ASTM D4994-89(1996)
(Practice)Standard Practice for Recovery of Viruses from Wastewater Sludges
Standard Practice for Recovery of Viruses from Wastewater Sludges
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 and 17.1. It is the user's responsibility to ensure the validity of this practice for untested matrices.
1.4 This standard may involve hazardous materials, operations, and equipment. This standard does not purport to address all of the safety problems 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.
1.5 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
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Standards Content (Sample)
Designation: D 4994 – 89 (Reapproved 1996)
AMERICAN SOCIETY FOR TESTING AND MATERIALS
100 Barr Harbor Dr., West Conshohocken, PA 19428
Reprinted from the Annual Book of ASTM Standards. Copyright ASTM
Standard Practice for
Recovery of Viruses from Wastewater Sludges
This standard is issued under the fixed designation D 4994; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (e) indicates an editorial change since the last revision or reapproval.
1. Scope 10 000 3 g, screw-capped 100-mL centrifuge bottles that can
withstand 10 000 3 g, and 250-mL screw-capped centrifuge
1.1 This practice is used for the recovery of viruses from
bottles capable of withstanding 2 500 3 g.
wastewater sludges and favors the enteroviruses.
5.2 pH Meter, measuring to an accuracy of at least 0.1 pH
1.2 Both procedures are applicable to raw, digested, and
unit, equipped with a combination-type electrode. Calibrate
dewatered sludges.
with standard buffers.
Sections
5.3 Filter Apparatus, for membrane sterilization, with
Procedure A—Adsorption 6 to 10
Procedure B—Sonication 11 to 15
47-mm diameter filter holder and 50-mL slip-tip syringe (see
7.7 for type of filter material).
1.3 This practice was tested on standardized sludges as
described in 10.1 and 17.1. It is the user’s responsibility to
6. Purity of Reagents
ensure the validity of this practice for untested matrices.
6.1 Purity of Reagents—Reagent grade chemicals shall be
1.4 This standard does not purport to address all of the
used in all tests. Unless otherwise indicated, it is intended that
safety concerns, if any, associated with its use. It is the
all reagents shall conform to the specifications of the Commit-
responsibility of the user of this standard to establish appro-
tee on Analytical Reagents of the American Chemical Society,
priate safety and health practices and determine the applica-
where such specifications are available. Other grades may be
bility of regulatory limitations prior to use.
used, provided it is first ascertained that the reagent is of
1.5 Only adequately trained personnel should be allowed to
sufficiently high purity to permit its use without lessening the
perform these procedures and should use safety precautions
accuracy of the determination.
recommended by the U.S. Public Health Service, Center for
6.2 Purity of Water—Unless otherwise indicated, references
Disease Control, for work with potentially hazardous biologi-
to water shall be understood to mean reagent water conforming
cal organisms.
to Specification D 1193, Type II.
2. Referenced Documents
PROCEDURE A—ADSORPTION
2.1 ASTM Standards:
D 1129 Terminology Relating to Water
7. Reagents and Materials
D 1193 Specification for Reagent Water
7.1 Aluminum Chloride Solution (12.07 g/L)—Dissolve
3. Terminology
12.07 g of aluminum chloride (AlCl ·6H O) in 500 mL of
3 2
3.1 Definitions—For definitions of terms used in this prac-
water and dilute to 1000 mL. Autoclave AlCl solution at
tice, refer to Terminology D 1129.
121°C for 15 min.
7.2 Buffered Beef Extract Solution—Dissolve 10 g of beef
4. Significance and Use
extract powder, 1.34gofNa HPO ·7H O, and 0.12 g of citric
2 4 2
4.1 Although many laboratories are presently isolating vi-
acid in 100 mL of water in a screw-cap flask by stirring for
ruses from sludge, a valid comparison of data generated has not
about2hona magnetic stirrer. Autoclave at 121°C for 15 min.
been possible because of the lack of a standard test method(s).
7.3 Disodium Hydrogen Phosphate Solution (4 g/100
mL)—Dissolve4gof disodium hydrogen phosphate
5. Apparatus
5.1 Centrifuge(s), refrigerated, capable of attaining
The Swinnex filter (No. SX0047000, available from Millipore Corp., 80 Ashby
Rd., Bedford, MA 01730, or equivalent, has been found suitable for this purpose.
Reagent Chemicals, American Chemical Society Specifications, American
This practice is under the jurisdiction of ASTM Committee D-19 on Water and
Chemical Society, Washington, DC. For suggestions on the testing of reagents not
is the direct responsibility of Subcommittee D19.24 on Water Microbiology.
listed by the American Chemical Society, see Analar Standards for Laboratory
Current edition approved Oct. 27, 1989. Published March 1990.
Richardson, J. H., and Barkley, W. E., Biological Safety in Microbiological and Chemicals, BDH Ltd., Poole, Dorset, U.K., and the United States Pharmacopeia
Biomedical Laboratories, 2nd. edition, U.S. Dept. of Health and Human Services, and National Formulary, U.S. Pharmaceutical Convention, Inc. (USPC), Rockville,
Public Health Service, Center for Disease Control, and National Institutes of Health MD.
and Human Services, 1988. Extract available from Grand Island Biological Corp., 3175 Staley Rd, Grand
Annual Book of ASTM Standards, Vol 11.01. Island, NY 14072, or equivalent, has been found suitable for this purpose.
