Disclosure to Promote the Right To Information

Whereas the Parliament of India has set out to provide a practical regime of right to information for citizens to secure access to information under the control of public authorities, in order to promote transparency and accountability in the working of every public authority, and whereas the attached publication of the Bureau of Indian Standards is of particular interest to the public, particularly disadvantaged communities and those engaged in the pursuit of education and knowledge, the attached public safety standard is made available to promote the timely dissemination of this information in an accurate manner to the public.

इंटरनेट मानक

“!ान $ एक न' भारत का +नम-ण”Satyanarayan Gangaram Pitroda

“Invent a New India Using Knowledge”

“प0रा1 को छोड न' 5 तरफ”Jawaharlal Nehru

“Step Out From the Old to the New”

“जान1 का अ+धकार, जी1 का अ+धकार”Mazdoor Kisan Shakti Sangathan

“The Right to Information, The Right to Live”

“!ान एक ऐसा खजाना > जो कभी च0राया नहB जा सकता है”Bhartṛhari—Nītiśatakam

“Knowledge is such a treasure which cannot be stolen”

“Invent a New India Using Knowledge”

है”ह”ह

IS 1622 (1981): Methods of sampling and microbiologicalexamination of water [CHD 32: Environmental Protection andWaste Management]

IS: 1622 -1981(Reaffirmed 1996)

Indian Standard

METHODS OF SAMPLING ANDMICROBIOLOGICAL EXAMINATION or WATER

(First Revision)

Fourth Reprint FEBRUARY 2003

UDC 663.63 : 543.39

co Copyright 1982

BUREAU OF INDIAN STANDARDSMANAK BHAVAN, 9 BAHADUR SHAH ZAFAR MARG

NEW DELHI 110002

Gr 10 October 1982

I. I 1622 • 1981

Indian Standard

METHODS OF SAMPLING ANDMICROBIOLOGICAL EXAMINATION OF WATER

( First Revision )

Water Sectional Committee. CDC 26

CluJi",....DR NILAY CSAUDSl1RI

Mnn6",MBMBER-SBOBBTABY ( .dltnlUlt, to

Dr Nila y Chaudhuri )SSRlj.S.CBAUDBRI

Sual H. G. O.BO ( Allmat", )CBIEI' WATBR ANALYST, KING INSTITt1r.,

~lADl\.8

SHRI]. D. CRUZSHBJ K. R. SAUU ( Altmlllt, )

DBPU'rY DIRECTOB ( CBBY ), ROSa, LUCKNOWCHEMIST & METALLURGI8T, WB8TJl:UN

RAILWAY, BOMBAY (AU"IUJI, )SRal M. V. DESAI

SHBI MANQAL SINOR ( Alt""." )SaBI O. L. DUQQAL

SURI R. VABADBAlf (Alt"nat, )SHRI B. K. DUTTA

SBRI N. RAMAOHANDRAN ( Alt"nat, )SsaI R. C. DWIVBDI

SHBI S. P. SAXIINA. ( Allmaat, )SaRI R. S. EXBOTBDB A-f. I. GURBAXA.:MJSaBI C. P.JAIN

SBBIJ. JRA (.4l"""', )SaRI K. K. KAKj,TB

SSBIJ. D.joY8r.oK (Alt""at,)DB P. K. MATHUBPROF R. S. MBHTA

SURI M. D. DAVJl1 (AllmJat, )~BMBaR-SECRBTABY

SHill S. K. MITRA

SHBI S. D. MUNDBBASaaI U. C. MANKAD ( Altmaat, )

SSRI D. V. S. MUBTBYSHBI P. K. BAN.RIBB ( .AII,rnal, )

8Bnl R. NATABA.JAN5MBI G. KRI8BNAYYA (AII,rnal, I )Da A. PaABHA][AB RAO (lfltlmtlt, II )

SHBI S. K. NBOQI

DR M. BANBRJJC.. ( All"""" )DR v. PACBAIYAPPANSHRI R. PABAKj,8rVAJI

SRBI M. V. NANOTI ( Allmaat,)SHal A. K. PODDAB

SRRIA. K.DA8 (AUwuu)58BI H. S. PUBI

SBBJ Q}JU'r RAJ ( All"""'" )

R,/W,,.,,,i"lCentral Board for the Prevention It Control of Water POJlutiOD.

New Delhi

Haryana State Board for the Prevention &. Control of WaterPollution, Chandigarh

Director of Public Health, Government of Tamil Nadu, Madras

Delhi Water Supply & Sewage Disposal Undertaking, New Delhi

Railway Board ( Ministry of Railways)

Indian Chemical Manufacturers" Association, Calcutta

Ballarpur Industries Ltd, Ballarpur

Ion Exchange ( India) Ltd, Bombay

U. P. Water Pollution Prevention & Control Board, Lucknow

Hindustan Organic Chemicals Ltd, RasayaniThe Tata Iron & Steel Co Ltd,Jamlhed~ur

Central Electricity Authority, New Delhi

Kerala State Board for Prevention & Control or Water Pollution,Trivandrum

Bhabha Atomic Research Centre, BombayGujarat Water Pollution Control Board, Gandhinagar

Maharashtra Prevention of Water PoJlution Board, BombayWest Bengal Prevention & Control of Water Pollution Board,

CalcuttaGeo·Miller & Co Pvt Ltd, Calcutta

M. P. State Prevearion & Control of Water Pollution Board , Bhopal

Bharat Heavy ElectricaIs Ltd, Hyderabad

Institution of Public Health Engineers India, Calcutta

The Fertiliser Association of India, New DelhiNational Environmental Engineering Research Institute ( CSIR ),.

Nagpur

Steel Authority of India Ltd, New Delhi

Punjab State Board for the Prevention lit Control orWater Pollution;Patiala

( Ctmtinu,donpag, 2 )

(f) Copyright 1982BUREAU OF INDIAN STANDARDS

Thll publicadoD 11 protected under the l"diGII CoJt:pi,AI Ad (XIV of 1957) and reproduction in whole or inpart by aDy mean. except with written permissioD of the publisher lhaU be deemed to be an infriulement orcopyrilbt UDdertbe laid Act.

II I 1622 • 1881

(Conl;nu,dfrom Is" I )

MmlbersSaar B. B. RAO ,

Da I. R.ADBAKBlaBBAH ( Mt",.." )SaRI S. liAN•••TB R.lO

SaRI C. H. GOVIWDA RAO (Al'"III''' )SHal G. S. RAYDB S. K. Roy

SJlBI P. K. CBAJ[BAVABTY ( .41''''''', )SURI K. RUDS.".

SUBI S. N. CaAKaABA.'!'1 ( ..411''''', )SRal A. K. SABA

SHIU P. BBATTAOBABYYA ( AI,."." )SHaJ R. M. SHAH

SHRIR.K.G.-DBI(AUmMU)SRRI P. R. SOftB

SHal S. P. Iy•• (AI""', )SaBI K. SURIYANABAYAllAJI

SliBI G. C. GAUTAII (AI,,,.,,)DB HARt BH~OWA.,

Director ( Chem )

R,pr,s,nti"1MiDlltry of Works It HouliDI

Karuataka State Board for Prevention It CoDtroJ of WaterPOllutiOD. Bansalore

The Fertilizer ( PlaoDiDI " DevelopmeDt) ludia Ltd SindriThe Alkali &. Chemica) CorpOration of India Ltd, Cal~utta

EDlineerl India Ltd. New Delhi

National Telt Houle, Calcutta

Tata Chemicals Ltd, Bombay

Excel Industries Ltd, ~mbay

All India Distillerl' AllOCiatioD, New Delhi

Director General, lSI ( EM-t#eio MM'" )

.ft S""lIJry. SBRI A. K. BAHL

AaliataDt Director ( Chem ), lSI

ianel for Methods of Test for Water and Efiluents, CQC 26 : PI

eo.....DB B. K. HAlfD.

M.."Saal Y. P. K.4a:A.B ( .41,,,,,,,, to

Dr B. K. Haada )5SBI S. K. BHATTAOBABJ'l"A

SaRI A.' M. Kl1LKABli1 ( ~11mUIt. )DR S. H. CL.BEDa P. K. MATHUBPaol' R. S. M.BTA

DR U. I. BBAft ( ~11mwJU)DB D. V. S. MURTHY

Da G. K. KBABB ( Altlrfldl' )Da S. P. PAlfDB

SaRI M. V. NdO'1'1 (Al''''', )DB N. M. PARBAD

DB s. a.J08SI ( AI""..,. )Ds I. RADBAKRU"AJfA

SHRI M. R. PAaTBA...TBY ( tAl",,,.,, )Saal G. S. RAYa••aBs••TATIQ

RBPRIISIIN'l'ATIV8RSpaI:8KNTA'l'IV8SDKI C. P. SHABDA

SURI M. S. DBI.ORA ( .41",.".,.)Da P. D. SRABMA

8aal D. C. M£~Bva ( MI.".,,)DR ( SliT) Z. R..TU••L

SURI K. C. ).IABmDBA ( AI",..,. )

Central Ground Water Board, New Delhi

IOD Exchange ( Ipdia ) Ltd. Bombay

Industrial Toxicology Releareb Centre ( aSIR), LucknowBbabba Atomic Research Centre, Bombay~uj.rat Water Pollution Control Board. GaDdhina.ar

M. P. State Prevention &. Control of Water Pollution Board, Bhopal

National Environmental Engineering Research Institute (aSIR).Nagpur

National Environmental Engioeeriol Research Jaltitate (CSIR ),Nagpur

Ministry of Worb at Housing

The Fertilizer ( Planninl It Development) India Ltd. SindriA. P. State Board (or Prevention and Control of Water Pollution,

Seeunderabad('.entral Board Cor the Prevention" Control of Water PollutioD.

New DelhiMabaraahtra Prevention of Water Pollution Board, BombayNational Insttrure or Oceanography ( aSIR ), BombaySbri Ram lnatitute for Industrial Research, belhi

HinduataD Copper Ltd, Khetri Copper Complex, Khelrin_lar

The IDstitute or Scimce, Bombay

2

JS I 1622 • 1981

CONTENTS

PAGE

O. FOREWORD 4

1. SCOPE 5

2. SAMPLING 52.1 SAMPLING POR BACTERIOLOGICAL EXAMINATION 52.2 SAMPLING FOR BIOLOGICAL EXAMINATION 6

3. BACTERIOLOGICAL EXAMINATION 73.1 GENERAL EQUIPMENT 73.2 STANDARD PLATE COUNT 83.3 TasT FOR COLIFORMS 93.4 TEST FOR FAECAL STREPTOCOCCI 143.5 TEST FOR CLOSTRIDIUM WELCHII 153.6 TEST FOR IRON BACTERIA 163.7 TEST FOR SULPHATE REDUCING BACTERIA 173.8 TEST POR SULPHUR BACTERIA 173.9 TEST FOR GELATIN LIQ,UEFYINO BACTERIA 173.10 TEST FOR SLIME FORMING BACTERIA 18

4. MICROSCOPIc EXAMINATION 184.0 OUTLINE OP THE METHOD 184.1 CONCENTRATION OF THE ORGANISMS 184.2 PROCEDURE POR ENUMERATION 19

ApPENDIX A PARTICULARS TO BE SUPPLIED ALONO WITH SAMPLES 21

APPBNDlx B TABLES OF MOST PROBABLE NUMBBRI 22

3

AMENOMENT NO. 1 JA~UARY 1985TO

15:1622-1981 METHOOS OF SAMPLING AND MICRO­BIOLOGICAL EXAMINATION OF WATER

(Fil'8t Revision)

(PagB 9. clause 3.2.4.3. lin. 2) ­Substitute '48' fo~ '24' and add the follovingNote after this clause:

'BOTE~The incubation may be carried outfor 72 hours where nece~sary'.

(CDC 26)

Printed at Simco Printing Press, Delhi

AMENDMENT NO. 2 DECEMBER 2000TO

IS 1621: 1981 METHODS OF SAMPLING ANDMICROBIOLOGICAL EXAMINATION OF WATER

( Fint Revisioll )

( Page 7, clause 3.1.1.1 ) - Insert the following new clause at tbe end of3.t.l.land renumber thesubsequent clauses:

'3.1.1.2 Lamina, flow air Ullit - Vertical or horizontal kind for use of pouringsterile nutrientmedia into sterile petridishes to avoidcontamination.'

( Page 8, clause 3.1.1.5 ) - Insert the following at the end of firstparagraph:

'If test tubes are used for culture work, these sball be plugged withnon-absorbent cotton or caps and shall be sterilized and autoclaved It 121°C for15 minutes.'

( Page 8, clause 3.%.1, line 11 ) - Substitute 'while higherplate counts givetbe earliest sign of contamination' for 'while rising.-----------pollution'.

( Page 9 t clause 3.2.3.1, line S ) - Substitut~ 'IS g of 19ar powder' forc1.5 percent Iglr powder'.

(Page 9, clause 3.1.4.1,line4 ) - Insertthe word 'sterile' before the words'petri disht

•

(Page 9, clause J.J.4.4, last senntence) - Substitute 'Record the numberof incubation and days IDd temperature of incubation' for 'Record thenumber of._-------- incubitiolll ' .

( Page 9, clause 3.3.1, line 19 ) - Insen 'Most Probable Number' beforetbe word ·MPN'.

[ Page 10, clause 3.J.l.1(b), line 22 ) - Substitute 'sterlize at 11SoC forabout 15 minutes(notexceeding 30 minutes) in the autoclave at 1.02 +0.03 kg!cm2 (15 + 0.5 psi) gauge pressure' lor 'sterlize at l1SoC for 10 minutes in theautoclave'.

