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 1167 (1965): Casein (Edible Quality) [FAD 19: DairyProducts and Equipment]

Gr'

IS : 1'17· 1111

Indian Standard

SPECIFICATION FORCASEIN (EDIBLE QUALITY)

( Revised)

Fourtb Reprint MARCH 2000

C Copyrl,,,, 1966

BVaEAtJ OF INDIAN STANDARDSMAtfAIt aRAVAN. , BAHADUR SHAH %APAR MAllO

NEW DBUlI 110002

May 1966

(~l"')xxx2010

IS: 1167 - 1965

Indian Standard

SPECIFICATION FORCASEIN (EDIBLE QUALITY)

(Revised)Dairy Industry Sectional Committee, AFDC 12

Chairman Re-presentingI)R K. C. SE~ Indian Dairy Science Association, Bangalore

~\1embersAGRICULTURAL M.~RKETJNG AD- Directorate of Marketing & Inspection (Ministry

VISER TO THE GOVERN!'IENT of Food & Agriculture), NagpurOF I:SDI.a.

SURI V. CHANDRAMOCLY (Alternate)SHRI B. R. BEDF.KAR Hindustan Milkfood Manufacturers Limited. Nabha

SHRI I. C. I~. D.a.BSON (A lIernate)SHRI C. Y. CUA~DR'" SEKHAR T. T. (Private) Limited, Bangalore

SURI S. S. ~L\~I (Alternate)SHRJ H. M. I)-,L.\Y Kaira District Co-operative Milk Producers' Union

Ltd, AnandJ)R ]. D. CONTRACTOR (Alternate)

SURI C. D. D ..\STOOR Larsen & Toubro Ltd, BombaySJlRI H. W. H.AMClIANIl.o\NJ (Alternate)

IlR N. X. DASTl'R National Dairy Research Institute, KamalDR C. P. ~\:'iA~T ..\KRISHN.~N f.Altt'rnale)

I>IRECTOR Directorate of Military Farms, Army Headquarters.\SSIST.'NT Dmxcroa, ~fILI-

TAKY FARMS (PLANNING) (Alternatl')EXECUTIVE J-IEALTH OFFICER Municipal Corporation, Bombay

~UNICIPAL ANALYST (Alternate)COl" A. G. FER~.\~DES Food Inspection Organization, Quartennaster

General's Branch, Army HeadquartersLT-COL X. G. C. IENGAR (Alternate';

SHRl (i. S. GoonoLE Dairy Development Commissioner, Governmentof Maharashtra

SURI Y. v. SAl_PEKAR (A lternatesOR K. K. IVA Ministry of Food & Agriculture

SMRI G. GOPINATH (Alternate)COL P. C. KHANNA Technical Standardization Committee. (Foodstufts)

(Ministry of Food & Agriculture)DR S. S. PHATAK (Alternate)

SHRI A. R. A. KRISHNAN Defence Production Organization [Ministry ofDefence (DG I)]

SHRI K. P. SINGII (Alte,nate)DR A. P. !\IAHADE\"AN Hindustan Lever Ltd, Bombay

DR K. K. G. MENON (Alte"nat~)

(Continue. on pag, 2)

BUREAU OF INDI-AN STANDARDSMANAK BHAVAN, 9 BAl-IADUR SHAH ZAFAR MARG

NEW DELl-II 110002

IS: u tJ7 ,. 1965

SHRI P. H. RAMANATHAN,Assistant Director (Agri &Food) (S,cyel4ry)

Centra) Committee for Food Standards (Ministryof Health)

SHRI P. JANARDANA AIVAR (Alter.tJQtt')DR M. SWAMINATHAl Central Food Technological Research Institute

(CSIR), ·MysoreSRR1 M. R. CH."-NDRASEKHARA (A 1I~,,,alt)

SRRI R. H. VARIAVA Polson Limited, BombaySaRI B. P. PALKHIWALLA (Alternale)

D. I. S. VERMA Dairy Technology Division, National DairyResearch Institute,' l<amal

SRRI M. R. SRINIVASAN (Alternate)SaRI JAMES N. WAltNER In personal capacity (AllaAabtJd .Agricultural

IHstilule. Allahabad)Director, lSI (Ex-officio M~",be,)

(C"rditlwd from pal' I)M,mbers Representing

MII=K COMMISSIONER Milk Commissioner, MadrasSHRI P. VISWANATHA MENON (Alternate)

SHRI S. N. MITRA Central Food Laboratory, CalcuttaSRRI B. K. MURTHY Indian Aluminium Co Ltd, Calcutta

SHRI N. GOPAL KRISHNAN (Alt~rnale)

SHRI E. E. NAEGELI Nestle's Products (India) Ltd, New DelhiSRRI F. J. RVAN (Alte,.nate)

SHRI J. PADMANABHAN The A. P. V. Engineering Co Ltd, Calcut~a

SRRI J. G. BaO\\rN (A /lerna!e)SRRI S. RAMASWAMY Directorate General of Technical DevelopmentSaRI v. H. SHAH Vulcan Trading Co Ltd, Bombay

SRRI A. JENSEN (Altentalt)na R. S. SRIVASTAVA

Producers'

National Dairy Research Institute, Kamal

Dairy Products Subcommittee, AFDC 12:.2COIItI,,,er

na v. R. BRALltRAOMe",l¥r,s

DR C. P. ANANTAKRlSHNAN National Dairy Research Institute, KamalDR A. T. DVDANJ (A lIe"" ale)

Da J. D.CONTIlACTOR Kaira District Co-operative MilkUnion Ltd, Anand

na 1. M. PATEL (Altt,nate)COL A. G. FERNAND~S Food Inspection Organization. Quartermaster

General's Branch Army HeadcuartersLT"'(.OL N. G. C. IENGAR (Alte,nate)