D 4994
(Na HPO ·7H O) in 100 mL of water and autoclave at 121°C and pour conditioned sludge into centrifuge bottle. To prevent
2 4 2
for 15 min. transfer of stir bar into centrifuge bottle when decanting
7.4 Hydrochloric Acid (1 + 1)—Add 1 volume of concen- sludge, hold another stir bar or magnet against bottom of
trated HCl (sp gr 1.19) to 1 volume of water. beaker. Remove sludge that adheres to stir bar in the beaker by
7.5 Hydrochloric Acid (1 + 9)—Add 1 volume of concen- manipulation with a stirring rod. If necessary, pour sludge
trated HCl (sp gr 1.19) to 9 volumes of water. several times from centrifuge bottle to beaker and back to
7.6 Sodium Hydroxide Solution (4 g/100 mL)—Dissolve 4.0 remove all sludge solids to bottle. Take care to avoid formation
g of dry sodium hydroxide (NaOH) in water and dilute to 100 of aerosols.
mL.
9.1.10 Replace and tighten cap on centrifuge bottle.
7.7 Filters, Disc, Membrane, 47-mm—3.0-, 0.45-, and
9.1.11 Centrifuge conditioned sludge at 2 500 3 g for 15
0.25-μm pore size which must be cut to proper size from sheet
min at 4°C. Discard supernatant.
filters. Disassemble filter holder. Place filter with 0.25-μm
9.2 Elution of Viruses from Sludge Solids:
pore size on support screen of filter holder and stack the
9.2.1 Add stir bar to the centrifuge bottle that contains
remaining filters on top in order of increasing pore size.
sedimented, conditioned sludge.
Reassemble and tighten filter holder. Filters stacked in-tandem
9.2.2 Add 100 mL of buffered beef extract solution to the
as described tend to clog more slowly when turbid material is
sedimented, conditioned sludge. The volume of buffered beef
filtered through them. Prepare several filter stacks.
extract solution used to elute viruses from the conditioned
sludge is equal to the original volume of the sample volume
8. Summary of Procedure
(see 9.1).
8.1 The adsorption procedure relies upon adsorption of
9.2.3 Replace and tighten cap on centrifuge bottle.
viruses from the liquid phase to the sludge solids, which are
9.2.4 Place centrifuge bottle on magnetic stirrer and stir at
concentrated by centrifugation. The supernatant is discarded.
speed sufficient to develop vortex. To minimize foaming
Viruses are desorbed from the solids by physicochemical
(which may inactivate viruses), do not mix faster than neces-
means and further concentrated by organic flocculation. De-
sary to develop vortex. Care must be taken to prevent bottle
contamination is accomplished by filtration.
from toppling. Stabilize bottle as necessary.
9. Procedure
9.2.5 Continue mixing for 30 min.
9.1 Conditioning of Sludge—In the absence of experience
9.2.6 Turn stirrer off and remove stir bar from centrifuge
that dictates otherwise, use 100-mL volumes for liquid sludges
bottle.
and 100-g quantities for digested, dewatered sludges.
9.2.7 Replace and tighten cap on centrifuge bottle and
9.1.1 Measure 100 mL of well-mixed sludge in a graduated
centrifuge conditioned sludge-eluate mixture at 10 000 3 g for
100-mL cylinder. Mix sludge vigorously immediately before it
30 min at 4°C.
is poured into cylinder because sludge solids, which contain
9.2.8 Remove cap from centrifuge bottle. Decant superna-
most of the viruses, begin to settle out immediately after
tant fluid (eluate) into beaker and discard sediment.
mixing stops.
9.2.9 Place a filter holder that contains a filter stack as
9.1.2 Place stir bar into a 250-mL beaker.
described in 7.7 on a 250-mL Erlenmeyer receiving flask.
9.1.3 Pour the 100-mL of measured sludge from the cylin-
9.2.10 Load 50-mL syringe with eluate.
der into the 250-mL beaker. If necessary, pour sludge several
9.2.11 Place tip of syringe into filter holder.
times from beaker to cylinder and back to remove all sludge
9.2.12 Force eluate through filter stack into 250-mL receiv-
solids to beaker. Take care to avoid formation of aerosols.
ing flask. Take care not to break off tip of syringe and to
9.1.4 Place beaker on magnetic stirrer, and stir at speed
minimize pressure on receiving flask, because such pressure
sufficient to develop vortex.
may splinter or topple the flask. If filter stack begins to clog
9.1.5 Add 1 mL of AlCl solution to sludge. Final concen-
badly, empty loaded syringe into beaker containing unfiltered
tration of AlCl in sludge is approximately 0.0005 M.
