ASTM F1095-88(1994)E01

Test Method for Rapid Enumeration of Bacteria in Electronics-Grade Purified Water Systems by Direct-Count Epifluorescence Microscopy (Withdrawn 2001)

SCOPE
1.1 This test method describes a procedure for counting total numbers of bacteria present in electronics-grade purified water systems. An on-line, large volume sampling technique is used with acridine orange epifluorescence microscopy. This procedure is applicable to industrial and environmental water systems with small amounts of detrital material.  
1.2 The analytical sensitivity associated with this method is dependent upon the sample volume. Sensitivities of ∧lt;20 cells/100 mL can be obtained on a routine basis (1).  A sample size of 5 L or more is required to obtain this sensitivity.  
1.3 The procedure is relatively rapid to perform: total bacteria counts can be obtained within 60 min from the start of sampling.  
1.4 Viable, nonviable, and nonculturable bacteria are stained by the acridine orange dye. This procedure does not, however, differentiate between these three groups of bacteria.  
1.5 Detrital material, algae, and fungi may also fluoresce when stained with the acridine orange.  
1.6 Performance of this procedure requires experience in light microscopy. The microscopist must be capable of distinguishing bacterial cells from other, fluorescing particulate matter. Refer to Mittelman et al. (1) for color photographs on cell morphology.  
1.7 This procedure is adaptable for use with image analysis equipment (2). The use of image analysis equipment will increase the rate at which the filter can be examined.  
1.8 The values stated in SI units are to be regarded as the standard. The values given in parentheses are for information only.  
1.9 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.

General Information

• Status: Withdrawn
• Publication Date: 16-Feb-1988
• Withdrawal Date: 09-Jun-2001
• Technical Committee: F01 - Electronics
• Drafting Committee: F01.10 - Contamination Control