GOVERNMENT ANALYST Government of MadrasDR K. K. IVA Ministry of Food & Agriculture

SHal R. GOPALAN (.Al,.,."al~)

SKRI B. R. KAPUR All India Ice ...Cream Manufacturers' Association,New Delhi

SOl K. L. SIBAL (Alle,"al,)DR ZAL R. KOTHAVALLA In personal capacity (8 Ali Aslltlr Road, B"",.'".e)DR A. P. MAHADBVAN Hindustan Lever Ltd, Bombay

Da K. K. G. MENON (Alle'''tlle)SR.l S. N. MITItA Central Food Laboratory, CalcuttaSaal S. RAMASWAMY Directorate General of Technical DevelopmentS••I JAMES N. WAltNER In personal capacity (..4UlJhAbatl Agri'tdt..ral

1n,slitute, A IlalJablJtl)

2

AMENDMENT NO. 1 DECEMBER 1989TO

IS : 1167 - 1965 SPECIFICATION FOR CASEIN( EDIBLE (tVALITY )

[ Pal' 4, Table I, Sf No. ( ii ), col 3 ] - Substitute' 3-0 <for .. 2"5 '.

[ Page 4, Table 1, Sf No. ( vii ), col 3 ] - Substitute' 6"0 • for' 5-6 •

(AFDC 34)

IS: 1167 - 1965

Indian Standard

SPECIFICATION FORCASEIN (EDIBLE QUALITY)

(Revised)

o. FOREWORD

0.1 This Indian Standard (Revised) was adopted by the Indian StandardsInstitution on 30 September 1965. after the draft finalized by the DairyIndustry Sectional Committee had been approved by the .Agricultural andFood Products Division Council.0.2 Casein is the chief protein of milk and it exists as a colloidal suspension.It is manufactured from wholesome skim milk by selective precipitation.and removal of whey, subsequent washing and drying of the precipitate.0.3 Since casein is a good protein nutritionally, it is used in the manufactureof protein foods for oral administration in cases of protein malnutrition,gastric disorders and other symptoms which call for protein supplementation.0.4 The Indian Standards Institution has already published a specificationfor- natural sour (lactic) casein 10r glue manufacture (IS: 850-1957) whichcovers the material produced by natural sour (lactic acid) precipitation,meant specially for glue manufacture. But there has been a long ielt need fora standard to cover caseinwhich is used for manufacture of edible products.0.5 The Indian Standard specification for casein (edible quality) (IS: 1167:­1YS7) was published in 1957. The ne~~ for revising the standard was feltbecause the original version covered material prepared bv acid precipitationmethod only. 'The scope has now been enlarged to ~ embrace productsprepared by acid precipitation or natural souring. The limit for moisturecontent has been increased so that the standard would be 'more realistic.Further. additional requirements, such as total acidity, free acidity andtotal bacterial count, have been introduced in this revision.0.6 For the purpose of deciding whether a particular requirement of thisstandard is complied with,- the final value, observed or calculated, express­ing the result of a test or analysis, shall be rounded off in accordance withIS: 2-1960·. The number of significant places retained in the rounded offvalue should be the same as that of the specified value in this standard.

·Rules for rounding 011 numerical values (revised).

3

IS: 1167 -1965

I. SCOPE1.1 This standard prescribes the require:nents and the methods of testfor casein of edible quality. .

2. REQUIREMENTS2.1 Casein shall be prepared from skim milk of either cow or buffalo or amixture of both. Casein shall be nearly white or pale cream in colour andshall have no undesirable odour or any foreign matter (see Appendix L).It shall be free from any added colour or preservative.

].] Particle Size - The size of the particles shall be such that 100 percentby weight of casein shall pass through SOO-micron IS Sieve (see IS: 460­1962·).

NOTE - The apejture of BS Sieve 30 is within the limits laid down for thespecified IS test sieve and may, therefore, be used as SOO-micron IS Sieve.

2.3 HYl1lenlc Requirements - Casein shall be manufactured and storedin premises built and maintained under hygienic conditions (see IS' 2491-1963t)·

2.4 The material shall also comply with the requirements given in Table 1.

TABLE I REQUIREMENTS' FOR CASEIN (EDIBLE QUALITY)'

SLNo.

(1)

CHARACTERISTIC

(2)

REgUIREMEN'l METHOD OFTEST (REF TO

ApPENDIX)

(3) (4)

i) Moistuft', percent by we-ight, M.~ii) Total asb, percent by weight (on dry

basis), Mllxiii) Acid insoluble ash, percent by weight (OD

dry basis) Maxiv) Fat, percent by weight (on dry basis),

M(J~

v) Nitrogen, percent by weight (on drybasis), Mitt

vi) Total acidity in terms of ml of 0·1 NNaOH/g -

vii) Free acidity in terms of ml of 0'1 NNaOH/IO I, Max

viii) Bacterial count, per gram, Maxix) Coliform count, per gram, MlJ~x) Mould count, per gram. Max

xi) .Rate of solubility

10·02·5

0·1

1·5

14·5

6 to 14

5·6

5000010SO

To conform totest

AB

C

D

E

F

G

HJ

KL

·Specification for test sieves (reflisetl) .tCode for _nitary conditions for food processing units. (Since reviled).

IS: 1167 - 1965

3. PACKING AND MARKING

3.1 Packlna - The material shall be packed in such a way as to protectit from contamination and deterioration. Polyethylene lined bags arerecommended.

3.1.1 Unless otherwise agreed to between the purchaser and the vendor,the packages shall be of 20 or 50 kg nett of material.

3.2 Marking - The following information shall be marked legibly andindelibly on each container:

a) Name of the material;b) Name and address of the manulacturer ;

c) Batch or code number; andd) Net weight.