eluate, fill syringe with air, and inject air into filter stack to
9.1.6 Place combination-type pH electrode into sludge and
force residual eluate from filters. Continue filtration procedure
adjust pH of sludge to 3.5 6 0.1 with HCl (1 + 1). If pH falls
with another filter holder and filter stack. Discard contaminated
below 3.5, readjust with NaOH solution (4 g/100 mL). If
filter holders and filter stacks. Repeat 9.2.9 through 9.2.12 as
sludge adheres to electrodes, clean electrodes by moving them
often as necessary to filter entire volume of eluate. Disas-
up and down gently in mixing sludge. pH meter must be
semble each filter holder and examine bottom filters to be
standardized at pH 4.
certain they have not ruptured. If a bottom filter has ruptured,
9.1.7 Continue mixing for 30 min. Check pH of the sludge
repeat 9.2.10 through 9.2.12 with new filter holders and filter
at frequent intervals. If the pH drifts up, readjust to 3.5 6 0.1
stacks.
with HCl (1 + 9). If the pH drifts down, readjust with NaOH
9.2.13 Refrigerate eluate immediately at 4°C, and maintain
solution (4 g/100 mL).
at that temperature until it is assayed for viruses (see 9.3). The
9.1.8 Turn stirrer off and remove pH electrode from sludge.
number of cell cultures necessary for the viral assay may be
9.1.9 Remove cap from a screw-capped centrifuge bottle
reduced by concentrating the viruses in the beef extract by the
organic flocculation procedure. Some loss of virus may occur
with this procedure. If viruses in eluates are to be concentrated,
Duo-Fine series sheet filters, available from Filterlite Corp., 2033 Green Spring
Dr., Timonium, MD 21093, or equivalent, have been found suitable for this purpose. proceed immediately to 9.4. If further concentration is not
D 4994
required and if assay for viruses cannot be undertaken within 8 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
flocculates.
9.3.1 At time of viral assay, rapidly thaw the frozen con-
9.4.14 Measure pH of dissolve flocculate. If pH is above or
centrate at 37°C and proceed with usual viral assay. At least
below 7.0 to 7.5, adjust to within this range with either HCl
10 % of the isolates should be confirmed by second passage.
(1 + 9) or NaOH solution (4 g/100 mL).
9.4 Procedure for Concentrating Viruses from Sludge Elu-
9.4.15 Refrigerate final concentrate immediately at 4°C, and
ates (Organic Flocculation Concentration)—It is preferable to
maintain at that temperature until assay for viruses is under-
assay eluted viruses in the beef extract eluate without concen-
taken. If assay for viruses cannot be undertaken within 8 h,
trating them because some loss of viruses may occur in
transfer dissolved precipitates to sterile sample bottles, cap
concentration. However, the numbers of cell cultures needed
tightly, and store immediately at − 70°C.
for assays may be reduced by concentrating the viruses in the
9.4.16 At the time of viral assay, rapidly thaw the frozen
eluate. Significant further loss of viruses may occur with the
concentrate at 37°C and proceed with usual viral assay. At least
currently available beef extract which may not produce suffi-
10 % of the isolates should be confirmed by second passage.
cient floc to adsorb all of the suspended virions.
9.4.1 Pour eluate from 9.2.13 into a graduated cylinder and
10. Precision and Bias
record the volume.
10.1 Eight independent laboratories participated in the
9.4.2 Pour eluate into 600-mL beaker.
evaluation of this recovery procedure for viruses in sludges.
9.4.3 For every 3 mL of beef extract eluate, add 7 mL of
Five standardized sludges were utilized in the study: (1)
sterile water to the 600-mL beaker. The concentration of beef
Anaerobic, high rate, digested (mesophilic), (2) Anaerobic,
extract is now 3 %. This dilution is necessary because 10 %
standard rate, digested (mesophilic), (3) Anaerobic, digested,
beef extract often does not process well by the organic
dewatered, (4) Aerobic, digested, and (5) Primary, undigested.
flocculation concentration procedure.
10.1.1 Sludge aliquots of each type were prepared by one
9.4.4 Pour the diluted, filtered beef extract into a graduated
laboratory and were shipped on-ice to participating laborato-
cylinder and record the total volume.
ries. Triplicate analyses were performed on each sludge within
9.4.5 Decant diluted filtered beef extract into 600-mL bea-
72 h after receipt by each laboratory utilizing its own equip-
ker and add a stir bar.
ment, media and reagents, and cell culture assay procedures.
9.4.6 Place beaker on magnetic stirrer and stir at a speed
Two sets of triplicate analyses were done on one day and a
sufficient to develop vortex. To minimize foaming (which may
third was done on the next day.
inactivate viruses), do not mix faster than necessary to develop
10.2 Bias—No bias statement is possible from the study
vortex.
data because each sludge was a natural material containing
9.4.7 Insert combination-type pH electrode into diluted,
only indigenous viruses. However, the following geometric
filtered beef extract and add HCl (1 + 9) slowly until pH of
means give some idea of the count ranges studied:
beef extract reaches 3.56 0.1. A flocculate or precipitate will
Geometric Mean Count
form. If pH drops below 3.4, add NaOH solutio
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