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NOTICE: This standard has either been superseded and replaced by a new version or
withdrawn.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.
e1
Designation: F 1095 – 88 (Reapproved 1994)
AMERICAN SOCIETY FOR TESTING AND MATERIALS
100 Barr Harbor Dr., West Conshohocken, PA 19428
Reprinted from the Annual Book of ASTM Standards. Copyright ASTM
Standard Test Method
Rapid Enumeration of Bacteria in Electronics-Grade Purified
Water Systems by Direct-Count Epifluorescence
Microscopy
This standard is issued under the fixed designation F 1095; 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.
e NOTE—Editorial changes were made throughout in September 1994.
1. Scope bility of regulatory limitations prior to use.
1.1 This test method describes a procedure for counting
2. Referenced Documents
total numbers of bacteria present in electronics-grade purified
2.1 ASTM Standards:
water systems. An on-line, large volume sampling technique is 3
D 1193 Specifications For Reagent Water
used with acridine orange epifluorescence microscopy. This
F 312 Methods for Microscopical Sizing and Counting
procedure is applicable to industrial and environmental water 4
Particles From Aerospace Fluids on Membrane Filters
systems with small amounts of detrital material.
3. Terminology
1.2 The analytical sensitivity associated with this method is
dependent upon the sample volume. Sensitivities of <20 3.1 Definitions:
3.1.1 colony forming unit—One or more bacteria, that when
cells/100 mL can be obtained on a routine basis (1). A sample
size of 5 L or more is required to obtain this sensitivity. cultured, form one colony on a membrane or agar surface.
3.1.2 detritus—organic debris from microorganisms; this
1.3 The procedure is relatively rapid to perform: total
includes but is not limited to cell wall fragments and extracel-
bacteria counts can be obtained within 60 min from the start of
lular proteins and polysaccharides.
sampling.
3.1.3 nonculturable bacteria—bacteria that cannot be
1.4 Viable, nonviable, and nonculturable bacteria are
grown or cultured under laboratory conditions.
stained by the acridine orange dye. This procedure does not,
3.1.4 nonviable bacteria—bacteria that are not living.
however, differentiate between these three groups of bacteria.
3.1.5 planktonic bacteria—bacteria that are free floating in
1.5 Detrital material, algae, and fungi may also fluoresce
a water system.
when stained with the acridine orange.
3.1.6 sessile bacteria—bacteria that are attached to the
1.6 Performance of this procedure requires experience in
walls of pipes, housings and other surfaces in water systems.
light microscopy. The microscopist must be capable of distin-
3.1.7 viable bacteria—bacteria that are living.
guishing bacterial cells from other, fluorescing particulate
matter. Refer to Mittelman et al. (1) for color photographs on
4. Significance and Use
cell morphology.
4.1 Bacterial contamination of electronics-grade purified
1.7 This procedure is adaptable for use with image analysis
water systems presents a serious threat to systems operations
equipment (2). The use of image analysis equipment will
and end product quality (3). Timely information concerning
increase the rate at which the filter can be examined.
bacterial numbers present in purified waters is essential to
1.8 The values stated in SI units are to be regarded as the
effective preventative maintenance and treatment programs.
standard. The values given in parentheses are for information
The currently employed cultural method (4) requires a 48 h
only.
incubation period for testing. In addition, the cultural method
1.9 This standard does not purport to address all of the
only detects viable bacteria that are capable of growth and
safety concerns, if any, associated with its use. It is the
subsequent colony forming unit production under the prevail-
responsibility of the user of this standard to establish appro-
ing cultural conditions. Nonviable bacteria and nonculturable
priate safety and health practices and determine the applica-
bacteria are thus not counted when the culture method is used.
4.2 Direct count epifluorescence microscopy is a rapid,
approximately one h total test time, and accurate, with resolu-
This test method is under the jurisdiction of ASTM Committee F-1 on
tion to a single cell, per microscopic viewing field technique
Electronics and is the direct responsibility of Subcommittee F01.10 on Contamina-
tion Control.
Current edition approved Feb. 17, 1988. Published April 1988.
2 3
The boldface numbers in parentheses refer to the list of references at the end of Annual Book of ASTM Standards, Vol 11.01.
this standard. Annual Book of ASTM Standards, Vol 14.02.
F 1095
for obtaining total bacterial counts in purified water systems. 6.5 Support Membranes, 5 μm, 25 mm, cellulose ester.
6.6 Vacuum Apparatus, vacuum pump, filtering flask/trap,
4.3 Both planktonic and sessile bacterial cells may be
and related tubing.
enumerated using this method (5).
6.7 Sample Collection Carboy, graduated to 10 L, with
4.4 The relatively low detrital content of purified waters
collection hoses.
results in few interferences with this technique.
6.8 Sonicator Bath, 150 by 300 by 100 mm (6 by 12 by 4
4.5 This procedure does not allow differentiation of viable
in.), 110 V, 125 W.
bacteria from non-viable bacteria.
6.9 Miscellaneous Supplies, flat tipped forceps, microscope
4.6 There is no necessary correlation between results ob-
slides, coverslips, pipets.
tained using the direct-count epifluorescence microscopy pro-
7. Reagents and Materials
cedure and the currently employed viable-count technique.
This is due both to variations in the ratio of viable to nonviable
7.1 Purity of Reagents—Reagent-grade chemicals shall be
cells as well as differences in the recovery of viable bacteria on
used in all tests. Unless otherwise indicated, it is intended that
culture media. Previous data (1, 6, 7) suggest that the method
all reagents conform to the specifications of the Committee on
described herein provides one to several orders of magnitude of
Analytical Reagents of the American Chemical Society, when
higher bacterial recoveries than does the viable-count method.
such specifications are available.
7.2 Purity of water—Unless otherwise indicated, references
5. Interferences
to water shall be understood to mean reagent water as defined
by Type 1A of Specification D 1193.
5.1 The acridine orange stain has an affinity for nucleic acid,
7.3 Irgalan Black.
and will stain microbial cells, living or dead. It will also stain
7.4 Acridine Orange Stain—N,N,N8N8-Tetramethyl-3,6-
some particulate material. The presence of non-cellular organic
acridineamine monohydrochloride.
material that can be stained will mask the presence of cells, and
7.5 Predarkened Polycarbonate Membrane Filters, 0.2
lead to erroneous results. Detrital levels are usually not at
μm, 25 mm. Plain polycarbonate membrane filters may also
levels that will cause errors in ultrapure water systems.
be darkened in the laboratory as described in Section 7.
5.2 Membrane filters that autofluoresce or bind the stain will
Polycarbonate filters are surface capture membranes and are
have too bright a background, and bacteria will not be seen.
recommended. Membranes cast from other polymers may not
5.3 Membrane filters that do not capture particles and cells
capture all the bacterial cells on the membrane surface.
on the surface will hide bacterial cells and particulates below
Membrane filters made from polymers other than darkened
the viewing field.
polycarbonate may exhibit either autofluorescence or binding
5.4 The acridine orange stain will lose the ability to fluo-
of the fluorescent dye to give unacceptably high background
resce with prolonged exposure to ultraviolet light. The stain
lighting. Bacterial cells in the membrane depth or on a highly
concentration in this procedure will allow sufficient exposure
fluorescing background result in reduced sensitivity.
time for normal counting. If the stained and mounted mem- 13
7.6 Immersion Oil, low fluorescing. (Cargille Type B or
brane is exposed to ultraviolet light for periods greater than
equivalent).
three to four min per effective viewing area, there is the
7.7 Isopropanol or Ethanol, 70 % volume per volume (v/v)
probability that the bacteria will not fluoresce brightly enough
in reagent water.
to be seen. Do not expose the stained and mounted membrane
7.8 Anionic Laboratory Detergent.
to ultraviolet light when not counting.
8. Preparation of Apparatus
5.5 Counting too few fields of view, or an inconsistent
8.1 Preclean the stainless-steel filter holder in an anionic
number of fields of view between samples will adversely effect
detergent cleaning solution with sonication for 30 min at room
the precision of this test.
6. Apparatus
Support membranes, available from Nuclepore Corp., (#140613) or equivalent,
have been found satisfactory for this purpose.
6.1 Epifluorescent Microscope, with 1003 fluorescence oil
Reagent Chemicals, American Chemical Society Specifications, American
immersion objective lens with an NA of 1.25.
Chemical Society, Washington, DC. For suggestions on the testing of reagents not
6.2 Eye Pieces,103, equipped with a micrometer reticle
listed by the American Chemical Society, see Analar Standards for Laboratory
Chemicals, BDH Ltd., Poole, Dorset, U.K., and the United States Pharmacopeia
(10 by 10).
and National Formulary, U.S. Pharmacopeial Convention, Inc. (USPC), Rockville,
6.3 High Pressure Mercury Lamp, 100 W with an exciter
MD.
filter, 500 nm dichroic filter and barrier filter.
Irgalan black, available from Ciba Geigy Corp., Dyestuff and Chemicals
Division, P.O. Box 18300, Greensboro, NC 27419 or equivalent, has been found
6.4 High-Pressure, Stainless Steel On-Line Filter Holder,
satisfactory for this purpose.
25 mm with a stainless steel screen support.
Acridine orange stain, available from Sigma Chemical, P.O. Box 14508, St.
Louis, MO 63178, has been found satisfactory for this purpose.
Predarkened polycarbonate membrane filters and stain available from Nucle-
Exciter filter, KV418-BG12, and barrier filter, OG515, available from Reichert pore Corp., (#991210) or equivalent, have been found satisfactory for this purpose.
Scientific Instruments (formerly American Optical) P.O. Box 123, Buffalo,
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