3.2.1 The product may also be marked with Standard mark.

3.1.1 The use of the Standard Mark is governed by the provisions of tbeBureau of Indian Standards Act, 1986 and tbe Rules and Regulations madethereunder. The details of conditions under which the licence for the use ofStandard Mlrk may be granted to manufacmrers or producers may be obtainedfrom the Bureauof IndianStandards.

4. SAMPLING

4.1 Representative samples of the material shall be drawn and tested forconformity to this standard as described in Appendix M.

5. TESTS

5.1 Tests shall be carried out as prescribed in the appropriate appendicesgiven in col .. of Table 1.

5.2 Quality of Reagents - Unless specified otherwise, pure chemicalsshall be employed in tests, and dist.illed water shall be used where the useof water as a reagent is intended.

NOTE -' Pure chemicals' shall mean chemicals that do not contain impuritieswhich affect the results of analysis.

5

18:1167-1965

APPENDIX A

[Table 1, Item (i)J

DETERMINATION OF MOISTURE

A-I. PROCEDURE

A-I.I Weigh accurately about 3 g of the material, powdered if necessary,into a flat bottomed glass or aluminium dish (with a cover) previously driedand weighed. Heat the dish containing the material (after uncovering)in an electric oven maintained at 100° to 102°C for about 3 hours. Cool ina desiccator and weigh wit.h the cover OIl. Repeat t.he process of drying,cooling and weighing at half hourly intervals, until the difference betweentwo consecutive wcighings is less than one milligram. Preserve the dishcontaining this dried material in a desiccator for the determination of totalash (B-l.1).

A-2. CALCULATION

_' 100(W1 - W 2)A-2.t Moisture, percent by weight = ----~::.'JV-

where"Vt ::::: weight in g of the dish with material before drying,Ii'2 =: weight in g of the dish with material after drying, andW =-:.: weight in g of the empty dish.

APPENDIX B[Table 1, Item (ii)]

DETERMINATION OF TOTAL ASH

B-1. PROCEDURE

B-l.1 Heat gently the crucible containing the dried material (A-l.l) onthe flame and then in a muffle furnace at 550 0 ± 20°C till grey ash results.Cool the crucible in a desiccator and weigh, Repeat the process ofheating in the muffle furnace for 30 minutes, cooling and weighing untilthe difference lx-twr-r-n t wo consecu t ivc \Vt'ighings is less than 1 mg.Note the lowest weight. Preserve the dish with the contents for thedetermination of acid insoluble ash (C-2.1).

6

IS: ll67 - 1965

B-2. CALCUL~~TION

. . . . . ,'.. 1no (1 r 1 .-- Jr )B-2.l Total ash, percent by we-ight (on dry basis) :=.. -'---ni-~- fJ' -----

2

wherel-f1 == wviuht in g of the crucible containing the ash,lr :.- weight in g of t he crucible, andJI'2 =:::-: weight in g of the crucible with the dried material taKen

for the test.

APPENDIX C

[Table 1, I tem (iii) j

DETERMINATION OF ACID INSOLUBLE ASII

c-r. REAGENT

C-l.1 Hydrochloric A id - approximatvlv 5 ;\

C-2. PROCEDURE

C-2.1 To the ash contained 111 t lu: porcelain dl:-,h {ll-l.l, add 25 ml hydro­chloric acid, cover with a \\·atch·~la~s and heat on a water-bath Ior 1(\minutes. Al low to coo] and filter the conu-nt- of t lu- dish through a What­man filter paper No. 4! or its equivalent. Wash the filter with water untilthe washings arc Iree from t hv acid. I~t'tllrtl the filt--r and thr rtsidu« h,the dish. Keep it in an electric air-oven maintuiur-d at 135" ± :r'C forabout 3 hours. Ignite ill a muffle furnace at 550') ± 2(r'C for OBt' hourCool the dish in a (it·sircator and \veigh. Repeat the proccs'-; of ignit ing inthe muffle furnace, cooling and \vl~jghin~ at half-hour intervals UHt il t lH'difference between two successive \veighillg.:; is ll'~s 1hall one JllilJig-'·;trll.Notfl the Iowest ,,·eight.

C-3. CAl,CUI~ATION

C-3.1 Acid insoluble ash, percent by weight (on drybasis)

100 (IF 2 ._- l.Jt",)-..--- nil·-~-fl-;--

whereIt· 2 :..:: weight in g of the porcelain dish with the acid insoluble ash;lr :::~ wr-ight in g of tilt' empt v purerlain dish. andIV! ;:-== \\,(light ill g of t he porcelain dish with the dried material taken

for the determination of total ash (8-t.l).

7

IS: 1167 - 1965

APPENDIX D[Table 1, Item (iv)J

DETERMINATION OF FA'r

n-i. APPARATUS

D-l.l Fat Extraction Apparatus - conforming to IS: 2311-1963*.

D-2. REAGENTS

D-2.1 Hydrochloric Acid - density 1·122 g/lnl at 20°C.

D-l.2 Ethyl EtherD-2.3 Petroleum Ether - redistilled slowly at a temperature below 65°C.

