a sample.
Processed sludges, soils, and
sediment samples are assayed in cell
culture for plaque-forming viruses. If
the inocula is observed to be or
suspected of being toxic to the cell
culture, or is so darkly colored as to
result in inaccurate plaque counts,
use method described in Chapter 8
(April 1986 revision) to reduce
interference.
The procedures in this chapter
require dispensing the entire sample
volume into a centrifuge bottle. If
bottles of sufficient capacity are
unavailable, the sample should be
divided and then recombined after
centrifugation.
Use aseptic techniques and sterile
materials and apparatus only.
Sterilize all contaminated materials
before discarding them (see Chapters
2 and 3).
2. Conditioning of
Suspended Solids
The procedure in this Section is
used only for processing of
suspended solids. Conditioning of
such samples (e.g. sludges) is
required as a means of adsorbing to
the solids those viruses that are
present in the liquid matrix. Non-
suspended solids, such as soil, are
not to be conditioned. Begin
processing these samples by
following the procedures given in
Section 3.
2.1 Preparation
2.1.1 Apparatus and Materials
(a) Refrigerated centrifuge
capable of attaining 2,500 x g
and screw-capped centrifuge
bottles.
Each sample centrifuged at
2,500 x g will consist of about
100 mL
(b) pH meter, measuring to an
accuracy of at least 0.1 pH
unit, equipped with a
combination-type electrode.
(c) Magnetic stirrer and stir bars.
2.1.2 Media and Reagents
(a) Hydrochloric acid (HCI) -
5 M.
(b) Aluminum chloride
(AICI3-6H2O) - 0.05 M.
Autoclave AICI3 solution at
121 "C for 15 minutes.
(c) Sodium hydroxide (NaOH) —
5 M.
2.2 Procedure
Flow diagram of procedure to
condition suspended solids is given
in Figure 7-1.
In the absence of experience that
dictates otherwise, use 100-mL
volumes per sample.
2.2.1 Measure 100 mL of well-
mixed sample in a graduated 100-mL
cylinder.
Sample must be mixed vigorously
immediately before it is poured into
graduated cylinder because solids,
which contain most of the viruses,
begin to settle out immediately after
mixing stops.
SUSPENDED SOLIDS (100 mL)
Mix suspension on
magnetic stirrer.
Add 1 mL of 0.05 M AICI3.
SALTED SOLIDS SUSPENSION
Continue mixing
suspension.
Adjust pH of salted
suspension to 3.5 ±0.1
with 5 M HCI.
Mix vigorously for 30
minutes.
pH-ADJUSTED SOLIDS SUSPENSION
Centrifuge salted, pH-
adjusted suspension at
2,500 x g for 15 minutes
at4°C.
Discard supernate.
Retain solids.
SOLIDS
Figure 7-1. Flow diagram of method
for conditioning sus-
pended solids.
2.2.2 Place stir bar into a 250-mL
beaker.
2.2.3 Pour the 100 mL of measured
sample from the graduated cylinder
into the 250-mL beaker.
It may be necessary to pour
sample several times back and forth
from beaker to graduated cylinder to
obtain all solids in the beaker.
CAUTION: Avoid formation of
aerosols. Slowly pour sample down
the inner wall of vessel to avoid
splatter.
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September 1989
2.2.4 Place beaker on magnetic
stirrer, cover loosely with aluminum
foil, and stir at speed sufficient to
develop vortex.
2.2.5 Add 1 ml of 0.05 M AICI3 to
mixing sample.
Final concentration ofAICIg in
sample is approximately 0.0005 M.
2.2.6 Place combination-type pH
electrode into mixing sample.
pH meter must be standardized at
pH4.
2.2.7 Adjust pH of sample to 3.5
±0.1 With 5 M HCl.
If pH falls below 3.4, readjust it
with 5 M NaOH. When solids adhere
to electrodes, clean electrodes by
moving them up and down gently in
mixing sample.
2.2.8 Continue mixing for 30
minutes.
The pH of the sample should be
checked at frequent intervals. If the
pH drifts up, readjust it to 3.5 ±0.1
with 5 M HCl. If the pH drifts down,
readjust it with 5 M NaOH.
2.2.9 Pour conditioned sample into
centrifuge bottle.
To prevent transfer of stir bar into
centrifuge bottle when decanting
sample, hold another stir bar or
magnet against bottom of beaker.
