ASTM D4994-19

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  
7 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, health, and environmental 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.  
1.7 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

General Information

• Status: Published
• Publication Date: 31-Mar-2019
• ICS: 13.060.30 - Sewage water
• Technical Committee: D19 - Water
• Drafting Committee: D19.24 - Water Microbiology

Relations

• Replaces: ASTM D4994-89(2014) - Standard Practice for Recovery of Viruses from Wastewater Sludges
• Effective Date: 01-Apr-2019
• Refers: ASTM D1129-13(2020)e2 - Standard Terminology Relating to Water
• Effective Date: 01-May-2020
• Refers: ASTM D1129-10 - Standard Terminology Relating to Water
• Effective Date: 01-Mar-2010
• Refers: ASTM D1129-06ae1 - Standard Terminology Relating to Water
• Effective Date: 01-Sep-2006
• Refers: ASTM D1129-06a - Standard Terminology Relating to Water
• Effective Date: 01-Sep-2006
• Refers: ASTM D1193-06 - Standard Specification for Reagent Water
• Effective Date: 01-Mar-2006
• Refers: ASTM D1129-06 - Standard Terminology Relating to Water
• Effective Date: 15-Feb-2006
• Refers: ASTM D1129-04e1 - Standard Terminology Relating to Water
• Effective Date: 01-Mar-2004
• Refers: ASTM D1129-04 - Standard Terminology Relating to Water
• Effective Date: 01-Mar-2004
• Refers: ASTM D1129-03a - Standard Terminology Relating to Water
• Effective Date: 10-Aug-2003
• Refers: ASTM D1129-03 - Standard Terminology Relating to Water
• Effective Date: 10-Mar-2003
• Refers: ASTM D1129-02a - Standard Terminology Relating to Water
• Effective Date: 10-Jul-2002
• Refers: ASTM D1129-01 - Standard Terminology Relating to Water
• Effective Date: 10-Jul-2002
• Refers: ASTM D1129-02 - Standard Terminology Relating to Water
• Effective Date: 10-Feb-2002
• Refers: ASTM D1129-99a - Standard Terminology Relating to Water
• Effective Date: 10-Feb-2002

Overview

ASTM D4994-19: Standard Practice for Recovery of Viruses from Wastewater Sludges is an internationally recognized standard developed by ASTM International. This practice provides laboratories with validated procedures for recovering viruses, particularly enteroviruses, from various types of wastewater sludges. It addresses a significant gap in the comparability and reproducibility of data generated by different facilities and researchers, enabling consistent analysis of virological contamination in raw, digested, and dewatered sludges.

By establishing standardized methods for virus isolation, ASTM D4994-19 supports public health monitoring, regulatory compliance, and environmental studies, making it an essential tool for water microbiology laboratories and professionals dealing with wastewater treatment and biosolids management.

Key Topics

• Standardized Virus Recovery: Presents methods to recover enteric viruses from wastewater sludges, enabling reliable virological analysis.
• Applicable Sludge Types: The practice applies to raw, digested, and dewatered sludges, covering a wide range of matrices encountered in the wastewater sector.
• Detailed Procedures: Provides two primary procedures for virus recovery:
  • Procedure A - Adsorption: Relies on adsorbing viruses to sludge solids, followed by desorption, concentration, and filtration.
  • Procedure B - Sonication: Uses sonication to release viruses from solids, followed by concentration and decontamination steps.
• Precision and Bias: Outlines the results of multi-laboratory evaluations to demonstrate the intralaboratory and total precision of the techniques across different sludge types.
• Safety Recommendations: Emphasizes the need for trained personnel and adherence to biosafety practices due to the handling of potentially hazardous biological materials.
• International Standardization: Developed in line with WTO TBT principles for global relevance and acceptance.

