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 2860 (1964): Methods of sampling and test for processedfruits and vegetables [FAD 10: Processed Fruits andVegetable Products]

REAFFIRMED , ./;.

IS : 2860 ·1984(Reaffirmed 2001)

Indian Standard REAFFIRMED- ,-,>, ZOC5'

METHODS OF SAMPLING AND TESTFOR PROCESSED FRUITS AND VEGETABLES

Second Reprint AUGUST 2005(Including Amendment No.1)

UDC 664.831.86 : 620.1

C Copyright 1965

BUREAU OF INDIAN STANDARDSMANAK BHAVAN, 9 BAHADUR SHAH ZAPAR MARO

NEW DELHI 110002

Gr 8 January 1965

xxxx 2008

xxxx

IS: 2860 • 19M

·1ndian Standard

METHODS OF SAMPLING AND TESTFOR PROCESSED FRUITS AND VEGETABLES

Fruits and Vegetables Sectional Committee, AFDC 23

CluJirmanSBBI R. T. MmCBANDANI

&pr,s,nting

Directorate of Marketing & Inspection (Ministry ofFood & Agriculture), Nagpur

Indian Council of A~cu1tural Research, New DelhiTechnical Standardization Committee (FoodatufJi)

( Miniltry of Food & Agricul ture )

Development Council for Food Processing Industries( Ministry of Industry & Supply)

Indian Agricultural Research Institute, New Delhijadavpur University, CalcuttaAgriculture Department, Government of Assam,

GauhatiAgriculture Department, Government of Maharash­

tra, PoonaState Horticultural Research Station, Government of

West Bengal, KrishnagarAgriculture Department, Government of Madras,

Madras

HOBTI011LTUJWJT

HOMTIOl7LTOBJIT

Da. G. S. SIDDUPA

HOftIOULT'C"&UT AND A8800IATBPaoJlWUOa 01' HOBTICULTUBB,AQalCULTUUL CoLLEO. &.Ra• .-cB IlfITITUTB, CoIK-.A'rO"a•• S. L. KATYAL

Col:,M. N. KVJrDt1

In personal capacity ( NasirlJur Farm, P.O. BaJuul"..garhFort, fatima)

Central Food Technological Research Institute,Mysore

8••1 H. C. BIUT.AGA. (.4llmUltl)Da. D. N. SBIV"T£VA • Directorate of Fruit Utilization, Government. of

Uttar Pradesh, Ranikhet

M""b"$S.NIOB MAlmmTINO OJirIClI:B

( FBPIT PBoDUCT8) (Alumat,to Shri R. T. Mirchandani)

SBBIP.D.BBABGAVA

DB. B. CHOUDHUaYDBI\ T. K. GBoaBHOBTIOULTURIST

Da. P. K. KTIIAL (Allmuau)Da. D. S. PATJIL Pure Products at Madhu Canning Private Ltd.,

BombaybaI~. V. PJlADlO8 Nagpur Orange Growen' Co-operat1w AuociatiOD

Ltd., Nagpur .,..aD! B~ B. SABDauAlfD. AU India Food Preservers' Association, Bombay

SBBJ R. N. GOYLII ( Al1lm4ll)Simi SATWANT SmQ.

( ContitwlJ ora /Jag' 2 )

BU_AU OF INDI"AN ST AN,DARDSYANAK. BHAV4.N, 9 BAHADUR SH~H ZAFAR MARG

NEW DELHI 110002

IS I 2860 • 1964

( Continuedfrom page I )

Members

DB.Y.K.SUBRAHMANYAM

SHBI L. ]. TAYLORVBGBTARLB: BOTANIST

DR.'D. V. KARMARKAB»Deputy Director (Agri & Food)

Representin,

Central Committee for Food Standards ( Ministry ofHealth ), New Delhi

Hindustan Lever Limited, Bombay'Agriculture Department, Government of Punjab,

ChandigarhDirector, lSI (&-offkio Memb,r)

S"rltarySURI G. S. VILltRU

Extra Assistant Director ( Agri & Food ), lSI

Processed Fruits and Vegetables Subcommittee, AFDC 23 : 3

Directorate of Marketing & Inspection (Ministry orFood & Agriculture), Nagpur

Tims Products Ltd., Calcutta

Midland Fruit and Vegetable Products, India,Mathura; and the Development Council for F~Processing Industries (Ministry of Industry' &.Supply) J

The Canning Industries Cochin Limited, Trichur

Da~ T. R. S8ADA ~ AI""",,, )

Convener

SSBI K. U. PATEL

MembnsAORICULTURAL MARKETING AD­

VISER TO THE GOV~RNMENTOJ'

INDIASRRI S. C. BHA'M'ACHARJYA.