D-3. PROCEDURE

D-3.1 Place 10 ml of hydrochloric acid in a tOO-nIl beaker. Carefullytransfer to the beaker about 5 g of the material accurately weighed. Addabout 10 ml of the hydrochloric acid so as to wet and wash down any parti­cles of the material that might. have adhered to the sides. Cautiously heatthe contents over a burner making sure that all particles of the materialadhering to the sides are dissolved in the acid. Finally boil for 10 minutes.Cool the beaker until a temperature of approximately 20°(: is reached.Quantitativl~ly transfer the contents to a Mojonnier flask or a Rohrig tubeusing about 10 ml of acid as wash liquid. Add 25 rnl of ethyl ether to thebeaker and transfer the contents of the Mojonnier flask or the ]-tohrig tube.Stopper with cork or a stopper of synthetic rubber unaffected by usualfat solvents, and shake vigorously for one minute. First add 25 rnl of pet­roleum ether to the beaker and then transfer it to the extraction flask ortube. Repeat vigorous shaking. Centrifuge the Mojonnier flask at about600 rotations per minute. If a Rohrig tube is used, let it stand until theupper liquid is practically clear. Decant the ether solution into a suitableflask or metal dish. Wash the lip and the stopper of extraction flask ortube with a mixture of equal parts of both the solvents and add the wash­ings t.o the weighing flask or metal dish. Repeat extraction of the liquidremaining in flask or tube twice, using 15 1111 of each solvent each time,Evaporate the solvent completely on a hot-plate or steam-bath at a tem­perature that does not cause spattering or bumping. Dry the fat in anoven at the temperature of boiling water to constant weight. Weigh thecooled flask or metal dish, using counterpoise duplicate container handledin the same manner. Remove the fat completely from the containerwith warm petroleum ether and weigh as before.

·Spccification for fat extraction apparatus fur milk and milk products l Sincereviled).

8

IS: 1167 - 1965

D-4. CALCULATION

, . " lOOOO(W.-W.)D-4.1 Fat, percent by weight (on dry basis) = --'ur-;--TiO(f=--m)-'where

U'1 = weight in g of contents in flask or metal dish before removal offat,

W 2 = weight in g of contents in flask or metal dish after removal of fat,Hla ::-::: weight in g of the material taken for the test) andm = percentage by weight of moisture in the material (A 2.1).

APPENDIX E[Table I, Item (,,)]

DETERMINATION OF NITROGEN

E-I. APPARATUS

E-I.l A recommended apparatus, as assembled, is shown in Fig, 1.

E-I.I.I Description - The apparatus consists of a round bottomedflask ..1 of 1 000 m l capacity fitted with a rubber stopper through whichpa.sses one end of the connecting bulbs tube B, The other end of the bulbtube R is connect~~d to the cor~dcn~er ~ which ,is ~ttached. by ~neans of arubber tube to a dip tube D which dips into the liquid contained In a beakerE of 250 ml capacity, .

E-2. REAGENTS

E-2.1 Anhydrous Sodium Sulphate

E-2.2 Copper Sulphate

E-2.3 Concentrated Sulphuric Acid - sp gr 1·84.

E-2.4 Sodium Hydroxide Solution - Dissolve about 225 g of sodiumhydroxide in 500 ml of water.

E-2.5 Standard Sulphuric Acid - approximately 0·1 N.

E-2.6 Methyl Red Indicator Solution - Dissolve one gram of methylred in 200Inl of rectified spirit (95 percent by volume).

E-2.7 Standard Sodium Hydroxide Solution - approximately 0·1 N.

9

IS: 1167 - 1965

FIG. 1 i\PPARATCS FOR DETER:\II~ATIO~ OF NITROGEN

E-3. PROCEDURE

E-3.1 Transfer carefully 0·2 g of the. material. accurately weighed. to aKjeldahl flask. taking precaution to see that particles of the material do notstick in the neck of the ftask. Add about 15 to 18 g of anhydrous sodiumsulphate. about 0·2 g of copper sulphate and 2S ml of concentrated sulphuricacid. Place the flask in an inclined position. Heat below "the boilingpoint of the acid until frothing ceases. Increase heat until the acid boilsvigorously and digest for 30 minutes after the mixture becomes clear and

10

IS: 1167 - 1965

pale green or colonrless. Wash down with the minimum quantity ofconcentrated sulc.iuric acid, particles, if any, sticking to the sides andcontinue digestion for 60 to 90 minutes. Cool the contents of theflask. Transfer quantitatively, using water, to the round bottom flaskA and dilute to 250 ml. Add with shaking, a few pieces of pumice stoneto prevent bumping. Add about 60 ml of sodium hydroxide solution ormore, if necessary (to make the solution alkaline) carefully througa theside of the flask so that it does not mix at once with the acid solution butforms a layer below it. Assemble the apparatus as shown in Fig. 1, takingcare that the dip tube D extends below the surface of a known quantity ofstandard sulphuric acid contained in the beaker E. Mix the contents ofthe flask by shaking and distil until all ammonia has passed over into thestandard sulphuric acid. Detach flask A from the condenser and shut offthe burner. Rinse the condenser thoroughly with water into the beakerE. Wash the dip tube D carefully so that all traces of the condensateare transferred to the beaker. When all the washings have drained intothe beaker E. add two or three drops of methyl red indicator and titratewith standard sodium hydroxide solution.

E-3.2 Carry out a blank using all reagents in the same quantities butwithout the material to be tested.

E-4. CALCULATION

E-4.1 Nitrogen percent by weight -- ~i!!J1! - AJ. N, - W (100 - m)

whereB = volume in ml of standard sodium hydroxide solution used to

neutralize the acid in the blank determination,A = volume in ml of standard sodium hydroxide solution used to

neutralize the excess of acid in the test with the material,N = normality of standard sodium hydroxide,W == weight in g of the material taken for the test, andm = percentage by weight. of moisture in the material (A-l.l).

APPENDIX F[Table 1, Item (vi)]

DETERMINATION OF TOTAL ACIDITY

F-t. REAGENTSF-l.l Standard Sodium Hydroxide Solution - 0·1 N.F-l.2 Standard Sulphuric Acid - 0·1 N.

11

IS: 1167 - 1965

1'-1.3 Bromotbymol Blue Indicator Solution - 0·04 percent (tv/v)in rectified spirit.

F-l. PROCEDURE

F-2.1 Transfer 2 g of the sample to a 2S0-ml Bask. Add 100 rnl of freshlyboiled and cooled distilled water and soak for 15 minutes. Add 50 ml of0·1 N sodium hydroxide solution and close the mouth of the flask. Heatin a water-bath at SO°C and shake until the sample has been completelydissolved. Titrate with standard sulphuric acid. using 1 ml of 0·04 percentsolution of bromothymol blue as indicator. Carry out the determinationomitting the casein and make a suitable correction. The acidity is expressedas the number of millilitres of 0·1 N sodium hydroxide solution required toneutralize 1·0 g of sample.