Applications

The primary value of ASTM D4994-19 lies in its practical use for:

• Public Health Surveillance: Supports investigations into virus prevalence in wastewater sludges, which is critical for assessing potential environmental and human health risks.
• Regulatory Monitoring: Assists wastewater treatment facilities and regulatory agencies in meeting environmental and biosolids disposal requirements regarding viral contamination.
• Research and Development: Enables scientists to compare results across studies and labs, improving data quality in environmental virology research.
• Process Validation: Used to validate and optimize sludge treatment and biosolids handling processes by quantifying virus removal or inactivation efficiency.
• Quality Assurance: Establishes a reproducible baseline procedure for interlaboratory comparisons, audits, and method validation.

Related Standards

Professionals using ASTM D4994-19 may also reference or integrate the following standards to ensure comprehensive water analysis and laboratory practice:

• ASTM D1129: Terminology Relating to Water, providing essential definitions for water-related testing.
• ASTM D1193: Specification for Reagent Water, ensuring water quality used in laboratory procedures meets required standards.
• Biological Safety Guidelines (e.g., recommendations from the U.S. CDC): For safe handling of infectious materials in laboratory environments.

---

Keywords: virus recovery, wastewater sludge, enterovirus, adsorption, sonication, biosolids, water microbiology, environmental monitoring, ASTM D4994-19, public health.

Standards Content (Sample)

