1、Designation: D932 15Standard Practice forFilamentous Iron Bacteria in Water and Water-FormedDeposits1This standard is issued under the fixed designation D932; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the year of last revisio
2、n. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon () indicates an editorial change since the last revision or reapproval.1. Scope1.1 This practice covers the determination of filamentousiron bacteria (FIB) by examination under the microscope. Thepractice provides
3、 for the identification of the following generaof 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 asstandard. No other units of measurement are included
4、in thisstandard.1.3 This standard does not purport to address the safetyconcerns, if any, associated with its use. It is the responsibilityof the user of this standard to establish appropriate safety andhealth practices and determine the applicability of regulatorylimitations prior to use.2. Referen
5、ced Documents2.1 ASTM Standards:2D887 Practices for Sampling Water-Formed DepositsD1129 Terminology Relating to WaterD1193 Specification for Reagent WaterD3370 Practices for Sampling Water from Closed ConduitsD5465 Practice for Determining Microbial Colony Countsfrom Waters Analyzed by Plating Metho
6、ds3. Terminology3.1 DefinitionsFor definitions of terms used in thispractice, refer to Terminology D1129.4. Summary of Test Method4.1 The iron bacteria are generally filamentous, typicallyfound in fresh water, and frequently surrounded by a sheathwhich is usually encrusted with iron or manganese, or
7、 both (1,2).3However, Starkey (3) reports another type which isclassified among the true bacteria. Detection and identificationis accomplished by microscopic examination of sediment fromthe sample.4.2 This practice provides a qualitative indication of thedensity of the filamentous iron bacteria and
8、the severity of theclogging problem in pipes caused by these bacteria.5. Significance and Use5.1 Filamentous iron bacteria is a general classification formicroorganisms that utilize ferrous iron as a source of energyand are characterized by the deposition of ferric hydroxide intheir mucilaginous she
9、aths. The process is continuous withthese growths, and over a period of time large accumulations ofslimy brown deposits can occur. Iron bacteria may clog waterlines, reduce heat transfer, and cause staining; objectionableodors may arise following death of the bacteria. The organicmatter in the water
10、 is consequently increased, and this in turnfavors the multiplication of other bacteria.6. Apparatus6.1 Centrifuge, complete with 250 mL conical bottles.6.2 Cover Glasses, round or square type, 19 mm (34 in.) indiameter.6.3 Filter Paper or Blotter.6.3.1 For 8.3.2.1 Grade 5 (nominal 2.5 m particle-si
11、zeretention).6.3.2 For 9.3 any absorbent paper medium will suffice.6.4 Containers, sterile 1 L glass or plastic (can be autocla-vable).6.5 Membrane Filter, 0.45 nominal pore size, with appro-priate filter-holding and vacuum assembly (see 9.2).6.6 Microscope that provides a magnification of 400 to100
12、0 and is complete with a suitable light source.Adark-fieldcondenser is desirable.1This practice is under the jurisdiction of ASTM Committee D19 on Water andis the direct responsibility of Subcommittee D19.24 on Water Microbiology.Current edition approved Feb. 1, 2015. Published March 2015. Originall
13、yapproved in 1947. Last previous edition approved in 2009 as D932 85 (2009).DOI: 10.1520/D0932-15.2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStandards volume information, refer to the standards Docu
14、ment Summary page onthe ASTM website.3The boldface numbers in parentheses refer to a list of references at the end ofthis standard.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States16.7 Pipets, Mohr-type, 10-mL, with an opening 3 to 4 mm
15、in diameter, for thick samples, and 1-mL Mohr-type pipets forthin samples or equivalent disposable plastic pipettes.6.8 Slides, glass, standard type, 25 by 76-mm (1 by 3 in.)with either plain or frosted end.6.9 Spatula, small and narrow, for handling thick samples.7. Reagents7.1 Purity of ReagentsRe
16、agent 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 Reagents of the American Chemical Society,where such specifications are available.4Other grades may beused, provided it is
17、first ascertained that the reagent is ofsufficiently high purity to permit its use without lessening theaccuracy of the determination.7.2 Purity of WaterUnless otherwise indicated, referencesto water shall be understood to mean reagent water conformingto Specification D1193, Type II.7.3 Huckers modi
18、fication of the Gram stain (4).7.3.1 Crystal Violet SolutionDissolve 2.0 g of crystalviolet (90 % dye content) in 20 mL of ethyl alcohol (95 %vv).7.3.2 Ammonium Oxylate SolutionDissolve 0.8 g of am-monium oxalate monohydrate (NH4)2C2O4H2O) in 80 mL ofwater.7.3.3 Ammonium Oxalate-Crystal Violet Solut
19、ionCombine crystal violet (2.3.1) and ammonium oxylate (2.3.2)solutions and mix well to ensure that the salts are dissolvedcompletely.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 SolutionPrepare Grams modification ofLugols solution (4) by dis
20、solving1gofiodine in a solutecontaining2gofpotassium iodide (KI) in 10 mL of water anddiluting the resulting solution to 300 mL with water.8. Sampling8.1 Collect the samples in accordance with either PracticesD887 or D3370, whichever is applicable.8.2 Obtain a 500-mL (1-pt) sample of water, using a
21、sterile1-L (1-qt) bottle.NOTE 1The bottle should not be more than half-filled because of theoxygen demand of suspended matter; filling the bottle may cause thesample 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 b
22、e visibleto the naked eye, samples should be concentrated beforestaining and microscopic examination.8.3.2 FiltrationUse a small side stream filter to collect thesample to be examined.8.3.2.1 Filter the water suspected of containing iron bacteriathrough a Grade 5 (nominal 2.5 m particle-size retenti
23、on)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 themaximum 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).
