ASTM D6734-01
(Test Method)Standard Test Method for Low Levels of Coliphages in Water
Standard Test Method for Low Levels of Coliphages in Water
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 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.
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Standards Content (Sample)
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Designation:D6734–01
Standard Test Method for
Low Levels of Coliphages in Water
This standard is issued under the fixed designation D 6734; 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 from the action of a single bacteriophage, microbiologists use
the term, PFU, to acknowledge that a plaque may have been
1.1 This test method covers the determination of coliphages
formed from the action of two or more bacteriophage in close
infective for E. coli C in water. The test method is simple,
proximity, which is indistinguishable from that formed by a
inexpensive, and yields an indication of water quality within
single phage.
6.5 h. This coliphage method can determine coliphages in
water down to 1 coliphage per volume of water sampled.
4. Summary of Test Method
1.2 The test method is applicable to natural fresh water
4.1 AmeasuredwatersampleisadjustedtopH6.0withHCl
samples and to settled, filtered or finished water samples.
or NaOH and filtered through a positively-charged filter. The
1.3 This standard does not purport to address all of the
coliphages trapped in the filter are eluted with Trypticase Soy
safety concerns, if any, associated with its use. It is the
Broth(TSB)atpH8.5.Thetotaleluateisdividedbetweenfour
responsibility of the user of this standard to establish appro-
Tubes of melted modified nutrient agar (MNA) and E. coli C
priate safety and health practices and determine the applica-
host culture is added to each tube. The contents of each-tube
bility of regulatory limitations prior to use.
are mixed and poured into a petri plate. The plates are
2. Referenced Documents incubated at 35°C for 6 h. The coliphages present infect the
host bacteria and form plaques.The total number of plaques on
2.1 ASTM Standards:
the four plates represents the number of coliphages in the
D 1129 Terminology Relating to Water
volume of water sample filtered.
D 1193 Specification for Reagent Water
D 3370 Practices for Sampling Water
5. Significance and Use
D 4201 Test Method for Coliphages in Water
5.1 Coliphage organisms may serve as indicators of fecal
3. Terminology contamination. The presence of coliphages in water in the
absence of a disinfectant indicates the probable presence of
3.1 For definitions of terms used in this test method, refer to
fecal contamination. The absolute relationship between the
Terminology D 1129.
number of coliforms and coliphages in natural waters has not
3.2 Description of Terms Specific to This Standard
been conclusively demonstrated. Coliphages are generally
3.2.1 bacterial lawn, n—confluent growth of bacteria cul-
more resistant than coliforms to chlorination and may have
tured on an agar plate.
some advantage over coliforms as an indicator of treatment
3.2.2 coliphage, n—bacterial virus capable of plaquing on
efficiency in disinfected waters. The detection of coliphages in
the wide-range E. coli host strain used in this assay.
a water sample depends upon the use of a sensitive host strain
3.2.3 plaque, n—the circular zone of clearing (lysis) of the
in the coliphage assay. Coliphages may be detected by this
visible growth of bacteria on a one or two layer agar plate,
concentration procedure in 6.5 h to provide important same-
caused by the action of one or more bacteriophage.
day information on the sanitary quality of water. The lower
3.2.4 plaque forming unit (PFU), n—thetermusedtoreport
detection limit of this concentration procedure is 1 coliphage
the number of plaques formed on an agar culture plate
per volume of water sample tested.
previously seeded with a microorganism susceptible to a
bacteriophage. Although theoretically, each plaque develops
6. Interferences
6.1 Highsaltconcentrations,suchasthesefoundinsalineor
This test method is under the jurisdiction of ASTM Committee D19 on Water
brackish water, interfere with this test method.
and is the direct responsibility of Subcommittee D19.24 on Water Microbiology.
6.2 Water sample turbidity in excess of 25 NTU (nepholo-
Current edition approved Nov. 10, 2001. Published January 2002.
metric turbidity units using Ratio Turbidimeter) results in
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 decreased plaque formation because bacterial viruses are
Standards volume information, refer to the standard’s Document Summary page on
the ASTM website.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.
D6734–01
trapped with the particulate matter in the Zesa Plus filter and
Agar 15.0 g
Final pH 7.3 6 0.2
are not completely eluted by TSB at pH 8.5.
