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    Tiêu chuẩn Việt Nam TCVN 6179-2:1996 (ISO 7150/2 : 1986) về Chất lượng nước - Xác định amoni - Phần 2

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    Tiêu chuẩn Việt Nam TCVN 6179-2:1996 (ISO 7150/2 : 1986) về Chất lượng nước - Xác định amoni - Phần 2

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    TCVN 6179-2-1986

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    windpower bt
    Tiêu chuẩn Việt Nam TCVN 6179-2:1996 (ISO 7150/2 : 1986) về Chất lượng nước - Xác định amoni - Phần 2

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    ‘Water quality — Determination of ammonium — Part 2: Automated spectrometric method Quelte de renu — Dosage de Vammonium — Partie 2: Méthode spect ue automatique First edition — 1986-12-15, UBe 643.342: 543.42 Ref. No, 1SO 7750/2-1986 (E) Descriptors ; ater, quay. chemical ani, determination ef content. xmmonium ian, soecrne naonealanaves Foreword 150 Ithe International Organization for Standardization) is @ worldwide federation of ‘ational standards bodies {ISO member bodies). The work of preparing Intemational ‘Standards is normally caied ont through ISO technical commitiees. Each member ‘ody interested in subject for which a technical committee has been established has the Fight to be ‘epresented on that committe. International organizations, govern ‘mental and non-governmental, in liaison with ISO, also take part in the work Draft International Standards edopted by the technical committees are circulated to the member bodies for approval before thei acceptance as international Standards by the !SO Council. They are approved in accordance with ISO procedures requiring at least 75 % approval By the member bodies voting Inernstionsl Standard SO 7150/2 was preoared by Technical Commitee 1SO/TC 147, Water quay. Users should nove that all International Standards undergo revision from time to time ‘and that any reference made herein to any other International Standard implies ts lest edition, ures otherwise stated. © Intemational Organization for Standardization. 1886 & Priotd in Site INTERNATIONAL STANDARD 1S0.7150/2-1986 (E) Water quality — Determination of ammonium — Part 2: Automated spectrometric method 1. Scope and field of apy 1.1. Substance determined ‘This part of ISO 7150 specfés an automated soectrometic ‘method for the determination af armmanium in water. NOTE ~ Fors manval soecrometric mntod for he dtemination ot ‘armen, see 180 7ISD 1.2. Type of sample ‘The method is apalcable tn the analysis of raw, potable and most waste waters. Application to excessively coloured or saline waters shal be preceded by distilain (see clause 10) For interferences, see clause & 13. Range ‘An ammonium nitrogen concentration, ayy of un to 50 mg/t can be determined using dais, oF up to 0,5 mg/l without alysis (soe clause 4) 1.4 Limit of detection” With diaisis, the imit of detection is oy = 0.03 ma/t Without dialysis itis oy 0.01 mgvt 2 Principle Spectromettic measurement at about 650 nm of the blue com- pound formed by reaction of ammonium with salicylate and hypochlorite ions in the presence of sodium nitrosopenta- cyanoteratelll (sodium nitroprusside! Hypochlorite ions are generated by the alkaline hydrolysis of 1.3-éichioro--sodio-1,3 Srazinanevione (sodium dichloroso cyanuratel. Reaction of the resulting ehioroamine with sodium salleyte takes place at oH 12,6 in the aresence of nitro: Drusside. Any chiorosmines present in the semple are quan: Utatvely determined a3 a consequence, Sodium citrate is acded to mask interference from cations, notably calekim and magnesium. [AI reactions are carried out automatically using continuous flow tecnniques.? The absorbance of the coloured compound is measured in 9 fow-ttwough spectrometer. ‘Two distinct analytical manifold configurations ae specified. (One incornorates a dialer binck ands suitable for the deter. rmination of ammonium nitzogen concentrations un te 80 mi ‘The other omits the daiyser and i suitable for the determin: tion of low level ammonium nitvogen concenteations UD to 0.5 mgt 3. Reagents During the analysis, use only reagents af recognized analvtical ‘rade ond only water arapared a5 described in 3.1 3.1. Water, ammoniumtres, orepared by one of the Follow: ing methods. 3.4.4 fon exchange method Pess distiled water through a colurm of strongly acide cation lxchange resin fin the hydrogen form) and coliet the eluate in a glass bore provided with a wellfiting glass stopper. Add bout 109 of the same resin to each lire of collected eluste for storage purposes. ‘Add 0,10 + 0.01 mi of sulfuic acid (e = 1.84 g/ml to 1000 £ 10 mi of cistiled water and redist in an all lass 20- Daratus Discard the fst 60 mi of cistilate, and then collect the distilatein a glass bore provided with 2 wel-fting lass ston- er. Add about 10g of strongly acc cation exchange resi (in ‘the hydrogen form to each lire of collected cstilate 3.2. Crate reagent 32.1. Preparation Dissove 40.0 + 0.59 of trisodium ciate dihydrate ICeHg0;Nay-2H,0) in about 950 mi of water (3.1) a 1 five ‘measuring evlnder, Dilute to 1 lie with wate (3.1), Umit of detection ealevared rom 3 fy, where f san axtimat, on a lest 9 degrees of freedom, ofthe wthin-batch standard devon of Dank solution vesopnaes Infomation fom the Unt Kingdom 2) HMSO. Mavrnas for Examination of Waters and Associares mata: Aur Seanmites C wins Fw Suramatic Anan the Laborato London, Hee Maieatys i inne OMe, 1973, ISO 7150/2-1946 (€) ‘Store the solution in a glass or pistes bottle, ‘This reagent is stable for atleast 3 weeks. 