( Alumau )SHBt P. D. BHARGAVA

DB. D. S. PATEL

SUBI M. I. DAVIDSHBI C. V. PAUL (Alternat,)

COL A. G. FERNANDES Army Headquarters ( Ministry of Defence )LT-COL N. G. C. ISNGAB (Alttrnat,)

DB. T. K. GROSZ jadaY\'ur University, CalcuttaSSRI GI&DBARI LALL ProviSion Merchants' Associatlon, DelhigaB, M. S. KOHLI Nagpur Orange Growers' Co-operative Association

Ltd.) NagpurTechnical Standardization Committee (Foodstuff.)

( Ministry of Food & Agriculture)Pure Products and Madhu Canning Private Ltd.,

Bombay81mI N. S. POCHXBANWALA . D & P Products Private Ltd., Bombay

SHRI L. J. D'SOUZA ( Alumat« )Da. G. S. SIDDAPPA Central Food Technological Research Institute,

MysoreSHal H. C. BSATNAGA.B (.4./tnnat,)

D•. Y. K. SUBBAHlUNYAK Central Committee for Food Standards (Miniatry orHealth), New Delhi

SHBI D. S. CHADHA ( A/141M" )SUBt L. j. TAYLOB Hindustan Lever Limited, Bombay

DB. H. C. SRIVASTAVA (Altnnau),DB. P. K. VUAYABAGIU.YAN Research at Development Organiaation ( Mini.try or

Defence )

2

AMENDMENT NO. 1 FEBRUARY 1995TO

IS 2860: 1964 METHODS OF SAMPLING AND TESTFOR PROCESSED FRUITS AND VEGETABLES

(Page 4, Table 1 ) - Add the following as 'Column 4':

'ACCEPTANCENUMBER~C)

(4)

o11

2233'

( Page6, clause3.4..5 ) - Add the following new clause after 3.4.!:

'3.4.6 The Jot shall be declared as acceptable for organoleptic characteristics,such as colour, flavour, defects, size and appearance, if the maximumDumberofdefectives in tbe sample does not exceed the permitted acceptance number (c)given in 0014oCTable 1.'

(FAOIO)Printed st: Sita AM Arts (P) Ud., New Delhi

IS: 2860 -1964

I ndian Standard

METHODS OF SAMPLING AND TESTFOR PROCESSED FRUITS AND VEGETABLES

o. FO R E W 0 R D

0.1 This Indian Standard was adopted by the Indian StandardsInstitution on 19 September 1964, after the draft finalized by the Fruitsand Vegetables Sectional Committee had been approved by the Agricul­tural and Food Products Division Council.

0.2 Different methods of analysis are at present being followed in differentlaboratories for testing the processed fruits and vegetables. Owing todifferences in the methods employed, there have been often differences ofopinion about the quality of the consignments. With the adoption of themethods prescribed in this standard, it is expected that differences in theresults due to adoption of different methods in different laboratories willbe reduced.

0.3 In reporting the result of a test or analysis made in accordance withthis standard, if the final value, observed or calculated, is to be roundedoff, it shall be done in accordance with IS : 2-1960*.

1. SCOPE

1.1 This standard prescribes the methods of sampling and test for processedfruits and vegetables.

2. Q,UALITY OF REAGENTS

2.1 Pure chemicals and distilled water (see IS: 1070 - 1960t ) shall beemployed in tests- Where a reagent of a special quality is required, ithas been specified in the detailed method.

NOTB - , Pure chemicals' shall mean chemicals that do not contain impurities whichaffect the experimental results.