Solids that adhere to stir bar in the
beaker may be removed by
manipulation with a pipette. It may be
necessary to pour sample several
times back and forth from centrifuge
bottle to beaker to obtain all solids in
the bottle.
CAUTION: Take care to avoid
formation of aerosols. Slowly pour
sample down the inner wall of vessel
to avoid splatter.
2.2.70 Centrifuge conditioned sample
at 2,500 x g for 15 minutes at 4°C.
2.2.77 Decant supernate into beaker
and discard.
2.2.12 Replace cap on centrifuge
bottle.
2.2.13 Elute viruses from solids by
following the procedure described in
Section 4.
3. Preparation of Non-
Suspended Solids
In the absence of experience that
dictates otherwise, use 100 g of
sample (e.g. soil).
3.1 Weigh two 100-g portions for
each test sample.
Test sample is transferred to a
large bottle and is thoroughly mixed
by manually shaking and tumbling
before it is weighed.
3.2 Place one of the 100-g portions
in a 250-mL beaker and cover loosely
with aluminum foil.
3.3 Elute viruses from the above
100-g sample by following the
procedure described in Section 4.
3.4 Place other 100-g portion in a
tared weighing pan.
3.5 Place pan and its contents in
an oven maintained at 103-105°C.
3.6 Heat sample to dryness.
3.7 Cool sample to room
temperature in a desiccator.
3.8 Weigh sample.
3.9 Record dry weight of sample.
3.10 Repeat Sections 3.5 through
3.9 until loss in weight is no more
than 4% of the previous weight.
Re-dry the sample for 1 hour
before repeating the cooling and
weighing cycle.
These data will be used in Chapter
10 (December 1987 revision) to
calculate viral content of non-
suspended solids in plaque forming
units per gram of dry weight.
4. Elution of Viruses from
Solids
4.1 Preparation
4.7.7 Apparatus and Materials
(a) Refrigerated centrifuge
capable of attaining 10,000 x
g and screw-capped
centrifuge bottles that can
withstand 10,000 x g.
A refrigerated centrifuge
capable of attaining 2,500 x g
and screw-cap centrifuge
bottles that can withstand
2,500 x g may be used for
samples such as soils, which
settle readily at lower
centrifugation speeds. If the
eluates resulting from such
centrifugation are not easily
forced through the membrane
filters used for sterilization
(see Step (e) in Section 4.1.1),
then centrifuge at 10,000 x g.
(b) pH rneter, measuring to an
accuracy of at least 0.1 pH
unit, equipped with a
combination-type electrode.
(c) Magnetic stirrer and stir bars.
(d) Membrane filter apparatus for
sterilization — 47-mm
diameter filter holder and 50-
mL slip-tip syringe (Millipore
Corp., Swinnex filter, No.
SX0047000, or equivalent for
filter holder only).
(e) Disc filters, 47-mm diameter
— 3.0-, 0.45-, and 0.25-um
pore size filters (Filterite
Corp., Duo-Fine series, or
equivalent). Filters may be cut
to the proper diameter from
sheet filters.
Disassemble Swinnex filter
holder. Place filter with 0.25-
ium pore size on support
screen of filter holder and
stack the remaining filters on
top in order of increasing
pore size. Reassemble and
tighten filter holder. Filters
stacked in tandem as
described tend to clog more
slowly when turbid material is
filtered through them.
Prepare several filter stacks.
4.1.2 Media and Reagents
(a) Disodium hydrogen
phosphate (Na2HPO4 •
7H2O).
(b) Citric acid.
(c) Beef extract powder (BBL
Microbiology Systems, or
equivalent).
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September 1989
Prepare buffered 10% beef
extract by dissolving 10 g
beef extract powder, 1.34 g
Na2HPO4-7H2O and 0.12 g
citric acid in 100 mL of
deionized distilled water.
Dissolve by stirring on a
magnetic stirrer. Autoclave
beef extract solution for 15
minutes at 121°C.
(d) Hydrochloric acid (HCI) -
5M.
' (e) Aluminum chloride (AICI3 •
6H20) - 0.05 M.
Autoclave AICI3 solution at
121 "C for 15 minutes.
Sodium hydroxide (NaOH) —
5M.