This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the
Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
Designation: D4994 − 19
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* 2. Referenced Documents
2.1 ASTM Standards:
1.1 This practice is used for the recovery of viruses from
D1129Terminology Relating to Water
wastewater sludges and favors the enteroviruses.
D1193Specification for Reagent Water
1.2 Both procedures are applicable to raw, digested, and
dewatered sludges.
3. Terminology
Sections
3.1 Definitions:
3.1.1 For definitions of terms used in this standard, refer to
Procedure A—Adsorption 7 to 10
Procedure B—Sonication 11 to 15 Terminology D1129.
1.3 This practice was tested on standardized sludges as
4. Significance and Use
described in 10.1. It is the user’s responsibility to ensure the
4.1 Although many laboratories are presently isolating vi-
validity of this practice for untested matrices.
rusesfromsludge,avalidcomparisonofdatageneratedhasnot
1.4 The values stated in SI units are to be regarded as
been possible because of the lack of a standard test method(s).
standard. No other units of measurement are included in this
standard. 5. Apparatus
5.1 Centrifuge(s), refrigerated, capable of attaining
1.5 This standard does not purport to address all of the
10000× g, screw-capped 100-mL centrifuge bottles that can
safety concerns, if any, associated with its use. It is the
withstand 10000× g, and 250-mL screw-capped centrifuge
responsibility of the user of this standard to establish appro-
bottles capable of withstanding 2500× g.
priate safety, health, and environmental practices and deter-
mine the applicability of regulatory limitations prior to use. 5.2 pH Meter, measuring to an accuracy of at least 0.1 pH
unit, equipped with a combination-type electrode. Calibrate
1.6 Only adequately trained personnel should be allowed to
with standard buffers.
perform these procedures and should use safety precautions
4,5
recommended by the U.S. Public Health Service, Center for 5.3 Filter Apparatus, for membrane sterilization, with
47-mm diameter filter holder and 50-mL slip-tip syringe (see
Disease Control, for work with potentially hazardous biologi-
7.7 for type of filter material).
cal organisms.
1.7 This international standard was developed in accor-
6. Purity of Reagents
dance with internationally recognized principles on standard-
6.1 Purity of Reagents—Reagent grade chemicals shall be
ization established in the Decision on Principles for the
used in all tests. Unless otherwise indicated, it is intended that
Development of International Standards, Guides and Recom-
all reagents shall conform to the specifications of the Commit-
mendations issued by the World Trade Organization Technical
tee onAnalytical Reagents of theAmerican Chemical Society,
Barriers to Trade (TBT) Committee.
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
This practice is under the jurisdiction ofASTM Committee D19 on Water and contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM
is the direct responsibility of Subcommittee D19.24 on Water Microbiology. Standards volume information, refer to the standard’s Document Summary page on
Current edition approved April 1, 2019. Published April 2019. Originally the ASTM website.
approved in 1989. Last previous edition approved in 2014 as D4494–89 (2014). The sole source of supply of the apparatus, Swinnex filter (No. SX0047000),
DOI: 10.1520/D4994-19. knowntothecommitteeatthistimeisMilliporeCorp.,80AshbyRd.,Bedford,MA
Richardson,J.H.,andBarkley,W.E., Biological Safety in Microbiological and 01730.
Biomedical Laboratories, 2nd edition, U.S. Dept. of Health and Human Services, If you are aware of alternative suppliers, please provide this information to
PublicHealthService,CenterforDiseaseControl,andNationalInstitutesofHealth ASTM International Headquarters. Your comments will receive careful consider-
and Human Services, 1988. ation at a meeting of the responsible technical committee, which you may attend.
*A Summary of Changes section appears at the end of this standard
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
D4994 − 19
where such specifications are available. Other grades may be means and further concentrated by organic flocculation. De-
used, provided it is first ascertained that the reagent is of contamination is accomplished by filtration.
sufficiently high purity to permit its use without lessening the
accuracy of the determination. 9. Procedure
6.2 Purity of Water—Unless otherwise indicated, references
9.1 Conditioning of Sludge—In the absence of experience
towatershallbeunderstoodtomeanreagentwaterconforming that dictates otherwise, use 100-mLvolumes for liquid sludges
to Specification D1193, Type II.
and 100-g quantities for digested, dewatered sludges.
9.1.1 Measure 100 mLof well-mixed sludge in a graduated
PROCEDURE A—ADSORPTION
100-mLcylinder. Mix sludge vigorously immediately before it
is poured into cylinder because sludge solids, which contain
7. Reagents and Materials
most of the viruses, begin to settle out immediately after
7.1 Aluminum Chloride Solution (12.07 g/L)—Dissolve
mixing stops.
12.07 g of aluminum chloride (AlCl ·6H O) in 500 mL of
3 2 9.1.2 Place stir bar into a 250-mL beaker.
water and dilute to 1000 mL. Autoclave AlCl solution at
3 9.1.3 Pour the 100-mL of measured sludge from the cylin-
121°C for 15 min.
der into the 250-mL beaker. If necessary, pour sludge several
times from beaker to cylinder and back to remove all sludge
7.2 Buffered Beef Extract Solution—Dissolve 10 g of beef
5,7
solids to beaker. Take care to avoid formation of aerosols.
extract powder, 1.34gofNa HPO ·7H O, and 0.12 g of
2 4 2
9.1.4 Place beaker on magnetic stirrer, and stir at speed