24、8.3.3.2 Centrifuge the subsamples at 9000 to 12 000gfor10 min.8.3.3.3 Decant the supernate from each 250-mL bottle.8.3.3.4 Resuspend the pellet from one centrifuge bottle into20 mL of phosphate buffer or physiological saline (PracticeD5465)8.3.3.5 Transfer the suspension (8.3.3.4) to a second, pelle
25、t-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 beenconsolidated into a single 20-mL suspension.8.4 Regardless of the method used to concentrate the solidsin the water, keep them moist until examined.8.5 Collect mud samples from the mud-wate
26、r interface inorder to obtain maximum bacterial populations.8.6 Transfer the deposit or mud samples to wide-mouthbottles and add sterile phosphate buffer or physiological saline(Practice D5465) to cover the deposits and maintain moistureuntil examined. Protect the samples from sunlight and hold at4C
27、 during transportation and storage.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 slide (6.8) and applya cover glass (6.2).9.1.1 Use a spatula (6.9) or wide-mouth pipet to tra
28、nsfer thesample to the slide.9.1.2 When flocs of material are encountered, Use a pipet;as the flocs settle to the tip when the pipet is held in a verticalposition, and concentrate in the first drop.9.1.3 In the case of very dilute solids or a water sample,concentrate the organisms by centrifuging (8
29、.3.3), pour off thesupernatant liquid, and repeat if necessary.9.1.4 Alternatively, filter a suitable volume (10 to 500 mL;based on estimated population density) through a 0.45-mmembrane filter in an appropriate membrane filtration assem-bly (6.5: holder, tubing, trap, flasks and vacuum pump).NOTE 2
30、For this test, it is not necessary to sterilize the filter assemblyfor each sample, but the assembly should be thoroughly cleaned betweentests.9.2 Examine the slide under the microscope to determine ifencrusted or colorless sheaths are present.9.2.1 Observe at least 20 microscope fields.9.2.2 Record
31、 the presence of the twisted stalks of Gallion-ella at this point, since treatment with acid in accordance with9.3 will dissolve the delicate stalks.4Reagent Chemicals, American Chemical Society Specifications, AmericanChemical Society, Washington, DC. For Suggestions on the testing of reagents notl
32、isted by the American Chemical Society, see Annual Standards for LaboratoryChemicals, BDH Ltd., Poole, Dorset, U.K., and the United States Pharmacopeiaand National Formulary, U.S. Pharmacopeial Convention, Inc. (USPC), Rockville,MD.D932 1529.3 Place a drop of HCl solution (7.4) at one side of thecov
33、er glass and draw it underneath by absorbing the liquid atthe opposite side by means of a filter paper or blotter (6.3.2).9.4 Continue this procedure until no more yellow ferricchloride is evident in the solution.NOTE 3In order to prevent the sample from being drawn to theabsorbent material, control
34、 the flow of the liquid.NOTE 4This treatment removes the iron deposited in the sheaths ofthe bacteria and allows the cells to be seen.9.5 In a similar manner, rinse the iodine solution (7.5) underthe cover glass until the color of the liquid becomes yellow orthe filter paper becomes colored.NOTE 5Th
35、e iodine stains the bacterial cells brown and makes themmore easily visible.9.6 Examine the slide under a microscope, using a high-power, dry objective, for the presence of Sphaerotilus,Crenothrix, Leptothrix, and Clonothrix. If used carefully, anoil-immersion lens may be helpful.9.6.1 Observe at le
36、ast 20 fields.9.7 Detection of Siderocapsa:9.7.1 Prepare a new slide by placing a drop of the sample ona clean slide and allowing it to air-dry.9.7.2 Stain the slide for 1 min with ammonium oxalate-crystal violet solution (7.3.3), wash it with water, and allow itto dry. Examine the slide under an oi