( Page 11, clause 3.3.1.1 ) -Insert the following noteafter J.3.I(b)(iv):

-NOTE -lupl" iodine is used I. morellatlad elhylaloobol is used u. decolourizer.'

I

Amend No. Zto IS 16%% : I"l

[ Pfl8t! 11, claus« 3.3.t.2(c)(iii) ) - Substitute 'Gram-staia' for 'gram ­stain' .

[ PG. 11,clause 3.3.1.2(c)(iv) ] - Substitute 'flOm the 19arsllnt' lor 'ontile 19arslant' .

[ PIIge 13, clause 3.3.5.Z(iii), I. 3 ] - Substitute 'cycloheXimide' for•cycioheximiDe,.

(Page IS, clawe3A.l.Z(b), lilae 12] - Substitute 'MPN'101 'MP '.

( Page 1St clause 3.4.2.2 , line 13 ) - Substitute ·10 I of Igar' lor'1 percent Iglr' .

(Page 16, clause 3.5.4.3 , line 7) - SutBtitute 'Wish, dry and examineunder oil immellioD, using I compouDd microicope'lor 'Wish, dry, mount andobserve uDder microscope'•

( Page 17, clause 3.1.2.1, line 10 ) - Substitute ·0.03 kg/cm2, lor·0.3 kg/cm2,.

(PlIge 17,clause 3.8.%.1, line 12 ) - Substitute 'about 15 minutes but notexceeding 30 minutes' lor '10 minutes).

(CHD 12)

Printed at Simco Printing Press, Delhi

2

AMENDMENT N(). 3 DECEMBER 2002T()

IS 1622: 1981 METHO(>S OF SAMPLING ANDMICROBIOLOGICAL EXAMINATION OF WATER

( First Revision )

( Page 13, clau..se 3.3.S.3(iii), line ) J - Substitute 'cyclohexamide' for'cyclohexarnine' .

( Amendment No.2, pl1Ls:e 2. lines 5 and 6 related to clause 3.3.5.2 ) -­Delete.

(CHD 32 )

Printed atSimco Printing Press, Delhi

AMENDMENT NO. 4 MAY 2003TO

IS 1622: 1981 METHODS OF'SAMPLING ANDMICROBIOLOGICAL EXAMINA·TION OF WATER

( Fint R"irion )

i Pag« 17, clause 3.7.4) -Substitute the following for theexisting:

'3.7.4 Detection ofSulpluJte Reducing Bacteria

Distribute 10 ml of double strength medium (double the quantity of mediumconstituents in I 000 ml distilled water) in 5 test tubes. Similarly, distribu~5 ml of single strength medium in 10test tubes. Sterilize at 1.02 ±0.03 kg/emgauge pressure (l5±O.S psi gauge pressure and 121°C temperatureapproximately) for 15 min.

Add the following samples aseptically as stated below andmix well:

Add 10 ml of the sample to 5 double strength sterile test tubes. AddI ml of the sample to 5 single strength sterile test tubes. Add 0.1 ml ofthe sample to 5 single strength sterile test tubes. Cover the surface ofeach test tube by sterile liquid paraffin to a depth of 1-2 mm. Incubatethe tubes at 28-30oC for5-7 days.

The production of black colour in medium will indicate the presence ofSulphate Reducing Bacteria (SRB).

After incubation, observe and note down the tubes showing black colour.Compare the tubes showing black colour from double strength andsingle strength media. With the help of McCrady's chart, quantify thenumber of SRB in thewater sample.

NOTE -If soil sample is tobe analysed, thea lake I 000 mg. 100 l1li.10ma in five tatlubes each. Add 10 ml of rhe double SIreDJ1h medium ia the I 000 mg tubes and SmI<ithe single strength medium in each of the rest 10 tat tubes. Pollow the rest of theprocedure asdescribed above."

( Page 25. Table 3 ) -Insert the following Table 4 after Table 3:

1

(CHD 32)

2Repropaphy Unit, 818. New Delbi.lnclia

reprelentinl the container and lull be transferred immediately tothoroughly clean and dry containers, sealed air-tight and labelled withparticulars given in A-I.5. The individual samples so obtained shall bedivided into three setl in I such a way that each set hal a samplerepresenting each selected container. One of these setl shall be markedfor the purchaser, the other for the vendor, and the third Cor the referee.

·A-3.2 Preparatloaof a Composite SllDple - From the material from eachselected container remaining after the individual sample has been taken.equal quantities of the material shall be taken and mixed together ,10 al to·Corm a composite sample weighing not less than 360 g. This composite.ampJe Ihall be divided into three. approximately equal parts an4 trans­ferred to clean and dry gla.1 contalnere and labelled with the particularsgiven in A-I.S. One of these composlte samples shall be marked for thepurchaser, the other for the vendor, and the third for the referee.

A-3.3 Refer.e Sa.plea -- Referee sample. shall consi.t of a set ofindividual samples (s,e A-3.1) and a composite sample (se. A-3.2)marked for this purpose and shall bear the seall of the purchaser andthe vendor. These shall be kept at a place agreed between the two.

A-4. NUMBER OF TESTS

A:..a.1 Telt. for requirements in respect of barley-grain content and proteinshall be conducted on each of "the samples constituting a set of individualtest samples ( se« A-3.1 ) .

.A~.2 Telts for the remaining characteristics) namely, moisture, totalash, acid insoluble ash, crude fibre and alcoholic acidity, shall beconducted on the composite sample ( see A-3.%).

A-5. CRITERIA FOR CONFORMITY

A-S.l The lot shall be considered satisfactory in respect of the require­ments or A-4.1, if each individual sample satisfies all the requirements.

A-S.2 The lot shall be considered satisfactory in respect of the require­ments of A-4.2, if the test results on the composite sample satisfy thecorresponding requirements.

A-5.3 The lot shall be declared to be in conformity with all the require­.menta of this specification, if it has been found satisfactory in accordancewith A-5.Z. •

(Pag,4, Claus« e-l.! ) - Substitute the following for the existingclause:

'C-l.l PreparatloD of Simple - Grind in a pestle and .mortarabout 100 g of the material so that at least 90 percent passes through

425·micrqn IS Sieve. Tra~.(er thi. prepared sample to • well.t~pperedglall bottle (or u~e .s indicated under C-I.2, '-3.1, G-2.1and H-2.1.

NO'I'II- In 0... 411-IPioron 18 8ieve (aonformiDI to 18: 480-1111) Ia nota.ailable. BS Tel' Sie.eae, A8TM 8ien '0 or T,l. Tel' 8Jeve II. _blob ba".their apertaree wi*in ,be limit. lpeoifted for tbi. 18 Bieve.ma), be UIICI.·

(Pal. 7, Cia•• H.L2 )-- Subltitute the foJlowinl for the exi.ti~1

clause:

c H-l.2 Sta.wd SotIl- HJtlroxlde Soilltl08 - 0·05 N.·

( PDI. 7. Cla",e H-l.3 ) - Substitute the following for the exiltingclause:

c H-I.3 PlaeDOlpbtllal.l. ladleator Solatlo. - Di.lolve 0'1 I ofphenolphthalein in 100 ml of 60 percent rectified spirit. t

(Pal. 7J Cia",. B-2.1 ) - Substitute the following for the ext.tinlclause: .

c 8-2.1 Weigh S g of the sample into a conical,la•• Itorpered lalkand add 50 ml of 90 percent alcohol (by volume previou.lyneutralized againle phenolphthalein. Stopper, shake and allowto stand for 24 houri, with oec••ionallhakiDg. Filter the alcoholicextract through a dry filter paper. Titrate 10 mI of the combinedalcoholic extract againlt the standard lodium hydroxide uliniphenolphthalein al indicator. Calculate the percentage of alcoholicacidity .1 sulphuric acid. •

•24-52 .4N' '12-25 .4N'~( Clau,e 8·3.1, line 3 ) - Sub.titute ._-- W lor W

4

aeprolraphy Unit, BIS} New Delhi, India

·8 I 1622. 1981

Indian Standard

METHODS OF SAMPLING ANDMICROBIOLOGICAL EXAMINATION OF WATER

( First Revision)

o. FOREWORD

0.2 This Indian Standard (First Revision)was adopted by the Indian Standards Institu­tion on 30 November 1981, after the draftfinalized by the Water Sectional Committeehad been approved by the Chemical DivisionCouncil.

0.2 Microbiological examination of waterincludes both bacteriological and biologicalexaminations. Bacteriological examination ofwater is necessary for determining its fitnessfor use for human consumption, and for usein industries such as food processing and dairy,photofilm, etc. Water used for drinking, foodprocessing ann dairying should be free fromfaecal or sewage contamination because micro­organisms causing water-borne diseases such astyphoid and paratyphoid fevers, food poison-ing, gastroenteritis, cholera; dysentery anddiarrhoea are excreted in the. faeces .ofindividuals suffering from the disease. Thedetection of these pathogenic organisms in asample of water is difficult and may not alwaysbe accomplished with certainty. Bacterialorganisms of the coliform and faecal strep­tococci groups, however, inhabit the intestinaltract of man and animals in great abundanceand are readily detectable. Hence their pre­ence in a sample of water is looked upon as anindication of the probable presence of intesti­nal pathogenic organisms, while their absencefrom wate~ usually precludes the presence ofsuch pathogens. Tests for the presence ofClostridium welch;; are also carried out onsamples of water for obtaining supplementaryevidence of faecal pollution.

0.2.1 The presence of organisms belongingto the groups such as iron bacteria, sulphurbacteria, sulphate reducing bacteria, slime­forming bacterial and gelatin liquefyingbacteria is undesirable in water used for drink­ing purposes, air-conditioning, paper manu­facture and many other industrial uses. Someof these organisms are known to causecorrosion.

0.2.2 Algae and other microscopic plantsand animal-life in water may cause odour and

taste problems and also affect suitability of thewater for use in various industries. However,this standard in-cludes only microscopic exami­nation and enumeration of these organisms.

0.3 This standard was first published in 1964.This revision incorporates the two amendmentsissued to IS : 1622-1964*, the membrane filtertechnique for coliforms and faecal streptococci,test for faecal coliforms, delayed incubationmethod for total coliforms and spore stainingtechnique for clostridium welchii.

0.4 This standard prescribes Iaboratory pre­paration of culture media. However, dehydra­ted media commercially available may also beemployed. Since preparation of culture mediaand solutions is a critical aspect of waterquality testing, the date of receipt of media( dehydrated powder medium) and the dateof opening should 'be recorded. Where practi­cal, 125 g bottles should be purchased to ensureminimum exposure.

0.4.1 ·When a new lot of media is used, thecontents should be tested for expected perfor­mance. It should be stored in a cool, dry placeaway from sunlight.

0.5 It is recommended that laboratory purewater suitable for microbiological applicationsshould be used as far al possible. For the pur­pose reference may be made to StandardMethods for the Examination of Water andWastewater. 1975 Ed 14. ( Geldreich, E. E. andCJark H. F. 1965 Distilled water suitability formicrobiological applications. ]. Milk FoodTechno! 28 : 351 )

0.6 In the preparation of this standard, consi­derable assistance has been derived from thefollowing publications, and the draft wasprepared by National Environmental Engineer­ing Research Institute, Nagpur:

International standards for drinking water.1971. World Health Organieation,Geneva.

Environmental microbiology - A courseoutline, NEERI. Nagpur,

• Methods of lamplinl and teat for microbiololical examlnatioD water used iD industry.

4

Bacteriological Examination of Watersupplies. Report No. 71, 1969. HerMajesty's Stationery Office, London.

Standard methods for the examination ofwater and wastewater. 1975 Ed 14.American Public Health Association;American Water Works Association; andWater Pollution Control FederationU.S.A.

1. SCOPE

1.1 This standard prescribes methods ofsampling and microbiological examination ofwater.

2. SAMPLING

2.1 Sampling for Bacteriological Exami­nation

2.1.1 Sampling Bottles - Samples for bacte­riological examination shall be collected inclean, sterilized, narrow mouthed neutralglass bottles of 250, 500, or 1 000 mJ capacity.The bottle shall have a ground glass stopperhaving an overlapping rim. The stopper shallbe relaxed by an intervening strip of paperbetween the stopper and the neck of the bottle.The stopper and the neck of the bottle shallbe protected by paper or parchment cover.The bottle shall be sterilized in hot air oven atIbO°C fa I' one hour or an autoclave at1'02 ± 0'03 kg/em! gauge pressure ( 15 ± 0-5psi gauge pressure, 120°0 temperature approxi­mately ) for 15 minutes. The sampling oottleshall not be opened except at the time ofsampling.

NOTE 1 - Discard bottles which have chips,cracks and etched surfaces. Before usc, bottlesshould be thoroughly cleaned with detergent andhot water, followed by a hot water rinse to removeall traer's of detergents. Then rinse them three timeswith laboratory pure water.

NOTE 2 - A chelating agent should be addedto sample bottles used to collect samples, suspectedto contain more than 0·01 mf{{l of heavy metalssuch as copper, lead, zinc. nickel, etc. Add 0'3 1111

of 15 percent EOTA - Na4 salt for each 125 m l ofsarnp le,

2.1.1.1 Dechlorination - If the water to besampled contains or is likely to contain chlorine,sodium thiosulphate shall be added to theclean, dry sampling bottles before sterilizationin an amount to provide an approximate con­centration of 100 mg/l in the sample. Thiscan be done by adding 0·5 ml of 5 percentthiosulphate solution to a 250-ml bottle.Sterilize in an autoclave.