citric acid in 100 mL of water in a screw-cap flask by stirring
sufficient to develop vortex.
for about2hona magnetic stirrer.Autoclave at 121°C for 15
9.1.5 Add 1 mL of AlCl solution to sludge. Final concen-
min. 3
tration of AlCl in sludge is approximately 0.0005 M.
7.3 Disodium Hydrogen Phosphate Solution(4g/100mL)—
9.1.6 Place combination-type pH electrode into sludge and
Dissolve4gof disodium hydrogen phosphate
adjust pH of sludge to 3.5 6 0.1 with HCl (1+1). If pH falls
(Na HPO ·7H O) in 100 mL of water and autoclave at 121°C
2 4 2
below 3.5, readjust with NaOH solution (4 g/100 mL). If
for 15 min.
sludge adheres to electrodes, clean electrodes by moving them
7.4 Hydrochloric Acid (1+1)—Add 1 volume of concen-
up and down gently in mixing sludge. pH meter must be
trated HCl (sp gr 1.19) to 1 volume of water.
standardized at pH 4.
9.1.7 Continue mixing for 30 min. Check pH of the sludge
7.5 Hydrochloric Acid (1+9)—Add 1 volume of concen-
at frequent intervals. If the pH drifts up, readjust to 3.5 6 0.1
trated HCl (sp gr 1.19) to 9 volumes of water.
with HCl (1+9). If the pH drifts down, readjust with NaOH
7.6 Sodium Hydroxide Solution(4g/100mL)—Dissolve4.0
solution (4 g/100 mL).
g of dry sodium hydroxide (NaOH) in water and dilute to 100
9.1.8 Turn stirrer off and remove pH electrode from sludge.
mL.
9.1.9 Remove cap from a screw-capped centrifuge bottle
7.7 Filters, Disc, Membrane, 47-mm—3.0-, 0.45-, and
and pour conditioned sludge into centrifuge bottle. To prevent
0.25-µm pore size which must be cut to proper size from sheet
transfer of stir bar into centrifuge bottle when decanting
5,8
filters. Disassemble filter holder. Place filter with 0.25-µm
sludge, hold another stir bar or magnet against bottom of
pore size on support screen of filter holder and stack the
beaker.Removesludgethatadherestostirbarinthebeakerby
remaining filters on top in order of increasing pore size.
manipulation with a stirring rod. If necessary, pour sludge
Reassemble and tighten filter holder. Filters stacked in-tandem
several times from centrifuge bottle to beaker and back to
as described tend to clog more slowly when turbid material is
removeallsludgesolidstobottle.Takecaretoavoidformation
filtered through them. Prepare several filter stacks.
of aerosols.
9.1.10 Replace and tighten cap on centrifuge bottle.
8. Summary of Procedure
9.1.11 Centrifugeconditionedsludgeat2500× gfor15min
8.1 The adsorption procedure relies upon adsorption of at 4°C. Discard supernatant.
viruses from the liquid phase to the sludge solids, which are
9.2 Elution of Viruses from Sludge Solids:
concentrated by centrifugation. The supernatant is discarded.
9.2.1 Add stir bar to the centrifuge bottle that contains
Viruses are desorbed from the solids by physicochemical
sedimented, conditioned sludge.
9.2.2 Add 100 mL of buffered beef extract solution to the
sedimented, conditioned sludge. The volume of buffered beef
Reagent Chemicals, American Chemical Society Specifications, American
extract solution used to elute viruses from the conditioned
Chemical Society, Washington, DC. For suggestions on the testing of reagents not
listed by the American Chemical Society, see Analar Standards for Laboratory
sludge is equal to the original volume of the sample volume
Chemicals, BDH Ltd., Poole, Dorset, U.K., and the United States Pharmacopeia
(see 9.1).
and National Formulary, U.S.PharmaceuticalConvention,Inc.(USPC),Rockville,
9.2.3 Replace and tighten cap on centrifuge bottle.
MD.
9.2.4 Place centrifuge bottle on magnetic stirrer and stir at
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
speed sufficient to develop vortex. To minimize foaming
14072.
(which may inactivate viruses), do not mix faster than neces-
Thesolesourceofsupplyoftheapparatus,Duo-Fineseriessheetfilters,known
sary to develop vortex. Care must be taken to prevent bottle
to the committee at this time is Filterlite Corp., 2033 Green Spring Dr., Timonium,
MD 21093. from toppling. Stabilize bottle as necessary.
D4994 − 19
9.2.5 Continue mixing for 30 min. 9.4.4 Pour the diluted, filtered beef extract into a graduated
cylinder and record the total volume.
9.2.6 Turn stirrer off and remove stir bar from centrifuge
bottle. 9.4.5 Decant diluted filtered beef extract into 600-mL bea-
ker and add a stir bar.
9.2.7 Replace and tighten cap on centrifuge bottle and
9.4.6 Place beaker on magnetic stirrer and stir at a speed
centrifuge conditioned sludge-eluate mixture at 10000× g for
30 min at 4°C. sufficient to develop vortex.To minimize foaming (which may
inactivateviruses),donotmixfasterthannecessarytodevelop
9.2.8 Remove cap from centrifuge bottle. Decant superna-
vortex.
tant fluid (eluate) into beaker and discard sediment.
9.4.7 Insert combination-type pH electrode into diluted,
9.2.9 Place a filter holder that contains a filter stack as
filtered beef extract and add HCl (1+9) slowly until pH of
described in 7.7 on a 250-mL Erlenmeyer receiving flask.
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
9.4.9 Replace and tighten caps on centrifuge bottles and
filter holders and filter stacks. Repeat 9.2.9 through 9.2.12 as
centrifuge the flocculated beef extract suspension at 2500× g
often as necessary to filter entire volume of eluate. Disas-
for 15 min at 4°C. Pour off and discard supernatants.
semble each filter holder and examine bottom filters to be
9.4.10 Place a small stir bar into each centrifuge bottle that
certain they have not ruptured. If a bottom filter has ruptured,
contains flocculate and replace covers loosely.
repeat 9.2.10 through 9.2.12 with new filter holders and filter
9.4.11 Measure a volume of Na HPO ·7H O solution equal
2 4 2
stacks.
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
...

