ASTM D4994-89(2002)

Standard Practice for Recovery of Viruses from Wastewater Sludges

SIGNIFICANCE AND USE
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.SectionsProcedure A-Adsorption6 to 10Procedure B-Sonication11 to 15
1.3 This practice was tested on standardized sludges as described in 10.1 and 17.1. It is the user's responsibility to ensure the validity of this practice for untested matrices.
1.4 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.
1.4 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, for work with potentially hazardous biological organisms.

General Information

• Status: Historical
• Publication Date: 26-Oct-1989
• ICS: 13.060.30 - Sewage water
• Technical Committee: D19 - Water
• Drafting Committee: D19.24 - Water Microbiology

Relations

• Replaces: ASTM D4994-89(1996) - Standard Practice for Recovery of Viruses from Wastewater Sludges
• Effective Date: 27-Oct-1989
• Replaced By: ASTM D4994-89(2009) - Standard Practice for Recovery of Viruses from Wastewater Sludges
• Effective Date: 01-May-2009

Standards Content (Sample)

NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.
Contact ASTM International (www.astm.org) for the latest information
Designation:D4994–89(Reapproved2002)
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 (e) indicates an editorial change since the last revision or reapproval.
1. Scope 4. Significance and Use
1.1 This practice is used for the recovery of viruses from 4.1 Although many laboratories are presently isolating vi-
wastewater sludges and favors the enteroviruses. rusesfromsludge,avalidcomparisonofdatageneratedhasnot
1.2 Both procedures are applicable to raw, digested, and been possible because of the lack of a standard test method(s).
dewatered sludges.
5. Apparatus
Sections
Procedure A—Adsorption 6 to 10
5.1 Centrifuge(s), refrigerated, capable of attaining
Procedure B—Sonication 11 to 15
10000 3 g, screw-capped 100-mL centrifuge bottles that can
1.3 This practice was tested on standardized sludges as withstand 10000 3 g, and 250-mL screw-capped centrifuge
described in 10.1 and 17.1. It is the user’s responsibility to bottles capable of withstanding 2500 3 g.
ensure the validity of this practice for untested matrices. 5.2 pH Meter, measuring to an accuracy of at least 0.1 pH
1.4 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- 5.3 Filter Apparatus, for membrane sterilization, with
priate safety and health practices and determine the applica- 47-mm diameter filter holder and 50-mL slip-tip syringe (see
bility of regulatory limitations prior to use. 7.7 for type of filter material).
1.5 Only adequately trained personnel should be allowed to
6. Purity of Reagents
perform these procedures and should use safety precautions
recommended by the U.S. Public Health Service, Center for 6.1 Purity of Reagents—Reagent grade chemicals shall be
Disease Control, for work with potentially hazardous biologi- used in all tests. Unless otherwise indicated, it is intended that
cal organisms. all reagents shall conform to the specifications of the Commit-
tee onAnalytical Reagents of theAmerican Chemical Society,
2. Referenced Documents 5
where such specifications are available. Other grades may be
2.1 ASTM Standards: used, provided it is first ascertained that the reagent is of
D1129 Terminology Relating to Water sufficiently high purity to permit its use without lessening the
D1193 Specification for Reagent Water accuracy of the determination.
6.2 Purity of Water—Unlessotherwiseindicated,references
3. Terminology
towatershallbeunderstoodtomeanreagentwaterconforming
3.1 Definitions—For definitions of terms used in this prac- to Specification D1193, Type II.
tice, refer to Terminology D1129.
1 4
This practice is under the jurisdiction ofASTM Committee D19 on Water and TheSwinnexfilter(No.SX0047000,availablefromMilliporeCorp.,80Ashby
is the direct responsibility of Subcommittee D19.24 on Water Microbiology. Rd., Bedford, MA 01730, or equivalent, has been found suitable for this purpose.
Current edition approved Oct. 27, 1989. Published March 1990. Reagent Chemicals, American Chemical Society Specifications, American
Richardson,J.H.,andBarkley,W.E., Biological Safety in Microbiological and Chemical Society, Washington, DC. For suggestions on the testing of reagents not
Biomedical Laboratories, 2nd. edition, U.S. Dept. of Health and Human Services, listed by the American Chemical Society, see Analar Standards for Laboratory
Public Health Service, Center for Disease Control, and National Institutes of Health Chemicals, BDH Ltd., Poole, Dorset, U.K., and the United States Pharmacopeia