3. SAMPLING

3.1 GeDeral Requirement. of Sampling

3.1.1 Sampling shall be done by a person agreed to between thepurchaser and the vendor and if desired by any of them, in the presence

• Rules for rounding off numerical values ( "vised).tSpecification for water, distilled quality ("ZJis,d).

3

IS I 2860 -1964

of" the purchaser (or his representative) and the vendor (or hisrepresentative ).

'.1.2 Samples shall be stored in such a manner that the temperature ofthe material does not vary unduly from the normal temperature.

3.2 Scale oflSamplla1

3.2.1 Lot - In any consignment, all the cans of the same size contain­ing material of the same type, style, and grade shall constitute a lot.

. 3.2.1.1 Samples shall be. examined from each lot separately forascertaining the conformity of the material to the respective specifications.

3.2.2 Selection of Sample - The number of packing cases to be selectedfrom a lot for drawing the sample cans shall depend on the size of the lotand shall be in accordance with col 1 and 2 of Table I.

TABLE 1 SCALE OP SAMPLING

No. OJ' CAlfS No. OJ' PACKING CAl. No. OJ' CANS TOIN 'lB. LOT TO BB OPENED . DB SBLBCTED

(1) (2) (3)

Up to 200 3 6201

"300 .. 8

301"

300 5 10

501 "SOO' ,6 J~

UOI " 1300 7.: 141SOl " S200 8 I'SS 201 and above 10 20

3.2.3 The packing cases shall be chosen at random from the lot and forthis purposesome random number table as agreed to between the purchaserand the vendor shall be used. In case such a table is not available, thefollowing procedure shall be adopted.

3.2.3.1 Arrange all the packing cases in a systematic manner andcount them as 1, 2, 3.......•....etc, up to r and so on. Every rth case socounted shall be withdrawn,

, being the integral part of-.!i.nwhere

N -= total number or cases In the lot, andII -= number of cases to be chosea.

IS I 2860 • 1964

If, turns out to be a iractional number, its value should be taken asequal to its integral part.

3.2.4 From each of the packing cases selected ( 3.2.3.1 ), t\VO cans shallbe drawn at random so as to get the total number of cans from the lot asshown in 'col 3 of Table 1.'

3.1.5 In addition to the cans selected as in 3.2.4, select 8 cans atrandom as far as possible from all the cases selected ( 3.2.3 ) for testing themicrobiological requirements.

3.3 Te.dDI of Sample.3.3.1 Each of the cans, selected as in 3.2.4, shall be tested individually

for all the requirements as laid down in the respective specifications,except the requirements for metallic impurities ( see 3.3.2 ) and the micro­biological requirements ( se« 3.3.3 ).

3.3.2 After testing the sample cans ( 3.2.4) for individual characteristics,the contents of all the cans shall be mixed to form a composite sample andthe composite sample so formed shall be tested for arsenic, lead, copper,zinc, tin and calcium.

3.3.3 Tests for Bacteriological Requirements3.3.3.1 Incubation at 37°C - Half of the cans selected as in 3.2.5 shall

be incubated at 37°C for not less than 7 days and subjected to bacterio­logical examination.

3.3.3.2 Incubation at 55°C - The remaining half of the cans shall beincubated at 55°0 for not less than 7 days and subjected to bacteriologicalexamination.

NOT. - In the C&I~ ofproceued fruits, the cans shall be incubated only at 37°C.

3.4 Criteria for Coalormlty3..1.1 For Vacuum and Head Space Rtqui"ments- The lot shall be declared

as conforming to the requirements for vacuum and head space, when eachof the sample cans (3.2.~) tested individually satisfies the 'requirementsgiven in the individual specification for the material.

3.4.2 For Draillld Wligh' - The average of the results of drained weightof each of the cans tested (3.2.4) shall satisfy the requirement given inthe individual specifications.

3.4.3· For Syrup and Brine - The test results for the characteristics ofsyrup or brine for each of the sample cans (3.2.4 ) tested, shall satisfy therequirements given in the individual specifications.