APPENDIX G[Table 1, Item (vii)]

DETERMINATION OF FREE ACIDITY

G-I. REAGENTS

G-l.1 Standard Sodium Hydroxide Solution - 0·1 N.-

G-l.2 Phenolphtbalein Indicator Solution - prepared by dissolving0·1 g of phenolphthalein in 100 ml of 60 percent rectified spirit(se, IS: 2263-1962*).

c-a. PROCEDURE

G-2.l Weigh 20 g of casein sample to within 0·1 g and transfer to a 500-mlbeaker containing 200 ml of boiling distilled water. Cover the beaker witha watch-glass while allowing the mixture to stand for 30 minutes withfrequent agitation. Filter the aqueous layer and pipette 50 ml of the coldfiltrate (corresponding to 5 g of casein) into a flask. Titrate with standardsodium hydroxide solution using phenolphthalein indicator. The freeacidity is expressed as the number of rnillilitres of O·t N sod ium hydroxidesolution required to neutralize 10 g of sample.

-.Methods of preparation of indicator solutions for volumetric a nalysis,

12

IS: 116' - 1965

APPENDIX H[Table 1, Item (viiij]

DETERMINATION OF BACTERIAL COUNT

9-1. APPARATUS

R-l.l Weiahina Scoop. Str.rtle - with count.er weight.

H-l.2 Bacterlologtcal Tr msfer Pipettes, Sterile -- accurately gradu­ated, with cotton plug in t he upper orifice.

H-I.3 Dilution Bottles. SterIle - made of heat resistant glass (prefer­ably borosilicate glass) of the following capacities:

a) 150 ml, with mark at 99 mllevel ; andb) 25 rnl, with mark at 9 mllevel.

H-I.4 Petri Dishes - with outside dish diameter 100 mrn, inside dishdiameter 91 mm, and depth 15 mm. The exterior and interior surfaces ofthe bottoms should be flat and free from bubbles, scratches or other defects.

H-I.5 Bacteriological Tubes, Sterile - 2S ml capacity with a markat the 10 ml level, with cot ton plugs.

B-2. REAGENTS

8-2.1 Dilution Waters - There shall be two types of dilut ion water,namely, Dilution Water 1 for usc with Medium 1 (9-2.2.1) and DilutionWater 2 for usc with Medium 2 (H-2.2.2). Sterilize dilution, water at121°C for 15 minutes before use.

H-2.1.1 Dilution Water 1 - Dissolve 34 g of potassium dihydrogen.phosphate (KIlzP04) in 500 ml of distilled water. adjust to pH 7-2 with 1 Nsodium hydroxide solution and make up to one lit re wit h distilled water.Dilute 1·25 ml offhis stock phosphate buffer solution with water to onelitre to obtain dilution water.

8-2.1.2 Dilution Wliter2 - shall have t.he following composition :

Sodium chloride 9 gPotassium chloride 0·42 gCalcium chloride 0·24 gSodium bicarbonate 0·2 gDistilled water 1 000 rnl

H-2.2 Me.dia - Either of the two media gIven III 8-2.2.1 and H-2.2.2shall be used.

13

Is: 116' -1965

8-2.2.1 Medi·um 1 - shall have the following composition:

Peptone 5-0 gYeast extract 2-5 gGlucose (dextrose) 1·0 gAgar I bacteriological grade 20·0 gDistilled wat.er I 000 lui

8-2.2.1.1 Preparation - Soak the materials (B-2.2.1) for 3 to 5minutes in cold water, then bring the mixture into complete solution withminimum delay by boiling above an asbestos centred wire gauze over aflame. Stir continuously and efficiently to avoid charring. Adjust thesolution to pIl 7·0 at SOt"lC with sodium hydroxide solution. Filter throughcotton pad till clear filtrate is obtained. Fill into bacteriological tubes to10-1uIIuark. Sterilize in an autoclave at 121°C for 20 minutes.

"-2.2.2 Medium 2 - shall have the following composition:

Yea~rcl 3 gPeptone 5 gAgar, bacteriological grade 20 gFresh whole milk 10 mlDistilled water 1000 ml

8-2.2.2.1 Preparation - Dissolve the yeastrel and the peptone indistilled water in the steamer and adjust the reaction at room temperatureto pH 7·4 using phenol red as the indicator. Wrap shreded agar in muslinand wash in running cold water for 15 minutes, Squeeze out the excesscold water and add the agar together with the freshly shaken milk to thebroth. Autoclave at 121°(: for 20 minutes and filter through paper pulp(see Note) in a Buchner funnel. (Egg should not be used for clearing.)Take the agar directly from the autoclave and filter hot, the whole appa­ratus being kept warm by a surrounding atmosphere of steam, Test thereaction of the filtrate at 50°C and adjust, if necessary, to pH 7,0, so thatthe final reaction at room temperature is pH 7·2.

Fill this medium into bacteriological tubes to Ifl-ml mark. Sterilizein an autoclave at 121°C for 20 minutes,

NOTE - The paper pulp is prepared by pulping small pieces of postlip or whiteheather paper in water by means of a pestle and mortar. A single layer of Chardinfilter paper should be laid on top of the Buchner funnel to prevent the pulp beingsucked through, and t.he pulp itself should then be packed down evenly on topof it. The filter when ready for use, should have a total depth of about l'S rom.