32.2 Wetting agent (ootional ‘The inclusion of @ wetting agent in this reagent in order to promote smooth flow in the system is optional. used, the wetting agent should be a proprietary detergent of the alky! benzenesulfonate type and should be added to give a concen tation of 1 mi 3.3. Salicylate reagent Dissolve 34.0 + 0.5 g of sodium salcyiate (CyHy0Na) in water (3.1) in a 1.000 ml one-mark volumevic fiask Then £364 0,400 + 0,005 g of sodium nitrosapentacvanofercatell inyarate {sodium nitroprusside, IFelCNI,NOINa,- 24,0} Dissolve the solid and then make up to the mark with water on Stored in an amber glass bottle, this reagent is stable for at least 2 weeks. 3.4 Sodium dichloroisocyanurate, solution, Dissolve 10,0 = 0,1 g of sodium hydroxide in 500 + 50 ml of water (8.1). Cool the solution to room temperature and, then ‘add 0.80 + 0.02 9 of sodium cichloroizocyarurate dihydrate (CN:0,C1,Na-2H,0) to the solution. Dissolve the sold and wansier the solution quentiatively to 1 000 mi one-mark volumetric flask. Moke up tothe mark with water (3.1) an rx wel Stored in an amber glass bottle at 4 °C, this reagent is stable for atleast 2 weeks 3.5. Selicytate/citrate mixed reagent (for use in the deter. ‘mination of ammonium ritrogen concentrations up tO 0.5 mg/0 35.1 Preparation Dissolve 34,0 + 0.1 g of sodium salicyiate (C,H,O5Nal and 40,0 # 05 g of risodtum citrate dydrate (CQH,O,Nay- 21,0) in about 950 mi of water (3.1) in a1 000ml one-merk volumetric. flask: ‘Then add 0,400 + 0,005 9 of socium nitrosopentacyanoteratell) hydrate {sodium nitroprusside, [Fe(CNIgNOINa,: 24,0}. Dissolve the eolid and then make up to the mark with water Stored in an amber glass botle, this reagent is stable for at ast 2 weeks. 352 Wetting agent (optional ‘A wetting agent (se 3.2.21 may be incorporated in this regent. 3.6 Ammonium nitrogen, standard solution, (y= 1.000 mg. Dissolve 3,818 + 0,008 g of ammonium chloride (dred at 105°C for at least 2} in about 800 mi of water (3.11 in 8 1000 mi onesmark volumevie lak. Make Up 19 the mark with 1 mi of this standard solution containg 1 ma of ammonium nitrogen. Stored in a stonoered glass bol, this solution is stabi for at least 1 month 3.7 Ammonium nitrogen, standard solution, 2y = 20 mail Piette 10 mi of the ammonium nitogen standard solution [3.6 ino a 500 mil one-mark volumetic flask. Make up to the 1 ml of this standard solution contains 0,02 mg ot ammonium ritrogen, Stored ina stopoered amber glass botle and kept at 2108 °C, {his solution is stable for 1 week, 4 Apparatus ‘Apparatus for this continuous flow method consist basicaly of te folowing. 4.1. Sample presentation unit (sampler 4.2. Multichannel peristaltic pump 4.3 Analytical cartridge (manifold), including puma tubes, mixing coils and cialvser unit. The design of the manifold depends on the intended range of ‘pplication. Figure 1 shows that for the determination of am mmonium nitrogen concentrations up to 50 mg/l. Figure 2 shows the manfold forthe determination of ammonium nitrogen con centration up to 0,5 mg/l. This is essentially a modification of figure 1 and permits greater sensitivity due to the larger sample flow rate, It isthe prefered configuration for the anaives of potable waters. For the analysis of low levels of ammonium the design of both systems can be improved by deaming ar used for segmentation through diute hyérochlorc aed in order to scrub any at- ‘mosphere ammonia. Errors from the seme source can slso;be reduced by capping the sample cups, ence fled with samples, With thin aluminium fol which wil be pierced as the sampling probe descends, ‘The manifold system shown in figure 2 ie unsuitable for ‘samples with a high suspended solids content unless the solids are first removed by settlement. centifuging or firation, Ensure that no ammonium is added or lost by any method of removal adopted. Altematvely dstilaion may be used (see clause 10, 4.4 Spectrometer capable of messuring absorbance at 650 nm, incorporating a flow coll with path length of atleast bmn 4.5 Recorder. 5 Sampling and samples Laboratory samples shall be collected in polyethylene or glass bottes. They should be analyeed as quickly 28 possible, or else stored at between 2 and 5 °C until analysed. Aciciication with sufurie aid to pH < 2 may also be used as an aid to preserva tion.- provided that possible contamination of the acidified sample by absorption of any atmospheric ammonia is avoided. 6 Procedure @.1 Starting operation Connect the system as shown in figure 1 or figure 2 depending fon the intended range of application. Follow the equisment manufacturer's general operating instructions where ap: propria With the sample probe at retin the wash receptacle solution, place al the reagent lines in ther respective reagent, start the ‘bump and switch on the spectrometer and recorder. Alow the system to equiforate for at least 20 min and during this period ‘check that the bubble pattern and hydraulic behaviour of the system is eatistactory. if not, aiminate difiuities before pro: ceeding 6.2. Initial sensitivity setting ‘when an scceprably smooth baseline trace is given on the ‘ecorder, aust the baseline cesponse to about 5 % of ful scale with the zero control and then wansfer the sample probe into the Oy, max C@Bbration solution where @y, max the greatest Cconcerivaton thatthe calibration is intended to cover. ‘When there is 8 positive stable response atthe measurement unit due to the colour produced from the ey, myx libration solution, aust this response with the scala expansion control 10 about 95 % of ful sale-——~ NOTE ~ The sample probe nats rembin in the mae Eaton sok ‘ion only or sucent time to ge @ andy reading ‘Then retun the sample probe to rest in the wash position, fist removing any waces of calitration solution from the outsige of the probe 6.3. Determination Ringe each sample cup at least once with the solution it ie to contain and then fit to the too, Load the samole turntable with cups containing the sample. blank and calibration stan dard in the order given in table 1 Repeat the sequence 6 to 38 unti al the samoles have been foaded, NOTE — When crosscontorination occurs benween two samples Wwisible an the snectometar ond