This document is not an ASTM standard and is intended only to provide the user of an ASTM standard an indication of what changes have been made to the previous version. Because
it may not be technically possible to adequately depict all changes accurately, ASTM recommends that users consult prior editions as appropriate. In all cases only the current version
of the standard as published by ASTM is to be considered the official document.
Designation: D4994 − 89 (Reapproved 2014) D4994 − 19
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. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope 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 A—Adsorption 7 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 safety, health, and healthenvironmental 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, for work with potentially hazardous biological
organisms.
1.7 This international standard was developed in accordance with internationally recognized principles on standardization
established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued
by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
2. Referenced Documents
2.1 ASTM Standards:
D1129 Terminology Relating to Water
D1193 Specification for Reagent Water
3. Terminology
3.1 Definitions—Definitions: For definitions of terms used in this practice, refer to Terminology D1129.
3.1.1 For definitions of terms used in this standard, refer to Terminology D1129.
4. 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).
5. Apparatus
5.1 Centrifuge(s), refrigerated, capable of attaining 10 000 × g, screw-capped 100-mL centrifuge bottles that can withstand
10 000 × g, and 250-mL screw-capped centrifuge bottles capable of withstanding 2 500 × 2500 × g.
This practice is under the jurisdiction of ASTM Committee D19 on Water and is the direct responsibility of Subcommittee D19.24 on Water Microbiology.
Current edition approved Jan. 1, 2014April 1, 2019. Published March 2014April 2019. Originally approved in 1989. Last previous edition approved in 20092014 as
D4494 – 89 (2009).(2014). DOI: 10.1520/D4994-89R14.10.1520/D4994-19.
Richardson, J. H., and Barkley, W. E., Biological Safety in Microbiological and Biomedical Laboratories, 2nd edition, U.S. Dept. of Health and Human Services, Public
Health Service, Center for Disease Control, and National Institutes of Health and Human Services, 1988.
For referenced ASTM standards, visit the ASTM website, www.astm.org, or contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM Standards
volume information, refer to the standard’s Document Summary page on the ASTM website.
*A Summary of Changes section appears at the end of this standard
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
D4994 − 19
5.2 pH Meter, measuring to an accuracy of at least 0.1 pH unit, equipped with a combination-type electrode. Calibrate with
standard buffers.
4,5
5.3 Filter Apparatus, for membrane sterilization, with 47-mm diameter filter holder and 50-mL slip-tip syringe (see 7.7 for
type of filter material).
6. Purity of Reagents
6.1 Purity of Reagents—Reagent grade chemicals shall be used in all tests. Unless otherwise indicated, it is intended that all
reagents shall conform to the specifications of the Committee on Analytical Reagents of the American Chemical Society, where
such specifications are available. Other grades may be used, provided it is first ascertained that the reagent is of sufficiently high
purity to permit its use without lessening the accuracy of the determination.
6.2 Purity of Water—Unless otherwise indicated, references to water shall be understood to mean reagent water conforming to
Specification D1193, Type II.
PROCEDURE A—ADSORPTION
7. Reagents and Materials
7.1 Aluminum Chloride Solution (12.07 g/L)—Dissolve 12.07 g of aluminum chloride (AlCl ·6H O) in 500 mL of water and
3 2
dilute to 1000 mL. Autoclave AlCl solution at 121°C for 15 min.
5,7
7.2 Buffered Beef Extract Solution—Dissolve 10 g of beef extract powder, 1.34 g of Na HPO ·7H O, and 0.12 g of citric acid
2 4 2
in 100 mL of water in a screw-cap flask by stirring for about 2 h on a magnetic stirrer. Autoclave at 121°C for 15 min.