37、l-immersion lens for thepresence of Siderocapsa, which will appear violet colored.9.7.2.1 Observe at least 20 fields.9.8 Table 1 and Figs. 1-10 (3) may be used to differentiatethe various types of filamentous iron bacteria. This practiceTABLE 1 Key for Identification of BacteriaFIG. 1 Siderocapsa tr
38、eubii. Multiple colonies surrounded by ferrichydrate. Magnification about 500 . Fig. 4 of Ref (5)D932 153provides an indication of the density of the iron bacteria andthe severity of the clogging problem in pipes caused by thesebacteria.10. Report10.1 Compute concentration factor of observed microsc
39、opefield.10.1.1 Calibrate the surface area of the microscope field.10.1.2 Compute concentration factor for volume placedonto microscope slide.10.1.3 Compute fraction of 10.1.2 observed per microscopefield.10.1.4 From 10.1.2 and 10.1.3, compute lower limit ofdetection (LLD) in filaments/mL, filaments
40、/g, or filaments/cm2of original sample.10.2 Computer either average percentage of coverage oraverage number of filaments of each type of filamentous ironbacterium per field.10.3 Report Present or Absent and LLD.10.3.1 If filaments are present, report relative abundance ofthe organisms present.10.3.1
41、.1 Report average percentage of coverage per fieldobserved, or10.3.1.2 Report average number of filaments counted perfield.NOTE 6When mixed population have been observed, preferably,report by taxon (for example, 10 % Crenothrix polyspora;30%Leptothrixochracea, etc.)FIG. 2 Gallionella major. Cells at
42、 the ends of excretion bands un-dergoing division. Magnification about 1180 . Fig. 3 of Ref (6)FIG. 3 Gallionella major. Curved cells at the ends of excretionbands. Magnification about 1120 . Fig. 6 of Ref (6)FIG. 4 Sphaerotilus dichotoma. Sketch showing false branching.Magnification about 230 . Fig
43、. 3b of Ref (7)D932 15410.3.1.3 Report Absent only after examination of severalslides.10.3.1.4 In accordance with 10.1.4, include LDL in Absentreport: for example, 1 filament/50 mL.11. Precision and Bias11.1 This standard is a qualitative type test. Consequentlyprecision and bias statements cannot b
44、e provided.12. Keywords12.1 biofouling; Clonothrix; Crenothrix; Dioymohelix; fila-mentous bacteria; Gallionella; iron bacteria; iron deposits;IRB; Leptothrix; microbiologically influenced corrosion; MIC;Siderocapsa; SphaerotilusFIG. 5 Crenothrix polyspora. Sketch showing details of falsebranching of
45、 cells within sheath. Magnification about 380 .Plate 1, Fig. A of Ref (8)D932 155FIG. 6 Crenothrix polyspora. Cells enclosed within a sheath of ferric hydrate and showing false branching. Magnification about 390 .Plate3,Fig.BofRef(8)D932 156FIG. 7 Leptothrix ochracea. Cells coming out of their sheat
46、h. Mag-nification about 2200 . Plate 4, Fig. 20 of Ref (9)FIG. 8 Leptothrix ochracea. Sheaths from an accumulation of pre-cipitated ferric hydrate in iron bearing water. Magnification about390 . Fig. 5 of Ref (7)D932 157FIG. 9 Clonothrix ferruginea. Sketch showing cells enclosed within sheath andfal
47、se branching. Magnification about 430 . Fig. 4 of Ref (7)D932 158REFERENCES(1) Bergey, D. H., Manual of Determinative Bacteriology, 8th Edition,Williams or through the ASTM website(www.astm.org). Permission rights to photocopy the standard may also be secured from the Copyright Clearance Center, 222Rosewood Drive, Danvers, MA 01923, Tel: (978) 646-2600; http:/ 10 Crenothrix polyspora. Conidia can be seen inside and coming out at ends of filaments. Magnification about 345 .Fig. 5 of Ref (9)D932 159