2.1.2 Sampling Procedure - The samples shallbe representative of the water to be tested and

.Rules for rounding off numerical values (revised).

s

IS I 1622 • 1981

Biological methods for monitoring theenvironment, EllA - 19i8.

0.7 In reporting the result of a test or analysismade in accordance with this standard, if thefinal value, observed or calculated, is to berounded off, it shall be done in accordancewith IS : 2-1960*.

they should be collected with utmost care toensure that no contamination occurs at thetime of collection or prior to examination.The sample bottle shall not be opened till thetime of filling. The stopper shall be removedwith care to eliminate soiling. During samp­ling, the stopper and the neck of the bottleshall not be touches and they shall Le protec­ted from contamination. The bottle shall beheld near the base, filled without rinsing, andthe stopper replaced immediately. Then thebrown paper wrapping should be tied to pro­tect the samples from contamination,

2.1.2.] Sampling from taps - Flame the tap( in case of plastic t.ap, apply alcohol or spirit,preferably rectified) and allow it to dry). Thetap shall be opened fully and the water allowedto run to waste for two to three minutes or fora sufficient time to permit clearing of theservice line. Thewlow from the tap shall thenbe restricted to permit filling the bottle with.out splashing. Leaking taps, which allowwater to flow over the outside of the tap shouldbe avoided as sampling points.

N()'r~ _.- If the tap is connected to an overheadstorage tank, this fact should be recorded in thesalnpling report.

2.1.2.2 Sampling direct from a SOUTCt - \\·henthe sample is to be collected directly from astream, river, Jake, reservoir, spring, or ashalJow well, it shall be representative of thewater that will be taken for supply to theconsumers. lienee a sample shall 110t be takentoo far from a point of draw-off or too close.Areas of relative stagnation in a stream shouldbe avoided. Samples {ronl a river, stream,lake, or a reservoir can often be taken by hold­ing the bottle in the hand near its base andplunging it neck downward. below the surface.The bottle shall then he turned until the neckpoints slightly upward, the ~outh beingdirected against the current. I f no currentexists, as in a reservoir, a current shall beartificially created by pushing the bottlehorizontally forward in a direction awayfrom the hand. I f it is not possible tocollect samples in this way, a weight may beattached to the base of the bottle which can

IS I 1622 • 1981

then be lowered into the water. In any case,damage to the bank should be guarded against,otherwise fouling of the water can occur.Special apparatus which permits mechanicalremoval of the stopper of the bottle below thesurface is required to collect samples from thedepths of a Jake or a reservoir. If the sampleis to be taken from a well, fitted with a hand­pump, Harne the mouth of the hand-pump( in case of a plastic mouth, apply alcohol orspirit, preferably rectified, and allow it to dry)and pump the water to waste for four to fiveminutes before the sample is collected. If thewell is fitted with a mechanical pump, thesample should be collected from a tap on thedischarge. (If the tap is connected toan overhead storage tank, this fact should berecorded in the sampling report.) If there is nopumping machinery, the sample can be collec­ted directly from the well in a sterilized bottlefitted with a weight at the base. In this case,care shall be taken to avoid contamination ofthe sample by any surface scum. Where it isnot possible to collect the sample directly intothe bottle, as for example where there is a highbank the sample may be obtained by means ofsuitable metal jug. The jug is sterilized bypouring into it a teaspoonful of methylatedspirit and tilting the jug in such a way that thespirit comes in contact with the entire innersurface of the jug, and igniting. The jug shallbe lowered to the required depth and thendrawn up and down two or three times beforeit is brought to the surface. It shall be rinsedout at least twice before the sample is taken.Should the jug come in contact with the bot­tom or skid along the surface so that it mayhave collected the surface film, the sample shallbe discarded, the jug re-sterilized and anothersample drawn. The water from the jug shallbe poured into the bottle and the glass stopperof the bottle be replaced, care being taken toavoid the cover being caught between thestopper and the neck of the bottle.

2.1.3 Size of the Sa",ple - The volume ofthe sample shall be sufficient for carrying outall the tests required. The sampling bottleshould not be filled up to the brim and 2to 3 ern space should be left for effectiveshaking of the bottle.

2.1.4 Preservation and Storage - The initialtime limit for starting analysis should be 1hour but not more than 6 hours after collectionof water samples. Under exceptional circums­tances the analysis should be commenced atleast within 30 hours and sample should bekept in dark at 1-4°0. If sampling and transittime requires more than 6 hours, temporaryfield laboratory should be set up or the delayedincubation procedure (se, 3.3.5) should beadopted if MF technique is used.

6

2.1.5 Identifyi", Data - All samples shall belegibly marked with the source of the sample,date and time of collection, and the name anddesignation of the person collecting the sample.As results of laboratory examination of thesample shall always be considered in conjunc­tion with the sanitary survey of the watersupply system, it is important that when sub­mitting a sample for analysis, complete andaccurate data of the nature and source of thesupply, topography of the water shed, possi­bility of pollution gaining access to the source,methods of treatment adopted, the condition ofthe distribution system, and such other infor­mation as would be relevant from sanitary view­point is furnished. It shall be ascertainedwhether the tap from where the sarnple iscollected is supplying water from a service pipedirectly connected with the main or with acistern or a storage tank. Specimen form forsuch information is given in Appendix A.

2.2 Sa.pliog Cor Biological Examination

2.2.1 General Considerations - The micros­copic organisms other than bacteria in waterinclude a large variety of algae, moulds orfungi, yeasts, protozoa, rotifers, crustacea, ani­malcula, etc many of which affect the qualityof water for drinking and industrial uses.Those organisms which occur free-floating inwater are collectively known as plankton, whilethose which inhabit the bottoms of the tanksand streams or are attached to the stones orother submerged objects are called periphyton,The examination of the nature and number ofthe organisms present in the sample is of use inunderstanding the nature of pollution, thecause of undesirable tastes and odours, slimegrowth, ecosystem imbalances, etc.

NOTE - The sampling report should mentionthe time, depth and frequency of sampling.

2.2.2 ProcedureJOT Sampling

2.2.2.1 The sample of natural plankton­producing water shall be collected in clean,neutral glass bottles of 2-litre capacity, fittedwith ground glass stopper. The bottle shallnot be filled completely and a small air spaceshall be Jeft below the stopper.

2.2.2.2 A concentrated sample or catch ofthe plankton organisms and other particulatesuspended matter in the water shall be collec­ted with the aid of a plankton net. The netshall be conical in shape, of suitable size, witha circular mouth and made of bolting silk clothwith more than 6 000 meshes per square centi­metre. The net shall be hauled through thewater in an oblique or horizontal directionfor a certain distance, lifted from the water,allowed to drain and the organisms in the netwashed down into a container by splashingwater on the outer surface of the net. The

catch shall then be made up to a knownvolume with the water. Nets provided withclosing devices shall be used for collectingsamples of plankton from different depths.

2.2.2.3 For quantification of the planktonit is recommended to strain known volume ofwater through plankton net. Number of orga­nisms caught in the catch may be used tocalculate back the original number per unitvolume of the sample.

2.2.2.4 Sampling phytoplankton with netsprovides data of limited value since the totalcount, volume, bio-mass and species composi­tion are not measurable. Because of selectivityof mesh size the smaller plankton ( nanoplank­tons) which may contribute as much as 60percent of the total bio-mass are not collected.In such situation sedimentation membranefiltration or centrifugation are recommended.

2.2.3 The samples shall be examine-d within2 to 3 hours after collection, when the orga­nisms are alive. If this is not possible, thesamples shall be preserved in ice or in therefrigerator ( 3 to 4°C) for a few days takingcare not to allow it to freeze. If the examina­tion is to be made later, the samples shall bepreserved as follows:

To each 100 ml of the sample, add about3 ml of 2 percent formaldehyde solution( made by diluting 5 ml of 37 - 40 percentaqueous formaldehyde solution to 100 ml withdistilled water ), O·S ml of 20 percent detergentsolution made by diluting 20 ml of liquiddetergent to 100 ml with distilled water and5 - 6 drops of copper sulphate solution 21 per­cent (vlv). This preservative maintains cellcolouration and is effective indefinitely. Storethe preserved sample in the dark.

3. BACTERIOLOGICAL EXAMINATION

3.1 General EquipmeDt

3.1.1 Equipment

3.1.1.1 General - It is essential for accurateand satisfactory laboratory work that goodequipment in proper working order be provi­ded. 'rhus, the minimum laboratory equip­me nt listed, must be available in an approvedlaboratory and all items should meet the mini­mum requirements given. Additional itemsof equipment not listed, will be required in anapproved laboratory and they should meetsimilar standards of quality and operation.

3.1.1.2 Incubators - Incubators shouldmaintain a uniform and constant temperature(35-37°C or 44-45°0) at all times in allparts. This can be accomplished by the useof a water-jacketed or anhydric type of incu­bator, with thermostatically controlled low­temperature electric heating units properly

7

18 I 1622 • 1181

insulated and located in or adjacent to wallsor floor of chamber, and preferably equippedwith mechanical means of circulating air.

Incubators should also be provided withshelves spaced to ensure uniformity of tempe­rature throughout the chamber. The insidedimensions of the chamber should be at least50 X 50 ern at the base and 60 em high, toaccommodate a maximum of 200 Petri dishes;2-5 em space should be provided betweenadjacent stacks of plates and between walls andstacks.

Accurate thermometers, with bulb conti­nuously immersed in liquid (glycerine, wateror mineral oil ), should be maintained withinthe incubator and daily readings of the tempe­ratures should be recorded. In addition, it isdesirable to maintain a maximum and mini­mum registering thermometer within theincubator on the middle shelf to record tempe­rature variations over a 24-hour period.Temperature variations within the incubatorfilled to maximum capacity should be determi­ned at intervals. It is recommended that arecording thermometer be installed in everyincubator whenever possible, so that a perma­nent record of temperature variations withinthe incubating chamber may be kept.

Incubators equipped with high-temperatureheating units are unsatisfactory, since suchsources of heat frequently cause localized over­heating. Incubators, so heated may be madeto operate satisfactorily by replacing the hightemperature units by suitable wiring, arrangedto operate at a lower temperature, and byinstaJIing mechanical air circulation. It isdesirable, where ordinary room temperaturesvery excessively, that laboratory incubators bekept in special rooms which may be main­tained at a few degrees below the recommen­ded incubator temperature.

3.1.1.3 Water-baths - Water-baths -are use­ful for carrying out the 44°C fermentation test.They should be capable of maintaining a tem­perature of 44°C.45°C. They should beequipped with mercury-toluol or other reliablethermostats for sensitive regulation of thetemperature, and should be adequately insula­ted against heat loss. An accurate thermometershould be provided, with its bulb placed at thelevel of the medium in the fermentation tubes.A continuous-recording thermometer is advisa­ble.

3.1.1.4 Sterilizers

a) Ovens - Hot-air sterilizing ovens shouldbe of sufficient size to prevent crowdingof the interior and constructed to giveuniform and adequate sterilizing tempe-rature" and equipped with suitablethermometers capable of registering

IS I 1622 • 1981

accurately in the range 160·180°0. Theuse of temperature-recording instrumentis optional.

b) Autoclaves - Autoclaves should be ofsize sufficient to prevent crowding of theinterior, and constructed to provide uni­form temperatures within chambers upto and including the sterilizing conditionsof 1 02 ± 0'03 kg/cml gauge pressure( 15 ± 0·5 psi gauge pressure, 120°0temperature approximately). Theyshould be equipped with pressure gau­ges, properly adjusted safety valves, andaccurate thermometers with bulb pro­perly located on exhaust line, so as toregister minimum temperature withinsterilizing chambers (temperature re­cording instrument optional). Inemergencies, a pressure-cooker may besubstituted for an autoclave, if resultshave previously been demonstrated to besatisfactory with this method.

3.1.1.5 Glassware - Clean glassware is cri­tical to ensure valid results. Previously usedor new glassware should be thoroughly cleanedwith phosphorus-free laboratory detergent andhot water, followed by at least three rinseswith laboratory pure water. Generally, pipet­tes, dilution bottles and petri dishes arerequired.

a) Pipettes - Pipettes may be of any con­venient size ( generally 1 ml or 10 ml )provided it is found by actual test thatthey deliver accurately the requiredamount in the manner in which they areused. The error of calibration shouldnot exceed 2·5 percent. Pipettes withunbroken tips and with graduationsdistinctively marked should be used.Pipettes with damaged tips should berepaired or discarded.

b) Dilution bottles - Bottles or tubes of re­sistant glass, preferably Pyrex, closedwith glass stoppers, rubber stoppers, orscrew caps equipped with liners that donot produce toxic or bacteriostatic com­pounds on sterilization, should be used.Cotton plugs shall not be used as clo­sures. Graduation levels should beindelibly marked on the side.

c) Petri dishes - Petri dishes 100 mm indiameter, with the side wall at least15 turn high, should be used with glassor porous tops, as preferred. The bot­toms of the dishes should be free frombubbles and scratches and should beflat, so that the medium will be of uni­form thickness throughout the plate.When available, sterilized disposableplastic petri dishes may be used as analternative.

8

3.1.1.6 Inoculating needle and loop - It shallbe 4 mm in diameter formed at one end of alength of 0·375 mm thick wire of nichrome,platinum or platinum-iridium alloy. I t shallbe 38 mm long from the loop to the holder.The holder consists of a thin metal rod ortube. It shall be sterilized by flaming.

