ASTM D4994-89(2014)
(Practice)Standard Practice for Recovery of Viruses from Wastewater Sludges
Standard Practice for Recovery of Viruses from Wastewater Sludges
SIGNIFICANCE AND USE
4.1 Although many laboratories are presently isolating viruses from sludge, a valid comparison of data generated has not been possible because of the lack of a standard test method(s).
SCOPE
1.1 This practice is used for the recovery of viruses from wastewater sludges and favors the enteroviruses.
1.2 Both procedures are applicable to raw, digested, and dewatered sludges.
Sections
Procedure A—Adsorption
6 to 10
Procedure B—Sonication
11 to 15
1.3 This practice was tested on standardized sludges as described in 10.1. It is the user's responsibility to ensure the validity of this practice for untested matrices.
1.4 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
1.5 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.
1.6 Only adequately trained personnel should be allowed to perform these procedures and should use safety precautions recommended by the U.S. Public Health Service, Center for Disease Control,2 for work with potentially hazardous biological organisms.
General Information
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Standards Content (Sample)
NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.
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Designation: D4994 − 89 (Reapproved 2014)
Standard Practice for
Recovery of Viruses from Wastewater Sludges
This standard is issued under the fixed designation D4994; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision.Anumber in parentheses indicates the year of last reapproval.A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope 3. Terminology
3.1 Definitions—For definitions of terms used in this
1.1 This practice is used for the recovery of viruses from
practice, refer to Terminology D1129.
wastewater sludges and favors the enteroviruses.
1.2 Both procedures are applicable to raw, digested, and
4. Significance and Use
dewatered sludges.
4.1 Although many laboratories are presently isolating vi-
Sections
rusesfromsludge,avalidcomparisonofdatageneratedhasnot
Procedure A—Adsorption 6 to 10
Procedure B—Sonication 11 to 15
been possible because of the lack of a standard test method(s).
1.3 This practice was tested on standardized sludges as
5. Apparatus
described in 10.1. It is the user’s responsibility to ensure the
validity of this practice for untested matrices.
5.1 Centrifuge(s), refrigerated, capable of attaining
10000× g, screw-capped 100-mL centrifuge bottles that can
1.4 The values stated in SI units are to be regarded as
withstand 10000× g, and 250-mL screw-capped centrifuge
standard. No other units of measurement are included in this
bottles capable of withstanding 2500× g.
standard.
5.2 pH Meter, measuring to an accuracy of at least 0.1 pH
1.5 This standard does not purport to address all of the
unit, equipped with a combination-type electrode. Calibrate
safety concerns, if any, associated with its use. It is the
with standard buffers.
responsibility of the user of this standard to establish appro-
4,5
priate safety and health practices and determine the applica-
5.3 Filter Apparatus, for membrane sterilization, with
bility of regulatory limitations prior to use. 47-mm diameter filter holder and 50-mL slip-tip syringe (see
7.7 for type of filter material).
1.6 Only adequately trained personnel should be allowed to
perform these procedures and should use safety precautions
6. Purity of Reagents
recommended by the U.S. Public Health Service, Center for
Disease Control, for work with potentially hazardous biologi-
6.1 Purity of Reagents—Reagent grade chemicals shall be
cal organisms.
used in all tests. Unless otherwise indicated, it is intended that
all reagents shall conform to the specifications of the Commit-
2. Referenced Documents
tee onAnalytical Reagents of theAmerican Chemical Society,
where such specifications are available. Other grades may be
2.1 ASTM Standards:
used, provided it is first ascertained that the reagent is of
D1129Terminology Relating to Water
sufficiently high purity to permit its use without lessening the
D1193Specification for Reagent Water
accuracy of the determination.
This practice is under the jurisdiction ofASTM Committee D19 on Water and
is the direct responsibility of Subcommittee D19.24 on Water Microbiology. The sole source of supply of the apparatus, Swinnex filter (No. SX0047000),
Current edition approved Jan. 1, 2014. Published March 2014. Originally knowntothecommitteeatthistimeisMilliporeCorp.,80AshbyRd.,Bedford,MA
approved in 1989. Last previous edition approved in 2009 as D4494–89 (2009). 01730.
