ASTM D932-20

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Designation: D932 − 15 D932 − 20
Standard Practice for
Filamentous Iron Bacteria in Water and Water-Formed
Deposits
This standard is issued under the fixed designation D932; 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
1.1 This practice covers the determination of filamentous iron bacteria (FIB) by examination under the microscope. The
microscopic examination. This practice provides for the identification of the following genera of bacteria found in water and
water-formed deposits: Siderocapsa,Gallionella (Dioymohelix),Sphaerotilus,Crenothrix,Leptothrix, and Clonothrix.
1.2 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
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 safety, health, and healthenvironmental practices and determine the
applicability of regulatory limitations prior to use.
1.4 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.
2. Referenced Documents
2.1 ASTM Standards:
D887 Practices for Sampling Water-Formed Deposits
D1129 Terminology Relating to Water
D1193 Specification for Reagent Water
D3370 Practices for Sampling Water from Flowing Process Streams
D5465 Practices for Determining Microbial Colony Counts from Waters Analyzed by Plating Methods
3. Terminology
3.1 Definitions—For definitions of terms used in this practice, refer to Terminology D1129. Definitions:
3.1.1 For definitions of terms used in this standard, refer to Terminology D1129.
4. Summary of Test MethodPractice
4.1 The iron bacteria are generally filamentous, typically found in fresh water, and frequently surrounded by a sheath which is
usually encrusted with iron or manganese, or both (1, 2). However, Starkey (3) reports another type which is classified among
the true bacteria. Detection and identification is accomplished by microscopic examination of sediment from the sample.
4.2 This practice provides a qualitative indication of the density of the filamentous iron bacteria and the severity of the clogging
problem in pipes caused by these bacteria.
5. Significance and Use
5.1 Filamentous iron bacteria is a general classification for microorganisms that utilize ferrous iron as a source of energy and
are characterized by the deposition of ferric hydroxide in their mucilaginous sheaths. The process is continuous with these growths,
and over a period of time large accumulations of slimy brown deposits can occur. Iron bacteria may clog water lines, reduce heat
This practice is under the jurisdiction of ASTM Committee D19 on Water and is the direct responsibility of Subcommittee D19.24 on Water Microbiology.
Current edition approved Feb. 1, 2015May 1, 2020. Published March 2015May 2020. Originally approved in 1947. Last previous edition approved in 20092015 as D932 –
85 (2009). 15. DOI: 10.1520/D0932-15.10.1520/D0932-20.
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 Standards
volume information, refer to the standard’sstandard’s Document Summary page on the ASTM website.
The boldface numbers in parentheses refer to a list of references at the end of this standard.
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D932 − 20
transfer, and cause staining; objectionable odors may arise following death of the bacteria. The organic matter in the water is
consequently increased, and this in turn favors the multiplication of other bacteria.
6. Apparatus
6.1 Centrifuge, complete with 250 mL conical bottles.
6.2 Cover Glasses, round or square type, 19 mm ( ⁄4 in.) in diameter.
6.3 Filter Paper or Blotter.
6.3.1 For 8.3.2.1 – Grade 5 (nominal 2.5 μm particle-size retention).
6.3.2 For 9.3 – any absorbent paper medium will suffice.
6.4 Containers, sterile 1 L glass or plastic (can be autoclavable).
6.5 Membrane Filter, 0.45 μ nominal pore size, with appropriate filter-holding and vacuum assembly (see 9.2).
6.6 Microscope that provides a magnification of 400 to 1000× and is complete with a suitable light source. A dark-field
condenser is desirable.
6.7 Pipets, Mohr-type, 10-mL, with an opening 3 to 4 mm in diameter, for thick samples, and 1-mL Mohr-type pipets for thin
samples or equivalent disposable plastic pipettes.
6.8 Slides, glass, standard type, 25 by 76-mm 76 mm (1 by 3 in.) with either plain or frosted end.
6.9 Spatula, small and narrow, for handling thick samples.
7. Reagents
7.1 Purity of Reagents—Reagent grade chemicals shall be used in all tests. Unless otherwise indicated, it is intended that all
reagents shall conform to the specifications of the Committee on Analytical Reagents of the American Chemical Society, where
such specifications are available. Other grades may be used, provided it is first ascertained that the reagent is of sufficiently high
purity to permit its use without lessening the accuracy of the determination.