3.1.1.7 Refrigerators - An approved labora­tory should have a refrigerator of sufficientcapacity for the required work load and capa­ble of maintaining a continuous temperaturebetween ooe and 5°C. An electrically opera­rated refrigeration unit provides the mostefficient service.

3.1.1.8 Colony eounter- An effective devicefor examining colonies, providing a magnifica­tion of 3 x , should be available. In general,a Quebec or similar colony counter will besuitable for this purpose.

3.2 Standard Plate Count

3.2.1 General - Standard plate count(which is an empirical method) serves toindicate the efficiency of certain processes inwater treatment, particularly coagulation,filtration and disinfection and the cleanlinessof the mains, reservoirs, etc. It provides anestimate of the general hygienic quality ofwater, which is important where large scalepreparation of food and drink is concerned.Low countsare of importance for avoiding foodspoilage, while rising plate counts give theearliest sign of pollution.

3.2.1.1 The standard plate count methodis a direct measurement of the viable aerobicand facultative anerobic bacteria in a waterenvironment capable of growth on the selectedplating medium. The procedure does not allowthe more fastideous aerobes or obligate anaero­bes to develop. Also the bacteria of possibleimportance in water such as Crino-thrix,sphaerotilus and actinomycetes wilJ not developwithin the incubation period specified for pot­able water. Clumps of organisms in the watersample which are not broken lip by shakingresult in under estimate of bacterial density.Since an aggregate of cells will appear as onecolony on the growth medium. The numberof types of bacteria that develop are influencedby the time and comparative temperatureof incubation, the pH of the medium, thelevel of the oxygen, the presence of specificnutrients on the growth medium competitionamong cells for nutrients, antibiosis, medation,etc.

3.2.2 In solid medium counting of orga­nisrns depends on the fact that living cells willproceed to multiply and in time will producesufficient progeny to form a colony visible tonaked eye. Since bacteria occur in water assingle cells, pairs, groups, chains or even denseclumps, not every individual living cell will

develop into a separate colony on incubation.Therefore, number of colonies appearing on aplate does not necessarily represent the totalnumber of organisms present in test volume.The results are expressed as number of coloniesper ml,

3.2.3 Medium and Reagtnt

3.2.3.1 Nutrient agar - Dissolve 1 g glucose,5·0 g of peptone and g·o g of beef extract in1000 ml of distilled water. Adjust the pH to7'2, distribute in required quantity and addI·S percent agar powder. Sterilize at 1-02±0·03 kg/em' gauge pressure (15 ± 0-5 psigauge pressure, 120'>0 temperature approxi­mately ) for 15 minutes in the autoclave.

3.2.3.2 Dilution uiaur

a) Buffered dilution wate, - To preparestock phosphate buffer solution, dissolve34 g of potassium dihydrogen phosphate( KH1POc ) in 500 ml of distilled water,adjust pH to 7-2 with sodium hydroxidesolution ( 1 N ) and dilute to 1 litre withdistilled water.

Add 1-25 ml of stock phosphate buffersolution to 1 litre of distilled wa ter,Dispense in amounts that will provide18 ± 0'4 ml or 9 ± 0-2 ml in 150 X25 rom or 150 X 18 mm test tubes res­pectively. Sterilize in autoclave at 1'02 ±0'03 kg/ems gauge pressure (15 ± 0-5psi gauge pressure, 120°C temperatureapproximately) for 15 minutes.

b) Quart" st"ngt!a ,ing,,'s solution - Dis­solve g-OO g of sodium chloride, 0·42 gof potassium chloride, 0·48 g of calciumchloride and 0-20 g of sodium bicarbo­nate in 1 litre of water. This solutionis known as Ringer's solution. Dilute500 ml of this solution to 2 litres toobtain quarter strength Ringer's solu­tion. Dispense in amounts that willprovide 18 ± 0·4 ml or 9 ±O"2 ml in150 X 25 mm or 150 X 18 mm testtubes respectively. Sterilize in autoclaveat 1'02 ± 0·03 kg/ems gauge pressure( 15 ± 0·5 psi gauge pressure, 120°Ctemperature approximately) for15 minutes.

3.2.4 Proeedur

3.2.4.1 Prepa,atio" and dilution - Shakethe samples about 25 times. Withdraw requiredportion with a sterile pipette. and introduceinto the petri dish or dilution tube.

3.2.4.2 Plating - Place 1·0 rnl, or 1·0 mlof other suitable dilution to be used for platingin the petri dish first. Then add to the petridish 10 to 15 ml of melted nutrient agarmedium at a temperature of 43 to 45°0

9

IS I 1622 • 1981

( tolerable to the skin). The nutrient agarand the sample shall be thoroughly mixedover the bottom of the petri dish by tilting androtating the dish several times, Allow theplate to solidify and place immediately in theincubator in an inverted position.

3.2.4.3 Incubalion - Incubate the platesat 37°C for 24 hours.

3.2.4.f Counting - In preparing plates,plant such amounts of water for dilution whichwill give from 30 to 300 colonies on a plate.Always have two or more plates for each dilu­tion. Report the result as the average ofall plates falling within limits. I t is notdesirable to plant more than 1·0 ml in a plate.If the colonies are more than 300 or less than30 from 1 ml sample, disregard it. In prac­tice, counts less than 30 occur when chlorina­ted water samples are plated. When thenumber of colonies is more than 300 in aplate, report the count at C TNC ' ( too nume­rous to count). Counting shall be done withan approved counting aid, such as colonycounter, Record the number of colonies tothe nearest 5 units per ml and report thetemperature of incubations.

3.3 Test for CoUforms

3.3.0 The coliform group includes aU of theaerobic and facultative anaerobic gram nega­tive, non-spore forming rod shaped bacteriawhich ferment lactose with gas formationwithin 48 hours at 37°0. The standard test forthe estimation of number of the coliformgroups may be carried out either by themultiple tube dilution test (presumptive test,confirmed test, or completed test) or by themembrane filter technique.

S.3.1 Multiple Tube Dilution Ttst ( MTD )­The presumptive. confirmed and completedtests are presented as total independent proce­dures. In using these procedures, the workermust know what is to be the stage at whichthe test is to be ended, and details of theprocedure throughout. Thus, if the workerknows that the test will be ended at the con­firmed test, he will stop at the confirmed teststage only. All the necessary information re­garding the sample should be recorded, It isconvenient to express the results of the exami­nation of replicate tubes and dilutions in termsof most probable number. This term is actu­ally an estimate based on certain probabilityformulae, The most satisfactory informationis obtained when the largest portion examinedshows no gas in all or a majority of the tubes.The MPN value for a given sample is obtainedby the use of MPN tables. Standard practicein water analysis is to plant five tubes for eachdilution and a minimum three different dilu­tions are employed. The results are to be

5g75 ml

IS I 1622 • 1981

recorded in the proper form. Table of MPNare given in Appendix B.

3.3.1.1 Media and reagentsa) Dilution water - se« 3.2.3.2.b) MacConkey broth - This is used as a

presumptive medium for the enumera­tion of coliform bacteria in watersamples. Its composition is an as under:

Peptone 20 gLactose 10 gSodium chloride 5 gBile salt 5 gDistilled water 1 000 ml

In place of bile lalt. which is a com­mercial product. sodium taurocholate orsodium tauroglycocholate may be used.

Dissolve all the ingredients and adjust thepH to 7"4. After adjusting the pH) add 1 mlof 1 percent alcoholic solution of bromocre­sol purple or 5 ml of 1 percent aqueous solutionof neutral red. This will be the single strengthmedium. Distribute 10 ml of the mediuminto 150 X 15 mm test lubes and add aDurham's tube (25 X 5 mrn ) in an invertedposition. Plug the tubes with non-absorbentcotton and sterilize at 115°0 for 10 minutes inthe autoclave. This medium is used for 1 mland the decimal dilutions of the water sample.For 10 ml and larger aliquots a double strengthmedium is used. For the double strengthmedium add the above ingredients in doublethe quantities in 1 OOll ml of distilled water.This medium is dispensed into 10 ml quanti­tie, in 150 X 18 mm test tubes added withDurham's tube and sterilized.

c) Briliiam Green bil«lactose broth ( BGB ) ­This medium is used asa confirmatorytest for coliforms as well as for faecalcoliforms, Its composition is as under:

Peptone 10 gLactose 10 gBile salt 20 gDistilled water 1 000 ml

Dissolve all the ingredients and adjust thepH to 7-4. Add 1 33 ml of 1 percent aqueoussolution of brilliant green indicator. Distri­bute 4 ml quantities into 150 X 12 mm testtubes and add a Durham's tube to each. Afterplu~gin~with non-absorbent cotton, sterilize at1·02 ± 0'03 kg/eml gauge pressure ( 15 ± 0·5psi gauge pressure, 120°0 temperature appro­xirnately ) for 15 minutes in the autoclave.

d) Pepton, water - This is used for indoletest or for preparing a liquid culture ofan organism. Its composition is asfollows:

Peptone 10 gSodium chloride 5 gDistilled water 1 000 ml

10

Dissolve all the ingredients. Adjust thepH to 7"4. Dispense 4 ml medium into 100 X12 mm tubes and plug with non-absorbentcotton. Sterilize in the autoclave at 1-02 ±0·03 kg/eml gauge pressure ( 15 ± 0·5 psigauge pressure, 120°C temperature approxi­mately ) for 15 minutes.

e) Mae Conkey ala, - The medium is usedfor the completed test or for IMViCclassification of coliforms. ItI composi­tion is as under:

Peptone 20 gLectose 10 gSodium chloride 5 gBile salt 5 g

Distilled water 1000 ml

Dissolve all the ingredients and adjust thepH to 7·4. Add 10 ml of 1 percent aqueoussolution of neutral red indicator and 15 g ofagar. Steam the medium for 15 to 30 minu­tes 10 that agar is dissolved properly andsterilize in autoclave at 1'02 ± 0·03 kg/em'gauge pressure ( 15 ± O-S psi gauge pressure,120°C temperature approximately) for 15minutes. After sterilization, cool to 45°0 andprepare the plates by pouring 15 ml of meltedagar per plate.

.\llow to solidify, invert and incubate at37°C for drying as well as for sterility test.

e) Nut,ient agar slants - Prepare the nut­rient agar as prescribed in 3.2.3.1.Dispense while in the melted conditionabout 10 ml quantity into each tube( 150 JDm X 15 mm). Sterilize in theautoclave at 1-02 ± 0·03 kg/eml gaugepressure (15 ± 0'5 psi gauge pressure.120°C temperature approximately) for15 minutes. After sterilization the slantsare prepared by keeping the tubes in aslanting position and allow them tosolidify. Unless they are to be used, theyshould be stored in a refrigerator.

g) KODtJ&·s "ag'''' - I t is used for indoletest. Its composition is as under:Paradimethyl aminobenzal­

dehydeAmyl alcohol or n-butanolConcentrated hydrochloric

acid 25 ml

Dissolve paradimethyl aminobenzaldehydein amyl alcohol and then add 25 ml of hydro­chloric acid. The reagent shall be yellowishin colour. Store in amber coloured glasastoppered bottle.

h) Grdm slaining "aglnlsi) Crystal violet is used as a primary

Itain.

Soluli01l A - Crystal violet( 85 percent dyecontent) 2 g

Ethyl alcohol( 95 percent ) 20 m1

Solution B - Ammoniumoxalate 0-8 g

water 80 ml

Mix solutions A and B in equal parts. Itis sometimes found, however, that this gives soconcentrated a stain that gram-negativeorganisms do not properly decolonize. Toavoid this, dilute solution A as much as tentimes. Use 20 ml of this diluted solution andmix with solution B.

ii) Lugol's iodine - Dissolve 1 g of iodinecrystals and 2 g of potassium iodidein 300 ml of distilled water.

iii) Safranin is used as a counter stain.Dissolve 25 g ofsafranin dye in 100 rotof 95 percent ethyl alcohol. Add10 ml of the solution to 100 ml ofdistilled water.

iv) Ethyl alcohol - 95 percent.

3.3.1.2 Procedure - Shake the water sam­ples thoroughly before making dilutions orbefore inoculation.

a) Presumptive test:

i) Use MacConkey broth. Inoculate aseries of fermentation tubes withappropriate measured quantities ofthe water to be tested. The concen­tration of nutritive ingredients in themixture should be sufficient andaccording to requirements. Ten mland above aliquots should be inocu­lated in double strength and 1 mland its dilution should be inoculatedinto single strength medium.

ii) Incubate all tubes at 37°C for 24 to48 hours. Examine each tube at theend of 24 ± 2 hours for gas produc­tion and if no gas has been formed,reiucubate for another 24 hours andat the end of 48 hours, examine again.Record the presence of or absence ofgas at each examination of the tubesregardless of the amount.

iii) Formation of the gas within 48 ± 3hours in any amount, in the innerfermentation tubes, constitutes a ·pos­sible presumptive test. The absenceof gas formation at the end of 48 ± 3hours of incubation constitutes anegative test.

h) Confirmtd test - The medium used forconfirmed test is brilliant green bilelactose broth ( BGB ).