and Human Services, 1988. and National Formulary, U.S. Pharmaceutical Convention, Inc. (USPC), Rockville,
Annual Book of ASTM Standards, Vol 11.01. MD.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.
D4994–89 (2002)
PROCEDURE A—ADSORPTION 9.1.4 Place beaker on magnetic stirrer, and stir at speed
sufficient to develop vortex.
7. Reagents and Materials
9.1.5 Add 1 mL of AlCl solution to sludge. Final concen-
tration of AlCl in sludge is approximately 0.0005 M.
7.1 Aluminum Chloride Solution (12.07 g/L)—Dissolve
9.1.6 Place combination-type pH electrode into sludge and
12.07 g of aluminum chloride (AlCl ·6H O) in 500 mL of
3 2
water and dilute to 1000 mL. Autoclave AlCl solution at adjust pH of sludge to 3.5 6 0.1 with HCl (1+1). If pH falls
121°C for 15 min. below 3.5, readjust with NaOH solution (4 g/100 mL). If
sludge adheres to electrodes, clean electrodes by moving them
7.2 Buffered Beef Extract Solution—Dissolve 10 g of beef
extractpowder, 1.34gofNa HPO ·7H O,and0.12gofcitric up and down gently in mixing sludge. pH meter must be
2 4 2
standardized at pH 4.
acid in 100 mL of water in a screw-cap flask by stirring for
about2honamagneticstirrer.Autoclaveat121°Cfor15min. 9.1.7 Continue mixing for 30 min. Check pH of the sludge
7.3 Disodium Hydrogen Phosphate Solution (4 g/100 mL)— at frequent intervals. If the pH drifts up, readjust to 3.5 6 0.1
Dissolve4gof disodium hydrogen phosphate with HCl (1+9). If the pH drifts down, readjust with NaOH
(Na HPO ·7H O) in 100 mL of water and autoclave at 121°C solution (4 g/100 mL).
2 4 2
for 15 min.
9.1.8 Turn stirrer off and remove pH electrode from sludge.
7.4 Hydrochloric Acid (1 + 1)—Add 1 volume of concen-
9.1.9 Remove cap from a screw-capped centrifuge bottle
trated HCl (sp gr 1.19) to 1 volume of water.
and pour conditioned sludge into centrifuge bottle. To prevent
7.5 Hydrochloric Acid (1 + 9)—Add 1 volume of concen-
transfer of stir bar into centrifuge bottle when decanting
trated HCl (sp gr 1.19) to 9 volumes of water.
sludge, hold another stir bar or magnet against bottom of
7.6 Sodium Hydroxide Solution (4 g/100 mL)—Dissolve4.0
beaker. Remove sludge that adheres to stir bar in the beaker by
g of dry sodium hydroxide (NaOH) in water and dilute to 100
manipulation with a stirring rod. If necessary, pour sludge
mL.
several times from centrifuge bottle to beaker and back to
7.7 Filters, Disc, Membrane, 47-mm—3.0-, 0.45-, and
removeallsludgesolidstobottle.Takecaretoavoidformation
0.25-µm pore size which must be cut to proper size from sheet
of aerosols.
filters. Disassemble filter holder. Place filter with 0.25-µm
9.1.10 Replace and tighten cap on centrifuge bottle.
pore size on support screen of filter holder and stack the
9.1.11 Centrifuge conditioned sludge at 2500 3 g for 15
remaining filters on top in order of increasing pore size.
min at 4°C. Discard supernatant.
Reassemble and tighten filter holder. Filters stacked in-tandem
9.2 Elution of Viruses from Sludge Solids:
as described tend to clog more slowly when turbid material is
9.2.1 Add stir bar to the centrifuge bottle that contains
filtered through them. Prepare several filter stacks.
sedimented, conditioned sludge.
9.2.2 Add 100 mL of buffered beef extract solution to the
8. Summary of Procedure
sedimented, conditioned sludge. The volume of buffered beef
8.1 The adsorption procedure relies upon adsorption of
extract solution used to elute viruses from the conditioned
viruses from the liquid phase to the sludge solids, which are
sludge is equal to the original volume of the sample volume
concentrated by centrifugation. The supernatant is discarded.
(see 9.1).
Viruses are desorbed from the solids by physicochemical
9.2.3 Replace and tighten cap on centrifuge bottle.
means and further concentrated by organic flocculation. De-
9.2.4 Place centrifuge bottle on magnetic stirrer and stir at
contamination is accomplished by filtration.
speed sufficient to develop vortex. To minimize foaming
(which may inactivate viruses), do not mix faster than neces-
9. Procedure
sary to develop vortex. Care must be taken to prevent bottle
9.1 Conditioning of Sludge—In the absence of experience
from toppling. Stabilize bottle as necessary.
that dictates otherwise, use 100-mLvolumes for liquid sludges
9.2.5 Continue mixing for 30 min.
and 100-g quantities for digested, dewatered sludges.
9.2.6 Turn stirrer off and remove stir bar from centrifuge
9.1.1 Measure 100 mLof well-mixed sludge in a graduated
bottle.
100-mLcylinder. Mix sludge vigorously immediately before it