recorder trace ar incompiete sere tion of censbeutwe sample resoonses| bom somal should De 1SO 7150/2-1986 (E) It necessary, eeacust the baseline response to about 8 % of full Scale deflection and start the sampling unit Table 1 — Order of loading of the turntable onturabie ee ToS | Callzavon solfons nassending oe ee 6 Sana7 | Worer lank 2.1) Biot? | Samois 13 | Cattraton soluions 12nd 20. | Water bla 2.1 211030 | Samples 31 | Caltration ston? szand3a | Wate lane 3.11 311038 _ | Caltration souitions in atcending order “Calbraton soluion” indicates one of the calbration solutions (ere 8.8) used In postions 1108, \When all the system sespanses due to the processed solutions have appeared on the recorder and a final baseline has been obtained, the recorder can be switched off. Then transfer all reagent fines to water and pure for atleast 15 min. 64 Calibration Prepare calibration solutions from either of the standard solu- tions (3.6 or 3.71 as shown in t2ble 2. Select the concentrations of the calnretion solutions aceording to the expected sample ‘ammonium concentrations and the manifold configuration in Load the five selected calibration solutions in positions 1 to Bof| the sample turntable as indicated in table 1 Table 2 — Volumes of standard olution for us In the preparation of the calibration solutions Volume of Standard Standard sotution | soraliution to Tiere ae a ‘mail TE ay = 1000 mai = @ 3 * 2 » a » 0 10 5 5 25 25 = Daa = T 0 os x 08 5 05 0 De 0 02 5 a4 25 08 Iso 7150/2 1986 16} [At tne end ofthe determination, messure the system response {a the caiation solutions from th recorder trace, taking the baseline trace obtained durina intial sensitivity setting (see 6.2) 26 the blank response valve Plot 2 granh of tesponee against ammonium sitrogen concen tration, oj. exoressedin miligrees per live. Ths graph should be linger and should pass through the cig. 7 Expression of rosults 7.1. Method of calculation Measure the system response tothe samles from the recorder ‘race. toking the baseline trace obtained during inital sanstivty setting (S82 6.2) as the blank response value. Read off the cor responding ammonium nitrogen concentration dy. expressed in ellggams Der live, from the calitration graph ifthe responses om the biank or calibration solutions included ‘on the turntable show any ditt of the baseline and/or calibra tion response, apply 2 correction for the change in sensitivity thus reveled See table 3 for conversion of ay to ammonis and ammonium Table 3 — Conversion table | ey [oma [on al | mat | malt | uml aa Tree 1 pane |r| bee tal cua ft | vase | say tet = tna om foam | 1 | ssa enngt = tno dow | oan | oars | “+ Example: ‘An ammoniurnion concentration. gy; 1 mg/l coresponds toa nitrogen concentaton of 0.777 mal. Table 4 — Reproducibility 7.2 Reproducibility Reproducibility standard deviations have been showa in table 6 8 Interferences ‘A range of substances often encountered in water samples has been tested for possible interference with this method. Of these ‘only iron and ethanolamine cause significant effects, Full details are given inthe annex In saline samples, interterence trom magnesium sreiptation ‘rises wen the complexing capacity of the citrate in the Feagents is exceeded. In this case preliminary cistilation is recessary (see clause 10), 9 Notes on procedure 9.1 General ‘The determination of low concentrations of ammonium is pr ticuary susceptible to bas caused by the presence of traces of amenonium in the analvtical environment. While careful aten- tion toa the instructions given in tis por of ISO 7180 shoule Imioimize this susceptibiy. the possibilty of biased results 16- ‘mains. A method for obtaining an indication of possible bas is row presented 9.2 Checking the accuracy of analytical results ‘When this method is fist used, obtain an estimate ofthe total standard deviation (with at least 9 degrees of freedom) on the termination of 8 contol ammonium nitrogen solution stan- ard with 2 concentration of approximately 60 % ofthat of the most concentrated calibration solution, ‘This control standard solution shall not be used for calibration purposes. stendard deviations* Reamonian Calibration | eproducioiny nitrogen ange ‘andar Semple concentration. oy my deviation, ¢ Sionaard satan Standard sluvon os Potable water Siandord sisson Standard eoution Potted river wer Sewage event Sewage etsen! 80 Dats tom the United Kingdom [Analyse one porion ofthe contol standard solution with every subsequant batch of detemunstions: perform ine cabrtion witha seis of ealbratio sluton (6.4 is essen hat the etermined concenvaion of this conto standard solution Fa within the concentration range owt an, en, isthe concenuation ofthe solution: 4s the predetermined standard deviation for the contol Standard solution. this eterion isnot ane in any baten of anys. the ‘reasons forthe bas thus revealed shall beinvestigate, tnd he boteh of analyses shat then be repeated. Aer at Ina 20 more determinations of this coal standord {olution have been made. wth all valves comping my the bove citer use those values to recaiulate te valve of for subsequent use. 10 Special cases I samoles are excessive coloured a stine such that ears are ‘arerance from hign cancenvations of magnetiem or croce 'stxely, a est smote anal be areoaces by itilation, The aro xoure gven in ISO £564 ena be followed. but neve thet cl tednn ofthe ota srailze mace in’ "9" hecrochor 20d. The sistiate anal rane nautsices ana mace 9 # {akan for dilation, #5, in maiives, sna alga be nota ISO 7150/2-1986 (€) The test sampia thus preosred can then be analysed as de- Serbed in clause 6. However, te result wl be the concer. Yon of ammoniam nitrogen i the test sample. The concent. ton in he origins samples given by the formula here ‘ris the result on the test sample: Viand Vy. are as defines above 11 Test report The test report shat include te flowing