DOI: 10.1520/D4994-89R14. If you are aware of alternative suppliers, please provide this information to
Richardson,J.H.,andBarkley,W.E., Biological Safety in Microbiological and ASTM International Headquarters. Your comments will receive careful consider-
Biomedical Laboratories, 2nd edition, U.S. Dept. of Health and Human Services, ation at a meeting of the responsible technical committee, which you may attend.
PublicHealthService,CenterforDiseaseControl,andNationalInstitutesofHealth Reagent Chemicals, American Chemical Society Specifications, American
and Human Services, 1988. Chemical Society, Washington, DC. For suggestions on the testing of reagents not
For referenced ASTM standards, visit the ASTM website, www.astm.org, or listed by the American Chemical Society, see Analar Standards for Laboratory
contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM Chemicals, BDH Ltd., Poole, Dorset, U.K., and the United States Pharmacopeia
Standards volume information, refer to the standard’s Document Summary page on and National Formulary,U.S.PharmaceuticalConvention,Inc.(USPC),Rockville,
the ASTM website. MD.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
D4994 − 89 (2014)
6.2 Purity of Water—Unless otherwise indicated, references is poured into cylinder because sludge solids, which contain
towatershallbeunderstoodtomeanreagentwaterconforming most of the viruses, begin to settle out immediately after
to Specification D1193, Type II. mixing stops.
9.1.2 Place stir bar into a 250-mL beaker.
PROCEDURE A—ADSORPTION
9.1.3 Pour the 100-mL of measured sludge from the cylin-
der into the 250-mL beaker. If necessary, pour sludge several
7. Reagents and Materials
times from beaker to cylinder and back to remove all sludge
7.1 Aluminum Chloride Solution (12.07 g/L)—Dissolve
solids to beaker. Take care to avoid formation of aerosols.
12.07 g of aluminum chloride (AlCl ·6H O) in 500 mL of
3 2
9.1.4 Place beaker on magnetic stirrer, and stir at speed
water and dilute to 1000 mL. Autoclave AlCl solution at
sufficient to develop vortex.
121°C for 15 min.
9.1.5 Add 1 mL of AlCl solution to sludge. Final concen-
7.2 Buffered Beef Extract Solution—Dissolve 10 g of beef
tration of AlCl in sludge is approximately 0.0005 M.
5,7
extract powder, 1.34gofNa HPO ·7H O, and 0.12 g of
2 4 2 9.1.6 Place combination-type pH electrode into sludge and
citric acid in 100 mL of water in a screw-cap flask by stirring
adjust pH of sludge to 3.5 6 0.1 with HCl (1+1). If pH falls
for about2hona magnetic stirrer.Autoclave at 121°C for 15
below 3.5, readjust with NaOH solution (4 g/100 mL). If
min.
sludge adheres to electrodes, clean electrodes by moving them
up and down gently in mixing sludge. pH meter must be
7.3 Disodium Hydrogen Phosphate Solution(4g/100mL)—
standardized at pH 4.
Dissolve4gof disodium hydrogen phosphate
9.1.7 Continue mixing for 30 min. Check pH of the sludge
(Na HPO ·7H O) in 100 mL of water and autoclave at 121°C
2 4 2
at frequent intervals. If the pH drifts up, readjust to 3.5 6 0.1
for 15 min.
with HCl (1+9). If the pH drifts down, readjust with NaOH
7.4 Hydrochloric Acid (1+1)—Add 1 volume of concen-
solution (4 g/100 mL).
trated HCl (sp gr 1.19) to 1 volume of water.