4.1.3 Procedure
Flow diagram of the virus elution
procedure is given in Figure 7-2.
(a) . Place stir bar into vessel
containing the solids (from
either Section 2.2.13 or
Section 3.3.)
Vessel may be a centrifuge
bottle (Section 2.2.13) or a
250-mL beaker (Section 3.3).
(b) Add 100 mL of buffered 10%
beef extract to the vessel
containing the solids.
(c) Place vessel on magnetic
stirrer, and stir at speed
sufficient to develop vortex.
To minimize foaming (which
may inactivate viruses), do not
mix faster than necessary to
develop vortex.
(d) Continue mixing for 30
minutes.
(e) If solids were processed in
beaker, pour contents into
centrifuge bottle.
To prevent transfer of stir
bar into centrifuge bottle
when decanting sample, hold
another stir bar or magnet
against bottom of beaker.
Solids that adhere to stir bar
in the beaker may be
removed by manipulation with
a pipette. It may be necessary
to pour sample several times
back and forth from
centrifuge bottle to beaker to
obtain all solids in the bottle.
SOLIDS
Add 100 mL of buffered
10% beef extract, adjust
to pH 7.0 ± 0.1 if
necessary.
Mix resuspended solids on
magnetic stirrer for 30
minutes to elute viruses.
RESUSPENDED SOLIDS
) '
ELUATE
Centrifuge resuspended
solids for 30 minutes at
4°C using a centrifugal
force of 2,500 x g for
readily settleable
mixtures (e.g. soil) and
10,000 xg for others
(e.g. sludge).
Discard solids.
Retain eluate (supemate).
Filter eluate through 47-
mm Filterite filter stack of
3.0-, 0.45- and 0.25-nm
with the 0.25-nm pore
size on support screen of
filter and remaining filters
on top in order of
increasing pore size.
FILTERED ELUATE
If concentration of viruses
is not necessary, see
Chapter 10 for virus
assay procedure.
If concentration of viruses
is necessary prior to
virus assay, proceed to
virus concentration
procedure outlined in
Figure 7-4.
ASSAY ELUATE FOR VIRUSES
Figure 7-2. Flow diagram of method
for elution of virus from
solids.
Use appropriate centrifuge
bottles for the centrifugal
force that will be applied.
CAUTION: Take care to
avoid formation of aerosols.
Slowly pour sample down the
inner wall of vessel to avoid
splatter.
(f) If solids were processed in
centrifuge bottle, remove stir
bar from bottle with long
forceps or a magnet retriever.
Determine if centrifuge
bottle is appropriate for the
centrifugal force that will be
applied.
(g) Centrifuge solids-eluate
mixture at either 10,000 x g or
2,500 x g for 30 minutes at
4°C.
Use a centrifugal force of
2,500 x g for mixtures such as
soil-eluate mixtures, which
settle readily at lower
centrifugation speeds. If the
eluates resulting from such
centrifugation are not easily
forced through the membrane
filters used for sterilization,
then centrifuge at 10,000 x g.
Sludge-eluate mixtures
generally require
centrifugation at 10,000 x g
for 30 minutes.
(h) Decant eluate into beaker and
discard solids.
(i) Place a filter holder that
contains a filter stack on a
250-mL Erlenmeyer receiving
flask.
0') Load 50-mL syringe with
eluate from Section 4.1.3,
Step (h).
(k) Place tip of syringe into filter
holder.
(I) Force eluate through filter
stack into 250-mL receiving
flask.
Take care not to break off
tip of syringe and to minimize
pressure on receiving flask
because such pressure may
crack or topple the flask. If
filter stack begins to clog
badly, empty loaded syringe
into beaker containing
unfiltered eluate, fill syringe
with air, and inject air into
filter stack to force residual
eluate from filters. Continue
filtration procedure with
another filter holder and filter
stack. Discard contaminated
filter holders and filter stacks.
Steps (i) thru (I) may be
repeated as often as
necessary to filter entire
volume of eluate.
Disassemble each filter
holder and examine bottom
filters to be certain they have
7-3
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September 1989
not ruptured. If a bottom filter
has ruptured, repeat Steps (f)
through (I) with new filter
holders and filter stacks.
