ASTM D8243-19

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: D8243 − 19
Standard Test Method for
Determination of APS Reductase to Estimate Sulfate
Reducing Bacterial Bioburdens in Water – Enzyme-Linked
Immunosorbent Assay Method
This standard is issued under the fixed designation D8243; 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 ization established in the Decision on Principles for the
Development of International Standards, Guides and Recom-
1.1 This test method provides a protocol for using enzyme-
mendations issued by the World Trade Organization Technical
linked immunosorbent assay (ELISA) technology to test water
Barriers to Trade (TBT) Committee.
samplesfortheenzymeadenosine5’-phosphosulfatereductase
(APSr) concentration.
2. Referenced Documents
1.1.1 APSr is present in all known sulfate reducing protists
2.1 ASTM Standards:
(SRP– sulfate reducing bacteria – SRB – and sulfate reducing
D1129Terminology Relating to Water
archaea – SRA).
D4412TestMethodsforSulfate-ReducingBacteriainWater
1.1.2 As reported in U.S. Patent 4,999,286, APS reductase
and Water-Formed Deposits
concentration can be used as a surrogate parameter for esti-
D5847Practice for Writing Quality Control Specifications
mating SRAbioburdens (Appendix X1 compares results from
for Standard Test Methods for Water Analysis
Test Methods D8243, D4412, and quantitative polymerase
D6300Practice for Determination of Precision and Bias
chain reaction – qPCR – testing).
Data for Use in Test Methods for Petroleum Products and
1.2 Thistestmethodhasbeenvalidatedintapwater,oilfield
Lubricants
-1 -1
producedwater(salinitiesrangingfrom100gL to600gL ),
D6499Test Method for Immunological Measurement of
and fuel-associated water (commonly referred to as bottoms-
AntigenicProteininHeveaNaturalRubber(HNR)andits
water).
Products
1.3 This test method detects APS reductase semi-
D7464Practice for Manual Sampling of Liquid Fuels, As-
quantitatively in the range of 0.001M to 0.1M – correlating to
sociated Materials and Fuel System Components for
2 6
10 SRP/mL to 10 SRP/mL.
Microbiological Testing
1.3.1 As described in Appendix X2 test method sensitivity
E691Practice for Conducting an Interlaboratory Study to
can be increased 10-fold to 100-fold. However, the precision
Determine the Precision of a Test Method
statistics provided in X apply only to 10-mL specimens.
E1601Practice for Conducting an Interlaboratory Study to
Evaluate the Performance of an Analytical Method
1.4 The values stated in SI units are to be regarded as
E1847Practice for Statistical Analysis of Toxicity Tests
standard. No other units of measurement are included in this
Conducted Under ASTM Guidelines
standard.
E1914Practice for Use of Terms Relating to the Develop-
1.5 This standard does not purport to address all of the
ment and Evaluation of Methods for Chemical Analysis
safety concerns, if any, associated with its use. It is the
(Withdrawn 2016)
responsibility of the user of this standard to establish appro-
2.2 Patents:
priate safety, health, and environmental practices and deter-
U.S. Patent 4,999,286Sulfate Reducing Bacteria Determi-
mine the applicability of regulatory limitations prior to use.
nation and Control, March 12, 1991
Some specific hazards statements are given in Section 9 on
Hazards.
1.6 This international standard was developed in accor-
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
dance with internationally recognized principles on standard-
contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
Standards volume information, refer to the standard’s Document Summary page on
the ASTM website.
1 3
This test method is under the jurisdiction ofASTM Committee D19 on Water The last approved version of this historical standard is referenced on
and is the direct responsibility of Subcommittee D19.24 on Water Microbiology. www.astm.org.
Current edition approved Jan. 1, 2019. Published February 2019. DOI: 10.1520/ Available from United States Patent and Trademark Office (USPTO), Madison
D8243-19. Building, 600 Dulany Street, Alexandria, VA 22314, https://www.uspto.gov.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
D8243 − 19
3. Terminology 4.7 At the end of the color-development period (that is, 15
min at 18 6 2°C), compare and match the color of the funnel
3.1 Definitions:
device’s filter membrane with the colors on the QuickChek
3.1.1 For definitions of terms used in this standard, refer to
color card (8.6).
Terminology D1129.
4.8 Report SRB/mL based on the color match observed in
3.1.2 adenosine 5’-phosphosulfate reductase (APS
4.7.
reductase), n—(EC 1.8.4.9 and CAS 9027-75-2) an enzyme
present on all known sulfate reducing bacteria and archaea,
5. Significance and Use
catalyzing the two-electron reduction of the sulfate group in
adenosine 5’-phosphosulfate (APS) to yield adenosine mono- 5.1 Sulfate reducing archaea and bacteria are known to
phosphate and sulfite.
contribute to microbiologically influenced corrosion.