9.1.8 Turn stirrer off and remove pH electrode from sludge.
7.5 Hydrochloric Acid (1+9)—Add 1 volume of concen-
9.1.9 Remove cap from a screw-capped centrifuge bottle
trated HCl (sp gr 1.19) to 9 volumes of water.
and pour conditioned sludge into centrifuge bottle. To prevent
7.6 Sodium Hydroxide Solution(4g/100mL)—Dissolve4.0 transfer of stir bar into centrifuge bottle when decanting
g of dry sodium hydroxide (NaOH) in water and dilute to 100
sludge, hold another stir bar or magnet against bottom of
mL. beaker.Removesludgethatadherestostirbarinthebeakerby
manipulation with a stirring rod. If necessary, pour sludge
7.7 Filters, Disc, Membrane, 47-mm—3.0-, 0.45-, and
several times from centrifuge bottle to beaker and back to
0.25-µm pore size which must be cut to proper size from sheet
5,8
removeallsludgesolidstobottle.Takecaretoavoidformation
filters. Disassemble filter holder. Place filter with 0.25-µm
of aerosols.
pore size on support screen of filter holder and stack the
9.1.10 Replace and tighten cap on centrifuge bottle.
remaining filters on top in order of increasing pore size.
9.1.11 Centrifugeconditionedsludgeat2500× gfor15min
Reassemble and tighten filter holder. Filters stacked in-tandem
at 4°C. Discard supernatant.
as described tend to clog more slowly when turbid material is
filtered through them. Prepare several filter stacks.
9.2 Elution of Viruses from Sludge Solids:
9.2.1 Add stir bar to the centrifuge bottle that contains
8. Summary of Procedure
sedimented, conditioned sludge.
8.1 The adsorption procedure relies upon adsorption of
9.2.2 Add 100 mL of buffered beef extract solution to the
viruses from the liquid phase to the sludge solids, which are
sedimented, conditioned sludge. The volume of buffered beef
concentrated by centrifugation. The supernatant is discarded.
extract solution used to elute viruses from the conditioned
Viruses are desorbed from the solids by physicochemical
sludge is equal to the original volume of the sample volume
means and further concentrated by organic flocculation. De-
(see 9.1).
contamination is accomplished by filtration.
9.2.3 Replace and tighten cap on centrifuge bottle.
9.2.4 Place centrifuge bottle on magnetic stirrer and stir at
9. Procedure
speed sufficient to develop vortex. To minimize foaming
9.1 Conditioning of Sludge—In the absence of experience
(which may inactivate viruses), do not mix faster than neces-
that dictates otherwise, use 100-mLvolumes for liquid sludges
sary to develop vortex. Care must be taken to prevent bottle
and 100-g quantities for digested, dewatered sludges.
from toppling. Stabilize bottle as necessary.
9.1.1 Measure 100 mLof well-mixed sludge in a graduated
9.2.5 Continue mixing for 30 min.
100-mLcylinder. Mix sludge vigorously immediately before it
9.2.6 Turn stirrer off and remove stir bar from centrifuge
bottle.
9.2.7 Replace and tighten cap on centrifuge bottle and
The sole source of supply of the apparatus, extract, known to the committee at
this time is Grand Island Biological Corp., 3175 Staley Rd, Grand Island, NY
centrifuge conditioned sludge-eluate mixture at 10000× g for
14072.
30 min at 4°C.
Thesolesourceofsupplyoftheapparatus,Duo-Fineseriessheetfilters,known
9.2.8 Remove cap from centrifuge bottle. Decant superna-
to the committee at this time is Filterlite Corp., 2033 Green Spring Dr., Timonium,
MD 21093. tant fluid (eluate) into beaker and discard sediment.
D4994 − 89 (2014)
9.2.9 Place a filter holder that contains a filter stack as 9.4.7 Insert combination-type pH electrode into diluted,
described in 7.7 on a 250-mL Erlenmeyer receiving flask. filtered beef extract and add HCl (1+9) slowly until pH of
beef extract reaches 3.5 6 0.1.Aflocculate or precipitate will
9.2.10 Load 50-mL syringe with eluate.
form.IfpHdropsbelow3.4,addNaOHsolution(4g/100mL)
9.2.11 Place tip of syringe into filter holder.
until pH is 3.5 6 0.1. Avoid reducing pH below 3.4 because
9.2.12 Force eluate through filter stack into 250-mLreceiv-
some inactivation of viruses may occur. Continue to stir for 30
ing flask. Take care not to break off tip of syringe and to
min.