3.4.4 For M,tallic Impuritils - The test results for the heavy metals onthe composite sample ( 3.3.2 ) shall satisfy the requirements prescribed forarsenic, lead, copper, zinc and tin.

"IS: 2860 - 1964

3.4.5 For Microbiological Requirements - For declaring the conformity ofthe lot to the microbiological requirements, the test results (3.3.3.1and 3.3.3.2 ) shall satisfy the requirements of the test.

4. EXAMINATION OF THE CAN

4.1 Exterior Condidon - Examine the sample cans (3.2.4) for dents,rust, perforations, seam distortions, etc, after removing the labels. Markthe cans with a code number. The condition of the can shall also beexamined for any leaking, panelling or swelling.

4.2 Interior Condition - After determining the drained weight (see 7 ),wash the can, clean and examine for any black discolouration marks,rusting ann pitting. Also examine the condition of the can.

5. DETERMINATION OF VACUUM OF THE CANS

5.1 The vacuum in the cans shall be determined with an electric record­ing type machine without opening the can. If such a machine is notavailable, a vacuum gauge of the piercing type shall be used. Reportvacuum in millimetres of mercury. Necessary corrections for altitude maybe made.

6. DETERMINATION OF HEAD SPACE

6.0 General- Either of the two methods, namely, Method 1 (see 6.1 )and Method 2 (see 6.2) may be used. Method 1 shall be the refereemethod in case of dispute.

6.1 Method 1

6.1.1 Apparatus6.1.1.1 Rotary cutter6.1.1.2 Measuring stick- a clean straight flat piece of wood ofsuitable

type not more thar, 3'2 mm in thickness and 6'4 mm in width.

6.1.2 Procedure - Cut out the lid on the edge of the end-plate partiallyby the rotary cutter, and lift the cut portion carefully so that the shape ofthe end-plate is not altered. Introduce the measuring stick in the can andmake certain that it goes straight in and rests flat against the bottom of thecan and against the body plate. Rest for a short time and lift the stickstraight. Find out the length of the stick which is wet, the space occupiedby the contents of the can. Empty out the contents of the can carefully andfill it completely with water. Again introduce a similar measuring stickand find out the length of the stick which is wet. The difference betweenthe readings gives the gross head space. Net head space is equal to grosshead space less 5 mm, Take four recordings. The average of the four net

6

.:~

IS : 2860 - 1964

head space recordings taken at different points shall be the head space otthe can.

1.2 Method 21.2.1 .~/Jfl1atus

6.2.1.1 Rotmyeutur6.2.1.2 Rulu - with millimetre divisions.

6.2.2 Proe,dur,- Cut out the lid on the edge of the end-plate with therotary cutter, and lift the cut portion carefully so that the shape of the end­plate is Dot altered. Take the measurement from the top of the double,seam to the surface of the liquid using a ruler as illustrated in Fig. 1. The·.~ageoffour measurements taken at different points shall be the headspace of the, can.

~\.. ,~-..- ......~.17-~ -r·,-~- ....

. .: ~ ,r: c·, '~:<---..". .. .'.-'J.. ~t

'.'~··-r?+~.~·II'~ 1. I , .. : .~_•• I •

.;'~'~;~'~'(~.~;. .'~T ~ .' ,'~'

""1 ., ;to'•• ~ .• ;. "'.-

1IfJ:4~~__~·.-iLi~ . - I ~

• ; :.. _ '7-:;''-'-'-.~."0\ :~~.~.....-:"~~. -- ....

t '., .. .

. ,,~,'·(~t.~,•.# .

-.~ i('-~'-:"

FIG. ,1 MEASt1BBIIBNT OJ' HEAD SPAOE IN CANS

7

IS ,·2860 - 1964

7. DRAINED WEIGHT

7.1 Apparatu8

7.1.1 1·70-mm IS Sieve ( Aperture 1·70 mm ) - Alternatively, BS Sieve 10or ASTM Sieve 12 or Tyler Sieve 10 may be used as they are equivalent to1·70-mm IS Sieve (see IS: 460-1962*).

