ASTM D6734-01(2009)
(Test Method)Standard Test Method for Low Levels of Coliphages in Water (Withdrawn 2015)
Standard Test Method for Low Levels of Coliphages in Water (Withdrawn 2015)
SIGNIFICANCE AND USE
Coliphage organisms may serve as indicators of fecal contamination. The presence of coliphages in water in the absence of a disinfectant indicates the probable presence of fecal contamination. The absolute relationship between the number of coliforms and coliphages in natural waters has not been conclusively demonstrated. Coliphages are generally more resistant than coliforms to chlorination and may have some advantage over coliforms as an indicator of treatment efficiency in disinfected waters. The detection of coliphages in a water sample depends upon the use of a sensitive host strain in the coliphage assay. Coliphages may be detected by this concentration procedure in 6.5 h to provide important same-day information on the sanitary quality of water. The lower detection limit of this concentration procedure is 1 coliphage per volume of water sample tested.
SCOPE
1.1 This test method covers the determination of coliphages infective for E. coli C in water. The test method is simple, inexpensive, and yields an indication of water quality within 6.5 h. This coliphage method can determine coliphages in water down to 1 coliphage per volume of water sampled.
1.2 The test method is applicable to natural fresh water samples and to settled, filtered or finished water samples.
1.3 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
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.
WITHDRAWN RATIONALE
This test method covers the determination of coliphages infective for E. coli C in water.
Formerly under the jurisdiction of Committee D19 on Water, this test method was withdrawn in December 2015 in accordance with section 10.6.3 of the Regulations Governing ASTM Technical Committees, which requires that standards shall be updated by the end of the eighth year since the last approval date.
General Information
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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NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.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: D6734 − 01(Reapproved 2009)
Standard Test Method for
Low Levels of Coliphages in Water
This standard is issued under the fixed designation D6734; 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 3.2.2 coliphage, n—bacterial virus capable of plaquing on
the wide-range E. coli host strain used in this assay.
1.1 This test method covers the determination of coliphages
3.2.3 plaque, n—the circular zone of clearing (lysis) of the
infective for E. coli C in water. The test method is simple,
visible growth of bacteria on a one or two layer agar plate,
inexpensive, and yields an indication of water quality within
caused by the action of one or more bacteriophage.
6.5 h. This coliphage method can determine coliphages in
water down to 1 coliphage per volume of water sampled.
3.2.4 plaque forming unit (PFU), n—thetermusedtoreport
the number of plaques formed on an agar culture plate
1.2 The test method is applicable to natural fresh water
previously seeded with a microorganism susceptible to a
samples and to settled, filtered or finished water samples.
bacteriophage. Although theoretically, each plaque develops
1.3 The values stated in SI units are to be regarded as
from the action of a single bacteriophage, microbiologists use
standard. No other units of measurement are included in this
the term, PFU, to acknowledge that a plaque may have been
standard.
formed from the action of two or more bacteriophage in close
1.4 This standard does not purport to address all of the
proximity, which is indistinguishable from that formed by a
safety concerns, if any, associated with its use. It is the
single phage.
responsibility of the user of this standard to establish appro-
4. Summary of Test Method
priate safety and health practices and determine the applica-
bility of regulatory limitations prior to use.
4.1 AmeasuredwatersampleisadjustedtopH6.0withHCl
or NaOH and filtered through a positively-charged filter. The
2. Referenced Documents
coliphages trapped in the filter are eluted with Trypticase Soy
Broth(TSB)atpH8.5.Thetotaleluateisdividedbetweenfour
2.1 ASTM Standards:
Tubes of melted modified nutrient agar (MNA) and E. coli C
D1129Terminology Relating to Water
host culture is added to each tube. The contents of each-tube
D1193Specification for Reagent Water
are mixed and poured into a petri plate. The plates are
D3370Practices for Sampling Water from Closed Conduits
incubated at 35°C for 6 h. The coliphages present infect the
D4201Test Method for Coliphages in Water (Withdrawn
hostbacteriaandformplaques.Thetotalnumberofplaqueson
2005)
the four plates represents the number of coliphages in the
volume of water sample filtered.
3. Terminology
3.1 Fordefinitionsoftermsusedinthistestmethod,referto 5. Significance and Use
Terminology D1129.
5.1 Coliphage organisms may serve as indicators of fecal
3.2 Definitions of Terms Specific to This Standard: contamination. The presence of coliphages in water in the
3.2.1 bacterial lawn, n—confluent growth of bacteria cul-
absence of a disinfectant indicates the probable presence of
tured on an agar plate. fecal contamination. The absolute relationship between the
number of coliforms and coliphages in natural waters has not
been conclusively demonstrated. Coliphages are generally
1 more resistant than coliforms to chlorination and may have
This test method is under the jurisdiction ofASTM Committee D19 on Water
and is the direct responsibility of Subcommittee D19.24 on Water Microbiology.
some advantage over coliforms as an indicator of treatment
Current edition approved April 1, 2009. Published April 2009. Originally
efficiency in disinfected waters.The detection of coliphages in
approved in 2001. Last previous edition approved in 2001 as D6734–01. DOI:
a water sample depends upon the use of a sensitive host strain
10.1520/D6734-01R09.
in the coliphage assay. Coliphages may be detected by this
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
concentration procedure in 6.5 h to provide important same-
Standards volume information, refer to the standard’s Document Summary page on
day information on the sanitary quality of water. The lower
the ASTM website.
detection limit of this concentration procedure is 1 coliphage
The last approved version of this historical standard is referenced on
www.astm.org. per volume of water sample tested.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
D6734 − 01 (2009)
6. Interferences teeonAnalyticalReagentsoftheAmericanChemicalSociety.
Other grades may be used, provided it is first ascertained that
6.1 Highsaltconcentrations,suchasthesefoundinsalineor
the reagent is of sufficiently high purity to permit its use
brackish water, interfere with this test method.
without decreasing the accuracy of the determination.