information ©) reference to this part of $0 7150, Ia information necessary for comolet ideation of datas of te storage and orecaraton ofthe aboratory samo before analysis sich manifold configuration {gure 1 or 21 nas used ry series: '$0 7180. of ragareea a8 covenas, ‘ogetner Yin any ci cumsunce rat may nave sect ta rau, waste | 82mm 02 si 0.23 [—craei9.2 s ©. Diisee i ee amole rate: up to $0 per hour ae Same to wash waar 9 1! rao: 2 en et ee TL 35205 min { 042 Ciro (2.2) 03 aie cob Stune®30 + 05 min) 0a Saboyite 2.31 Coll ne (20 0.5 min ox Cichorioevanarate (34) - Cole 7tone (23 = S min) Wsie—e—] 3.00 Exhaust co fon ene i "mm bore Fomest Comer cole with mr mixing Sorctometsr 65) nm .. Recorder ft delay tos ae | Figure Same cae: uo t 50 per hou Pome SSemle to wash water (31 ato: 2:2 ir om Lae J 60 | Same ois 20 ue —e 0.32 | —salevine/eivate 38 i30F osm 70 we 0.22 | —cichorssoevanurate (3.4) (252 Smal -—————> wore 1.00 Exhaust bebubbie | . Spectrometer 650 nm. Recorder, 7 (Cols 18 mn smear 2 mm bore Other col with sma mixing tnd clay times are Figure 2 180 7150/2-1986 (E) Annex The effect of other substances on on* ‘The effect of other substances on tis method, using the manifold configuration shown in figure 2 (ammonium niroben concent tion range up to 0,5 mg/l are shown in table 5 Table 5 ~ The effect of other substances on oy, Tantantnon of ubwnce Tia ot waboanes on Submance ‘ere etal ya ; a naa a Sodom ca Pa 20 oom ~o010 Sedum iogenabonie ct 100 +0010 som Socio ste ome +200 vom too Secon erbopheshate enn ito com com Sedum slene i ‘0 oes : Sedum rv 3 0 oon Secu rite 0 -oars Pet he o ® “oes Poti ye toe ‘0 are acu conde eo 1 0m Maren ete cS 0 ora vent ute as 0 <0 vent a on 2 tom Manguest ate fon j e soos oo tne as ® oa concrete es 0 note Utad sere 5s . “ous 00s ‘tons on P oes toms ee cea ° oom soon Ecerteine ee 1 +200 coon ethane conn 1 oom 00m + Data tom the United Kingdom "+ lone eheges if any, have been omined “Mother substances do not imertte, the 9 8% confidence its would be CConcanrain, ey igi) 2.00 a0 95 % confidenceliitsimgili = 0018 + 0.085 TC INTERNATIONAL Iso STANDARD 9308-1 First edition 1980-10-01, Water quality — Detection and enumeration of coliform organisms, thermotolerant coliform organisms and presumptive Escherichia coli — Part 1: Membrane filtration method Qualité de l'eau — Recherche et dénombrement des organismes coliformes, des organismes coliformes thermotolérants et des Escherichia coli présumés — Partie 1: Méthode de filtration sur membrane Reference number 180 9308-1:1880(E) 180 9308-1:1990(E) Foreword ISO (the International Organization for Standardization) is @ worldwide federation of national standards bodies (/SO member bodies). The work of preparing International Standards is normally carried out through ISO technical committees. Each member body interested in a subject for which @ technical committee has been established has the right to be represented on that committee. International organizations, govern- mental and non-governmental, in liaison with ISO, also take part In the work, ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization. Draft International Standards adopted by the technical committees are circulated to the member bodies for voting. Publication as an Interna- tional Standard requires approval by at least 75 % of the member bodies casting a vote. International Standard ISO 9308-1 was prepared by Technical Committee ISO/TC 147, Water quality. 10 9308 consists of the following parts, under the general title Water quality — Detection and enumeration of coliform organisms, thermotolerant coliform organisms and presumptive Escherichia coli — Part 1: Membrane filtration method — Part 2: Multiple tube (most probable number) method Annexes A and B of this part of ISO 9308 are for information only. © 180_ 1990 A rights reserved. No part ofthis publication may be reproduced or utlzed in any form fr by any means, slecranic or mechanical, inlusing photocopying end micrtim, witout Bermiscion in writing from the pubisher. International Organization for Standardization Case Postale 5 + CH-1211 Genave 20 * Switzerland Printed in Switzeriend Introduction The presence and extent of faecal pollution is an important factor in assessing the quality of a body of water. Examination of water samples for the presence of members of the coliform group of organisms”, which normally inhabit the bowel of man and other warm-blooded animals. provides an Indication of such pollution. As the ability of some members. Gf the coliform group of organism to survive in water is limited, their numbers can also be used to estimate the degree of recent faecal pol- lution 1}, See annex A for further microbiological information relevant to water ex- amination for the coliform group of organisms. INTERNATIONAL STANDARD 10 9308-1:1990(E) Water quality — Detection and enumeration of coliform organisms, thermotolerant coliform organisms and presumptive Escherichia coli — Part Membrane filtration method 1 Scope This part of ISO 9308 specifies a method for the de- tection and enumeration in water of coliform organisms, thermotolerant coliform organisms and presumptive Escherichia coli (presumptive £. coli) after filtration through a membrane, subsequent culture on a differential lactose medium (seo 10 7704) and calculation of their numbers in the sample, This method can be applied 10 all types of water except where the presence of suspended matter in- terferes with filtration or large numbers of other ‘organisms may interfere with growth The choice of tests used in the detection and confir- mation of the coliform group of organisms, including E. coll, can be regarded as part of a continuous se- quence, The extent of confirmation wilh a particular sample depends partly on the nature of the water and partly on the reasons for the examination. In practice, the detection in water of presumptive E.coli as defined in 3.9 usually provides an indi- cation of recent faecal pollution. 