3.1.3 categorical data, n—variates that take on a limited
5.2 Sulfate-reducing bacteria are widely distributed in ma-
number of distinct values. E1847
rine and fresh water muds which, in consequence, frequently
are laden with the hydrogen sulfide produced by these organ-
3.1.4 chromogen, n—a chemical or compound that reacts
isms during dissimilatory sulfate reduction.
with an enzyme to produce a colored end-product, used to
detect the presence of a substance of interest.
5.3 Traditional, culture-dependent methods such as those
describedinTestMethodsD4412,prescribeincubationperiods
3.1.5 detection limit, n—the smallest net signal (or the
of as long as 21 days before assigning a below detection limit
derived property value, constituent mass fraction, etc.) ob-
(BDL)scoretoaspecimen.Moreover,itiswellknownthatnot
tained by a given measurement procedure, that can be distin-
all SRP will proliferate in the nutrient media specified in Test
guished from the background signal at a specified confidence
Methods D4412.
level. E1914
5.4 This test method uses ELISA technology to provide
3.1.6 enzyme linked immunosorbent assay (ELISA), n—an
semi-quantitative, culture-independent, SRP bioburden test
immunological test method to quantify antigen or antibody
results in less than 30 min.
levels using an enzyme as the detection mechanism. D6499
5.4.1 Because all the reagents and supplies used are non-
3.2 Symbols:
hazardousandprepackagedforsingletestuse,thistestmethod
APSr—APS reductase
does not require any apparatus other than a laboratory timer.
BDL—below detection limit
Consequently, it can be performed at or near the point of
GHS—Globally Harmonized System of Classification and
sample collection.
Labeling of Chemicals
5.4.2 The opportunity to minimize the delay between
SRA—sulfate reducing archaea
sample collection, testing, and results availability translates
SRB—sulfate reducing bacteria
into timely use of the data to drive preventive and corrective
SRP—sulfate reducing protists
SRB control measures.
4. Summary of Test Method
6. Interferences
4.1 A 10-mL water specimen is filtered through a diatoma-
6.1 SRP population densities >10 cells/mL can cause an
ceousearthfilter(8.3)thatcapturesmicrobialcells.Thefiltrate overload effect that can cause the population density to be
is discarded.
underestimated.
4.2 Thewashbottle’scap(8.5)isreplacedwiththefiltercap
7. Apparatus
from 4.1. The bottle is inverted and the 2 mLof wash solution
7.1 Timer, laboratory
is filtered through the filter medium to eliminate interferences.
4.3 The filter cap is removed from the wash bottle and
8. Reagents and Materials
placed onto the bottle containing the rehydrated lysing reagent
8.1 Bottle, dropper, containing 2-mL lysing reagent rehy-
(8.2). The filter cake is dispersed from the cap into the lysing
dration fluid.
reagent and allowed to stand for 2 min.
8.2 Bottle, lysing reagent, proprietary, freeze-dried.
4.4 The filter cap is replaced with the final filter cap (8.7)
and the lysed solution is filtered into the immunoreagent vial
(8.9). The filtrate-immunoreagent mixture is allowed to stand 5
QuickChek is a trademark of Modern Water, Inc, New Castle, DE, https://
for 2 min. www.modernwater.com,andisthesolesourceofsupplyknowntothecommitteeat
this time. If you are aware of alternative suppliers, please provide this information
4.5 Thelysate-immunoreagentmixtureisdispensedintothe
to ASTM International Headquarters. Your comments will receive careful consid-
erationatameetingoftheresponsibletechnicalcommittee, whichyoumayattend.
funnel device (8.8).
The sole source of supply of the proprietary chromogen liquid, final filter,
4.6 After all of the fluid has passed through the funnel
immunoreagent, lysing reagent, rehydrating fluid, sample bottle with filter cap, test
membrane device, and wash solution is Modern Water, Inc, New Castle, DE,
device’s filter membrane, the contents of the chromogen
https://www.modernwater.com. If you are aware of alternative suppliers, please
reagent dropper vial (8.5) are dispensed onto the funnel
provide this information toASTM International Headquarters.Your comments will
device’s filter membrane. A laboratory-timer (7.1), set to the
receive careful consideration at a meeting of the responsible technical committee,
appropriate time (15 min at 18 6 2°C) is started. which you may attend.