"-3. PROCEDURE

0-3.1 Dilution - Weigh 11 g of the material from the sample marked· For Bacteriological Tests' (M-3.1) using a sterile spatula and suspendin 99 1111 of dilution water at 45°C. Agitate mildly, soak for 1 to 3 minutesand then agitate vigorously to avoid churning out the fat. Prepare

14

IS: 1167 - 1965

dilutions of this so as to give bet wor-n 30 and .lOU colonies per plate(see H-3.~). Add one millilit.rc of suitable dilutions in t riplicate to thesterile petri dishes. Use at least 5 dilutions.

8-3.2 Pouring Plates - Melt agar medium (H-2.2) in bacteriologicaltubes and keep at 48° to 50°C. Introduce this medium aseptically, at42° to 44°C, into the petri dishea and mix by rotating and tilting disheswithout spreading over the edges. Spread the mixture evenly over thebottom of the plate. Allow to so1i(lify.

8-3.3 Incubation - Invert the plates and incubate at 37°C for 48 hours.

H-3.4 Counting - Count the colonies with the aid of magnification underuniform and properly controlled illumination. Count only those plateswith 30 to 300 colonies, including pin point size colonies. .

8-3.5 Computation - Compute the bacterial count per gram from thedilutions used.

NOTE - All precautions shall be observed to prevent microbiological conta­mination throughout the test.

APPENDIX J[Table 1) Item (ix)]

DETERl\fINATION OF COLIFORM COUNT

.r-i. GENERAL

J -1.1 Coliform Bacteria - Coliform bacteria includes all aerobic andfacultative anaerobic, Gram-negative. nonspore --- forming bacteria whichferment lactose with the production of acid and gas. A positive presumptivetest is indicated by the formation of acid and gas within 48 hours at 35° to37°C in a fermentation tube containing lactose bile salt broth, Alternative­ly, the development of dark red colonies at least O'5 mm in diameter in asolid medium (violet and rr-d bile agar) within 20 to 24 hours at 35° to 37°Cmay be considered as a positive evidence of the presence of coliform bacteria.Violet red bile agar is one of t he standard media used for determinationof general types of coliform organisms including those of faecal origin inwater .. milk and other materials of sanitary importance.

J-l. APPARATUS

J-2.1 Weighing Scoop, Sterile - with counter weight.

J-2.2 Bacteriological Transfer Pipettes, Sterile - accurately gra­duated, with cotton plug in the upper orifice.

15

3·0 ~7·0 g1·5 g

10·0 g5-0 g

20·0 g

IS: 1167 - 1965

J-l.3 Dilution Bottles, Sterile - made of heat-resistant glass (prefer­ably borosilicate glass) closed with rubber stoppers, preferably screw capswith new friction-fit liners for making them leak-proof and of the followingcapacities:

a) 1SO ml, with mark at 99 mllevel ; andb) 2S ml, with mark at 9 mllevel.

J-2.4 Petri Dishes -\vith outside dish diameter 100 mm, inside dishdiameter 91 mm, and depth 15 mm, The exterior and interior surfaces ofthe bottom should be flat and free from bubbles, scratches or other defects,which would interfere with counting of colonies.

J-l.5 Baeterioloaical Tubes, Sterile - 25 ml capacity with a mark atthe lO-mllevel, with cotton plugs. ..

J-2.& Tubes having Inverted Vials - Durham tubes.

J-3. REAGENTS

J-3.1 Dilution Water - Dissolve 34 g of potassium dihydrogen phosphate(KH.PO,) in 500 ml of distilled water. adjust to pH 7-4 with 1 N sodiumhydroxide solution and make up to one litre with distilled water. Dilute1·25 ml of this stock phosphate buffer solution with distilled water to onelitre to obtain dilution water.

J-3.2 Medium - Violet red bile agar shall be the medium, the compositionof which shall be as follows:

Yest extractPeptoneSodium taurocholateLactoseSodium chlorideAgar-agar

Indicator:

Neutral red 0-03 gCrystal violet 0·002 gWater 1 000 mlFinal pH 7·4±O·t

J-3.2.1 Preparation and Sterilization of Medium. - Soak the mate­rials (J-3.2) for 3 to 5 minutes in cold water, then bring the mixture intocomplete solution with minimum delay by boiling above an asbestos-centredwire gauze, over a flame. Stir continuously and efficiently to avoid charr­ing. Adjust the solution to pH 7·4±O-t at 50°C with sodium hydroxidesolution. Filter through cotton pad till clear filtrate is obtained. Fillthe filtrate into bacteriological tubes to to-rot mark. Sterilize in anautoclave at 121°C for 15 minutes.

16

IS: I1b1 - 1965

J-4. PROCEDURE

J -4.1 Dilution - Using aseptic precautions and appropriate meansthoroughly.mix each sample until representative portions may be removed,Heat 99 ml of sterile 2 percent sodium citrate solution into a sterile con­tainer (preferably 150 ml wide-mouth screw-cap jars) to 45°C for dispersingand diluting casein. Promptly add 11 g of casein and triturate it withsterile glass rod until thoroughly mixed. Allow the mixture to cool at roomtemperature. Prepare suitable dilutions of this and add one millilitre ofsuitable dilutions in triplicate to the sterile petri dishes.

J-4.2 Pouring Plates - Melt the medium (J-3.2.1) in bacteriologicaltubes and keep at 48° to 50°C. Int.roduce this medium aseptically at 42°to 44°C into the petri dishes and mix by rotating and tilting dishes withoutspreading over the edges. Spread the mixture evenly over the bottom ofthe plate. Allow to solidify, After solidification of medium in plate, addcover layer of medium.

J ..4.3 Incubation - Invert t.he plates and incubate at 35° to 37°C for24 hours.

J-4.4 Counting - Count the dark red colonies which have a diameter of0·5 rom or over.

J-4.5 Computation - Compute the coliform count per gram from thedilutions used (J-4.1).

NOTE t - 1n case of doubt regarding the colonies developed 011 violet redbile agar, representative colonies are picked and transferred to lactose hile saltbroth in tubes hav i ng inverted vials. Production of acid and gas is confirmatoryfor coliform organisms.