2 Normative references The following standards contalfi provisions which, through reference in this text, constitute provisions Of this part of ISO 9308. At the time of publication, the editions indicated were valid. All standards are subject to revision, and parties to agreements based fon this part of ISO 9908 are encouraged to investi- gate the possibility of applying the most recent edi- tions of the standards indicated below. Members of IEC and ISO maintain registers of currently valid International Standards 180 3696:1987, Water for analytical laboratory use — ‘Specification and test methods. 180 $667-1:1980, Water quality — Sampling ~ Part {: Guidance on the design of sampling pro- ‘grammes. 180 $667-2:1982, Water quality — Sampling — Part 2: Guidance on sampling techniques. 180 5667-3:1985, Water quality — Sampling — Part 3: Guidance on the preservation and handling of samples. 180 6887:1983, Microbiology — General guidance for the preparation of dilutions for microbiological ex- amination, 180 7704:1985, Water quality — Evaluation of mem- brane filters used for microbiological analyses. 180 8199:1988, Water quality — General guide to the enumeration of micro-organisms by culture. 3. Definitions For the purposes of this part of ISO 9308, the follow- ing definitions apply. 3.4 coliform organisms: Organisms capable of forming colonies aerobically at er 35 °C + 05 °C or 37 *C +05 °C on a selective and differential lactose culture medium with the production of acid (and aldehyde) within 24 h. 3.2 thermotolerant coliform organisms: Coliform organisms as described in 3.1 which have the same ISO 9308-1:1990(E) fermentative properties within 24h, at 44°C 0,25 "Cor 445 °C + 0.25 °C either NOTE 1 As gas production is not detectable on mem- branes, the organisms obtained by membrane filtration are not necessarily the same as those detected by the ‘multiple tube [most probable number (MPK}] method, 3.3. presumptive Escherichia coli (presumptive E.coli): Thermotolerant coliform organisms as de- scribed in 3.2 which also produce gas from lactose {and mannitol) as well as indole from tryptophan within 24h, at either 44°C +025 °C or 445 °C + 0,25 °C. 4 Principle Filtration of a test portion of the sample through @ membrane which retains the organisms; placing the membrane on either a selective lactose agar culture medium or on an absorbent pad saturated with a selective liquid medium containing lactose. Incubation of the membrane for 24h at either 35 °C or 37 °C for the detection of coliform organisms, or alternatively at 44*C for the presence | of thermotolerant coliform organisms. Direct count of characteristic colonies formed on the membrane; subculture of some of these colonies for confirmatory tests for gas and for indole production. Calculation of the numbers of colifarm organisms, thermotolerant coliform organisms and presumptive E. coli likely to be present in 100 mi of the sample. 5 Diluent, culture media and reagents 5.1 Basic materials Use ingredients of uniform quality and chemicals of analytical grade for the preparation of culture media and reagents and follow the instructions given in annex B. For information on storage see ISO 8199, Alternatively, use dehydrated complete media and follow strictly the manufacturer's instructions. For the preparation of media, use glass-distlled water or de-ionized water free from substances which might inhibit bacterial growth under the con- ditions of the test, and in accordance with ISO 3698, 5.2 Diluent For making sample dilutions, use one of the diluents recommended in annex B. Prepare the diluent ac- cording to the instructions given in annex B. 5.3 Isolation media Use one or more of the following culture media el- ther in solid form with agar or as a broth for satu- rating absorbent pads. Instructions for preparing the media are given in annex 8. 534 532 533 54 55 538 337 Lactose TTC agar with Tergitot 7! Lactose agar with Tergitol 7 Membrane enriched Teepot broth! Membrane lauryl sulfate broth Endo medium LES Endo agar ) mFC medium 5.4 Confirmatory media Use one or more of the following, 341 ‘Medium for gas production Lactose peptone water. 54.2 Medium for indole production Tryptone water. 543 Single-tube medium for both gas and indole production Lauryl tryptose mannitol broth with tryptophan, 5.5 Reagents 554 55.2 Kovacs’ reagent for Indole Oxidase reagent for the oxidase test 6 Apparatus Usual microbiological laboratory equipment, includ- 61 Hot-air oven for dry-heat sterilization and an autoclave, Apart from apparatus supplied sterile, glassware and other equipment shall be sterilized according to the instructions given in ISO 8199, 2) Tergitcl 7, Teepot broth are examples of suitable products available commercially, This information is given for the Convenience of users of this part of 180 9308 and does not constitute an endorsement by ISO of these products, 6.2 Incubator or ws controlled at either 37 °C £05 °C. sr bath, thermostatically 35°C £05 °C or 6.3 Incubator or water bath, thermostatically controlled at eilher 44°C $0.25 °C or 445°C & 0.25 °C. 6.4 pH meter. 6.5 Apparatus for membrane filtration. 6.8 Membrane filters, usually about 47mm or 50mm in diameter, with filtration characteristics equivalent to a raled nominal pore diameter of 0,45 um. If not obtained sterile, they shall be steri- lized according to the manufacturer's instructions. 6.7 Forceps, for handling membranes. 7 Sampling Take the samples and deliver them to the laboratory in accordance with ISO 8199, ISO 5667-1, |SO 5667-2 and ISO 5887-3, @ Procedure 81 Preparation of the sample, filtration and inoculation of media For preparation of the sample, making dilutions, fil tration and inoculation of isolation media, follow the instructions given in ISO 8199 and ISO 6887. 