7.1.1.1 For determining the drained weight in case of tomatoes.4·75-mm IS Sieve shall be used.

7.2 v p r ocedur e - Carefully weigh the clean and dry sieve and empty thecontents of the can into the sieve in such a manner as to distribute theproduct evenly. ·Without disturbing the product, incline the sieve so as tofacilitate drainage. Allow to drain for two minutes, and weigh the sieveagain along with the product. The drained weight shall bethe weight ofthe product and sieve less the weight of the sieve.

7.2.1 A sieve of size 20·3 X 20·3 em shall be used for A.21 cans orsmaller. In case of cans of size larger than A.2i, a sieve of size30·5 X 30·5 em shall be used.

8. DETERMINATION OF pH OF CUT-OUT SYRUP

8.1 Determination of pH - The pH of the cut-out syrup shall be deter­mined by either the colorimetric (comparator) method or the potentio­metric method.

8.1.1 Colorimetric (Comparator) Method - The method is based on thefact that certain indicators show definite changes of colour, depending onthe pH of the sample tested. Accuracy higher than 0·2 pH unit may not beexpected from this method.

Various models are available in the market for the colorimetricdetermination of pH consisting basically of a comparator, standard disc tobe used with each indicator; standard indicator solution, l-rnl graduated

. pipette and cells or test-tubes in duplicate. The detailed instructions formaking the test are furnished with each set which shall be followed,Usually, 10 ml of the syrup is used and 0-2 ml of a standard indicatorsolution is added. Another cell containing the syrup \·,.ith')ut indicatorsolution serves as a control for matching the colour, The blank tube isplaced on the left, and the syrup containing the indicator on the right inthe slots provided in the comparator. The standard disc is rotated to getan even match. The determination is carried out in the davlizht or withgood daylight lamp. The indicators most suitable for determining the pHof syrups calorimetrically are methyl o,ange pH 3·1 to 4·4" methyl red(pH 4·3 to 6·3 j and bromocresol purple . pH 5·-1- to i·O~.

• Specification for test sieves ~ ,,~i!td ;.

8

IS : 2860 • 1964

8.1.2 Potentiometric Method - This is the most accurate and reliablemethod for measuring pH. Several types of apparatus are available for theelectrometric measurement either working with the help of dry cells,battery (bench types) or directly from the mains, where DC supply is alsoavailable. With each instrument, the instructions of the maker shall befollowed.

8.1.2.1 Apparatus

a) Syrup half cell- Place in a 25-mI beaker or a wide mouth test­tube enough syrup to cover the glass electrode.

b) The standard saturated calomel half cell - The calomel electrodeconsists of a layer of purified mercury, covered wi th a mixture ofmercury and calomel, above which is a saturated solution of potas­sium chloride saturated with calomel. The mixture of mercuryand calomel is prepared by grinding them into a paste with apestle and mortar. The mixture is washed with saturated potas­sium chloride solution by decantation several times. I t is thenshaken with the remainder of potassium chloride solution. Whensaturated with calomel, the solution is decanted and' kept in astoppered bottle for further use.

There are many forms of electrode vessels available to set up th-ehalf cell. The connection between the mercury and the potentiometer ismade by a platinum wire. The two half cells are conveniently connectedby an agar-agar bridge (thin glass tube bent into a U-shape and filledwith agar solution containing 5 g of agar-agar per 100 ml of saturatedpotassium chloride solution). The bridge shall be kept in a saturatedpotassium chloride solution when not used.

8.1.2.2 Procedur« - Standardize the instrument against a buffer solu­tion of known pH and check against another buffer of different pH. Then

•connect the saturated calomel and syrup half cells to the potentiometer.Balance the potentiometer to take the reading.

Equilibrium occurs almost instantaneously and no 'drift is experi­enced. The pH is indicated directly on the instrument.

NOH - When a batch of samples is to be examined, remove the agar-agar bridgeafter testing one sample, wash the end dipping into the syrup carefully with glass:distilled water and replace in the next sample.

9. DETERMINATION OF SPECIFIC GRAVITY

9.1 Apparatu8

9.1.1 Specific Gravity Boul«

:9.2 Procedure - Clean and thoroughly dry the specific gravity bottle andweigh it. Fill it up to the mark with freshly boiled and cooled water,which hu been maintained at a temperature of 20° ± 1°0 and weigh.