11

18 11622 • 1981

i) Submit all primary fermentationtubes showing any amount of gas atthe end of24 hours incubation to theconfirmed test, If additional primaryfermentation tubes show gas at theend of 48 hours incubation, these tooshall be submitted to the confirmedtest. Use a sterile metal loop 3 to4 mm in diameter to transfer ~ne ortwo loopful of medium from the pre­sumptive positive tubes to a tube ofBGB .. broth. .When making suchtransfers, gently shake the tube first ormix by rotating. Incubate the inocu­lated tubes at 37°C for 48 ± 3 hours.

ii) The formation of gas in any amountin the Durham's tubes of BOB tubeat any time within 48 ± 3 hoursconstitutes a positive confirmed test.

e) Completed testi) It may be applied to positive BOB

tubes. Shake the tube, and streakwith the help of a loop on the Mac­~onkey agar plates as soon as possible10 such a way so as to get discretecolonies. Incubate the plates at 37°Cfor 24 ± 2 hours.

ii) From each plate pick up typical oratypical colonies and inoculate lactosebroth and nutrient agar slants.Incubate at 37°C for 24 to 48 hours.

iii) Nutrient agar slants can be used forgram:-stain. If organisms are gramnegative, non-spore forming bacilliand if gas is produced in laclose broththe test is considered completed andthe presence of coliform organisms isdemonstrated.

iv) Gram-stain technique - Prepare a thinsmear of the growth on the agarslant on a clean glass slide. Air dry,fix by passing the slide through aflame, and stain for 1 minute withammonium oxalate-crystal violetsolution. Wash the slide in .waterimmerse in Lugol's iodine solution fo;1 minute. Wash the slide in water,blot dry; decolor ize with ethylal~ohc:>1 for 30 seconds, using gentleagitauon, Blot and cover with coun­ter stain for 10 seconds !with safranin,then wash, dry and examine underoil immersion.CelJs whi~h d~co]orize. and acceptthe safranin stain are pink in colourand defined as gram-negative inreaction. Cells which do not de­colorize but retain the crystal violetstain, are deep blue in colour and aredefined as gram-positive.

3.3.1.3 Computing and recording of MPN­The number of positive findings of coliform

Dissolve the above ingredients in 1 000 mlof distilled water containing 20 ml of ethylalcohol ( 95 percent). Heat the medium toboiling point. Do not heat for a long time ordo not submit to steam under pressure. Thefinal pH ahould be between 7.1 to 7.3.

3.3.2.6 Proeedur« - Saturate the pad in asmall petridish with M. Endo broth .and placethe filter on it. Remove excessive medium bytilting. Invert the plate. and incubate at 37°0for 24 hours under humid chamber. Allcolonies which produce a dark red colony witha metallic shine within 24 hour incubation areconsidered members of coliform group and

4·375 g1-375 g

0·05 g0·1 g2-1 g1·05 g

dihydrogen

3.3.2.5 Medium - Medium used for enu­merating coliform! by membrane filter tech­nique is known as M. Endo broth. Thecomposition of the medium is given below:

Tryptone or polypeptone 10·0 gThiopeptone or thiotone 5·0 gCasitone or Trypticase S·O gYeast extract 1·5 gLactose 12·5 gSodium chloride 5·0 gDipotassium hydrogen

phosphatePotassium

phosphateSodium lauryl sulphateSodium desoxycholateSodium sulphiteBasic fuchsin

oximately ) for 15 minutes. Aftersterilization, inmediately release steamin the autoclave by opening the outlet,

3.3.2~3 Selection of sampl, site - The sizeof the sample is governed by the expectedbacterial density. An ideal quantity shouldresult in the growth of 20 colonies and notmore than 200 colonies of all types. Always,filter sample in duplicate. If water is heavilycontaminated use less quantity ofwater. Whenless than 20 ml is to be filtered, dilute the portionto a minimum of 30 ml before filtration.

3.3.2.4 Filtration of sampl, - Using sterileforceps, place a sterile filter over the porouspl~te ?r stainless steel mesh of the apparatus,grid lade up. Place the funnel unit carefullyover the receptacle and lock it in place. Thenpass the sample through the filter undervacuum. Rinse the filter by filtration, two tothree times with 20 to 30 ml of sterile bufferwater. Unlock the assembly. remove the filterby sterile forceps and place it on the sterile pador agar with a rolling motion to avoid theentrapment of air.

3.3.2.2 D,s"iption of MF assembly

a) There are many varieties of MF assem­blies. The common one is milliporestandard hydrosol filter holder. Mostcomponents are made of stainless steel.These are locking ring, fine mesh stain­less steel screen for supporting the filtermembrane and the funnel assembly.

b) Only those filter membranes may beused which have been found, throughcomplete laboratory tests certified bymanufacturer, to provide full bacterialretention, stability in use, freedom fromchemicals immical to the growth anddevelopment ofbacteria, and satisfactoryspeed of filteration, They should pre­ferably be gridmarked in such a waythat bacterial growth is neither inhibi­ted nor stimulated along the grid lines.The membrane filters of 47 mm in dia­meter and 0·45 micron pore size is used.

-c) Absorbent pads for nutrients should con­sist of discs or filter paper or other mat­erials known to be of high quality andfree of sulphites or other substances thatcould inhibit bacterial growth. Theseshould be approximately 45 mm in dia­meter and of thickness sufficient toabsorb l·a to 2·2 ml of nutrient. .

4) Sterilization of filter assemblyt filters andabsorbent pads is carried out at 1-02 ±0·03 ks;/cm

'gauge pressure ( 15 ± 0 5 psi

gauge pressure, 120°0 temperature appr-

group organisms (either presumptive, confir­med, or completed) resulting from themultiple portion decimal dilution plantingshould be computed as combination of thepositives and recorded in terms of the MostProbable Number. The Most Probable Num­ber for a variety of planting series and resultsis given in Appendix B.

3.3.2 Membra", Filt" ( MF) Technique

3.3.2.1 Ouuin« of the method - The mem­brane filter technique in water analysis isbecoming more and more popular due to itsadvantages over the multi tube dilution t~ch­

nique. Results are obtained within 24 or48 hours as compared to 48 to 96 hours bymulti tube dilution technique. Much largervolume and hence more representative samplecan be tested. Results are obtained withmuch greater precision and require less labora­tory space, equipment is not bulky andinvolv~s less labour. The limitations of thistechnique are few. Samples with high turbi­dity and less indicator bacterial count will bedifficult to examine. Samples having highnumber of non-indicator organisms will giveJess count.

12

IS I 1622 - 1981

3.3.5.3 Media

NOTE - Cycloheximide is used for sam ples thathave shown problems of overgrowth with fungi. Itis a powerful skin irritant and should be handledwith care.

filter. The filter retaining the micro-organismsis placed on an absorbent pad saturated withM.Endo preservative medium or M-ColiformHolding Broth in a tight-lidded petridish andtransported from field site to the labora tory,The holding medium maintains the viabilityof the coliform organisms and generally doesnot permit visible growth during transport. Inthe laboratory the filter is transferred toM-Endo growth medium and incubated at35°0 for 18-24 h. Sheen colonies are countedas total coliforms/IOO ml,

Add 48 g of this medium to 1 litre of labor­atory pure water containing 20 ml of 95 percentethanol. Dentured alcohol should not be used.Heat in boiling water bath for solution, Storeprepared medium in the dark at 4cC. Discardthe unused medium after 96 hours.

ii) Sodium 6an~o(Jt, solution - Dissolve 12 gof sodium banzoate in about 85 ml oflaboratory pure water, then bring to100 ml final volume. Sterilize by auto­cJaving or filteration, Discard the solu­tion after 6 months.

iii) Cycloh,ximide solution (optional) - Pre­pare an aqueous solution containing1'25 grams of cycloheximinej lOtl mllaboratory pure water. Store solutionin referigerator and discard after6 months.

0'075 g

1'05 g10'0 gi-s g

5'0 g1"375 g

10-0 g

5·0 g

12·5 g

4·375 ghydrogen

a) M·Endo holding medium is preparedby adding 3-2 ml of solution benzoatesolution (12 percent) per 100 ml ofM-Coliform broth, prepared as Rivenbelow. 4 ml of the cycloheximide solu­tion may be added if required,

i) M-Colifo,m broth

Tryptone or polypeptoneCasitone or TrypticaseLactose

Dipotassiumphosphate

Sodium lauryl sulphate

Basic fuchsinThiopeptone or thiotoneYeast extract

Sodium chloridePotassium dihydrogen

phosphateSodium desoxycholate

are counted. ~he count is made by a lowpower stereo microscope.

Coliform coloniesColiform density counted X 100(coliforms/100 ml) == Volume in ml of the

sample filtered

3.3.3 Test for Fo,eal Coliform.r

3.3.3.1 G,n,ral - This procedure is used todifferentiate coli forms of faecal origin fromthose of non-faecal origin. Faecal coliforrns arethose coli forms which can ferment lactose at~4'5°C within 24 ± ~ ~ours with the produc­tion of gas. Use brilliant green bile lactosebroth medium for this test.

3.3.3.2 Subculture all presumptive positivetubes of the coliform test, at the end of 24 and48 hours into BGB medium (St, 3.3.1.1 e )and incubate at 44"5°0 for 24 hours in a water­bath. Gas formation within 24 hours is con­sidered a positive reaction for faecal coliforms.

3.3.~ Testfor E. Coli

3.3.4.1 E. Coli is one of the members offaecal coliforms which ferments lactose withthe production of gas at 44·5°C within 24 hours.as well as produce indole from tryptophoneat 44-5°0 within 24 hours. Subculture fromall the positive tubes of BGB broth at 44'5°C( faecal coliforms ) into tubes of peptone water.Incubate at 44-5°0 for 24 ± 2 hours. At theend of the incubation period. test for indoleproduction by adding a few drops of Kovac'sreagent. Positive test will give pink colourwhile negative test will give yellow colour.

3.3.5 D'layed Incubation Method ( Total coli­forms)

3.3.5.1 G,n"al - The delayed incubationMF method is useful in survey, monitoring oremergency situations when the single step coli­f?rm test cannot be performed at the samplingsite or when time and temperature limits forsample storage cannot be met. The advantagesare: a) The method permits confirmation andbiochemical identification of organisms asnecessary; and b) The method eliminates fieldprocessing and equipment needs.

The coliform bacteria can be kept for up to72 hours with little effect on final counts.However, it is desirable that the holding periodshould be kept to the minimum. The applic­ability of the delayed incubation procedure forspecific water source should be determined bycomparative test procedures with conventionalmethods, The delayed incubation procedureis not a substitute for the immediate incubationtest and should be used only when otheralternatives are not applicable.

3.3.5.2 Principle - A specific volume ofwater sample is filtered through membrane

13

3·0 g

1·0 g0·5 g3·0 g1·0 g1-2 g

II I 1622 ·1911

b) M-Coliform holding medium (LESholding medium) may be used as analternative holding medium. To preparethe medium, add the following reagentsin 1 litre of laboratory pure water andmix ( do not heat ) to dissolve:

Composition:

Tryptone or TrypticasePeptone

Dipotassium hydrogenphosphate

SulphanilamideCycloheximideM-Endo Broth MFSodium benzoateParamioobenzoic acid

3.3.5.. -t Procedu,,·- Saturate the sterileabsorbent pads with about 2·0 ml of M-EndoHolding Medium or LES Holding Medium,prepared as outlined above. Pour off excessbroth. Using a sterile forceps, place a Diem­brane filter on the filter base, grid side up_Attach the funnel to the base of the filter unit;the membrane filter is now held between thefunnel and base. Shake the sample vigorouslyabout 25 times and measure into the funnelwith the vacuum off. If the sample is less than10 ml, add 10 ml of sterile dilution water to themembrane filter before adding the sample. Thesample volume should be such as to producecounts of 20·80 coliform colonies. Filter thesample through the membrane and rinse thesides of the funnel walls at least twice with 20­30 ro1 sterile dilution water. Turn off thevacuum and remove the funnel from the baseof the filter unit. With flame sterilizedforceps remove the filter from the filterbaseand place grid side up 00 an absorbent padsaturated with M-Endo Holding Medium ofLES Holding Medium, using a rolling actionat one edge. Exercise care to avoid trappingair bubbles under the membrane. Place topon petri dish and proceed with filtration ofnext volume. Clearly mark the lid of eachpetri dish, indicating location, time of collec­tion, time of incubation, sample number andsample volume. Use a waterproof felt tipmarker or grease pencil.

Inspect each membrane in the petri dish foruniform contact with the saturated pad. If airbubbles are present under the filter ( indicatedby bulges) remove filter with sterile forcepsand roll onto the absorbent pad again. Sealthe petri dish by firmly pressing down the top.Place the culture dish in shipping containerand send it to the examining laboratory. Atthe examining laboratory remove the mem­brane from the holding medium, Place it inanother dish containing M.Endo Broth or agar

)4

medium and complete testing as alreadydescribed.

3.3.6 D,lay,d IneulJation Tlst for FilleafColiform,

3.3.6.1 The delayed incubation procedurefor faecal coliforms is same as tllat for totalcolif~rms, as ~iven .in 3.3.5 except that M.VFCholding medium IS used. The composition ofthe medium is given below:

Casitone, Vitamin Free 0·2 gSodium benzoate 4·0 gSulphanilamide 0-5 g

Final pH 6·7 ± 0·2

p"paration - Add 4·7 g of medium (as.given above) per litre oflaboratory pure watercontaining 10 ml of 95 percent ethanol. Dena­ture~ alcohol sho~.dd no~ be used. Htat slightlyto dissolve the .Ingredlents, then sterilize bymembrane filtration ( 0'22 I'm ) Store preparedmedium at 4°C. Discard after 1 month.