8.1.1 For coliform organisms, filter the required volume of the sample, or a dilution of it, through one membrane. Place on the medium chosen, ensuring that no air is trapped underneath it 8.4.2. For thermotolerant coliform organisms, filter the required volume of the sample, or a dilution of it, through one membrane, Place on the medium chosen, ensuring that no air is trapped underneath it NOTE 2 The volume of sample filtered should be the same as In 8.1.1 8.2. Incubation of membranes 24 For coliform organisms, incubate the mem- brane for 18h to 24h at either 35 °C + 0,5 °C or 37°C + 05 °C 8.2.2 For thermotolerant coliform organisms, incu- bale the membrane for 18h to 24h at either 44°C £025 °Cor 445 °C + 0,25 °C. NoTEs 3. The same medium can generally be used for both coliform organisms and thermotolerant coliform Organisms, but mFC medium should be used only at 44°C, and Endo and LES Endo media should be used at 35 °Cor 37°C. 4A preliminary period at a lower temperature such as 430°C for the firet 4h of incubation is recommended to resuscitate stressed organisms, especially in the exam- ination of drinking water. 8.3 Examination of membranes 8.3.1. Coliform organisms Examine the membranes and count as presumptive coliform organisms all colonies, irrespective of size, which show, after incubation at 35 °C or 37 °C, the following characteristics, = On lactose TTC agar with Tergitol (5.3.1): a yel- low. orange or brick red colouration with a yellow central halo in the medium under the membrane. — on laclose agar with Tergitol 7 (5.3.2): a yellow central halo in the medium under the membrane. = On membrane enriched Teepo! broth (5.9.3): @ yellow colour extending on to the membrane. — On membrane lauryl sulfate broth (5.3.4): a yel- low colour extending on to the membrane. ~ On Endo agar or broth (5.3.5): 2 dark red colour with a golden-green metallic sheen. = On LES Endo agar (5.3.6): a dark red colour with a golden-green metallic sheen, 8.3.2. Thermotolerant coliform orgat ms Regard as presumptive thermotolerant coliform ‘organisms all colonies which show, after incubation at 44°C, the same colonial characteristics as those described in 8.3.1. With mEC medium (5.9.7), such colonies are blue in colour. 8.4 Confirmatory tests IIs Important to note that the counts of colonies on membranes at 35 °C or 37 °C and at 44°C are only presumptive coliform results. Since gas production Is not detected, there is also an additional presumption that the organisms forming colonies can also produce gas from lactose. For the exam- ination of raw or partly-treated waters, this may be sufficient, but for potable supplies and other cir- 150 9308-1:1990(E) cumstances, itis important to carry out confirmatory tests, preferably on pure subcultures. 8.4.1 Subculture, Incubation and examination A.A Coliform organisms To confirm the membrane results, subculture each colony (8.3.1) or a representative number of them to tubes of lactose peptone water (5.4.1) and incubate at 35 °C or 37 °C for 48 h: gas production within this period confirms the presence of coliform organisms. 1.2 Thermotolerant coliform organisms and presumptive E. coli For thermotolerant coliform organisms and presumptive E. coli on membranes, whether incu- bated al 44 °C or at 35 °C or 37 °C, subculture each colony (8.3.2) or a representative number of them, to tubes of lactose peptone water and tryptone water and incubate at 44 °C for 24h, Gas production in lactose peptone water confirms the presence of thermotolerant coliform organisms, and develop- ment of 2 red colour at the surface of the tryptone water culture after the addition of 0,2 ml to 0,3 ml of Kovacs’ reagent (5.5.1) confirms the presence of presumptive E. coli Notes 5 The use of lauryl tryptose mannitol broth with tryptophan allows both gas and indole production to be demonstrated in a single tube. 8 The detection of presumplive E. coll is regarded as isfactory evidence of faecal pollution. However, further tests for the confirmation of €. coll may be carried out If considered necessary (see 8.5) 7. When subculturing from colonies on the membrane to tubes of confirmatory madia, its preferable to subculture algo to a plate of nutrient agar medium for the oxidase fest. 85 Oxidase test ‘Some bacteria found in water may conform to the efinition of coliform organisms in most respects, but are able to produce gas from lactose only at temperatures below 7 °C. They therefore give neg- ative results in the standard confirmatory tests for coliform organisms and their presence in water is not usually regarded as significant. Aeromonas spe- cles, which occur naturally in water, have an opti- mum growth temperature in the range 30°C to 35°C but may nevertheless produce acid and gas {rom lactose at 37 *C. They are distinguishable from thé coliform group by a positive oxidase reaction, 8.5.1 Carry out the oxidase test with pure subcul- tures of lactose-fermenting organisms, grown on nutrient agar medium, as follows: — place 2 to 3 drops of freshly prepared oxidase reagent (5.5.2) on a filler paper in a Petri dish; — with 2 glass rod, swab stick or platinum (not nichrome) wire loop, smear some of the growth fon the prepared filter paper (see note 7) = regard the appearance of a deep blue-purple colour within 10 s as a positive reaction NOTE 8 On each occasion that the oxidase reagent is used, control tests should be conducted with cul tures of an organism known to give a positive reaction (Pseudomonas aeruginosa) and a negative reaction (Ecol n of results 9. Expres: From the numbers of characteristic colonies counted fon the membranes and taking account of the results of the confirmatory tests performed, calculate the numbers of coliform organisms, thermotolerant coliform organisms and presumptive E. coli present in 100 ml of the sample in accordance with ISO 8199, 8.4, according to the following equation: AXNXV,XE GC BxM where Cis the confirmed colony count per 100 mi 4 is the number of colonies actually con- firmed: Bis the number of colonies subcultured for confirmation; N Is the number of characteristic colonies on the membrane (8.3.1 and 8.3.2); ¥, is the test volume of water sample file tered (8.1.1 and 8.1.2); V, is the reference volume for expression of results (100 ml); Fis the ditution factor. 