minimize pressure on receiving flask, because such pressure
9.4.8 Turn stirrer off, remove electrode from beaker, and
may splinter or topple the flask. If filter stack begins to clog
distribute contents of beaker evenly among centrifuge bottles.
badly, empty loaded syringe into beaker containing unfiltered
To prevent transfer of stir bar into a centrifuge bottle, hold
eluate, fill syringe with air, and inject air into filter stack to
another stir bar or magnet against bottom of beaker when
force residual eluate from filters. Continue filtration procedure
decanting contents.
withanotherfilterholderandfilterstack.Discardcontaminated
filter holders and filter stacks. Repeat 9.2.9 through 9.2.12 as 9.4.9 Replace and tighten caps on centrifuge bottles and
often as necessary to filter entire volume of eluate. Disas- centrifuge the flocculated beef extract suspension at 2500× g
semble each filter holder and examine bottom filters to be for 15 min at 4°C. Pour off and discard supernatants.
certain they have not ruptured. If a bottom filter has ruptured,
9.4.10 Place a small stir bar into each centrifuge bottle that
repeat 9.2.10 through 9.2.12 with new filter holders and filter
contains flocculate and replace covers loosely.
stacks.
9.4.11 Measure a volume of Na HPO ·7H O solution equal
2 4 2
9.2.13 Refrigerate eluate immediately at 4°C, and maintain
to ⁄20 of the volume recorded in 9.4.4. Divide this volume
at that temperature until it is assayed for viruses (see 9.3). The
equally among the flocculates in the centrifuge bottles.
number of cell cultures necessary for the viral assay may be
9.4.12 Replaceandtighten-downcapsoncentrifugebottles,
reduced by concentrating the viruses in the beef extract by the
and place each on a magnetic stirrer. Stir flocculates slowly
organic flocculation procedure. Some loss of virus may occur
until dissolved completely. Support bottles as necessary to
withthisprocedure.Ifvirusesineluatesaretobeconcentrated,
prevent toppling. Avoid foaming which may inactivate or
proceed immediately to 9.4. If further concentration is not
aerosolizeviruses.Flocculatesmaybepartiallydissipatedwith
requiredandifassayforvirusescannotbeundertakenwithin8
spatula before or during stirring procedure.
h, distribute eluate into sterile sample bottles, cap tightly, and
9.4.13 Remove caps from centrifuge bottles and combine
store immediately at−70°C.
the dissolved flocculates in a small beaker. To prevent transfer
9.3 Viral Assay: of stir bars into beaker, hold another stir bar or magnet against
the bottom of centrifuge bottle when decanting dissolved
9.3.1 At time of viral assay, rapidly thaw the frozen con-
flocculates.
centrate at 37°C and proceed with usual viral assay. At least
10% of the isolates should be confirmed by second passage. 9.4.14 Measure pH of dissolve flocculate. If pH is above or
below 7.0 to 7.5, adjust to within this range with either HCl
9.4 Procedure for Concentrating Viruses from Sludge Elu-
(1+9) or NaOH solution (4 g/100 mL).
ates (Organic Flocculation Concentration)—It is preferable to
9.4.15 Refrigeratefinalconcentrateimmediatelyat4°C,and
assay eluted viruses in the beef extract eluate without concen-
maintain at that temperature until assay for viruses is under-
trating them because some loss of viruses may occur in
taken. If assay for viruses cannot be undertaken within 8 h,
concentration. However, the numbers of cell cultures needed
transfer dissolved precipitates to sterile sample bottles, cap
for assays may be reduced by concentrating the viruses in the
tightly, and store immediately at–70°C.
eluate. Significant further loss of viruses may occur with the
9.4.16 At the time of viral assay, rapidly thaw the frozen
currently available beef extract which may not produce suffi-
concentrateat37°Candproceedwithusualviralassay.Atleast
cient floc to adsorb all of the suspended virions.
10% of t
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