3.4 Test for Faecal Streptococci - The testterms faecal streptococci and enterococci have~een used somewhat synonymously by many10 re~en~ years. The faecal streptococci group­are indicators of faecal pollution of water~ecau~e the. general habitat of these organisms.IS the Intestine of man and animals. They aregram positive cocci and ferment glucose withthe production of acid only and are capableof growing in the presence of 40 percent bileand at ~5~C. On the basis of newer concepts­of specranon of faecal streptococci, it issuggested that the terms faecal streptococciand Lancefields group D streptococcus beconsidered synonymous. The standard test forthe estimation of number of the faecal strep­tococci may be carried out either by the multipletube dilution technique or by the membranefilter technique.

3.f.l Multipl, Tube Dilution Technique­Multiple tube dilution technique employs Pre­sumptive test procedures and confirmed testprocedures.

3.4.1.1 Mtdia

a) Dilution wall' - see 3.2.3.2_

b) Az.ide dext,os, broth ( ADB ) - This is apresumptive test medium used for enu­merating faecal streptococci in the watersamples. Dissolves the following ingre­dients and adjust the pH to 7.3:

Tryptone or Polypeptone 15 gBeef extract 4·5 gGlucose 7-5 gSodium chloride 7·5 gSodium azide 0-2 gDistilled water 1 000 ml

0·4 g1 000 ml

20 g5g2g4g

0·4 g1 000 ml,

Dispense 6 to 7 ml of medium into 150 X15 mm test tubes and plug with non-absorbent-cotton. Sterilize in the autoclave at 1·02 ± 0·03kg/em' gauge pressure (15 ± 0·5 psi gaugepressure, 120°0 temperature approximately) for15 minutes. This a single strength medium andused for 1 ml aJiquotes and decimal dilutionwhen 10 ml sample or more has to be inocula­·ted use double strength medium. This isprepared by using double the quantities givenabovein 1000 ml of water. 10 ml of this doublestrength medium is put into each 150 X 18 mmtelt tube.

c) Ethyl vialt' azide broth ( EVA ) - Con­firmatory medium used for enumeratingfaecal streptococci is as follows:

Tryptone or biosate 20 gGlucose 5 gSodium chloride 5 gDi-potassium hydrogen 2·7 g

phosphatePotassium di-hydrogen

phosphateSodium azideDistilled water

Dissolve all the ingredients and adjustthe pH to 7.1. Add 1 ml of 0·083 percentalcoholic solution of ethyl violet. Dispense10 ml medium into 150 X 18 mm testtubes and plug with non-absorbent cotton.Sterilize in the autoclave at 1'02 ±0·03 kg/em- gauge pressure ( 15 ± O·S psigauge pressure, 120°0 temperature approxi­mately ) for 15 minutes.

3.4.1.2 Procedure - Shake the water sampleihoroughly before making dilution or before.inoculation.

a) Presumptive test - Inoculate a series oftubes ofazide dextrose broth with appro­priate graduated quantities of the waterto be tested ( follow the same procedureas given for coliforms ). Incubate inocul­ated tubes at 37°0_ Examine each tubeat the end of 24 hours for the presenceof turbidity. If no definite turbidity ispresent reinoculate and read at the endof 48 hours.

b) Confirmed test - All azide dextrose brothtubes showing turbidity after 24 or48 hours incubation must be subjectedto the confirmed test. Transfer threeloopfuls of growth from each azidedexlrose broth to ethy1 violet azide brothtubes. Incubate the inoculated tubes for48 hours at 37°0. The presence of stre­ptococci is indicated by the formationof a purple button at the bottom of thetube, or occassionaJly by a dense tur­bidity. Find out the MP value from

15

18 I 1622 - 1981

Appendix B. Record the result asnumbers per 100 ml of the sample.

3.4.2 Membrane Filte, Technique

_ 3.4.2.1 For outline of the method, descrip­tion of the MF assembly, selection of thesample size and filtration of the sample,se« 3.3.2.

3.4.2.2 Medium - Medium used for enu­meration of faecal streptococci by membranefilter technique is known as M enterococcusagar and is given below:

TryptoneYeast extractGluc.)seDi-potassium hydrogen

phosphateSodium azideDistilled water

Dissolve all the ingredients and adjust thepH to 7.2. Add 1 percent agar and heatsufficiently to dissolve agar. After slight cool­ing, add 1 ml of 1 percent sterile solution of2, 3, 5 triphenyl tetrazoJium chloride per100 ml of the medium. Pour the plates( 15 mm X 60 mm) by adding 10 ml of theagar. Allow to solidify and use fresh plates.

3.4.2.3 Procedure - Instead of pad, use asolid agar medium. Pour approximately 10 mlof M enterococcus agar into 60 mm petridishes, Allow to harden and pJace the filter onit. Invert and incubate at 37°C for 48 hoursunder humid chamber. Count all red and pinkcolonies with the help of stereomicroscope.Express the count as number of faecal stre­ptococci per 100 ml of water ( see 3.3.2.6 ).

NOTE - Since the requirement of med ium foreach sarnple is very Iitrle, dehydrated powdermedium is recommended.

3.5 Test for Clo.tridiulD Welchii

3.5.1 Gmeral>: Clostridium welchii are largerod-shaped. non-motile anaerobic bacteriawhich form spores which are relatively resistantto heat, drying and ordinary bacterial agents.

3.5.2 Medium

3.5.2.1 Litmus milk medium - Keep freshraw milk of low bacterial content in the refriger­ator for 18 ± 1 hours so that the cream mayseparate. Remove the cream and add 10percentlitmus solution to the milk to give a purplishblue 'colour. Distribute in tubes in 10 mlquantities. Add a mixture (melting pointapproximately 45°C) of equal parts of paraffinwax and petroleum jelly to form a layer a bout2 to 5 rom thick of the surface of the medium.Steam for 30 minutes on 3 successive days.Test for sterility by incubation at 37·0° ±O-soC for 48 ± 3 hours.

11 11622 • 1111

3.5.3 P"e,dur, - Inoculate varying quan­tities of the sample into bottles or tubes con­taining freshly boiled (in a water-bath) andrapidly cool litmus milk: medium. Heat thetubes in a controlled water-bath at 80 ± 10 0for 15 minutes, remove from water bath, coolto approximately 37°0 and place the tubes inan incubator at 37 ± 0'5°C for 5 days. Examinethe tubes every day for signs of stormy fermen­tation which indicates a positive test. Furtherconfirmation may be done by the spore stainingtechnique.

3.5.4 Confirmation by Spor, Staining Teehniqu«

3.5.4.1 General - Stormy fermentation is apresumptive indication for the presence ofclostridium uulchii; spore staining technique canbe used for confirmation. The positive testeliminates doubt about non-spore forming rodsand cocci and the position of the spore givesadded information, for example, terminal, sub.terminal, central etc.

3.5.4.2 R,agtnts

a) Ziehl Neelsen's Carbo! Fuchsin (ZNCF) ­Its composition is given below:Basic Fuchsin 5 gPhenol 25 gEthanol ( 95 percent) 50 mlDistilled water 500 ml

b) Sulphuric acid - 0·5 percent.c) Mtthyl,n, blu« - 1 percent.

3.5.4.3 Proc,dur, - Prepare a smear andfix it. Stain with ZNCF and beat the prepara­tion until steam rises. Wash with water andtreat with 0'5 percent sulphuric acid for 1 to2 minutes. Wash with water and counter stainwith 1 percent aqueous methylene blue for3 minutes. Wash, dry, mount and observeunder microscope. The cell will. have bulgingin the middle due to central spore. The sporestains are bright red and the protoplasm ofbacilli blu e.

3.6 Test for IrOD Bacteria

3.6.1 Gentral- Iron bacteria are consideredto be capable of withdrawing iron present intheir aqueous habitat and of depositing it inthe form of hydrated ferric hydroxide on or intheir mucilaginous secretion. Their presencemay cause pitting and tuberculations in pipesand render the water unsuitable for domesticand industrial purposes. Bacteria of this type,to obtain energy, oxidize ferrous to ferric ironwhich is precipitated as ferric hydrate. Ironmay be obtained from the pipe itself or fromthe water being carried. The amount of ferrichydrate deposited is very large in comparisonwith the enclosed cells. The main types areGallionella ferruginea, uptolhri%. CrelJothrix Poly-

16

spo,a. Spha"olilus "atans and TbioblJ&illus fe,ro­oxidants. L,ptolh,i1l and Crenolla,ix are filamentous.forms which deposit iron in their sheaths andthe family Gallion,llaconsists of stalked bacteria.

3.6.2 Proe,dur, - It is generally sufficientto ascertain the presence or absence of theseorganisms in a water sample. For this purpose,the centrifuged deposit from the sample spreadover a glass slide shall be examined under amicroscope. The ferric deposits which mayobscure the structure of the organisms may beremoved by adding dilute hydrochloric acidto the smear on the slide and staining theorganisms with Lugol's iodine solution (pre­pared by dissolving 1 g of iodine and 2 g ofpotassium iodide in 300 ml of water).

3.6.3 Cultivation of Iron Bacteria - Whennecessary the iron bacteria in the sample shallbe cultivated and enriched by the methodgiven in 3.6.3.1 and 3.6.3.2.

3.6.3.1 Gallionella

a) Medium - Mix equal volumes of 10per­cent solution of ferrous sulphate in waterand 3 percent solution of agar at 45°0.Distribute in screw ca p tubes and sterilize.in the autoclave at 1'02 ± 0'03 kg/em-gauge pressure (15 ± 0'5 psi gaugepressure, 120°0 temperature approxi­mately ) for 20 minutes. Then preparethe agar slants. Dis solve 1'0 g ofammonium chloride, 0'5 g of dipotassiumphosphate, 0'2 g of magnesium sulphateand 0'1 g of calcium chloride in 1 litreof water. Sterilize in the autoclave for25 minutes at 1'02 ± 0'03 kg/em l gaugepressure ( 15 ± 0'5 psi gauge pressure,120°0 temperature approximately).Bubble carbondioxide through this.medium for 10 to 15 seconds. Place ineach agar tube a quantity of this liquidmedium sufficient to cover the agarcompletely.

b) Proctdur, - Inoculate the tubes contain­ing the medium with a drop of thesuspension of the organisms in the centri­fuged deposit of the sample and incubateat room temperature. Examine after 18to 36 hours. White deposits on the sidesof the culture tube indicate the presenceof Gal/;onella colonies. Pick the depositwith a sterile needle, spread over a slide,and examine under the microscope as­in 3.6.2.

3.6.3.2 Llplothri~

a) Mldium - Dissolve 1-0 g of ammoniumsulphate, 0'5 g of magnesium sulphate,0'1 g dibasic potassium phosphate, and0·02 g of calcium nitrate in 1 litre of water.Place 100 ml quantities in conical flasks

and sterilize at 1-02 ± 0 03 kg/em.gauge pressure (15 ± 0 5 psi gaugepressure, 120) C temperature approxi­mutely ) in an autoclave for 20 minutes.Place in each flask 0 05 g of sterilizediron filings.

b) Procedure - Inoculate the medium pre­pared above with a suspension of thecentrifuged deposit of the sample andincubate at room temperature. Examineafter three days under the microscope asin 3.6.2.

3.7 Test for Sulphate Reducing Bacteria

3.7 General- The sulphate reducing bacteriaeffect a direct reduction of sulphates, Theyare widely distributed in nature and arecommon in soils, canals and lake waters,sewage, marine sediment, etc. The water usedfor sealing petroleum or gas tanks generallyharbour these bacteria. They may also bepresent in water cooling systems of industrialplants. These bacteria cause blackening ofpulp in a paper mill, and corrode concretesewage pipes and pipe surfaces. The mostcommon of these organisms is Desutphooibriodesulphuricans, Some strains are rnesophilicand grow best at 25 to 40°0 while others arethermophilic and grow at 45 to 60°C. Theyare curved rods, and are strictiy anaerobic.

3.7.2 lJpparalus

3.7.2.1 Culture bottle - 60·0 ml glass stop­pered bottle, sterilized in hot-air sterilizer,

3.7.3 Medium - Dissolve 10 g tryptone, 1 gsodium sulphite and 10 011 of 5 percent ferriccitrate solution in 1 000 ml of distilled water.Sterile in an autoclave at 1'02 ± 0-03 kg{cm 2

gauge pressure ( 15 ± o· 5 psi gauge pressure,120°C temperature approxirnatcly ) for15 minutes.

3.7.4 Using multiple dilution techniquedis­tribute 10 ml of the medium in each test tubeand sterilize at 1-02 ± 0'03 kg{cn1 2 gauze pres­sure ( 15 ± O·5 psi gauge pressure, 1~O°C tem­perature approximately) for 15 minutes. Add1 ml of the sarnple or its dilution asepticallyto each tube and cover the surface of the liquidby sterile liquid paraffin to a depth of 1 to2 mID. Incubate the tube at 28 to 30°("" for 2to 3 days, The production of black colour inthe medium will indicate the presence ofsulphate reducing bacteria.

3.8 Test for Sulphur Bacteria

3.8.1 General - The organisms belongingto the group sulphur bacteria are autotrophicbacteria which oxidize elemental sulphur orreduce sulphur compounds, obtaining theircarbon requirements from carbon dioxide.They are undesirable in water used in manyndustrial processes, and the acid produced

17

IS I 1622 • 1981

during their metabolism may be destructiveto concrete and other structures, T'hiobacittusthioparus and Thiobacillus thiooxidans arc themore common forms belonging to this group.