The number of cell cultures
necessary for the viral assay
may be reduced by
concentrating the viruses in
the beef extract by the
organic ftocculation
procedure of Katzenelson
(reference cited in Section
5.1). Some loss of viruses
may occur with this
procedure. If viruses in
eluates are to be
concentrated, proceed
immediately to Section 5. If
concentration is not required,
proceed to Step (m).
(m) Refrigerate eluate immediately
at 4°C, and maintain it at that
temperature until it is assayed
for viruses.
// assay for viruses cannot
be undertaken within eight
hours, store eluate
immediately at -70°C.
5. Concentration of
Viruses from Eluates
5.1 Organic Ftocculation
Concentration Procedure of
Katzenelson (Katzenelson, E., B.
Fattal, and T. Hostovesky, Appl.
Environ. Af/crob/o/.32:638,1976)
It is preferable to assay eluted
viruses in the beef extract eluate
without concentrating them because
some toss of viruses may occur in
concentration. However, the number
of cell cultures needed for assays
may be reduced by concentrating the
viruses in the eluate.
Floe formation capacity of the
powdered beef extract reagent must
be pretested. Some powdered beef
extracts may not produce sufficient
floe resulting in significantly reduced
virus recoveries. Where it has been
predetermined that insufficient floe is
formed, the reagent is fortified with
floe from paste beef extract.
Procedure for preparing the
additional floe is described in
Section 5.1.3.
5.1.1 Apparatus and Materials
(a) Magnetic stirrer and stir bars.
(b) pH meter, measuring to an
accuracy of at least 0.1 pH
unit, equipped with a
combination-type electrode.
(c) Refrigerated centrifuge
capable of attaining 2,500 x g
and screw-capped centrifuge
bottles.
Each sample centrifuged at
2,500 x g will consist of about
330 mL
5.1.2 Media and Reagents
(a) Disodium hydrogen
phosphate (Na2HPO4 -7H2O)
-0.15M.
(b) Hydrochloric acid (HCI) -
1 M.
(c) Sodium hydroxide (NaOH) —
1 M.
(d) Paste beef extract (Difco
Laboratory, Bacto beef extract
or equivalent) — 3%.
Pasfe beef extract is not
needed if the 10% powdered
beef extract reagent used for
the elution process in Section
4.1.2, Step (c) produces
sufficient floe when
processed by the organic
flocculation concentration
procedure of Katzenelson.
Prepare the 3% paste beef
extract stock solution by
dissolving 30 g of paste beef
extract in 1,000 mL deionized
distilled water. Autoclave the
stock solution at 121° C for 15
minutes and use at room
temperature. From this stock
solution, one 330-mL aliquot
is removed for each sample
requiring supplementation
with paste beef floe. Although
the paste beef extract stock
solution may be stored at4°C
for an extended time period, it
is advisable to prepare
solutions on a weekly basis,
thereby lessening the
possibility of microbial
contamination.
5.1.3 Preparation of Floe from Paste
Beef Extract Reagent
Prepare required paste beef floe
before proceeding to Section 5.1.4
for those samples in which the
powdered beef extract reagents had
been determined to produce
insufficient floe when processed by
the organic flocculation
concentration procedure.
Flow diagram of procedure to
prepare reagent floe is given in
Figure 7-3.
(a) Place stir bar in 600-mL
beaker.
(b) Pour 330 mL of a 3% paste
beef extract stock solution into
beaker and cover loosely with
aluminum foil.
PASTE BEEF EXTRACT REAGENT
Autoclave at 121°C for 15
minutes (If stored, cool
and hold at 4°C).
Use at room temperature.
STERILE PASTE BEEF EXTRACT
Add a 330-mL portion of
the paste beef stock to a
beaker containing stir
bar.
Place beaker on magnetic
stirrer and mix.
Adjust pH of paste beef
stock to 3.5 ±0.1 with
1 M HCI.
Continue mixing for 30
minutes.
FLOCCULATED PASTE BEEF
EXTRACT
Centrifuge flocculated
paste beef extract at
2,500 x g for 15 minutes
at 4°C.
Discard supernate.
Retain floe.
PASTE BEEF EXTRACT FLOC
Filtered eluate that
requires floe supple-
mentation (See Figure
7-4) is added to floe.
Disperse manually using a
pipette until floe is
dissolved.
FILTERED ELUATE PASTE FLOC
SOLUTION
Figure 7-3. Flow diagram of method
for preparation of floe
from paste beef extract
reagent.