NOTE 2 .._.~ 1\11 precautions shall be observed to prevent microbiological conta­mination throughout the test.

APPENDIX K

[Table 1, Item (x)J

DETERMINATION OF MOULIl COUNT

x-i. GENERAL

K-l.l Mould -l-Iigh mould count is not desirable and indicates thatcasein is manufactured under improper plant sanitary control. It is alsoa result of improper packing and faulty storagt'. The develupment ofcolonies in a solid medium (potato-dextrose-agar at pH 3·5) within 5 daysat 22°(. should be considered as a positive evidence of moulds.

17

IS: 1161- lCJ65

K-l. APPARATUS

K-l.1 Weighing Scoop, Sterile - with counter weight.

K-2.1 Bacterlolol1lcal Transfer Pipettes, Sterile - accurately gradu­ated, with cotton plug in the upper orifice.

K-2.3 Dilution Bottles, Sterile - made of heat-resistant glass (preferablyborosilicate glass) closed with rubber stoppers, preferably screw caps withnew friction-fit liners for making them leak-proof and of the followingcapacities:

a) 150 ml, with mark at 99 mllevel; andb) 25 ml, with mark at 9 mlleveI.

K-2.4 Petri Dishes - with outside dish diameter 100 mm, inside dishdiameter 91 rnm, and depth 15 mm, The exterior and interior surfaces ofthe hottom should be flat and free from bubbles, scratches or other defects,which would interfere with counting of colonies.

K-l.5 Bactertologlcal Tubes, Sterile - 25 ml capacity with a mark at10 mllevel, with cotton plugs.

K-3. REAGENTS

K-3.1 Dilution Water - Dissolve 34 g of potassium dihydrogen phosphate(KH1P04) in 500 rnl of distilled water, adjust to pH 7·4 with 1 N sodiumhydroxide solution and make up to one litre with distilled water. Dilute1·25 ml of this stock phosphate buffer solution with distilled water to onelit.re to obtain dilution water.

K-3.2 Medium - Pot.ato glucose agar (acidified) shall be the medium,the composition of which shall be as follows:

Potato infusion From 200 g of white potatoes (see Note)Agar 15 gGlucose 20 gDistilled Water 1 000 rnl

NOTE - Suspend 200 g of sliced potatoes in distilled water sufficient to coverthe material. Boil for one hour and filter through cheese cloth.

K-3.2.1 Preparation and ..~te,ilization of Medium - Heat the mix­ture (K-3.2) to boiling to dissolve the ingredients. Distribute into tubesor flasks and autoclave for 15 minutes at 121°e. Melt in flowing steamor boiling water, and cool. Adjust reaction in each container to pH 3·S±O·twith sterile 10 percent tartaric or lactic acid. As remelting of acidifiedmedium may destroy its solidifying properties, adjust only amount neededfor immediate plating. Amount of acid required for adjustment in anyone flask of same batch of medium ordinarily will establish amount neededin each of the other flask containing equal quantities.

18

IS: t 16'1 - 19t-5

For colorimetric adjustment, use bromophenol blue and titrate 5 m1of medium with dilute acid prepared by adding 1 ml of sterile 10 percentstock solution to 19 rnl of water. The number of millilitres of dilute acidused to titrate to pH 3·5 will represent the amount of stock solution thatshall be added to 100 ml of medium.

K-4. PROCEDURE

K-4.1 Dilution - Using aseptic precautions and appropriate means,thoroughly mix each sample until representative portions may be removed,Heat lJY ml of sterile 2 percent sodium citrate solution into a sterile container(preferably 150 111 I wide-mouth screw-cap jars) to 45°(: for dispersing anddiluting cast-in, Prompt ly a<1(1 1t g of cast-in and triturate it with sterileglass nul until thoroughly mixed. Allow the mixture to cool at room tC111­

perature, Prepare suitable dilutions of this and add one millilitre ofsuitable dilutions in triplicate to the sterile petri dishes.

K-4.2 Pouring Plates --1'{(-1t the medium (K-3.2.1) in bacteriologicaltubes and keep at 4X') to 50('C. Jntroduce this medium aseptically at 42 0

to 44"( into the petri dishes and mix by rotating and tilting dishes withoutspreading over the edges. Spread the mixture evenly over the hottomof the plate. ....\110\\· to solidify. After solidification of the medium inplate, add cover layer of medium.

K-4.3 Incubation - Invert the plates and incubate at 22°C for 5 days.

K-4.4 CountinQ- If moulds are developing in large numbers, count plateson the third day of incubation and then recount on the fifth day.

K-4.5 Computation - Compute the mould count per gram from thedilutions used (see K-4.1).

APPENDIX L

[1"able 1J ItC11t (xi)]

DETERMINATION OF RATE OF SOI4UBILITY

t-r. APPARATUS

L-l.1 Glass Stirring Rods -- wit tl flat tr-ued ends.

L-I.2 Centrffuge Tubes-(~nni('al, apprux imatr-lv 170 mm long and25 1l1n1 diametr-r , graduah-d at 50 ml and at 0·1 ml intervals- between 0and 1 InI and at 0·2 III 1 intervals between 1 and 3 rnl.

19

IS: 11" - 1965

L-l.3 Centrlfuae -:- with swing-out buckets and, capable of a speed togive approximately 900revolutions per minute.

L-2. REAGENTS

L-].l Borax Solution - dissolve 20 g of sodium tetraborate (Na.B,O"10B.O) in water and dilute to 1 000 mi.

L-].] Water - pH 6·0 at 20°C.

