ASTM D932-85(2002)

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Designation: D 932 – 85 (Reapproved 2002)
Standard Test Method for
Iron Bacteria in Water and Water-Formed Deposits
This standard is issued under the fixed designation D 932; 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 5. Significance and Use
1.1 This test method covers the determination of iron 5.1 Iron bacteria is a general classification for microorgan-
bacteria by examination under the microscope. The method isms that utilize ferrous iron as a source of energy and are
provides for the identification of the following genera of characterized by the deposition of ferric hydroxide in their
bacteria found in water and water-formed deposits: Sidero- mucilaginous sheaths. The process is continuous with these
capsa, Gallionella (Dioymohelix), Sphaerotilus, Crenothrix, growths, and over a period of time large accumulations of
Leptothrix, and Clonothrix. slimey brown deposits can occur. Iron bacteria may clog water
1.2 This standard does not purport to address the safety lines, reduce heat transfer, and cause staining; objectionable
concerns, if any, associated with its use. It is the responsibility odors may arise following death of the bacteria. The organic
of the user of this standard to establish appropriate safety and matter in the water is consequently increased, and this in turn
health practices and determine the applicability of regulatory favors the multiplication of other bacteria.
limitations prior to use.
6. Apparatus
2. Referenced Documents
6.1 Centrifuge, complete with conical tubes.
2.1 ASTM Standards: 6.2 Microscope that provides a magnification of 400 to
D 887 Practices for Sampling Water-Formed Deposits 10003 and is complete with a suitable light source. A
D 1129 Terminology Relating to Water dark-field condenser is desirable.
D 1193 Specification for Reagent Water 6.3 Pipets, Mohr-type, 10-mL, with an opening 3 to 4 mm
D 3370 Practices for Sampling Water from Closed Con- in diameter, for thick samples, and 1-mL Mohr-type pipets for
duits thin samples.
6.4 Spatula, small and narrow, for handling thick samples.
3. Terminology
6.5 Membrane Filter, with appropriate filter-holding assem-
3.1 Definitions—For definitions of terms used in this test
bly (see 9.2).
method, refer to Terminology D 1129.
7. Reagents
4. Summary of Test Method
7.1 Purity of Reagents—Reagent grade chemicals shall be
4.1 The iron bacteria are generally filamentous, typically used in all tests. Unless otherwise indicated, it is intended that
found in fresh water, and frequently surrounded by a sheath
all reagents shall conform to the specifications of the Commit-
which is usually encrusted with iron or manganese, or both (1, tee onAnalytical Reagents of theAmerican Chemical Society,
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2). However, Starkey (3) reports another type which is classi-
where such specifications are available. Other grades may be
fied among the true bacteria. Detection and identification is used, provided it is first ascertained that the reagent is of
accomplished by microscopical examination of sediment from
sufficiently high purity to permit its use without lessening the
the sample. Table 1 and Figs. 1-10 (3) may be used to accuracy of the determination.
differentiate the various types. This test method provides an
7.2 Purity of Water— Unless otherwise indicated, refer-
indication of the density of the iron bacteria and the severity of ences to water shall be understood to mean reagent water
the clogging problem in pipes caused by these bacteria.
conforming to Specification D 1193, Type II.
7.3 Ammonium Oxalate-Crystal Violet Solution—Prepare
1 Hucker’s modification of the Gram stain (4) by mixing a
This test method is under the jurisdiction ofASTM Committee D-19 on Water
and is the direct responsibility of Subcommittee D19.24 on Water Microbiology.
Current edition approved Aug. 30, 1985. Published October 1985. Originally
1 5
published as D 932 – 47 T. Last previous edition D 932 – 72 (1984)e . “Reagent Chemicals,American Chemical Society Specifications,”Am. Chemi-
Annual Book of ASTM Standards, Vol 11.02. cal Soc., Washington, DC. For suggestions on the testing of reagents not listed by
Annual Book of ASTM Standards, Vol 11.01. theAmerican Chemical Society, see “Reagent Chemicals and Standards,” by Joseph
The boldface numbers in parentheses refer to the list of references at the end of Rosin, D. Van Nostrand Co., Inc., New York, NY, and the “United States
this test method. Pharmacopeia.”
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.
D 932 – 85 (2002)
TABLE 1 Key for Identification of Bacteria
FIG. 1 Siderocapsa treubii. Multiple colonies surrounded by ferric
FIG. 2 Gallionella major. Cells at the ends of excretion bands
hydrate. Magnification about 500 3.Fig.4ofRef (5)
undergoing division. Magnification about 1180 3.Fig.3ofRef
(6)
D 932 – 85 (2002)
FIG. 3 Gallionella major. Curved cells at the ends of excretion
bands. Magnification about 1120 3.Fig.6ofRef (6)
FIG. 5 Crenothrix polyspora. Sketch showing details of false
branching of cells within sheath. Magnification about 380 3 .
Plate 1, Fig. A of Ref (8)
7.4 Hydrochloric Acid (1 + 4)—Mix 1 volume of hydro-
chloric acid (HCl, sp gr 1.19) with 4 volumes of water.
7.5 Iodine Solution— Prepare Gram’s modification of
Lugol’s solution (4) by dissolving1gof iodine in a solute
containing2gof potassium iodide (KI) in 10 mLof water and
diluting the resulting solution to 300 mL with water.
7.6 Filter Paper or Blotter.
7.7 Slides, standard type, 25 by 76-mm (1 by 3 in.) with
either plain or frosted end.
7.8 Cover Glasses, round or square type, 19 mm ( ⁄4 in.) in
diameter.
8. Sampling
8.1 Collect the samples in accordance with either Practices
D 887 or D 3370, whichever is applicable.
8.2 Obtain a 500-mL (1-pt) sample of water, using a sterile
1-L(1-qt) bottle. 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 If the number of iron bacteria are very low or that they
FIG. 4 Sphaerotilus dichotoma. Sketch showing false branching.
Magnification about 230 3.Fig.3b of Ref (7) are just becoming established in the system, use a small side
stream filter to collect the sample to be examined. The water
solution of 2.0 g of crystal violet (90 % dye content) in 20 mL suspected of containing iron bacteri
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