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September 1989
(c) Place beaker on magnetic
stirrer, and stir at a speed
sufficient to develop vortex.
(d) Insert combination-type pH
electrode into paste beef
extract stock solution.
pH meter must be
standardized at pH 4.
(e) Add 1 M HCI to flask slowly
until pH of beef extract
reaches 3.5 ±0.1.
A precipitate will form.
(f) Remove electrode from
beaker, and pour contents of
beaker into a 1,000-mL
centrifuge bottle.
To prevent transfer of stir
bar into a centrifuge bottle,
hold another stir bar or
magnet against bottom of
beaker when decanting
contents.
(g) Centrifuge precipitated beef
extract suspensions at 2,500
x g for 15 minutes at 4°C.
(h) Pour off and discard
supernate.
(i) Retain floe in centrifuge bottle
at 4°C for subsequent mixing
with the non-flocculating
buffered beef extract.
5.1.4 Procedure
Flow diagram of the virus
concentration procedure is given in
Figure 7-4.
(a) Pour filtered eluate from
Section 4.1.3, Step (I) into
graduated cylinder, and
record volume.
(b) Pour filtered eluate into 600-
mL beaker and cover loosely
with aluminum foil.
(c) For every 3 mL of beef extract
eluate, add 7 mL of deionized
distilled water to the 600-mL
beaker.
The concentration of beef
extract is now 3%. This
dilution is necessary because
10% beef extract often does
not process well by the
organic flocculation
concentration procedure.
(d) Record the total volume of the
diluted, filtered beef extract.
Proceed to Step (h) only if
the powdered beef extract
FILTERED ELUATE
Add sufficient volume of
deionized distilled water
to filtered eluate to
reduce concentration of
beef extract from 10% to
3%. Record total volume
of the diluted beef
extract.
Mix previously prepared
paste floe (Figure 7-3)
with those filtered
eluates in which the
powdered beef extract
reagent has been
determined to produce
insufficient floe when
processed by the organic
flocculation procedure.
DILUTED, FILTERED ELUATE
Mix diluted eluate on
magnetic stirrer.
Adjust pH of eluate (3%
beef extract) to 3.5
±0.1 with 1MHCI. A
precipitate (floe) will
form. Continue mixing
for 30 minutes.
FLOCCULATED ELUATE
Centrifuge flocculated
eluate at 2,500 x g for
15 minutes at4°C.
Discard supernate.
Retain floe.
FLOG FROM ELUATE
Add 0.15 M Na2HPO4to
floe, using l/20th of the
recorded volume of the
diluted 3% beef extract.
Mix suspended floe on
magnetic stirrer until floe
dissolves.
Adjust with 1 M HCI or
1 M NaOH if pH is
below 7.0 or above 7.5.
DISSOLVED FLOC
See Chapter 10 for virus
assay procedure.
ASSAY DISSOLVED FLOC FOR
VIRUSES
Figure 7-4.
Flow diagram of method
tor concentration of vir-
uses from beef extract
eluate.
reagent used for the virus
elution process is known to
form sufficient floe to
undertake eluate processing
by the organic flocculation
concentration procedure
without addition of paste floe.
Where the addition of paste
floe is required, add the
diluted beef extract to the floe
as described in Steps (e)
through (g) before
proceeding to Step (h).
(e) Pour extract from beaker into
centrifuge bottle containing
floe from Section 5.1.3, Step
0).
(f) Disperse floe manually using
a pipette until it is dissolved in
the extract.
(g) Pour contents into a 600-mL
beaker.
(h) Place stir bar in beaker that
contains diluted, filtered beef
extract from either Step (d) or
Step (g).
(i) Place beaker on magnetic
stirrer, cover loosely with
aluminum foil, and stir at a
speed sufficient to develop
vortex.
To minimize foaming (which
may inactivate viruses), do not
mix faster than necessary to
develop vortex.
G') Insert combination-type pH
electrode into diluted, filtered
beef extract.
pH meter must be
standardized at pH 4.
(k) Add 1 M HCI to flask slowly
until pH of beef extract
reaches 3.5 ±0.1.
A precipitate will form. If pH
is accidentally reduced below
3.4, add 1 M NaOH until pH is
3.5 ± 0.1. Avoid reducing pH
below 3.4 because some
inactivation of viruses may
occur.
