An American National Standard

Designation: D 3863 – 87 (Reapproved 2003)

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 1
This standard is issued under the fixed designation D 3863; 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 3.2.2 vacuum—for the procedure used, a source of suction

1.1 This test method covers a procedure to test membrane that can produce a reading of 500 to 600 mm Hg on a vacuum
filters for their ability to retain bacteria whose diameter is equal gage.
to or slightly larger than membrane filters with pore size rated 4. Summary of Test Method
at 0.40 to 0.45 µm.
1.2 The procedures described are for the use of user 4.1 This test method is based on the cultivation of organ-
laboratories as differentiated from manufacturers’ laboratories. isms whose diameters are equal to or slightly larger than pores
1.3 This standard does not purport to address all of the of the membrane filter to be tested and then filtering a specific
safety concerns, if any, associated with its use. It is the aliquot containing organisms through the membrane followed
responsibility of the user of this standard to establish appro- by an examination of the filtrate after incubation for sterility. A
priate safety and health practices and determine the applica- sterile filtrate indicates complete retention of the organism and
bility of regulatory limitations prior to use. validates the ability of the membrane to retain bacteria equal to
or slightly larger than the stated pore size.
2. Referenced Documents
5. Significance and Use
2.1 ASTM Standards: 2
D 1129 Terminology Relating to Water 5.1 Microbiological water testing procedures using mem-
D 1193 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 method refer to could have serious repercussions due to the presence of
Terminology D 1129. unrecognized potential health hazards in the water being tested,
3.2 Definitions of Terms Specific to This Standard: especially in drinking water.
3.2.1 Gram’s stain—a routine bacterial stain that divides 5.2 This procedure as devised will enable the user to test
bacteria into two categories, depending on whether they can be each membrane filter lot number for its ability to retain all
decolorized with acetone, alcohol, or aniline oil after staining bacteria equal to, or larger than, the stated membrane pore size.
with one of the rosaniline dyes such as crystal violet, methyl
6. Apparatus
violet, or gentian violet and treating with iodine. Those that
resist decolorization remain blue or violet and are designated 6.1 Membrane Filtration Units, six.
Gram-positive; those that are decolorized and take up the red 6.2 Vacuum Source with trap vessel.
counterstain, such as neutral red, safranin, or dilute carbol 6.3 Filtering Flasks, 1-L, with vacuum tubing into which a
fuchsin are termed Gram-negative. glass tube and a Y-tube have been incorporated as in Fig. 1. The
free end of the Y-tube is connected by tubing to a sterile
bacterial air vent. The tubing to air vent is clamped shut during
1
filtration and released after filtration.
This test method is under the jurisdiction of ASTM Committee D19 on Water
and is the direct responsibility of Subcommittee D19.08 on Membranes and Ion 6.4 Forceps, blunt-nosed, and small beaker of 95 % ethanol.
Exchange Materials. 6.5 Incubator, 20 to 25°C.
Current edition approved March 27, 1987. Published July 1987.
2
6.6 Pinch-Cock Clamps.
For referenced ASTM standards, visit the ASTM website, [Link], or
contact ASTM Customer Service at service@[Link]. For Annual Book of ASTM
6.7 Autoclave or Other Sterilizing Equipment.
Standards volume information, refer to the standard’s Document Summary page on 6.8 Appropriate Equipment for producing reagent grade
the ASTM website. water.

Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.

1
 D 3863 – 87 (2003)

FIG. 1 Apparatus Required for Testing Retention Characteristics of Membrane Filters

