ASTM D8429-21

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: D8429 − 21
Standard Test Method for
1,2
Legionella pneumophila in Water Samples Using Legiolert
This standard is issued under the fixed designation D8429; 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 priate safety, health, and environmental practices and deter-
mine the applicability of regulatory limitations prior to use.
1.1 This test method describes a simple procedure for the
1.4 This international standard was developed in accor-
detection of Legionella pneumophila (L. pneumophila)in
dance with internationally recognized principles on standard-
potable water and non-potable waters (cooling towers, for
ization established in the Decision on Principles for the
example). This procedure describes a liquid culture method
Development of International Standards, Guides and Recom-
based on a bacterial enzyme technology. The detection of L.
mendations issued by the World Trade Organization Technical
pneumophila is signaled through the utilization of a substrate
Barriers to Trade (TBT) Committee.
present in the Legiolert reagent. L. pneumophila cells grow
rapidly and reproduce using the rich supply of amino acids,
2. Referenced Documents
vitamins and other nutrients present in the Legiolert reagent.
2.1 ASTM Standards:
Actively growing strains of L. pneumophila use the added
D1129 Terminology Relating to Water
substrate to produce a brown color indicator or produce turbid
D1193 Specification for Reagent Water
growth with or without brown coloration. Legiolert can detect
D2777 Practice for Determination of Precision and Bias of
this bacterial species at the following minimum concentrations
Applicable Test Methods of Committee D19 on Water
based on the protocol employed:
D3370 Practices for Sampling Water from Flowing Process
1.1.1 Potable Water:
Streams
1.1.1.1 ≥1 organism / 100 mL at 7 days for 100 mL potable
D5952 Guide for the Inspection of Water Systems for
protocol.
Legionella and the Investigation of Possible Outbreaks of
1.1.1.2 ≥1 organism / 10 mL at 7 days for 10 mL potable
Legionellosis (Legionnaires’ Disease or Pontiac Fever)
protocol.
D6503 Test Method for Enterococci in Water Using Enter-
1.1.2 Non-potable Water:
olert
1.1.2.1 ≥1 organism / 1.0 mL at 7 days for 1.0 mL
non-potable protocol.
3. Terminology
1.1.2.2 ≥1 organism / 0.1 mL at 7 days for 0.1 mL
3.1 Definitions:
non-potable protocol.
3.1.1 For definitions of terms used in this standard, refer to
1.1.3 This test method can be used for potable (drinking)
Terminology D1129.
waters and non-potable waters such as cooling tower waters
3.1.2 Legionella, n—a bacterial genus containing over 50
(1). It is the user’s responsibility to ensure the validity of this
species and at least 71 serogroups; abbreviated to the first
test method for waters of untested matrices.
initialwhenusedrelatedlywithaspeciesname,forexample,L.
1.2 The values stated in SI units are to be regarded as
pneumophila.
standard. No other units of measurement are included in this
3.1.3 most probable number (MPN), n—a statistical method
standard.
for determining bacterial concentration based on the Poisson
1.3 This standard does not purport to address all of the
distribution.
safety concerns, if any, associated with its use. It is the
3.2 Definitions of Terms Specific to This Standard:
responsibility of the user of this standard to establish appro-
3.2.1 Legionella pneumophila, n—in the context of this
method,apathogenicgram-negativebacteriathatproducesany
degree of brown coloration or turbid growth with or without
Legiolert is a trademark of IDEXX Laboratories, Inc., Westbrook, ME.
brown coloration in the presence of the Legiolert reagent.
This test method 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 Dec. 1, 2021. Published December 2021. DOI: For referenced ASTM standards, visit the ASTM website, www.astm.org, or
10.1520/D8429-21. contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
The boldface numbers in parentheses refer to a list of references at the end of Standards volume information, refer to the standard’s Document Summary page on
this standard. the ASTM website.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
D8429 − 21
3.2.2 Quanti-Tray/Legiolert, n—a system for the quantifica- out based on risk assessments or on local, state, or federal
tion of Legionella pneumophila. It consists of a Quanti-Tray requirements or guidelines.
5,6
Sealer PLUS and 96-multi well Quanti-Trays that express L.
6. Interferences
pneumophila counts as MPN per volume of sample.
3.2.2.1 Discussion—For drinking water or similar waters
6.1 Hardnesscaninterferewiththeuseof100mLofpotable
where the volume is typically 100 mL, the system can
water sample and the addition of the Legiolert Supplement is
enumerate up to 2272.6/100 mL or for small sample volumes
required to prevent interference (12.2.1.3).
such as the 0.1 mL protocol for non-potable water the system
6.2 For non-potable, the Legiolert Pretreatment reagent is
can enumerate up to 22726/1 mL.
required to be added to the sample (12.2.1.4).