L-2.3 Sodium Hydroxide Solution - 1-0 N_

L-3. PROCEDURE

L-3.1 Weigh 6 g of the casein sample in a test-tube containing a glassstirring rod. Add 36 ml of borax solution in the test-tube, stir and mixthe contents with the rod. Add a volume of 0-1 N sodium hydroxide solu­tion equivalent to the • Free acidity J, (see 0-1.1). Heat the tube in awater-bath at 70°±1°C with continuous mixing of the contents for thefirst 5 minutes, and then at an interval of every 5 minutes for a period of40 minutes. If the casein dissolves completely, note the time of completesolution. If after 4S minutes the casein is not completely dissolved, transferthe contents of the test-tube to a centrifuge tube, wash the test tube withwater at 70°C and collect the washings until the volume in the centrifugetube becomes 50 ml. Centrifuge at a speed of about 900 rev/min for 10minutes. Decant the supernatant liquid, including any light materialwhich may be present above the compact, lower layer of sediment. Add40 ml of cold water, mix and centrifuge again for 10 minutes at 900 rev/min.Read the volume of sediment excluding any light material above the compactlayer.

L-4. REPORT

L-4.1 For conformity, the casein sample shall not have sediment which isindication of the presence of insoluble material.

APPENDIX M(Clause 4.1)

SAMPLING OF EDIBLE CASEIN

M-l. GENERAL REQUIREMENTS OF SAMPLING

M-l.O In drawing, preparing storing and handling samples the followingprecautions and protections shall be observed.

20

IS: 1167 - 1965

M-I.l Samples shall be taken in a protected place not exposed to dampair, dust or soot.

M-l.2 The sampling instrument shall be clean and dry when used. Whentaking samples for bacteriological examination, it shall also be sterile.

M-l.3 The samples, the material being sampled, the sampling instrumentand the containers for sampling shall be protected from adventitious conta­mination.

M-l.4 The samples shall be placed in clean and dry glass containers. Thesample containers shall be of such a size that they are almost completelyfilled by the sample. The sample containers shall, in addition, be sterilewhen they are used for samples for bacteriological examination.

M-l.!5 Each sample container shall be sealed air-tight after filling andmarked with full details of sampling, batch or code number, name of themanufacturer and other important particulars of the lot.

M-I.6 The samples shall be stored in such a manner that the temperatureof the material does not vary unduly from the normal temperature andthat they are protected from light.

M-l.7 Sampling shall be done by a person agreed to between the purchaserand the vendor and in the presence of the purchaser (or his representative)and the vendor (or his representative).

M-2. SCALE OF SAMPLING

M-2.1 Lot - In a single consignment all the containers containing thematerial of the same grade and the same batch of manufacture shall consti­tute a lot.

M-].2 Sample shall be tested for each lot for ascertaining the conformityof the material for the requirements of this specification.

M-l.3 The number of containers to be selected from a Jot shall depend onthe size of the lot and shall be in accordance with col 1 and 2 of Table 2.

M-l.4 These containers shall be chosen at random and for this purposea random number table as agreed to between the purchaser and the vendorshall be used. In case such a table is not available the following procedureshall be adopted:

Starting from any container, count all the containers in one orderas 1,2,3-, up to r and so on. Every rth container so counted shallbe withdrawn, where, is the integral part of N]», N being the numberof containers in the lot and n the number of containers to be selected.

M-3. PREPARATION OF SAMPLES

M-3.1 Individual Samples for Baetertologlcal Examination - Drawwith in appropriate sampling instrument, which is sterile, about 300 g

21

IS: 1167 - 1965

TABLE 1 NUMBER OF CONTAINERS TO BE SELECTEDFOR SAMPLING

(CIIJ"s, M-2.3)

NUMBER OFCONTAINEas IN

THB LOTN

12 to 89" 25

26 II 5051 ,. 300

301 II 500501 and over

NUMBER OFCONTAINERS TO

BE SELECTED

•1234568

of the material from each selected container by taking small portions ofthe material from at least 10 different parts of the container. Thoroughlymix all Portions of the material drawn from the same container. Takeabout 100 g of the material and divide this material into three equalparts taking care not 10 bring any bacterial contamination. Each partso obtained shall c-onstitute an individual sample representing the consign­ment and shan be transferred to sterile glass containers which shall besealed air-tight and labelled ,..."ith the particulars described in M-I.5 andthe words f For Bacteriological Tests'. The individual samples so obtainedshall be divided into three sets in such a "ray that each set has a samplerepresenting each selected container. One of these sets shall be markedfor the purchaser, another for the vendor and the third for the' referee.

M-3.2 Composite Samples for Other Tests - From the mixed materialfrom each selected container remaining after the individual sample hasbeen taken, equal quantities of the material from each container, shall betaken and mixed together so as to give material representative of the lotand weighing not less than 200 g. This material shall be divided into threeequal parts. Each part so obtained shall constitute a composite samplerepresenting the lot and shall be transferred to a clean, dry container madeof glass or tinplate and labelled with the particulars given in M-l.5. Oneof these composite samples shall be for the purchaser, another tor th~ vendorand the third for the referee.

M-3.3 Referee Samples - Referee samples shall consist of a set ofindividual samples (M-3.1) and a composite sample (M-3.2) marked forthis purpose and shall bear the seals of the purchaser and the vendor. Theseshall be kept at a place and under conditions agreed to between the pur­chaser and the vendor.

22

IS: 1167 - 1965

M-4. NUMBER OF TESTS

M-4.1 Tests for the determination of bacterial count shall he conductedseparately on each of the samples constituting a set of individual samples(M-3.1).

M-4.2 Tests for the determination of t 11(~ remaining characteristics shallbe conducted on the composite sample (M-3.2).

M-S. CRITERIA FOR CONFORMITY

M-5.1 The lot shall btl considered as conforming to the requirement of thisspecification when :

a) each of the test results for bacterial count sat.sty the requirementsspecified in Table 1 (Sl No. viii) I and

b) all the test results on the composite sample satisfy the respectiverequirements as specified in this specification.

23

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