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 Broth Cultures of Serratia marcescens, 18 6 2 h. changeable and henceforth referred to as agar medium, formu-
6.11.6 Sterile Membrane Filters—Test membranes. lated, prepared, and dispensed in accordance with the manu-
6.11.7 Petri Dishes, 50-mm, containing 6 to 8-mL of agar. facturers’ specifications.
7.7 Tryptone Soya and Tryptic Soy Broth—are interchange-
7. Reagents and Materials able and henceforth referred to as broth medium, formulated,
7.1 Purity of Reagents—Reagent grade chemicals shall be prepared, and dispensed in accordance with the manufacturers’
used in all tests. Unless otherwise indicated, it is intended that specifications.
all reagents shall conform to the specifications of the Commit-
tee on Analytical Reagents of the American Chemical Society, 8. Procedure
where such specifications are available.3 Other grades may be 8.1 Place 140 mL of double-strength broth into six 1-L
used, provided it is first ascertained that the reagent is of vacuum flasks with the attached vacuum tubing, Y-tube, and
sufficiently high purity to permit its use without lessening the bacterial air vent. Wrap in kraft paper and sterilize by auto-
accuracy of the determination. claving at 121°C for 15 min.
7.2 Purity of Water— Unless otherwise indicated, reference 8.2 Aseptically assemble membrane filtration apparatus
to water shall be understood to mean reagent water conforming onto each flask, connect to the vacuum source, and aseptically
to Specification D 1193, Type II, with 0.2-µm membrane place the test membrane filter into the a filter holder and secure.
filtration. In addition, suitability tests for determining the Place the clamp on tubing between the Y-tube and sterile
bactericidal properties of the reagent grade water should be bacterial air vent.
performed. 8.3 Pour the culture suspension (100 mL) into a filter funnel
7.3 Bacterial Suspension—Add 1 mL of an 18 6 2-h broth and turn on the vacuum.
culture of Serratia marcescens to 99 mL of 0.1 % peptone 8.4 After the suspension has been filtered, wash down the
water, to obtain a working concentration of approximately 103 sides of the funnel with two 20-mL peptone water rinses and
to 104 organisms per millilitre. Prepare five bottles. Incubator immediately turn off the vacuum when the last drop of peptone
temperature of suspension is 25°C. water has been filtered. Release the clamp between Y-tube and
7.4 Peptone Water (0.1 %)—Prepare a 10 % stock solution bacterial air vent to allow air in the flask to equilibrate. After
of peptone in water. Dilute a measured volume of the 10 % equilibration has taken place, place the clamp on the vacuum
tubing in front of the glass tube at point ( 1) and remove the
glass tube and the rest of vacuum tubing.
3
Reagent Chemicals, American Chemical Society Specification, American 8.5 Repeat the process using sterile equipment and sterile
Chemical Society, Washington, DC. For suggestions on the testing of reagents not 0.1 % peptone as a negative control inoculum.
listed by the American Chemical Society, see Analar Standards for Laboratory
8.6 Aseptically remove the membrane filtration apparatus
Chemicals, BDH Ltd., Poole, Dorset, UK, and the United States Pharmacopeia and
National Formulary, U.S. Pharmaceutical Convention, Inc. (USPC), Rockville, and place the sterile stopper in the flask and then incubate the
MD. stoppered flask for 48 h at 25°C.

2
 D 3863 – 87 (2003)
8.7 Using flamed forceps, aseptically remove the membrane 8.10 Test a minimum of five randomly selected membranes
from the membrane-filter holder and place a filter on agar from five randomly selected packages. Take the control mem-
(50-mm petri dish) for 48 h at 25°C and check the membranes brane from the same package as one of the test membranes.
for growth outside of filtering area. Growth outside of the
filtering area will indicate a faulty filtering apparatus and may 9. Precision and Bias
result in a false positive test.
8.8 Note any signs of turbidity in the liquid medium as an 9.1 Since this is a positive or negative test, precision and
indication of growth and thus failure of the membrane to retain bias statements are not applicable to this procedure.
bacteria larger than 0.45 µm. Confirm turbid broths by streak-
ing to an agar plate and check for strain purity. Apply Gram’s 10. Keywords
stain test and perform biochemical tests if in doubt. 10.1 filter; membrane; microbiological; water quality
8.9 Examine control broth culture after 48 h. If any sign of
turbidity occurs, this indicates technique failure and the test
and control procedures should be repeated.

ANNEX

(Mandatory Information)

A1. INTERPRETATION OF TEST RESULTS

A1.1 Failures in Retention of Test Organism—The appear- retention should be examined prior to its use. Membrane
ance of test organisms downstream of the test membrane filter filtration units that employ a positive seal such as those fitted
in a retention test may be due to one or more of three possible with a sealing O-ring are ideal for this application. Once
factors: ( 1) inherent failure of the membrane, (2) failure of the equipment is qualified to ensure proper sealing, it may then be
membrane filtration unit to form a proper seal, or damage to the reserved exclusively for membrane-retention testing.
membrane from improper handling during the test. Before
concluding that a membrane filter is inherently faulty, items (2) A1.3 Damage Due to Improper Handling—Exercise care at
and (3) must be considered as possible causes of an apparent all times when manipulating dry membranes. While handling
failure. with smooth-tip filter forceps, examine membranes visually
A1.2 Membrane Filtration Apparatus—The performance of prior to testing. Discard any filters showing visible flaws such
equipment used to evaluate membrane filters for bacterial as cracked edges or holes.

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