3.8.2 Test for T. thioparus

3.8.2.1 Medium - Dissolve 10'0 g of sodiumthiosulphate pentahydrate, 2'0 g of dibasicpotassium phosphate, 0'1 g of magnesiumsulphate heptahydrate, 0·1 g of calciumchloride, 0·1 g of ammonium chloride, O·O~ gof ferric chloride hexahydrate and 0 02 g ofmanganese sulphate in 1 litre of water, Place50 ml quantities in sterilized 250 ml conicalflasks and sterilize by autoclaving at 1-02 ±0-3 kg/cm2 gauge pressure ( 15 ± 0'5 psi gaugepressure, 120°C temperature approximately)for 10 minutes.

3.8.2.2 Procedure - Inoculate a shallowlayer of the medium with a known volume ofthe sample (50 ml or 10 ml or less) andincubate at about 30cC for 2 to 3 days. In thepresence of T. thioparus bacteria the surface ofthe inoculated medium becomes covered withsulphur from the autotrophic oxidation of thethiosul phate,

3.8.3 Test JOT T. Thiooxidans

3.8.3.1 Medium - Dissolve 0'2 g of ammo­nium sulphate, U'5 g of magnesium sulphateheptahydrate, 3·0 g of monobasic potassiumphosphate, 0-25 g of calcium chloride ando 01 g of ferrous sulphate in 1 litre of water,Weigh 1-0 g of elemental sulphur in a 250-rnlflask and add to it 100 ml of this solution.

NOTJ": - "Ferrous sulphate solution should best er i iiz od bv filtration through a m iflipore rn ern br arrefi1t('r of 0-45 l-lffi and then mixed a se-pt ir a l l v to therest of the basal medium, Heat ing, st e-am ing orautoclaving of ferrous sulphate solut ion m av re.su ltin oxidation and hydrolysis of the salt. So lu: ion ofpot ass iu m phosphate should be ster il iz cd sep a ratr-Ivand then added aseptically to the basal lTH'c.huni.Sulphur should be ster ilized by str-arru rnr for30 minutes on three successive days be [ore- addin~

to the rucd iurn Rest of the rn cd iu m shou ld bestvri lizvd in a steam sterilizer for 30 ru inu t es on3 successive days.

3.8.3.2 Procedure- I noculate flasks contain­ing the medium with 10 ml or Jess of thesample, and incubate at 25 to 3<)c C for 4 to 5days. The sulphur sinks to the bottom, thereaction of the medium decreases to pI-I 2'0and in the presence of 1", thiooxidans themedium will become turbid.

3.9 Test for Gelatin Liquefying Bacteria

3.9.1 General - Certain micro-organisms arecapable of producing proteolytic Iermentswhich digest and liquefy gelatin. The pre-onceof these organisms in process water is of impor­tance in industries such as the rnanufacturr- ofphotographic films, edible gelatin, glue and Infood processing.

IS I 1622 • 1981

3.9.2 Medium - Dissolve 3 g of beef extract,5 g of peptone and 120 g of gelatin in 1 litreof water over a water-bath. Cool to about50°C and distribute into tubes in quantities of10 to 15 ml. Sterilize in the autoclave for20 minutes at 1·02 ± 0·03 kg/eml gauge pressure( 15 ± O'S psi gauge pressure, 120°C temper­ature approximately). The final reaction ofthe medium shall be pH 6.8.

3.9.3 Procedure

3.9.3.1 Place 1 ml of the well shaken samplein a sterile petri dish. Add the molten mediumat 30'0 ± 0·2°C and mix thoroughly bycareful rotation and to and fro movement ofthe dish placed on a flat table. Not more than20 minutes shall elapse between placing thesample in the petri dish and adding themedium, Incubate the dish at 20·0 ± O'2°Cand examine every day for seven days forevidence of liquefaction of the medium aroundthe colonies of micro-organisms. If the atmo­spheric temperature is above 20°C the exami­nation of the dish should be made as soon asit is taken from the incubator before the mediastarts t hawing. The negative dishes shall beincubated further up to a period of at least21 days. It is preferable to inoculate at least5 dishes for each sample.

3.9.3.2 Confirmation - If further confirma­tion of the gelatin liquefying property of theorganisms is desired, the following procedureshaJl be adopted:

a) Preparaiion of nutrient gelatin medium­Dissolve 3 g of beefextract, 5 g of sodiumchloride and 109 of peptone in 1 litre ofwater. To this solution, add 120 g ofgelatin, dissolve in a water-bath, coolto about 5()OC and adjust to pH 7.5. Add109 of egg albumen, mix thoroughly,heat for half an hour and filter whilehot. Distribute in 10 ml quantities inpreviously autocJaved tubes and sterilizehv steaming for 20 minutes on 3 succes­sive days.

b) Place the gelatin medium tubes at 20'0 ±O'2°C for the medium to harden andtake out only just before use. Pick out asuspected colony from the petri dishculture obtained in 3.9.3.1 using astraight needle and prepare a stabinoculation in the tube. Incubate thetubes at 20.0 ± O·2°C and examine forevidence of liquefaction as jnt3.9.3.1.

3.9.3.3 If a 20°C incubator is net available,the test for presence of gelatin liquefyingorganisms may be carried out by inoculating1 ml of the sample into a tube of nutrientgelatin medium and incubating at 31'0 ±O·2°C. Examine every day for seven days forevidence of liquefaction by placing the tube incooled water (below 20°C) and observing

18

whether the medium hardens or not. Anuninoculated tube of nutrient gelatin mediumshall also be incubated and tested as a control.

3.10 Test for SUme FormlDI Bacteria

3.10.1 G,neral - A large variety of non­pathogenic bacteria is carried by water whichis of vital interest to bacteriologists and waterengineers because these may produce slimewhich will adhere to structures and increaseeither by growth or by collecting and holdinginsoluble debris from water supply. The pre­sence of these slime forming organisms isespecially undesirable to condenser systems,paper mills, food processing plants, etc, Theseare mixtures of various types of bacteria. Thetime and labour involved in making bacterialisolations and counts on laboratory media fromindustrial slimes may not, however, be justifiedby the information gained. A clear picture ofthe slime may be obtained by direct microscopicexamination of the material.

3.10.2 Procedure

3.10.2.1 Place on a clear glass slide a smallamount of the sample and spread it evenly.Cover with a cover glass and examine underthe low power of the microscope for largeforms, such as algae and mould and record.

3.10..2.2 Prepare another mount as aboveand stain with Lugol's iodine solution (see 3.6.2).Examine under the high power of the micros­cope for filamentous bacteria. Dry and fixthe smear, stain by the Gram method andobserve under the oil immersion. A variety oforganisms may be observed of which one ortwo types may be prominent. Record the­observations.

4. MICROSCOPIC EXAMINATION

4·0 OutliDe of the Method - The sarnpleshall be examined microscopically as early aspossible after collection. Qualitative assessmentby identifying the organisms shall be madebefore preserving the sample. Quantitativeassessment of the individual organisms may bemade after preservation. In general indentifica­tion of the organisms up to their generic namesis enough. Further identification to specieslevel is only essential under special circum­stances and can only be done by a speciallytrained personnel. Quantitative examinationby direct counting can be done if organismsare sufficiently numerous. Otherwise theorganisms should be concentrated in a smallvolume of the sample as prescribed in 4.1.

4.1 CODceDtratioD of the OrlaDi8m8

4.1.1 The Sedgwick Raft,r M,thod

4.1.1.1 Equipment

a) Filter funnel- Cylindrical, with diameter5 em at the top, a straight side for about20 em and narrowing over a distance ofabout 5 em to a bore of 1 cm diameterand terminating in a straight portion of1 em diameter about 6 em in length.The capacity shall be about 500 ml. Asingle-holed rubber stopper shall befitted tightly into the bottom. A smallglass U -tube is inserted in the stopperwith the outer end extending about0'2 em above the inner end of thestopper.

b) Filtering s41ld- Washed white sand,passing 250 micron IS Sieve and retainedon 125-micron IS Sieve.

c) Cloth discs - About 1 em in diameter.These shall preferably be cut frombolting silk cloth having 80 meshesjcms,alternately nylon or linen cloth maybe used.

4.1.1.2 Procedure - Place a cloth dise aftermoistening it on the rubber stopper of thefunnel and insert the stopper firmly III the lowerend of the funnel. Introduce a small amountof filtering sand into the funnel to form a layernot less than 12 mm thick on the top of therubber stopper. Place the funnel in an uprightposition in a suitable support, introduce a smallquantity of .water for settling the sand andallow it to drain through the sand. Mix thesample under test gently but thoroughly andadd a measured portion ( 500 to 1 000 ml ) tothe funnel without disturbing the sand. Thisis done by commencing the addition of thesample before all the liquid has drainedthrough. Continue filtration of the sample,returning the first 100 ml to the funnel, wash­ing down the sides of the funnel with a streamof the filtrate from time to time for dislodgingany organisms adhering to the sides, until thewater level reaches that of the outer end of theU-tube. Then remove the U-tube and allowthe remaining liquid to drain completely.Place a small beaker under the funnel andremove the stopper, catching the sand in thebeaker. Flush the inside wall of the funnelwith 5 to 10 ml of 3 to 5 percent formalin inwater collecting it in the beaker. Shake thebeaker gently for separating the organismsfrom the sand particles, allow time for thesand particles to settle, and decant the suspen­sion of the organisms into a second beaker.Wash the sand with 5 ml of dilute formalin anddecant into the second beaker as before.Measure the total volume of the concentrateand make up to a known volume, usually amultiple of five,

f . · 1'1Degree 0 concentration == -V;

19

IS I 1622 • 1981

where

V1 c:: volume in ml ofsampJe filtered, and

VI = volume in ml of the concentrate.

4.1.2 Centrifug«kfethod

f.I.2.1 Equipment

a) Centrifuge- Electrically 0 per ate d,holding 20 ml tubes and with a speed of2 500 to 3 000 rev/min.

b) Centrifuge tubes - 10 ml capacity withthe "lower end tapered and graduated.

4.1.2.2 Procedure - Fill the centrifuge tubesto the mark with the well mixed sample andcentrifuge at 2 500 to 3 000 rev/min for10 minutes. All matter in suspension is drivento the bottom of the tubes. Carefully pour offthe clear supernatant liquid up to a little abovethe 0'1 rnl mark in the narrow portion of thetube, taking care not to disturb the sediment.Remove the liquid to exactly the level of the0'1 ml mark with a pipette and after expellingthe liquid, use the pipette for mixing thedeposit in the tube thoroughly with the remain­ing liquid. Pool the concentrates thus obtainedin the centrifuge tubes and make up to a knownvolume. Add a trace of formalin for preven­ting the movement of mobile organisms thatmay be present.

4.1.2.3 If the organisms in the originalsample are few in number, it will be necessaryto use a large volume of the sample for obtain­ing a suitable concentrate. For this purposea Foerst type of continuous centr ifuge havinga speed of 20000 rev/min may be used. In thisapparatus the sample is fed continuously into arevolving bowl and the organisms are depositedin the angle formed by the junction of the sidewall of the bowl with the bottom. After centri­fugation, the compacted deposit is gentlybrushed loose from the bowl, mixed with thesmall amount of water in the bowl and trans­ferred to a beaker and made up to a knownvolume.

4.1.3 Millipore Filter

4.1.3.1 Phytoplankton concentration maybe made through filtering a known volumethrough a rnillipore filter assembly and thenumber of cells trapped on the filter papermay be counted by direct observation with acompound microscope.

4.1.4 In case of heavy water samples havinghigh phytoplankton count initial concentrationof the sample is not necessary, However,known volume may be strained through theplankton net into a collection vessel forzooplankton assessment.

4.2 Procedure for Enumeration4.2.1 Equipment

IS I 1622 • 1981

4.2.1.1 Compound microscope - Provided with7·S X and 10 X oculars and 16 rom objectiveshall be used. A binocular microscope ispreferable.

4.2.1.2 ~'Vhipple ocular micrometer - It is acircular glass disc which fits exactly into theinterior of the ocular. I t shall bear an accura­tely ruled square subdivided into 25 equalsquares. One of the small squares in the centrewill be further subdivided into 25 smallersquares.

4.2.1.3 Stage micrometer - It consists of athin glass disc mounted permanently upon aglass slide, An accurate linear scale, usually1 rnrn divided into hundredths, is etched onthe underside of t he disc.

4.2.1.4 Counting cell - It is a glass or brassrectangle, 50 X 20 X 1 rom, scaled to a glassmicroscope slide. It has a capacity of 1 ml andencloses an area of 1 000 mrns. A rectangularcover glass large enough to cover the cell isrequired.

4.2.2 Procedure

4.2.2.1 Standardization of tile fVllipple micro­meter - Place the micrometer ruling side down­wards in the ocular of the microscope, Placethe stage micrometer on the stage of the micro­SCOl.JC and, using the 16 mm objective, focus toget a clear image of the rulings. Adjust thedraw tube of the microscope so that one sideof the ocular micrometer covers exactly 1 mmon the stage micrometer. Record the tubelength of the microscope for future reference.In this position, the area enclosed by the largesquare of the ocular micrometer is 1 rnmson the stage, that enclosed by each of the100 interior squares is 0'01 mrnt and thatenclosed by one of the smallest squaresis 0'000 4 roms. One side of the smallest squareis 0·02 mm or 20 I'm in length.