D8243 − 19
8.3 Bottle, sample, with proprietary filter cap, 10 mL. 13.1.3 Replace lysing reagent bottle cap, and gently swirl
bottle for 15 sec to facilitate reagent rehydration.
8.4 Bottle,wash,containing:washbuffer,2mL,proprietary.
13.2 Collect Specimen:
8.5 Chromogen, proprietary, in dropper vial.
13.2.1 Shake sample vigorously for 30 sec to ensure that it
8.6 Color card, QuickChek.
is mixed properly.
8.7 Filter, final, dispensing cap, proprietary.
13.2.2 Remove dropper cap from sample bottle (8.3).
13.2.3 Dispense 10 mL of sample into sample bottle (8.3)
8.8 Funnel device, proprietary.
and replace dropper cap.
8.9 Immunoreagent, proprietary, in vial.
13.2.4 Invert sample bottle (13.2.3) and shake for 15 sec to
8.10 Wash reagent, final, in dropper vial.
disperse filter cake.
13.2.5 Keeping sample bottle (13.2.4) inverted, wait 15 sec
9. Hazards
to allow filter cake to settle into dropper tip.
9.1 Hydrogen sulfide can accumulate in any air space in 13.2.6 Apply steady pressure to squeeze sample bottle
samplecontainersfilledwithSRB-contaminatedwater.Hydro-
(13.2.5) to filter sample.
gen sulfide is flammable (GHS02), corrosive (GHS05), and
13.2.6.1 Dispense filtrate into a waste container.
toxic (GHS06). Appropriate precautions shall be taken when
13.2.6.2 Maintain pressure on sample bottle until the entire
handlingsampleslikelytocontaindetectableSRBpopulations.
10-mL specimen has been filtered.
NOTE 1—While performing the filtration step, do not release pressure.
10. Sampling, Test Specimens, and Test Units
Doingsowillcreateapartialvacuuminthesamplebottle.Thiswillpermit
10.1 Samples shall be collected and handled in accordance
air to enter the bottle, causing the filter cake to re-disperse into the
with guidance provided in Practice D7464. specimen and potentially cause SRP to be dispensed with the filtrate.
NOTE2—Avacuumdevicemaybeusedtofacilitatefiltration.However,
10.1.1 Because microbes continue to be metabolically ac-
the test results used to obtain the precision statistics provided for this test
tive in collected samples, microbiological parameters are
method were based on manual squeezing in accordance with 13.2.6.2.
perishable.
13.3 Wash Specimen:
10.1.2 Optimally, samples shall be tested within 4 h after
13.3.1 Remove cap from wash buffer bottle (8.4).
collection.
13.3.2 Remove filter cap from sample bottle (13.2.6).
10.1.3 Acceptable test results can be obtained if testing is
startedwithin24honsamplesthathavebeeneitherkeptonice 13.3.3 Place cap (13.3.2) onto wash buffer (8.4).
or refrigerated since collection. 13.3.4 Invert wash buffer bottle (13.3.3) and shake for 15
10.1.4 As the delay between sampling and testing increases sec to disperse filter cake.
beyond 24 h the relationship between test results and SRP 13.3.5 Keepingwashbufferbottle(13.3.4)inverted,wait15
bioburdens in the systems from which samples were collected sec to allow filter cake to settle into dropper tip.
becomes increasingly tenuous. 13.3.6 Apply steady pressure to squeeze wash buffer bottle
(13.3.5) to filter sample.
10.2 The standard specimen for this test method is 10 mL.
13.3.7 Dispense filtrate into a waste container.
10.3 Thesampleunitsizeisnotcritical,butaminimumunit
13.3.8 Maintain pressure on sample bottle until the entire
size of 100 mL is recommended for replicate testing.
2-mL wash buffer has been filtered.
11. Calibration and Standardization
13.4 Lyse Cells:
13.4.1 Remove cap from lysing reagent bottle (13.1.3).
11.1 Nocalibrationorstandardizationisneededforthistest
13.4.2 Remove dropper tip from wash buffer bottle (13.3.8)
method. However, if a user is concerned about obtaining false
and place it onto lysing reagent bottle (13.4.1).
negative results, reagent response can be tested in accordance
13.4.3 Flick the side of the lysing bottle/filter cap firmly
with the protocol provided in Appendix X2.
with your index finger.
12. Conditioning
13.4.3.1 If the filter cake does not become dislodged, invert
the bottle and flick it firmly again.