10 Test report ‘The test report shall contain the following informa- tion a) a reference to this part of ISO 8308; b) all details necessary for complete Identification of the sample; ©) the technique and isolation medium used 4) the confirmatory media and tests used: e) the time, temperature and conditions of incu- bation; ISO 9308-1:1990(E) f) the resulls expressed in accordance with clause 9: 9) any other information relevant to the method, 1990(E) Annex A (informative) Further microbiological information relevant to water examination for the coliform group of organisms For routine water examination purposes, the coliform group of organisms may be described gen- erally in. microbiological, though not taxonomic, terms as follows, Coliform organisms are Gram-negative, non- sporing, oxidase-negative, rod-shaped bacteria, which are capable of aerobic and facultatively anaerobic growth in the presence of bile-salts (or other surface-active agents with similar growth- Inhibiting properties). They are also able to ferment lactose (and mannitol) with the production of acid, gas and aldehyde within 48 h, when incubated at a temperature of 35 °C to 37 °C. Thermotolerant coliform organisms are coliform organisms which exhibit the same fermentative and biochemical properties when incubated at a tem- perature of 44 °C to 44,5 °C. Presumptive E coli are thermotolerant coliform ‘organisms which are also able to produce indole from tryptophan, E.coli may be regarded as presumplive E. coli which also give @ positive result in the methyl red test and can decarboxylate L-glutamic acid, but which are not able to produce acetyl methyl carbinol, utilize citrate as the sole source of carbon (or fo grow in potassium cyanide (KCN) broth. ISO 9308-1:1990(E) Annex B (informative) Culture media, reagents and diluents Isolation media Lactose TTC agar with Tergitol 7 Melt the basal medium and cool to between 45 °C. and 50°C. Add the TIC and Tergitol 7 solutions aseptically, mixing thoroughly but avoiding the for- mation of bubbles after each addition. Distribute in Petri dishes to a depth of about 5 mm. If not for im- mediate use, store at 4 °C in the dark for not longer than 10 days. Lactose agar with Tergitol 7 Basal medium Lactose 209 Peptone 109 Yeast extract 89 Meat extract 59 Bromothymol blue 0.05 9 Agar 16g to 25g) Distilled water 41000 mi 41) Depending on the gel strength of the agar. Dissolve the ingredients In boiling water. if neces: sary, adjust the pH so that after sterilization it Is 7.2 & 0,2. Distribute the medium in bottles in vol- umes of 100 ml and sterilize in the autoclave at 421 °C 1°°C for 15 min, TTC solution 2, 9, S-Triphenyh-tetrazolium chloride (TTC) Distilled water 0.05 400 mi Dissolve the TTC in some of the water and make up to 100 ml. Steriize by membrane filtration through ‘a membrane of pore size < 0,2 um. Tergitol 7 solution Tergitol 7 O29 Distilled water 1 Dissolve the Tergitol 7 in some of the water and make up to 100 mi. Sterilize in the autoclave at 421 °C + 1 °C for 15 min Complete medium Basal medium 100 mi TTC solution 5 mi Tergitol 7 solution 5 mi Pepione 5 Yeast extract 3g Lactose 409 Tergitol 7 0.1 mi Bromothymol blue 0,025 9 Agar 159 Distilled water 41000 mi Dissolve the Ingredients in the water. Dispense in bottles in volumes of 100ml and autoclave at 421°C 41°C: for 15min. The final pH is 6,9 + 0,2. For use, melt_and distribute in Petri dishes to a depth of about 5 mm. Store at 4 °C in the dark for not longer than 10 days. Membrane enriched Teepol broth Peptone a Yeast extract 69 Lactose 309 Phenol red [0.4 % (mim)] aqueous solution 50 mi Teepol 610 4mi Distilled water 4000 mi ‘Add the peptone and yeast extract to the water and steam to dissolve. Add the lactose, phenol red and Teepol afterwards and mix gently to avoid froth. The final pH shall be 7.4 to 7.5 and it may be necessary to adjust the pH to about 7.6 before sterilization in order to achieve this. Distribute in screw-capped bottles and autoclave at 110 °C to 115 *C for 10 min. 1S0 9308-1:1990(E) Membrane lauryl sulfate broth Peptone 409 Yeast extract 59 Lactose 309 Phenol red [0,4 % (m/m)] aqueous solution 50 mt Sodium lauryl sulfate, high purity 1g Distilled water 1000 ml ‘Add the ingredients to the water and mix gently to avoid froth. The final pH should be 7.4 to 7,5 and It may be necessary to adjust the pH to about 7.6 be- fore sterilization to achieve this, Distribuie in screw-capped bottles and autoclave at 110 °C to 415 °C for 10 min Endo medium WARNING — Basic fuchsin is suspected of being a carcinogen. Tryptose Thiopeptone Trypticase (casitone or tryptone) Yeast extract Lactose Sodium chloride Dipotassium hydrogen phosphate Potassium dihydrogen phosphate Sodium lauryl sulfate Sodium desoxycholate Sodium sulfate Basic fuchsin Distilled water Dissolve ingredients in the water containing 20 mi of 95 % (V/V) ethanol. Do not autociave but heat to boiling, remove promptly and cool to between 45 *C and 50°C. Final pH shall be 7,2 + 0,2. Store finished medium at 4 °C in the dark and discard unused me- dium after 4 days. NOTE 9 This medium may be solidified by the addition Of 12 g per litre to 15.g per litre agar before boiling. LES Endo agar WARNING — Basic fuchsin Is suspected of being a carcinogen. Yeast extract Trypticase (casitone or tryptone) Thiopeptone ‘Tryptose Lactose Dipotassium hydrogen phosphate Potassium dihydrogen phosphate Sodium chloride Sodium desoxycholate Sodium laury! sulfate Sodium sulfite Basic fuchsin Agar Distilled water Dissolve ingredients in