1、Designation: D 932 85 (Reapproved 2002)Standard Test Method forIron Bacteria in Water and Water-Formed Deposits1This standard is issued under the fixed designation D 932; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the year of
2、last revision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon (e) indicates an editorial change since the last revision or reapproval.1. Scope1.1 This test method covers the determination of ironbacteria by examination under the microscope. The methodprovides for
3、 the identification of the following genera ofbacteria found in water and water-formed deposits: Sidero-capsa, Gallionella (Dioymohelix), Sphaerotilus, Crenothrix,Leptothrix, and Clonothrix.1.2 This standard does not purport to address the safetyconcerns, if any, associated with its use. It is the r
4、esponsibilityof the user of this standard to establish appropriate safety andhealth practices and determine the applicability of regulatorylimitations prior to use.2. Referenced Documents2.1 ASTM Standards:D 887 Practices for Sampling Water-Formed Deposits2D 1129 Terminology Relating to Water3D 1193
5、 Specification for Reagent Water3D 3370 Practices for Sampling Water from Closed Con-duits33. Terminology3.1 DefinitionsFor definitions of terms used in this testmethod, refer to Terminology D 1129.4. Summary of Test Method4.1 The iron bacteria are generally filamentous, typicallyfound in fresh wate
6、r, and frequently surrounded by a sheathwhich is usually encrusted with iron or manganese, or both (1,2).4However, Starkey (3) reports another type which is classi-fied among the true bacteria. Detection and identification isaccomplished by microscopical examination of sediment fromthe sample. Table
7、 1 and Figs. 1-10 (3) may be used todifferentiate the various types. This test method provides anindication of the density of the iron bacteria and the severity ofthe clogging problem in pipes caused by these bacteria.5. Significance and Use5.1 Iron bacteria is a general classification for microorga
8、n-isms that utilize ferrous iron as a source of energy and arecharacterized by the deposition of ferric hydroxide in theirmucilaginous sheaths. The process is continuous with thesegrowths, and over a period of time large accumulations ofslimey brown deposits can occur. Iron bacteria may clog waterli
9、nes, reduce heat transfer, and cause staining; objectionableodors may arise following death of the bacteria. The organicmatter in the water is consequently increased, and this in turnfavors the multiplication of other bacteria.6. Apparatus6.1 Centrifuge, complete with conical tubes.6.2 Microscope th
10、at provides a magnification of 400 to10003 and is complete with a suitable light source. Adark-field condenser is desirable.6.3 Pipets, Mohr-type, 10-mL, with an opening 3 to 4 mmin diameter, for thick samples, and 1-mL Mohr-type pipets forthin samples.6.4 Spatula, small and narrow, for handling thi
11、ck samples.6.5 Membrane Filter, with appropriate filter-holding assem-bly (see 9.2).7. Reagents7.1 Purity of ReagentsReagent grade chemicals shall beused in all tests. Unless otherwise indicated, it is intended thatall reagents shall conform to the specifications of the Commit-tee on Analytical Reag
12、ents of the American Chemical Society,where such specifications are available.5Other grades may beused, provided it is first ascertained that the reagent is ofsufficiently high purity to permit its use without lessening theaccuracy of the determination.7.2 Purity of Water Unless otherwise indicated,
13、 refer-ences to water shall be understood to mean reagent waterconforming to Specification D 1193, Type II.7.3 Ammonium Oxalate-Crystal Violet SolutionPrepareHuckers modification of the Gram stain (4) by mixing a1This test method is under the jurisdiction of ASTM Committee D-19 on Waterand is the di
14、rect responsibility of Subcommittee D19.24 on Water Microbiology.Current edition approved Aug. 30, 1985. Published October 1985. Originallypublished as D 932 47 T. Last previous edition D 932 72 (1984)e1.2Annual Book of ASTM Standards, Vol 11.02.3Annual Book of ASTM Standards, Vol 11.01.4The boldfac
15、e numbers in parentheses refer to the list of references at the end ofthis test method.5“Reagent Chemicals, American Chemical Society Specifications,” Am. Chemi-cal Soc., Washington, DC. For suggestions on the testing of reagents not listed bythe American Chemical Society, see “Reagent Chemicals and
16、 Standards,” by JosephRosin, D. Van Nostrand Co., Inc., New York, NY, and the “United StatesPharmacopeia.”1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.TABLE 1 Key for Identification of BacteriaFIG. 1 Siderocapsa treubii. Multiple
17、colonies surrounded by ferrichydrate. Magnification about 500 3 .Fig.4ofRef(5)FIG. 2 Gallionella major. Cells at the ends of excretion bandsundergoing division. Magnification about 1180 3 .Fig.3ofRef(6)D 932 85 (2002)2solution of 2.0 g of crystal violet (90 % dye content) in 20 mLof ethyl alcohol (9
18、5 % with a solution of 0.8 g of ammoniumoxalate monohydrate (NH4)2C2O4H2O) in 80 mL of water.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 Grams modification ofLugols solution (4) by dissolving1gofiodine in a sol
19、utecontaining2gofpotassium iodide (KI) in 10 mL of water anddiluting 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.) witheither plain or frosted end.7.8 Cover Glasses, round or square type, 19 mm (34 in.) indiameter.8. Sampl
20、ing8.1 Collect the samples in accordance with either PracticesD 887 or D 3370, whichever is applicable.8.2 Obtain a 500-mL (1-pt) sample of water, using a sterile1-L (1-qt) bottle. The bottle should not be more than half-filledbecause of the oxygen demand of suspended matter; filling thebottle may c
21、ause the sample to become anaerobic.8.3 If the number of iron bacteria are very low or that theyare just becoming established in the system, use a small sidestream filter to collect the sample to be examined. The watersuspected of containing iron bacteria should be filtered througha highly retentive
22、 filter paper (or some other comparablemedia) for 24 h. Centrifugation or membrane filtration isFIG. 3 Gallionella major. Curved cells at the ends of excretionbands. Magnification about 1120 3 .Fig.6ofRef(6)FIG. 4 Sphaerotilus dichotoma. Sketch showing false branching.Magnification about 230 3 .Fig.