7.3 Disodium Hydrogen Phosphate Solution (4 g/100 mL)—Dissolve 4 g of disodium hydrogen phosphate (Na HPO ·7H O) in
2 4 2
100 mL of water and autoclave at 121°C for 15 min.
7.4 Hydrochloric Acid (1 + 1)—Add 1 volume of concentrated HCl (sp gr 1.19) to 1 volume of water.
7.5 Hydrochloric Acid (1 + 9)—Add 1 volume of concentrated HCl (sp gr 1.19) to 9 volumes of water.
7.6 Sodium Hydroxide Solution (4 g/100 mL)—Dissolve 4.0 g of dry sodium hydroxide (NaOH) in water and dilute to 100 mL.
5,8
7.7 Filters, Disc, Membrane, 47-mm—3.0-, 0.45-, and 0.25-μm pore size which must be cut to proper size from sheet filters.
Disassemble filter holder. Place filter with 0.25-μm 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.
8. Summary of Procedure
8.1 The adsorption procedure relies upon adsorption of viruses from the liquid phase to the sludge solids, which are
concentrated by centrifugation. The supernatant is discarded. Viruses are desorbed from the solids by physicochemical means and
further concentrated by organic flocculation. Decontamination is accomplished by filtration.
9. Procedure
9.1 Conditioning of Sludge—In the absence of experience that dictates otherwise, use 100-mL volumes for liquid sludges and
100-g quantities for digested, dewatered sludges.
9.1.1 Measure 100 mL of well-mixed sludge in a graduated 100-mL cylinder. Mix sludge vigorously immediately before it is
poured into cylinder because sludge solids, which contain most of the viruses, begin to settle out immediately after mixing stops.
9.1.2 Place stir bar into a 250-mL beaker.
9.1.3 Pour the 100-mL of measured sludge from the cylinder into the 250-mL beaker. If necessary, pour sludge several times
from beaker to cylinder and back to remove all sludge solids to beaker. Take care to avoid formation of aerosols.
9.1.4 Place beaker on magnetic stirrer, and stir at speed sufficient to develop vortex.
9.1.5 Add 1 mL of AlCl solution to sludge. Final concentration of AlCl in sludge is approximately 0.0005 M.
3 3
9.1.6 Place combination-type pH electrode into sludge and adjust pH of sludge to 3.5 6 0.1 with HCl (1 + 1). If pH falls below
3.5, readjust with NaOH solution (4 g/100 mL). If sludge adheres to electrodes, clean electrodes by moving them up and down
gently in mixing sludge. pH meter must be standardized at pH 4.
The sole source of supply of the apparatus, Swinnex filter (No. SX0047000), known to the committee at this time is Millipore Corp., 80 Ashby Rd., Bedford, MA 01730.
If you are aware of alternative suppliers, please provide this information to ASTM International Headquarters. Your comments will receive careful consideration at a
meeting of the responsible technical committee, which you may attend.
Reagent Chemicals, American Chemical Society Specifications, American Chemical Society, Washington, DC. For suggestions on the testing of reagents not listed by
the American Chemical Society, see Analar Standards for Laboratory Chemicals, BDH Ltd., Poole, Dorset, U.K., and the United States Pharmacopeia and National
Formulary, U.S. Pharmaceutical Convention, Inc. (USPC), Rockville, MD.
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 14072.
The sole source of supply of the apparatus, Duo-Fine series sheet filters, known to the committee at this time is Filterlite Corp., 2033 Green Spring Dr., Timonium, MD
21093.
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9.1.7 Continue mixing for 30 min. Check pH of the sludge at frequent intervals. If the pH drifts up, readjust to 3.5 6 0.1 with
HCl (1 + 9). If the pH drifts down, readjust with NaOH solution (4 g/100 mL).
9.1.8 Turn stirrer off and remove pH electrode from sludge.
9.1.9 Remove cap from a screw-capped centrifuge bottle and pour conditioned sludge into centrifuge bottle. To prevent transfer
of stir bar into centrifuge bottle when decanting sludge, hold another stir bar or magnet against bottom of beaker. Remove sludge
that adheres to stir bar in the beaker by manipulation with a stirring rod. If necessary, pour sludge several times from centrifuge
