ASTM D3863-22

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: D3863 − 22
Standard Test Method for
Retention Characteristics of 0.40 to 0.45 μm Membrane
Filters Used in Routine Filtration Procedures for the
Evaluation of Microbiological Water Quality
This standard is issued under the fixed designation D3863; 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 3.2 Definitions of Terms Specific to This Standard:
3.2.1 Gram’s stain—a routine bacterial stain that divides
1.1 This test method covers a procedure to test membrane
bacteria into two categories, depending on whether they can be
filters for their ability to retain bacteria whose diameter is equal
decolorized with acetone, alcohol, or aniline oil after staining
to or slightly larger than membrane filters with pore size rated
with one of the rosaniline dyes such as crystal violet, methyl
at 0.40 to 0.45 μm.
violet, or gentian violet and treating with iodine. Those that
1.2 The procedures described are for the use of user
resist decolorization remain blue or violet and are designated
laboratories as differentiated from manufacturers’ laboratories.
Gram-positive; those that are decolorized and take up the red
1.3 The values stated in SI units are to be regarded as counterstain, such as neutral red, safranin, or dilute carbol
fuchsin are termed Gram-negative.
standard. No other units of measurement are included in this
standard.
3.2.2 vacuum—for the procedure used, a source of suction
that can produce a reading of 500 to 600 mm Hg on a vacuum
1.4 This standard does not purport to address all of the
safety concerns, if any, associated with its use. It is the gage.
responsibility of the user of this standard to establish appro-
4. Summary of Test Method
priate safety, health, and environmental practices and deter-
mine the applicability of regulatory limitations prior to use.
4.1 This test method is based on the cultivation of organ-
1.5 This international standard was developed in accor-
isms whose diameters are equal to or slightly larger than pores
dance with internationally recognized principles on standard-
of the membrane filter to be tested and then filtering a specific
ization established in the Decision on Principles for the
aliquot containing organisms through the membrane followed
Development of International Standards, Guides and Recom-
by an examination of the filtrate after incubation for sterility. A
mendations issued by the World Trade Organization Technical
sterile filtrate indicates complete retention of the organism and
Barriers to Trade (TBT) Committee.
validates the ability of the membrane to retain bacteria equal to
or slightly larger than the stated pore size.
2. Referenced Documents
2.1 ASTM Standards: 5. Significance and Use
D1129 Terminology Relating to Water
5.1 Microbiological water testing procedures using mem-
D1193 Specification for Reagent Water
brane filtration are based on the premise that all bacteria within
a specific size range will be retained by the membrane filter
3. Terminology
used. If the membrane filter does not retain these bacteria, false
3.1 Definitions:
negative results or lowered density estimates may occur that
3.1.1 For definitions of terms used in this standard, refer to
could have serious repercussions due to the presence of
Terminology D1129.
unrecognized potential health hazards in the water being tested,
especially in drinking water.
This test method is under the jurisdiction of ASTM Committee D19 on Water
5.2 This procedure as devised will enable the user to test
and is the direct responsibility of Subcommittee D19.24 on Water Microbiology.
each membrane filter lot number for its ability to retain all
Current edition approved Jan. 1, 2022. Published January 2022. Originally
approved in 1987. Last previous edition approved in 2011 as D3863 – 87 (2011)
bacteria equal to, or larger than, the stated membrane pore size.
which was withdrawn July 2020 and reinstated in January 2022. DOI: 10.1520/
D3863-22.
6. Apparatus
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
6.1 Membrane Filtration Units, six.
Standards volume information, refer to the standard’s Document Summary page on
the ASTM website. 6.2 Vacuum Source with trap vessel.
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D3863 − 22
6.3 Filtering Flasks, 1 L, with vacuum tubing into which a 7.2 Purity of Water—Unless otherwise indicated, reference
glass tube and a Y-tube have been incorporated as in Fig. 1. The to water shall be understood to mean reagent water conforming
free end of the Y-tube is connected by tubing to a sterile to Specification D1193, Type II, with 0.2 μm membrane
bacterial air vent. The tubing to air vent is clamped shut during filtration. In addition, suitability tests for determining the
filtration and released after filtration. bactericidal properties of the reagent grade water should be
performed.
6.4 Forceps, blunt-nosed, and small beaker of 95 % ethanol.
7.3 Bacterial Suspension—Add 1 mL of an 18 6 2 h broth
6.5 Incubator, 20 to 25 °C.
culture of Serratia marcescens to 99 mL of 0.1 % peptone
6.6 Pinch-Cock Clamps.
water, to obtain a working concentration of approximately 10
6.7 Autoclave or Other Sterilizing Equipment. to 10 organisms per millilitre. Prepare five bottles. Incubator
temperature of suspension is 25 °C.
6.8 Appropriate Equipment for producing reagent grade
water. 7.4 Peptone Water (0.1 %)—Prepare a 10 % stock solution
of peptone in water. Dilute a measured volume of the 10 %
6.9 Appropriate Laboratory Glassware.
stock solution to obtain final solution of 0.1 % peptone in
6.10 Sterile Rubber Stoppers, to fit 1 L filtering flask.
required amount. Sterilize at 121 °C for 15 min.
6.11 Expendables:
7.5 Test Organism—Serratia marcescens ATCC strain
6.11.1 Double-Strength Broth, 140 mL aliquots.
14756, also called FDA strain PC1 1107. Red pigmented
6.11.2 Sterile Pipets, 1 and 10 mL.
Serratia marcescens strains other than the above may also be
6.11.3 Sterile 0.1 % Peptone in 99 mL quantities.
used.
6.11.4 Sterile 0.1 % Peptone as rinse water.
7.6 Tryptic Soy Agar and Tryptone Soya Agar—are inter-
6.11.5 Br
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