3.2.2.2 Discussion—To increase the systems counting range
asampledilutioncanbeutilized,buttoobtainthecorrectMPN
7. Apparatus
valueandlowerandupperconfidencelimits,thesevaluesmust
be multiplied by the dilution factor. 7.1 Incubator, capable of maintaining a temperature of 39
°C 6 0.5 °C (for potable water samples).
3.2.3 snap pack, n—a package containing Legiolert reagent
for testing 100 mL sample volumes or dilutions thereof,
7.2 Incubator, capable of maintaining a temperature of 37
quantitatively by means of the Quanti-Tray/Legiolert system.
°C 6 0.5 °C (for non-potable water samples).
7.3 Quanti-Tray Sealer PLUS.
4. Summary of Test Method
4.1 Bacteria are grown in a defined liquid culture medium
8. Reagents and Materials
containing the Legiolert reagent.
8.1 Purity of Water—Unless otherwise indicated, references
4.2 The reagent is added to the sample, sealed in a Quanti-
towatershallbeunderstoodtomeanreagentwaterconforming
Tray, and then incubated, in the presence of humidity, for 7
to Specification D1193, Type IV. Sterilize water by either
days at either at 39 °C 6 0.5 °C or 37 °C 6 0.5 °C for potable
autoclaving or by sterile filtration (0.22-micron filter filtered
or non-potable water samples, respectively.
water).
4.3 L. pneumophila is detected if any of the Quanti-Tray
8.2 Legiolert Test Kit:
wells exhibit growth indicated as a brown color change and/or
8.2.1 The Legiolert reagent is a commercially available
turbidity.
substrate provided in a snap pack format and is suitable for
single samples.
4.4 Test results are reported as MPN per volume of sample
(that is, MPN/mL).
8.3 Quanti-Tray/Legiolert:
8.3.1 For MPN results, the Legiolert regent must be used in
5. Significance and Use
conjunction with the Quanti-Tray/Legiolert 96-well trays.
5.1 This test provides an easy and reliable method for the Quanti-Tray/Legiolert is commercially available, and one tray
is suitable for a single sample.
detection of L. pneumophila in potable and non-potable waters
in 7 days.
8.4 Legiolert Supplement Kit:
8.4.1 The Legiolert Supplement is a commercially available
5.2 Routine monitoring for L. pneumophila determines
kit containing of (1) a canister of dip strip tests designed to
whether implemented control measures are effective, such as
determine the total calcium/magnesium hardness in a water
those outlined in a water safety program (2).
sample and (2) a 100 mL vessel containing supplement
5.2.1 Water system management is necessary to maintain L.
pneumophila concentrations below hazardous levels. Through solution powder. The hardness strips are used to determine the
total hardness of the sample and the supplement solution, once
routine measurement of L. pneumophila levels, a monitoring
program can ensure that control measures are effective and the powder is reconstituted with 100 mL of sterile deionized
water,isaddedtothesamplebasedonthedeterminedsample’s
implemented when necessary in response to increasing levels.
Water samples may be examined for L. pneumophila during hardness.
epidemiological investigations as part of local authority,
8.5 Legiolert Pretreatment Kit:
industrial, or hospital programs, or in order to validate treat-
8.5.1 The Legiolert Pretreatment is a commercially avail-
ment control methods. Routine sampling could also be carried
able kit containing of four vessels containing a dry powder to
be reconstituted in sterile water.
8.6 Quality Control Strains:
Quanti-Tray is a trademark of IDEXX Laboratories, Inc., Westbrook, ME.
ThesolesourceofsupplyofQuanti-TraySealerPLUSknowntothecommittee 8.6.1 IDEXX-QC Legionella pneumophila or equivalent.
at this time is IDEXX Laboratories, Inc. If you are aware of alternative suppliers,
please provide this information to ASTM International Headquarters. Your com-
ments will receive careful consideration at a meeting of the responsible technical
2 8
committee, which you may attend. The sole source of supply of IDEXX-QC Legionella pneumophila, UN3373-
The sole source of supply of Quanti-Tray/Legiolert supplies and reagents WQC-LP98-009287-00, known to the committee at this time is IDEXX
known to the committee at this time is IDEXX Laboratories, Inc. If you are aware Laboratories, Inc. If you are aware of alternative suppliers, please provide this
of alternative suppliers, please provide this information to ASTM International information to ASTM International Headquarters. Your comments will receive
Headquarters.Your comments will receive careful consideration at a meeting of the careful consideration at a meeting of the responsible technical committee, which
responsible technical committee, which you may attend. you may attend.