7.2 Purity of Water—Unless otherwise indicated, references to water shall be understood to mean reagent water conforming to
Specification D1193, Type II.
7.3 Hucker’s modification of the Gram stain (4).
v
7.3.1 Crystal Violet Solution—Dissolve 2.0 g of crystal violet (90 % dye content) in 20 mL of ethyl alcohol (95 % ⁄v).
7.3.2 Ammonium Oxylate Solution—Dissolve 0.8 g of ammonium oxalate monohydrate (NH ) C O •H O) in 80 mL of water.
4 2 2 4 2
7.3.3 Ammonium Oxalate-Crystal Violet Solution—Combine crystal violet (2.3.1)(7.3.1) and ammonium oxylate (2.3.2)(7.3.2)
solutions and mix well to ensure that the salts are dissolved completely.
7.4 3N Acid (1 + 4)—Mix 1 volume of hydrochloric acid (HCl, sp gr 1.19) with 4 volumes of water.
7.5 Iodine Solution—Prepare Gram’sGram’s modification of Lugol’sLugol’s solution (4) by dissolving 1 g of iodine in a solute
containing 2 g of potassium iodide (KI) in 10 mL of water and diluting the resulting solution to 300 mL with water.
8. Sampling
8.1 Collect the samples in accordance with either Practices D887 or D3370, whichever is applicable.
8.2 Obtain a 500-mL (1-pt) sample of water, using a sterile 1-L (1-qt) bottle.
NOTE 1—The bottle should not be more than half-filled because of the oxygen demand of suspended matter; filling the bottle may cause the sample
to become anaerobic.
8.3 Sample concentration by following either 8.3.2 or 8.3.3.
8.3.1 If the population is not sufficiently dense to be visible to the naked eye, samples should be concentrated before staining
and microscopic examination.
8.3.2 Filtration—Use a small side stream filter to collect the sample to be examined.
8.3.2.1 Filter the water suspected of containing iron bacteria through a Grade 5 (nominal 2.5 μm particle-size retention) filter
paper (6.3.1 or some other comparable media) for 24 h.
8.3.2.2 Adjust the side-stream filter flow rate to match the maximum filtration capacity of the filter medium used.
8.3.3 Centrifugation:
8.3.3.1 Divide the 500 mL sample (8.2) equally, by weight, among four 250 mL centrifuge bottles (6.1).
Reagent Chemicals, American Chemical Society Specifications,ACS Reagent Chemicals, Specifications and Procedures for Reagents and Standard-Grade Reference
Materials, American Chemical Society, Washington, DC. For Suggestionssuggestions on the testing of reagents not listed by the American Chemical Society, see
AnnualAnalar Standards for Laboratory Chemicals, BDH Ltd., Poole, Dorset, U.K., and the United States Pharmacopeia and National Formulary, U.S. Pharmacopeial
Convention, Inc. (USPC), Rockville, MD.
D932 − 20
8.3.3.2 Centrifuge the subsamples at 9000 to 12 000 × g for 10 min.
8.3.3.3 Decant the supernate from each 250-mL bottle.
8.3.3.4 Resuspend the pellet from one centrifuge bottle into 20 mL of phosphate buffer or physiological saline (Practice(Prac-
tices D5465)).
8.3.3.5 Transfer the suspension (8.3.3.4) to a second, pellet-containing centrifuge bottle and repeat 8.3.3.4.
8.3.3.6 Repeat 8.3.3.4 and 8.3.3.5 until all pellets and been consolidated into a single 20-mL suspension.
8.4 Regardless of the method used to concentrate the solids in the water, keep them moist until examined.
8.5 Collect mud samples from the mud-water interface in order to obtain maximum bacterial populations.
8.6 Transfer the deposit or mud samples to wide-mouth bottles and add sterile phosphate buffer or physiological saline
(Practice(Practices D5465) to cover the deposits and maintain moisture until examined. Protect the samples from sunlight and hold
at 4°C during transportation and storage.
8.7 As soon as possible after collection of the solids, microscopically examine them for the presence of iron bacteria.
9. Procedure
9.1 Place a portion of the sample on the slide (6.8) and apply a cover glass (6.2).
9.1.1 Use a spatula (6.9) or wide-mouth pipet to transfer the sample to the slide.
9.1.2 When flocs of mater
...