4.2.2.2 Place the counting cell on a levelsurface. Shake the concentrated sample (see 4.1.1or ".1.2) in the beaker gently but thoroughlyand with a pipette, transfer the sample to thecount ing cell filling- it completely. Place aclean cover glass over the cell by sliding itgt'lllly from one end taking care not to encloseauv air bubble. A)]0"' five minutes for theon~anistns to settle and examine the ccll underthe microscope equipped with the calibrated\'Vrlipple ocular microrneter. Select at randomone area which is entirely within the cell andcount all the organisms within the large squareof the ocular micrometer. If an organisms ispartly within and partly outside the countingsquare, estimate and record the fractionalportion within the square. The microscopeshaII be focussed through the entire depth ofthe cell for detecting and counting all theorganisms. Count also particles of debris and

2C

other matter and record them under thatheading. Count the organisms in ten fieldsmoving the cell appropriately. Record thenumber of organisms of each kind counted ina suitable form. No special form is prescribedbut in general, the form used should providespace for recording information about thesource of the sample, time and date of collec­tion, date of the examination, laboratory serialnumber. and name of the examiner. I t shallbe ruled to facilitate entry of results when tenfields are counted.

4.2.3 Calculation - Calculate the number oforganisms per millilitre of the original sampleas follows:

Number of organismsjrnlof original sample = 1 000 VI X N

-~~Jxn-

where

V'l = volume in rnl of concentratedsample taken for the test.

.lv = total number of organisms counted,V = volume in ml of original sample

taken for concentration andfiltered, and

n = number of fields counted.. 1 000 l~l

4.2.3.1 The value of the expression ·_-V·····is commonly referred to as the Sedgwick Rafterfactor.

4.2.3.2 Reporting i11 cubic standard units - Formost purposes of biological examination repor­ting in terms of number of organisms of eachkind in 1 ml of the sample will be sufficient.When the size of the organisms has also to beestimated, the system of reporting in cubicstandard units shall be adopted, the cubicstandard unit is 0·000 008 mrns or 8 000 14m3,the length of one side of which is 0'02 mm, Forobtaining the volume of the organisms, themicroscope shall be equipped with a graduatedmicrometer head and vernier on the fineadjustment. Measure the length and breadthof each organisms at the time the count ismade, with the aid of the ocular micrometer,noting the squares or partial squares which theorganisms cross. One side of the square is0-02 mm. Focus carefully the topmost andbottom most surface of the organisms anddetermine its thickness from the vertical dist­ance through which the objective moved byreading on the graduated fine adjustmentscrew. Organisms which are uniform in size,such as diatoms, may be counted individuallyand then converted to volume by multi plyingthe total number counted by a constant factorexpressing their size. Calculate the total volumeof the organisms of kind per miJlilitre of theoriginal sample and express the result as cubicstandard unit per millilitre,

IS I 1622 • 1981

4.2.4 Drop M,'hodfor Co"",i", Phytopianklon cell count per drop. After placing the drop ona clean slide drop gently a 22 mm square

4.2.4.1 Direct microscopic examination is cover glass. Take care that there are no airnecessary for qualitative enumeration and for bubbles under the cover glass. Place the slidefurther quantitative assessment of phytoplank- on the microscope stage and bring the middleton in terms of cell numbers. Since delicate of one edge of the cover glass into view withcells such as small flagellates are almost unre- high power objective. Move the slide from onecognisable in a fixed state a direct examination side to the other counting the number of cellsof the sample in fresh condition is ideal, if in that transect. Carry out this operationnecessary after centrifugation. quickly so as to avoid drying of the drop and

...2....2 Procldu.r, - Take a drop, small consequent formation of air cavities under theenough to fit under a square cover glass of cover glass. Repeat the operation, counting22 mm size and put on a clean microscopic another transect well separated from the first.slide. Standardize the drop with a long drawn, Count another drop in the same way. If thepreferably with a pasture pipette and always results are very different from the first, countuse a uniform sized drop. To minimize experi- a third drop. Repeat this till uniform count ismental error in drop selection count more achieved. Calculate the number of cells pernumber of drops for obtaining uniformity in drop using the following formula:

T 1 b 11 Id Area of the cover glass X individual counts recorded per transect

ota num er ce s rop- Area of one transect

Count per rnl can be calculated by such drops that make one milliJitre.multiplying the count/drop with number of

APPENDIX A( Clause 2.1.5 )

PARTICULARS TO BE SUPPLIED .ALONG WITH SAMPLES

A-I. While submitting samples. the followingparticulars shall be supplied along with sample:

a) Name and address of person requestingthe examination;

b) Narne, designation or other identificationparticulars of the person drawing sam­ples;

c) Date and time ofcollection and despatch;d) Reasons for examination and whether it

is a routine sample or otherwise;e) Source of water (well, spring, stream,

public supply, etc );f) Exact place from which sample was

taken. I f from a tap whether the samplewas drawn through a cistern: or directlyfrom the mains;

g) The method of purification and sterili­zation used, if any; details of dose ofchemicals, point ofapplication, quantitytreated, etc;

h) Temperature of the sample;j) Weather at the time of collection and

particulars of recent rainfall;k) Whether the water becomes affected in

appearance, odour or taste after heavyrains;

m) If the sample has been taken from awell, then:

21

1) Depth of well) and of water surfacefrom ground level;

2) Whether covered or uncovered, andnature, material. and construction ofthe cover;

3) Whether newly constructed or withany recent alterations which mightaffect the condition of the water;

4) Type of construction - i) bricks setdry or in cement; ii) cement orcylinder lined) and whether puddledoutside the lining; j ii) depth oflining; iv) whether bricked aboveground surface, if so, height ofcoping; v) presence and extent ofapron; and vi) method of pumpingor other means of raising water;

5) Proximity of drains. cesspools orother possible sources of pollution,and distance from source;

6) Any discoloration of the sides of thewell or other visible indication ofpollution;

7) Nature of subsoil and water-bearingstratum.

NOTE - When available. a section or drawingof the well and general surroundings should befurnished.

18 I 1622 • 1981

n) If the sample hal been taken from aspring, then:1) Stratum from which it issuesj2) Whether the sample has been taken

direct from the spring or from acollecting chamber. If from thelatter, the mode of construction ofchamber;

p) If the sample has been taken from ariver or stream. then:1) Depth below surface at which the

sample was taken;

2) \Vhether the sample was taken fromthe middle or side;

3) Whether the level of water is aboveor below the average;

4) Condition. of weather at the time ofsampling and particulars of anyrecent rainfall or flood eonditloas;

5) Observations with reference to anypossible sources of polJution in the'vicinity and approximate distancefrom sampling point; and

q) Results of field tests made on the sample..

,____- __A~

50..ml Tubes lO-ml Tubes

(1) (2) (3)

0 1 10 2 20 3 4

0 4 51 0 21 1 31 2 61 3 9

1 4- 16

APPENDIX B

( Clause 3.3.1.3 )

TABLES OF MOST PROBABLE NUMBERS

TABLE 1 MOST PROBABLE NUMBER ( MPH) OF ORGANISMS PER 100 mlOF SAMPLEAND CONFIDENCE UMITS USING 1 TUBE OF 50 .1 AND 5 TUBES OF 10 mI

NVMUKn OF POSITIVE MOlT PaoBABLB NUMB!:R ( MPN ) LIMITS WITHIN WHIOH MPNTCDES PER 100 ml .. PBR 100 ml CAN LIE,--- -..J+.. -----.

Lower Limit Upper Limit

(4) (5)

<0'5 4<0·5 6<O·S 11

1 13

<O-S 6<O·S 9

1 152 214 40

22

IS : 1622 • 1981

TABLE 2 MOlT .a08ABLB NUMBBR ( MPH ) OF ORGANISMS PER 100 aaI O' SAMPLE ANDCONftDBNCB LIMITS USING I TUBB 01' 50 mI, 5 TUBBS OF 10 _I AND 5 TOBES OF I ml

Nv_••• 01' P081TIVB MoaT PaoBABLB NUKBBB LIMITS WITHIN WHICH MPNTUB" ( MPN ) PB. 100 ml PEa 100 ml CAN LIB

~. . A

-~ r-.,J..___~

So-ml Tubes 10-ml Tubes I-ml Tubes Lower Limit Upper Limit

(1) (2) (3) (4) (5) (6)0 ° 1 1 <0'5 40 0 2 2 <0-5 60 1 0 1 <05 40 1 1 2 <05 60 1 2 s <0'5 80 2 0 2 <0-5 6

° 2 I S <0'5 80 2 ~! 4- <0-5 11

° 3 0 :3 <0'5 80 :3 1 5 <0'5 130 4 0 5 <0'5 131 0 0 1 <0-5 41 ° 1 3 <0'5 81 ° 2 4 <O·S 111 0 :3 6 <0'5 151 1 0 3 <0-5 81 1 1 5 <0-5 13I 1 2 7 1 171 1 3 9 2 211 2 0 5 <0-5 131 2 1 7 171 2 2 10 3 231 2 :3 12 3 281 3 ° 8 2 191 3 1 It :3 261 3 2 14 4 341 :3 3 18 5 531 3 4 21 6 661 4 0 13 4- 311 4 1 17 5 471 4- 2 22 7 691 4 :3 28 8 851 4 4- 35 12 1011 4 5 43 15 1171 5 0 24- 8 751 5 1 35 12 1011 5 2 54 18 1381 5 3 92 27 2171 5 4 161 39 450

23

III 1&22 • 1981

TABLB 3 MOlT PRO.ABU NUMaBR: MPN ) 01' ORGANIIMS PRESENT PER 100.1 01' SAMPLBAND CONflDBNCB UMlTI 11S1NO 5 TlJBU or 10 mi. 5 TOB.S 01' 1 aaI AND 5 TUB" 01' O-IID!

NVIIB.. owPOII'1'IV. MOlT PBO.BBL. Nv•••• LtIlIT! WITBIX WHIO. MPNTUB•• ( MPN ) Po 100ml P •• 100 ml <lur LI.

t

__A~ ,...--- A

\IO-m! Tubes I-ml Tube. O·l-ml Tube. Lower Limit Upper Limit

(I) (2) (3) (4) (5) (6)

0 0 I 2 <0-5 70 0 2 4 <0-5 110 1 0 2 <O-s 70 1 I 4- <O-S 110 1 2 6 <0·5 J50 2 0 4 <O-S 110 2 1 6 <O-S IS0 3 0 6 <0-5 151 0 0 2 <O-S 7I 0 1 4 <O'S II1 0 2 6 <0'5 151 0 S 8 1 19) 1 0 4 <0'5 It1 1 1 6 <0'5 151 1 2 8 1 191 2 0 6 <0'5 15I 2 1 8 t 191 2 2 10 2 231 3 0 8 1 191 3 1 10 2 231 4 0 11 2 252 0 0 5 <0'5 132 0 1 7 1 172 0 2 9 2 212 0 3 12 3 282 1 0 7 1 17

2 1 1 9 2 212 1 2 12 s 28

2 2 0 9 2 212 2 1 12 S 282 2 2 14 4 84-

2 3 0 12 S 28

2 3 1 14 4- 34-

:l 4 0 15 4 37

3 0 0 8 1 193 0 1 11 2 253 0 2 13 3 31

3 1 0 11 2 253 1 1 14 4- 34

3 1 2 17 5 463 1 3 20 6 60

3 2 0 14- 4 34-

3 2 1 17 5 46

3 2 2 20 6 60

3 3 0 17 5 46

3 3 1 21 1 63

3 4 0 21 7 63

3 4 1 24 8 72

3 .5 0 25 8 75

4 0 0 13 3 31

4 0 1 17 5 46

4- 0 2 21 7 63

4 0 3 25 8 75

(eo"ti".tl )

24.

IS I 1622 • 1981

TABLE 3 MOST PROBABLE NUKIIER( MPN ) or ORGANI~MSPRESENT PER 100 ml OF SAMPLEAND CONFIDENCE LIMITS USING 5 TUBES OF 10 m1 J 5 TUBES OF 1 ml AND 5 TUBES OP O-l ml - Conld

NUMBER 01' POSITIVB MOST PROBABLE NUMBER LIMIT8 WITHIN WHICH MPNTUBER ( MPN ) PER 100 ml PEa 100 ml CAN LIE,..._______.A

~--~ fA-____~

IO-ml Tubes I-ml Tubes O"I-ml Tubes Lower Limit Upper Limit

(1) (2) (3) (4) (5) (6)

4- 1 0 17 5 464 1 1 21 7 634- 1 2 26 9 784 2 0 22 7 674 2 1 :.::6 9 784- 2 2 32 11 914 3 0 27 9 804- 3 1 33 II 934- 3 2 39 13 1064 4 0 34 12 964- 4 1 40 14- 1084 5 0 41 14- 110

4 5 1 48 16 124

5 0 0 23 7 70

5 0 1 31 11 895 0 2 413 15 114

5 0 3 58 19 144

5 0 4 i6 24- 180

5 1 0 ~J3 11 93

5 1 1 46 16 1205 1 2 63 21 154

5 1 3 84- 26 197

5 2 0 49 17 126

5 2 1 70 23 1685 2 2 94 28 219

5 :l 3 120 33 281

5 2 4 148 38 366

5 2 5 177 44- 515

5 3 0 79 25 187

5 3 1 109 31 2535 3 2 141 37 343

5 3 3 J75 44 503

5 3 4- 212 53 (it)9

5 3 5 2~)3 77 7UB5 4- 0 130 :J5 302

5 4- J 172 ·1:l 480

5 4 2 221 57 698

5 4- 3 278 90 849

5 4 4 345 117 999

5 4- 5 426 1·15 lIt,)

f") 5 0 140 liH 75~1

5 5 1 34-U 118 100.1

5 5 2 542 180 ) 405

2S

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