6.3 Analysis for coliphage can be performed on settled and
8.4.2 Preparation—Add 40 g or the dehydrated medium to
wastewaters filtered waters, disinfected waters or wastewaters;
1 L of water and mix well. Heat while stirring on a hot plate.
however, the relationship between coliphage and coliform
Boilfor1minoruntilcompletelydissolved.Dispense8-10mL
bacteria will be different from that observed in natural fresh
quantitiesintoscrew-capculturetubes.Autoclavefor15minat
waters. Coliphage are less efficiently removed by settling and
121°C (15 lbs pressure). Remove from autoclave while still
filtration than coliforms, and coliphage are generally more
molten and incline tubes at appropriate angle for slants. Let
resistant than coliforms to chlorination.
cool to harden.
8.5 Trypticase (Tryptic) Soy Broth (TSB) and Glycerol
7. Apparatus
8.5.1 Composition per Litre:
7.1 Water Bath,46 6 1°C.
Pancreatic Digest of Casein 17.0 g
7.2 Incubator,35 6 0.5°C. Papaic Digest of Soybean Meal 3.0 g
Sodium Chloride (NaCl) 5.0 g
7.3 Petri Plates, glass or plastic, sterile, 100 3 15 mm.
Dipotassium Phosphate (K PO ) 2.5 g
2 4
7.4 Pipets, sterile T.D. bacteriological or Mohr, glass or
Dextrose 2.5 g
plastic, 1 and 5 mL.
8.5.2 Preparation—Add 30 g of the dehydrated medium
7.5 Test Tubes, with airtight caps or screw caps, 16 3 125
and 100 mLof glycerol to 900 mLof water. Mix well and heat
mm and 25 3 150 mm.
gently to dissolve in a hot water bath. Dispense 5 mL volumes
7.6 Platinum Transfer Loop, 3 mm loop.
into 16 mm screw-cap test tubes and 50 mL volumes into 125
7.7 Sterile Vials,12 3 75 mm with crimp or screw caps.
mLErlenmyer flasks.Autoclave for 15 min at 121°C. Final pH
7.8 Spectrophotometer, suitable for absorbance measure-
7.3 6 0.2.
ments at 520 nm.
8.6 pH Adjusted Tryptic Soy Broth
7.9 Freezer, with manual defrost.
3 8.6.1 Preparation—Add 30 g of dehydrated Tryptic Soy
7.10 Filters, Zeta Plus 60S positively charged 47 mm.
Broth to 1 L of water. Mix well and heat gently in a hot water
7.11 Membrane Filtration Units, (filter base and funnel),
bathtodissolve.AddinNaOHdrop-wisetoraisethepHto8.5.
reusable glass, plastic or stainless steel units wrapped with
Dispense in 200 mL volumes in 250 mL screw-cap flasks and
aluminum foil or kraft paper and sterilized, or disposable,
autoclave for 30 min at 121°C.
sterile, plastic units.
8.7 Modified Nutrient Agar (MNA):
7.12 Vacuum Pump, capable of creating 15 psi pressure for
8.7.1 Composition per Litre:
filtration of wager.
Nutrient Agar 23.0 g
7.13 Vacuum Flasks, sterile 1 L.
Nutrient broth 8.0 g
7.14 Turbidimeter, Hach ratio turbidimeter or equivalent.
Strontium Nitrate, Sr (NO)0.23g
Ammonium Nitrate, NH NO 1.76 g
4 3
8. Reagents and Materials Sodium Chloride, NaCl 5.0 g
8.1 Purity of Reagents—Reagent grade chemicals shall be
8.7.2 Preparation—Add the ingredients to 1 Lof water and
used in all tests. Unless otherwise indicated, it is intended that
mixwell.Heatinboilingwaterbathuntildissolvedcompletely.
all reagents shall conform to the specifications of the commit-
Dispense5.5mLvolumesinto16 3125mmscrew-capculture
tee onAnalytical Reagents of theAmerican Chemical Society.
tubes and autoclave for 15 min at 121°C.
Other grades may be used, provided it is first ascertained that
the reagent is of sufficiently high purity to permit its use
9. Sampling
without decreasing the accuracy of the determination.
9.1 Collect 1 L water samples in accordance with Practice
8.2 Purity of Water—Unless otherwise indicated, references
D 3370.
to water shall be understood to mean reagent water conforming
to Specification D 1193, Type II.
10. Procedure
8.3 Host Culture—Escherichia coli C, ATCC No. 13706.
10.1 Frozen Host Preparation:
8.4 Trytpicase Soy Agar (TSA)
10.1.1 Inoculate5mLsterileTSBina16 3125mmculture
8.4.1 Composition per Litre:
tube with the E. coli C host culture from an agar slant or ag
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