9.2.7 Replace and tighten cap on centrifuge bottle and
is poured into cylinder because sludge solids, which contain
centrifugeconditionedsludge-eluatemixtureat10000 3 gfor
most of the viruses, begin to settle out immediately after
30 min at 4°C.
mixing stops.
9.2.8 Remove cap from centrifuge bottle. Decant superna-
9.1.2 Place stir bar into a 250-mL beaker.
tant fluid (eluate) into beaker and discard sediment.
9.1.3 Pour the 100-mL of measured sludge from the cylin-
9.2.9 Place a filter holder that contains a filter stack as
der into the 250-mL beaker. If necessary, pour sludge several
described in 7.7 on a 250-mL Erlenmeyer receiving flask.
times from beaker to cylinder and back to remove all sludge
9.2.10 Load 50-mL syringe with eluate.
solids to beaker. Take care to avoid formation of aerosols.
9.2.11 Place tip of syringe into filter holder.
9.2.12 Force eluate through filter stack into 250-mLreceiv-
ing flask. Take care not to break off tip of syringe and to
Extract available from Grand Island Biological Corp., 3175 Staley Rd, Grand
minimize pressure on receiving flask, because such pressure
Island, NY 14072, or equivalent, has been found suitable for this purpose.
may splinter or topple the flask. If filter stack begins to clog
Duo-Fineseriessheetfilters,availablefromFilterliteCorp.,2033GreenSpring
Dr.,Timonium,MD21093,orequivalent,havebeenfoundsuitableforthispurpose. badly, empty loaded syringe into beaker containing unfiltered
D4994–89 (2002)
eluate, fill syringe with air, and inject air into filter stack to To prevent transfer of stir bar into a centrifuge bottle, hold
force residual eluate from filters. Continue filtration procedure another stir bar or magnet against bottom of beaker when
withanotherfilterholderandfilterstack.Discardcontaminated decanting contents.
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 3 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
to ⁄20 of the volume recorded in 9.4.4. Divide this volume
9.2.13 Refrigerate eluate immediately at 4°C, and maintain
equally among the flocculates in the centrifuge bottles.
at that temperature until it is assayed for viruses (see 9.3). The
9.4.12 Replaceandtighten-downcapsoncentrifugebottles,
number of cell cultures necessary for the viral assay may be
and place each on a magnetic stirrer. Stir flocculates slowly
reduced by concentrating the viruses in the beef extract by the
until dissolved completely. Support bottles as necessary to
organic flocculation procedure. Some loss of virus may occur
prevent toppling. Avoid foaming which may inactivate or
withthisprocedure.Ifvirusesineluatesaretobeconcentrated,
aerosolizeviruses.Flocculatesmaybepartiallydissipatedwith
proceed immediately to 9.4. If further concentration is not
spatula before or during stirring procedure.
requiredandifassayforvirusescannotbeundertakenwithin8
9.4.13 Remove caps from centrifuge bottles and combine
h, distribute eluate into sterile sample bottles, cap tightly, and
the dissolved flocculates in a small beaker. To prevent transfer
store immediately at−70°C.
of stir bars into beaker, hold another stir bar or magnet against
9.3 Viral Assay:
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
9.4.14 Measure pH of dissolve flocculate. If pH is above or
10% of the isolates should be confirmed by second passage.
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 the isolates should be confirmed by second passage.
9.4.1 Pour eluate from 9.2.13 into a graduated cylinder and
record the volume.
10. Precision and Bias
9.4.2 Pour eluate into 600-mL beaker.
10.1 Eight independent laboratories participated in the
9.4.3 For every 3 mL of beef extract eluate, add 7 mL of
evaluation of this recovery procedure for viruses in sludges.
sterile water to the 600-mL beaker. The concentration of beef
Five standardized sludges were utilized in the study: (1)
extract is now 3%. This dilution is necessary because 10%
Anaerobic, high rate, digested (mesophilic), (2) Anaerobic,
beef extract often does not process well by the organic
standard rate, digested (mesophilic), (3) Anaerobic, digested,
flocculation concentration procedure.
dewatered, (4)Aerobic, digested, and (5) Primary, undigested.
9.4.4 Pour the diluted, filtered beef extract into a graduated
10.1.1 Sludge aliquots of each type were prepared by one
cylinder and record the total volume.
laboratory and were shipped on-ice to participating laborato-
ries.Triplicate analyses were performed on each sludge within
9.4.5 Decant diluted filtered beef extract into 600-mL bea-
...