bottle to beaker and back to remove all sludge solids to bottle. Take care to avoid formation of aerosols.
9.1.10 Replace and tighten cap on centrifuge bottle.
9.1.11 Centrifuge conditioned sludge at 2500 × g for 15 min at 4°C. Discard supernatant.
9.2 Elution of Viruses from Sludge Solids:
9.2.1 Add stir bar to the centrifuge bottle that contains sedimented, conditioned sludge.
9.2.2 Add 100 mL of buffered beef extract solution to the sedimented, conditioned sludge. The volume of buffered beef extract
solution used to elute viruses from the conditioned sludge is equal to the original volume of the sample volume (see 9.1).
9.2.3 Replace and tighten cap on centrifuge bottle.
9.2.4 Place centrifuge bottle 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. Care must be taken to prevent bottle from toppling. Stabilize
bottle as necessary.
9.2.5 Continue mixing for 30 min.
9.2.6 Turn stirrer off and remove stir bar from centrifuge bottle.
9.2.7 Replace and tighten cap on centrifuge bottle and centrifuge conditioned sludge-eluate mixture at 10 000 × g for 30 min
at 4°C.
9.2.8 Remove cap from centrifuge bottle. Decant supernatant fluid (eluate) into beaker and discard sediment.
9.2.9 Place a filter holder that contains a filter stack as described in 7.7 on a 250-mL Erlenmeyer receiving flask.
9.2.10 Load 50-mL syringe with eluate.
9.2.11 Place tip of syringe into filter holder.
9.2.12 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 splinter 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.
Repeat 9.2.9 through 9.2.12 as often as necessary to filter entire volume of eluate. Disassemble each filter holder and examine
bottom filters to be certain they have not ruptured. If a bottom filter has ruptured, repeat 9.2.10 through 9.2.12 with new filter
holders and filter stacks.
9.2.13 Refrigerate eluate immediately at 4°C, and maintain at that temperature until it is assayed for viruses (see 9.3). The
number of cell cultures necessary for the viral assay may be 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, proceed
immediately to 9.4. If further concentration is not required and if assay for viruses cannot be undertaken within 8 h, distribute
eluate into sterile sample bottles, cap tightly, and store immediately at −70°C.
9.3 Viral Assay:
9.3.1 At time of viral assay, rapidly thaw the frozen concentrate at 37°C and proceed with usual viral assay. At least 10 % of
the isolates should be confirmed by second passage.
9.4 Procedure for Concentrating Viruses from Sludge Eluates (Organic Flocculation Concentration)—It is preferable to assay
eluted viruses in the beef extract eluate without concentrating them because some loss of viruses may occur in concentration.
However, the numbers of cell cultures needed for assays may be reduced by concentrating the viruses in the eluate. Significant
further loss of viruses may occur with the currently available beef extract which may not produce sufficient floc to adsorb all of
the suspended virions.
9.4.1 Pour eluate from 9.2.13 into a graduated cylinder and record the volume.
9.4.2 Pour eluate into 600-mL beaker.
9.4.3 For every 3 mL of beef extract eluate, add 7 mL of sterile 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.
9.4.4 Pour the diluted, filtered beef extract into a graduated cylinder and record the total volume.
9.4.5 Decant diluted filtered beef extract into 600-mL beaker and add a stir bar.
9.4.6 Place beaker on magnetic stirrer 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.
9.4.7 Insert combination-type pH electrode into diluted, filtered beef extract and add HCl (1 + 9) slowly until pH of beef extract
reaches 3.5 6 0.1. A flocculate or precipitate will form. If pH drops below 3.4, add NaOH solution (4 g/100 mL) until pH is 3.5
6 0.1. Avoid reducing pH below 3.4 because some inactivation of viruses may occur. Continue to stir for 30 min.
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9.4.8 Turn stirrer off, remove electrode from beaker, and distribute contents of beaker evenly among centrifuge bottles. To
pre
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