the water containing 20 mi of 85 % (¥/V) ethanol. Do not autoclave but heat to boiling, cool to between 45°C and 50 °C and dis- pense in 4 ml volumes in small Petri dishes (60 mm diameter). Final pH shall be 7.2 + 0,2. Store plates at 4°C in the dark and discard unused medium after 2 weeks. Do not expose to direct sunlight. mFC medium Trplose 1006 Proteose peptone No. 3 or polypeptone 5.09 Yeast extract 309 Sodium chloride 509 Lactose 1259 Bile salts No. 3 or bile salts mix- ture 159 Aniline blue tg Distilied water +1000 mi Rehydrate in the distilled water containing 10 ml of 4% rosolic acid in 0,2 mol/l NaOH. Heat the me- dium to the boiling point, promptly remove from heat and cool to below 45 °C to 50 °C, Do not sterilize by autoclaving. Final pH shall be 7,4 + 0,2. Store the finished media at 2 °C to 10°C and discard any un- used medium afler 96h Notes 10 This medium may be solidified by the addition of 1.2 % to 1,5 % agar before boiling 11 Rosolic acid will decompose if sterlized by autoclaving. The stock solution should be stored in the dark at 2°C to 10 °C and be discarded afler 2 weeks, oF sooner if ite colour changes from dark red to muddy brown. Confirmatory media Lactose peptone water (for gas production) Peptone 109 Sodium chloride 59 Lactose 109 Phenol red [0.4 % (n/n)] aqueous solution 25 mi (or Andrade’s indicator) (40 m Distilled water 4000 mi Dissolve the ingredients in the water and adjust to pH 7,5 + 0,2. Add the phenol red indicator and dis- tribute in § mI volumes into tubes containing in: verted inner fermentation (Durham) tubes, Alternatively, adjust to pH 8,8 to 7,0 and add the ‘Andrade’s indicator. Autoclave at 110 °C for 10 min. Alternatively, steam for 20 min on each of three successive days. Test for sterility by incubation at 37 °C for 24h. It is important to ensure that, after autoclaving and before use, the Durham tube is completely filled with medium. Otherwise a false positive result for gas production may be obtained, Andrade’s indicator This is prepared by dissolving 0,5 9 of acid fuchsin in 100 mi of distilled water. Add 17 ml of sodium hy- droxide solution (1 mol/l) and leave at room tem- peralure overnight. The solution should be straw coloured the following morning. If it is as at all brownish, add a little more sodium hydroxide sol- ution and allow to stand again. This solution is strongly alkaline, and consequently media to which Ils added should be adjusted previously to a pH of about 6.8. Tryptone water (for indole reaction) Certain peptones which give satisfactory results in test at 35°C or 37°C are not satisfactory for the indole test at 44 °C. Tryptone has been found satis- factory and is recommended Tryptone 209 Sodium chloride 5 Distilled water 1000 mi Dissolve the ingredients in the water and adjust to pH 7.5. Distribute in § mi volumes and autoclave at 145 °C for 10 min, ISO 9308-1:1990(E) NOTE 12 The addition of 0,1 % (mim) 0 or OL tryptophan may Improve the performance of the medium, Lauryl tryptose mannitol broth with tryptophan (Single-tube medium for both gas and indole pro- duction.) Tryptose 209 Mannitol 59 Sodium chioride 59 Dipotassium hydrogen phosphate 2,75 9 Potassium dihydrogen phosphate 2,75 g Sodium lauryl sulfate Ota L(~) Tryptophan og Distilled water 1000 mi Add the Iryptose, sodium chloride, mannitol, phos- phates and tryptophan to the water and warm to dissolve. Add the sodium lauryl sulfate and mix genily to avoid froth. Adjust to pH 6,8 + 0.2. Dis- tribute in § ml volumes in tubes containing an in- verted inner fermentation (Durham) tube. Autoclave at 115 °C for 10 min, Reagents, Kovacs’ reagent for indole p-dimethylaminobenzaldehyde 5g ‘Amyl alcohol (free from organic bases) 75 mi Hydrochloric acid (p = 1,18 g/ml) 25 mi Dissolve the aldehyde in the amyl alcohol. Add the concentrated acid with care, Protect from light and store at 4°C. NOTE 13. The reagent should be light yellow to light brown In colour; some samples of amyl alcohol are un- satisfactory, and give a dark colour with aldehyde. Oxidase reagent Tetramethyl-p-phenylenediamine hydrochloride ong Distilled water 410 mi ‘This reagent does not keep and it must therefore be freshly prepared for use in small amounts each time that itis needed, ISO 9309-1:1990(E) Diluents Peptone diluent (0,1 %) Peptone 109 Distilled water 41000 mt Dissolve the peptone in about 950 mt of the water, Adjust the pH with sodium hydroxide solution or hydrochloric acid (1 mol/l), so that after sterilization it will be 7,0 + 0,1. Make up to 1000 ml with the water, dispense in convenient volumes and autociave at 121 °C £ 1 °C for 15 min, Peptone galine solution Peptone. Sodium chloride 859 Distilied water 41000 mt Dissolve the constituents in about 950 ml of the wa- ter by boiling. Adjust the pH with sodium hydroxide solution or hydrochloric acid (1 mol/), so that afer sterilization It will be 7,0 + 0,1, Make up to 1000 mi with the water, dispense in convenient volumes and autoclave at 121 °C + 1 °C for 15 min, Ringer's solution — quarter strength ‘Sodium chloride 2.250 Potassium chloride 0405 9 Calcium chloride, anhydrous 029 Sodium hydrogen carbonate 0.05 9 Distilled water 4000 mi Dissolve the ingredients and dispense in convenient volumes. Sterilize by autoclaving at 121 °C + 1 °C for 15 min. Final pH should be 7,0 + 0,1. 0 Phosphate buffer solution Potassium dihydrogen phosphate 42,5 mg Magnesium chloride 190 mg Distilled water 4000 mt Preparation a) Phosphate solution Dissolve 34 g of the phosphate in 500 ml of dis- tilled water. Adjust to pH 7,2 0,5 with sodium hydroxide solution (1 mol/i) ‘and make up to 1000 mi with distilled water. b) Magnesium chloride solution Dissolve 38 g of magnesium chloride in 1009 ml of distilled water. Final solution For use, add 1,25 ml of phosphate solution and 5.0 mi of magnesium chloride solution to 1000 mi of,

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