12.1 Enzyme reaction rates are temperature dependent.
13.4.4 After the filter cake is dislodged, strike the base on a
Therefore, all samples and reagents shall be permitted to
flat surface to make the filter cake fall into the lysing fluid.
equilibratetoambienttemperaturebeforetesting.Asexplained
13.4.5 If any of the cake remains in the filter cap, tip the
in 13.7.3, the time allowed for ELISAreaction color develop-
bottleupsidedownandallowthefluidtorunintothefiltercap.
ment is temperature dependent.
NOTE 3—When the fluid will go all the way to the plug in the cap, all
13. Procedure
the filter cake has been transferred.
13.1 Rehydrate Lysing Reagent:
13.4.6 Swirllysingbottle(13.4.5)for15sectomixthefilter
13.1.1 Remove caps from rehydrating solution dropper
cake and lysing reagent well.
bottle (8.1) and lysing reagent bottle (8.2).
NOTE 4—Be careful not to mix so aggressively as to cause the filter
13.1.2 Invert rehydrating solution dropper bottle (8.1) over
cake dispersion to foam.
lysing reagent bottle (8.2) and squeeze the latter to dispense
rehydrating solution into lysing reagent bottle. 13.4.7 Remove filter cap and let lysing vial stand for 2 min.
D8243 − 19
13.4.8 Snapfinalfilterdroppertop(8.7)intoplaceonlysing
bottle (13.4.7).
13.5 React Analyte with Immunoreagent:
13.5.1 Remove the rubber cap from the glass immunore-
agent vial (8.9).
13.5.2 Invert the lysing vial (13.4.8) over the immunore-
agentvial(13.5.1)andsqueezetheliquidfromthelysingbottle
into the immunoreagent vial.
NOTE 5—Between 10 and 20 drops of fluid should flow through the
final filter into the immunoreagent vial.
13.5.3 Stopsqueezingthelysingbottlewhenalltheliquidis
expelled, and foam appears at the filter tip.
13.5.4 Mix the liquid in the immunoreagent vial gently and
let it incubate for two minutes.
NOTE 6—During this incubation period, the antibody-coated particles
bind with the APS reductase in solution.
NOTE 7—The prescribed 2 min is nominal. Incubation intervals
between 2 min and 4 min do not affect test results.
13.6 Concentrate Antigen-Antibody Complexed Particles:
13.6.1 Decant the contents of the immunoreagent vial
(13.5.4) onto the funnel device’s membrane (8.8).
13.6.2 Allow all fluid to be drawn through the funnel
FIG. 1 QuickChek Color Card Next to Filter Device Membrane to
device’s membrane.
Facilitate SRP Bioburden Scoring
NOTE 8—In the funnel device (8.8) the filter membrane rests on an
absorbent bed. The absorbent draws the fluid through the membrane. The
time required for all of the fluid to drain through the funnel device’s
14. Calculation or Interpretation of Results
membrane can vary from 2 min to 5 min.
13.6.3 Remove yellow cap from final wash reagent vial
14.1 For 10-mL specimens, no calculations are required.
(8.10) and squeeze vial to dispense wash reagent onto mem- The reported SRP/mL is read directly from the QuickChek
brane.
color card (8.6) in accordance with 13.8.3.
13.6.4 Allow all fluid to be drawn through the funnel
14.2 Interpretation:
device’s membrane.
14.2.1 SRPupper control limits can vary, depending on the
13.7 Color Development:
system from which samples are collected.
13.7.1 Remove blue cap from chromogen vial (8.5).
14.2.2 In many industrial applications, any SRP bioburden
13.7.2 Invert vial and squeeze its sides to dispense chromo-
that is greater than BDL triggers follow-up action.
gen onto funnel device’s membrane (13.6.4).
14.2.3 Some applications set the upper control limit at <10
13.7.3 Set lab timer to appropriate time in accordance with
SRP/mL.
Table 1.
15. Reporting
13.7.4 Allow membrane color to develop for period set in
13.7.3.
15.1 Recognizing that SRP/mL can range from <10
13.7.4.1 Do not perform this test method if the ambient
SRP/mLto>10 SRP/mL,Log transformationfacilitatesdata
temperature is <16°C or >38°C.
entry and process control plotting.
15.1.1 To transform test results, compute Log of the
13.8 Observe Color:
observed cells/mL as in Eq 1:
13.8.1 Place QuickChek color card (8.6) near funnel device
membrane as shown in Fig. 1. 3
Log ~10 SRP ⁄ mL!53Log SRP⁄mL (1)
10 10
13.8.2 Align color card (8.6) with membrane to match
15.1.2 After Lo
...