(I) Continue to stir for 30
minutes.
(m) Remove electrode from
beaker, and pour contents of
beaker into a 1,000-mL
centrifuge bottle.
To prevent transfer of stir
bar into a centrifuge bottle,
7-5
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September 1989
hold another stir bar or
magnet against bottom of
beaker when decanting
contents.
(n) Centrifuge precipitated beef
extract suspensions at 2,500 x
g for 15 minutes at 4°C.
(o) Pour off and discard
supernate.
(p) Place a stir bar into centrifuge
bottle that contains precipitate.
(q) Add to centrifuge bottle a
volume of 0.15 M
Na2HPO4-7H20 equal to 1/20
of the volume recorded in
Step (d).
The volume of 0.15 M
Na2HP04-7H2O in which the
precipitate will be dissolved is
equal to 5 mL for each 100
mL of diluted beef extract.
(r) Place centrifuge bottle on a
magnetic stirrer, and stir
precipitate slowly until
precipitate has dissolved
completely.
Support bottle as
necessary to prevent
toppling. Avoid foaming which
may inactivate or aerosolize
viruses. Precipitate may be
partially dissipated with
spatula before or during
stirring procedure.
(s) Measure pH of dissolved
precipitates.
If pH is above or below 7.0-
7.5, adjust to that range with
either 1 M HCI or 1 M NaOH.
(t) Refrigerate dissolved
precipitates immediately at
4"C and maintain at that
temperature until assay for
viruses is undertaken.
If assay for viruses cannot
be undertaken within eight
hours, store dissolved
precipitates immediately at
-70"C.
(u) Assay for viruses in
accordance with instructions
given in Chapter 10
(December 1987 revision).
6. Bibliography
Berg, G., D. Berman, and R. S.
Safferman. 1982. A Method for
Concentrating Viruses Recovered
from Sewage Sludges. Can. J.
Microbiol. 28:553-556.
Berg, G., and D. R. Dahling. 1980.
Method for Recovering Viruses
from River Water Solids. Appl.
Environ. Microbiol. 39:850-853.
Berman, D., G. Berg, and R. S.
Safferman. 1981. A Method for
Recovering Viruses from Sludges.
J. Virol. Methods. 3:283-291.
Bitton, G. 1987. Recovering Viruses
from Soils and Aquatic Sediments,
p. 68-75. In: G. Berg (ed), Methods
for Recovering Viruses from the
Environment. CRC Press, Inc., Boca
Raton, FL.
Brashear, D. A., and R. L Ward.
1982. Comparison of Methods for
Recovering Indigenous Viruses
from Raw Wastewater Sludge. Appl.
Environ. Microbiol. 43:1413-1418.
Farrah, S. R. 1982. Isolation of
Viruses Associated with Sludge
Particles, p. 161-170. In: C.P. Gerba
and S. M. Goyal (eds), Methods in
Environmental Virology. Marcel
Dekker, Inc., New York, NY.
Gerba, C. P. 1982. Detection of
Viruses in Soil and Aquatic
Sediments, p. 151-160. In: C.P.
Gerba and S. M. Goyal (eds),
Methods in Environmental Virology.
Marcel Dekker, Inc., New York, NY.
Goddard, M. R., J. Bates, and M.
Butler. 1981. Recovery of
Indigenous Enteroviruses from Raw
and Digested Sewage Sludges.
Appl. Environ. Microbiol. 42:1023-
1028.
Hurst, C. J. 1987. Recovering Viruses
from Sewage Sludges and from
Solids in Water, p. 25-51. In: G.
Berg (ed), Methods for Recovering
Viruses from the Environment. CRC
Press, Inc., Boca Raton, FL.
Katzenelson, E., B. Fattal, and T.
Hostovesky. 1976. Organic
Flocculation: an Efficient Second-
Step Concentration Method for the
Detection of Viruses in Tap Water.
Appl. Environ. Microbiol.32:638-639.
Nielsen, A., and B. Lydholm. 1980.
Methods for the Isolation of Virus
from Raw and Digested Wastewater
Sludge. Water Res. 14:175-178.
Pancorbo, O. C., P. R. Scheuerman,
S. R. Farrah, and G. Bitton. 1981.
Effect of Sludge Type on Poliovirus
Association with and Recovery from
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