D8429 − 21
8.6.2 At least one target organisms: Legionella 11.2.1 IDEXX-QC Legionella pneumophila—Consists of
pneumophila, American Type Culture Collection (ATCC) three of each Legionella pneumophila (positive control), and
33152/World Trade Center for Microorganisms (WDCM) Enterococcus faecalis (negative control).
00107 or ATCC 33156/WDCM00180, or both. 11.2.1.1 Removevial(s)fromfreezer.Equilibrateatambient
8.6.3 At least one non-target organism: Enterococcus temperature for 15 minutes. Open a vial and aseptically
faecalis, ATCC 29212/WDCM 00087. transfer the pellet to an appropriately labeled vessel of 100 mL
of sterile, nonbuffered, oxidant-free water in a sterile vessel.
8.7 Optional Reagents:
Swirl the sample and allow to stand for 5 minutes. The pellet
8.7.1 Butterfield’s/phosphate buffer.
should completely dissolve. After it is dissolved, mix by
8.7.2 Peptone water, 0.1 %.
inverting the vessel 10 times. Use within 30 minutes of
8.7.3 Water, oxidant free, nonbuffered, sterile.
hydration.Allstepsmustbeperformedatambienttemperature.
8.8 Sterile 100 mL vessels, with or without sodium thiosul-
Review the kit’s lot activity information on the certificate of
fate.Any water containing an oxidizing agent such as chlorine
analysis to determine if a dilution of hydrated sample is
must be appropriately neutralized with sodium thiosulfate.
required.
8.9 Sterile microtubes and sterile tubes, capable of holding 11.2.2 For each of the American Type Culture Collection
(ATCC) or World Data Centre for Microorganisms (WDCM)
≥1.5 mL of sample or ≥5.0 mL of sample.
bacterial strains:
9. Precautions
11.2.2.1 Target Organisms—Legionella pneumophila,
9.1 The analyst must observe the normal good laboratory ATCC 33152/WDCM 00107 or ATCC 33156/WDCM00180,
practices and safety procedures required in a microbiology or both, streak the culture onto a BCYE plate and incubate at
laboratory while preparing, using, and disposing of cultures 35 °C 6 0.5 °C for 48 h to 72 h.
and reagents and materials and while operating sterilization or 11.2.2.2 Non-target organism—Enterococcus faecalis
other equipment. ATCC 29212/WDCM 00087; streak onto a blood agar plate
and incubate at 35 °C + 0.5 °C for 18h to 24 h.
9.2 Risk of harm is caused by the inhalation of aerosolized
11.2.2.3 For each bacterial strain inoculate a sterile vessel
L. pneumophila and it is therefore advisable to assess all
containing 100 mL of sterile diluent (DI water, phosphate/
techniques for their ability to produce aerosols (3).
Butterfield’s buffer or 0.1 % peptone water) with 10 CFU/
9.3 According to the CDC’s Biosafety in Microbiological 3
sample to 103 CFU/sample target organisms and 10 CFU/
and Biomedical Laboratories, standard bio-safety level 2
sample to 10 CFU/sample non-target organisms.
(BSL-2) practices may be performed when routinely process-
11.2.2.4 See 12.3.2.1 – 12.3.2.5 for Quanti-Tray/Legiolert
ing environmental water samples for Legionella (4). Labora-
enumeration procedure and result interpretation.
tories testing for L. pneumophila may follow the same pre-
cautions they have in place for other BSL-2 bacteria.
12. Procedure
12.1 Potable Water Sample Preparation (environmental
10. Sampling
samples only):
10.1 Collect samples as described in detail in the USEPA
12.1.1 10 mL Protocol:
Microbiological Methods Manual (5) and in accordance with
12.1.1.1 Add 90 mLof sterile diluent (DI water, phosphate/
Practices D3370.
Butterfield’s buffer, or 0.1 % peptone water) to a sterile vessel
10.2 Sample Storage, Temperature and Handling
without sodium thiosulfate.
Conditions—Iceorrefrigeratesamplesatatemperatureof2°C
12.1.1.2 Add 10 mL of water sample.
to 8 °C during transit to the laboratory. Use insulated contain-
12.1.1.3 Bring sample to ambient temperature.
erstoensurepropermaintenanceofstoragetemperatures.Take
12.1.1.4 Carefully separate one snap pack from the strip.
care that samples containers are not totally immersed in water
12.1.1.5 Tap snap pack to ensure that all of the reagent is
during transit.
towards the bottom of the pack.
12.1.1.6 Open the pack by snapping back the top of the
10.3 Handling Time Limitations—Examine samples as soon
score line. Do not touch the ope
...