23、3b of Ref (7)FIG. 5 Crenothrix polyspora. Sketch showing details of falsebranching of cells within sheath. Magnification about 380 3 .Plate 1, Fig. A of Ref (8)D 932 85 (2002)3satisfactory also. The flow rate of the water should be at themaximum filtering capacity of the material employed.8.4 Regard
24、less of the method used to concentrate the solidsin the water, keep them moist until examined.8.5 Mud samples should be collected from the mud-waterinterface for maximum bacterial populations.8.6 Transfer the deposit or mud samples to wide-mouthbottles and add clean, chlorine-free water to cover the
25、 depositsand maintain moisture until examined. Protect the samplesfrom sunlight and hold at 4C during transportation andstorage.8.7 As soon as possible after collection of the solids,microscopically examine them for the presence of iron bacte-ria.9. Procedure9.1 Place a portion of the sample on the
26、slide and apply acover glass. A spatula or wide-mouth pipet can be used totransfer the sample to the slide. Use a pipet when flocs ofmaterial are encountered, as the flocs settle to the tip when thepipet is held in a vertical position, and concentrate in the firstdrop. In the case of very dilute sol
27、ids or a water sample,concentrate the organisms by centrifuging, pour off the super-natant liquid, and repeat if necessary.9.2 An alternative procedure is to filter a suitable volume ofthe dilute solids or the entire water sample through a 0.45-mmembrane filter in an appropriate membrane filtration
28、assem-bly (holder, tubing, trap, flasks and vacuum pump). For this testit is not necessary to sterilize the filter assembly for eachsample, but the assembly should be thoroughly cleaned be-tween tests.9.3 Examine the slide under the microscope to determine ifencrusted or colorless sheaths are presen
29、t. Note the presence ofthe twisted stalks of Gallionella at this point, since treatmentwith acid in accordance with 9.4 will dissolve the delicatestalks.FIG. 6 Crenothrix polyspora. Cells enclosed within a sheath of ferric hydrate and showing false branching. Magnification about 390 3 .Plate3,Fig.Bo
30、fRef(8)D 932 85 (2002)49.4 Place the HCl (1 + 4) at one side of the cover glass anddraw it underneath by absorbing the liquid at the opposite sideby means of a filter paper or blotter. Continue this procedureuntil no more yellow ferric chloride is evident in the solution.Take care that the flow of t
31、he liquid is not fast, or the samplemay be drawn to the absorbent material. This treatmentremoves the iron deposited in the sheaths of the bacteria andallows the cells to be seen.9.5 In a similar manner, rinse the iodine solution under thecover glass until the color of the liquid becomes yellow or t
32、hefilter paper becomes colored. The iodine stains the bacterialcells brown and makes them more easily visible.9.6 Examine the slide under a microscope, using a high-power, dry objective, for the presence of Sphaerotilus, Creno-thrix, Leptothrix, and Clonothrix. If used carefully, an oil-immersion le
33、ns may be helpful.9.7 Prepare a new slide by placing a drop of the sample ona clean slide and allowing it to air-dry. Then stain it for 1 minwith ammonium oxalate-crystal violet solution, wash it withwater, and allow it to dry. Examine the slide under anoil-immersion lens for the presence of Sideroc
34、apsa, which willappear violet colored.10. Report10.1 The report shall state “Present” or“ Not found, prob-ably absent.” Make a statement as to the relative abundance ofthe organisms present. Make a negative report only afterexamination of several slides.11. Precision and Bias11.1 Since this standard
35、 is a qualitative type test, precisionand bias statements cannot be provided.FIG. 7 Leptothrix ochracea. Cells coming out of their sheath.Magnification about 2200 3 . Plate 4, Fig. 20 of Ref (9)FIG. 8 Leptothrix ochracea. Sheaths from an accumulation ofprecipitated ferric hydrate in iron bearing wat
36、er. Magnificationabout 390 3 .Fig.5ofRef(7)D 932 85 (2002)5FIG. 9 Clonothrix ferruginea. Sketch showing cells enclosed within sheath andfalse branching. Magnification about 430 3 .Fig.4ofRef(7)D 932 85 (2002)6REFERENCES(1) Bergey, D. H., Manual of Determinative Bacteriology, 8th Edition,Williams or through the ASTM website(www.astm.org).FIG. 10 Crenothrix polyspora. Conidia can be seen inside and coming out at ends of filaments. Magnification about 345 3 .Fig.5ofRef(9)D 932 85 (2002)7