6.2 Water sample turbidity in excess of 25 NTU (nepholo-
8.2 Purity of Water—Unless otherwise indicated, references
metric turbidity units using Ratio Turbidimeter) results in
towatershallbeunderstoodtomeanreagentwaterconforming
decreased plaque formation because bacterial viruses are
to Specification D1193, Type II.
trapped with the particulate matter in the Zesa Plus filter and
are not completely eluted by TSB at pH 8.5.
8.3 Host Culture— Escherichia coli C, ATCC No. 13706.
6.3 Analysis for coliphage can be performed on settled and 8.4 Trytpicase Soy Agar (TSA)
wastewaters filtered waters, disinfected waters or wastewaters;
8.4.1 Composition per Litre:
however, the relationship between coliphage and coliform
Pancreatic Digest of Casein 15.0 g
bacteria will be different from that observed in natural fresh
Papaic Digest of Soybean Meal 5.0 g
Sodium Chloride 5.0 g
waters. Coliphage are less efficiently removed by settling and
Agar 15.0 g
filtration than coliforms, and coliphage are generally more
Final pH 7.3 ± 0.2
resistant than coliforms to chlorination.
8.4.2 Preparation—Add 40 g or the dehydrated medium to
1 L of water and mix well. Heat while stirring on a hot plate.
7. Apparatus
Boilfor1minoruntilcompletelydissolved.Dispense8-10mL
7.1 Water Bath, 46 6 1°C.
quantitiesintoscrew-capculturetubes.Autoclavefor15minat
121°C (15 lbs pressure). Remove from autoclave while still
7.2 Incubator, 35 6 0.5°C.
molten and incline tubes at appropriate angle for slants. Let
7.3 Petri Plates, glass or plastic, sterile, 100 × 15 mm.
cool to harden.
7.4 Pipets, sterile T.D. bacteriological or Mohr, glass or
8.5 Trypticase (Tryptic) Soy Broth (TSB) and Glycerol
plastic, 1 and 5 mL.
8.5.1 Composition per Litre:
7.5 Test Tubes, with airtight caps or screw caps, 16 × 125
Pancreatic Digest of Casein 17.0 g
Papaic Digest of Soybean Meal 3.0 g
mm and 25 × 150 mm.
Sodium Chloride (NaCl) 5.0 g
Dipotassium Phosphate (K PO ) 2.5 g
2 4
7.6 Platinum Transfer Loop, 3 mm loop.
Dextrose 2.5 g
7.7 Sterile Vials, 12 × 75 mm with crimp or screw caps.
8.5.2 Preparation—Add30gofthedehydratedmediumand
100 mL of glycerol to 900 mL of water. Mix well and heat
7.8 Spectrophotometer, suitable for absorbance measure-
gently to dissolve in a hot water bath. Dispense 5 mLvolumes
ments at 520 nm.
into 16 mm screw-cap test tubes and 50 mL volumes into 125
7.9 Freezer, with manual defrost.
mLErlenmyerflasks.Autoclavefor15minat121°C.FinalpH
7.3 6 0.2.
7.10 Filters, Zeta Plus 60S positively charged 47 mm.
8.6 pH Adjusted Tryptic Soy Broth
7.11 Membrane Filtration Units, (filter base and funnel),
reusable glass, plastic or stainless steel units wrapped with
8.6.1 Preparation—Add 30 g of dehydrated Tryptic Soy
aluminum foil or kraft paper and sterilized, or disposable,
Broth to 1 Lof water. Mix well and heat gently in a hot water
sterile, plastic units.
bathtodissolve.AddinNaOHdrop-wisetoraisethepHto8.5.
Dispense in 200 mL volumes in 250 mL screw-cap flasks and
7.12 Vacuum Pump, capable of creating 15 psi pressure for
autoclave for 30 min at 121°C.
filtration of wager.
8.7 Modified Nutrient Agar (MNA) :
7.13 Vacuum Flasks, sterile 1 L.
8.7.1 Composition per Litre:
7.14 Turbidimeter, Hach ratio turbidimeter or equivalent.
8. Reagents and Materials
Reagent Chemicals, American Chemical Society Specifications, American
Chemical Society, Washington, DC. For Suggestions on the testing of reagents not
8.1 Purity of Reagents—Reagent grade chemicals shall be
listed by the American Chemical Society, see Annual Standards for Laboratory
used in all tests. Unless otherwise indicated, it is intended that
Chemicals, BDH Ltd., Poole, Dorset, U.K., and the United States Pharmacopeia
all reagents shall conform to the specifications of the commit-
and National Formulary, U.S. Pharmacopeial Convention, Inc. (USPC), Rockville,
MD.
The sole source of supply of the material known to the committee at this time
is American Type Culture Collection, Rockville, MD 20854. If you are aware of
The sole source of supply of the apparatus known to the committee at this time alternative suppliers, please provide this information to ASTM Internat
...
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