9

IS 12860 -1964

Remove the water, dry the bottle again and fill ~t with the material main­tained at the same temperature. Weigh the bottle again.

9.3 CalculadoD

9.3.1 Specify the temperature of-testing.

9.3.2 Calculate as follows:

Specific gravity at 20o/20°C - ~=~

whereC = weight in g of the specific gr,vity bottle with the

material,A == weight in g of the empty specific gravity bottle, andB = weight in g of the specific gravity bottle with water.

9.3.2.1 To find out the 'degrees Brix, the table given in Appendix Ashall be used.

10. ACIDITY OF CUT-OUT SYRUP

10.1 Apparata.

10.1.1 Erl,nmeyer Flask - 'of 250 m1 capacity.

10.2 aealeDt8

10.2.1 Standard Soaium Hydroxid« Solution- 0·1 N.

10.2.2 Phlnolphthallin Indicator Sol"'ion- prepared by dissolving0·1 g of phenolphthalein in 100 ml of 60 percent rectified spirit(s" IS: 323·195,9* ).

10.3 Proeeclare - Weigh 10 g of the syrup into the Erlenmeyer flask, adda few millilitres of water and mix thoroughly. Titrate against the standardsodium hydroxide solution Ulin~ 0-3 ml phenolphthalein indicator101ution till a faint pink colour, persISting for 30 seconds, appears. Calculatethe Percentage acidity in terms of citric acid from the relationship, 1 ml of0·1 N sodium hydroxide i. equivalent to 0-006 4 g of citric acid anhydrous.

11. DpmtMlNATlON OF SODIUM CHLORIDE IN BRINE

11.0 Geaenl-Eitherofthetwomethods, namely, Method 1 (,"11.1)and Method 2 (SII 11.2) may be-used, Method 1 shall be the rerereemethod in cue of dispute•

•S~~cadoD (or rectified spirit (,IPiIM).

10

IS I 2860 • 19M'

11.1 Method 1

11.1.1 Apparatusa) Graduatedjlask - of 100 ml capacity.

b) Erlenmeyer flask - of 300 ml capacity.

11.1.2 Reagentsa) Ethyl alcoholb) Concentrated nitric acidc) Standard silver nitrate solution - 0'1 N.d) Ferric alum indicator solution - a saturated solution of ferric ammo­

nium sulphate [FeNHt (SO, )2' 12HaO].e) Standard ammonium thiocyanate solution - 0'1 N.

11.1.3 Procedure - Weigh exactly 5'0 g of the brine and transferto a 100-ml graduated flask with roughly 50 ml of 80 percent alcohol.Shake well to suspend all insoluble material. Add 1 ml of concentratednitric acid, and with pipette add excess of known volume of 0'1 N silvernitrate solution. Dilute to 100 ml with alcohol. Then transfer the mixtureto the centrifuge bottle and centrifuge for five minutes at approximately1·800 rev/min. -

Pipette 50 ml of the supernatant liquid into a 300-ml Erlenmeyerflask and to it add 2 ml of concentrated nitric acid and 2 ml of ferric alumindicator solution. Titrate with the standard ammonium thiocyanatesolution to a permanent light brown colour.

11.1.4 Calculation - Sodium chloride, in the brine, percent by weight11'6 ( VINl - VIN.)== W---

whereVI =:s volume in millilitres of the standard silver nitrate

solution,Nl == normality of the standard silver nitrate solution,V. == volume in millilitres of the standard ammonium thiocya­

nate solution used,N I == normality of the standard ammonium thiocyanate used,

andW - weight in g of the brine in the aliquot.

11.2 Method 211.2.1 R"ag,nts

a) Standard sotlium laydroxid, solution - 0-1 N.b) Standard silvIr nilTa" solution - 0'1 N.

11

IS : 2860 - 1964

c) Phenolphthalein indicator solution - prepared by dissolving 0·1 g Ofl

phenolphthalein in 100 ml of 60 percent rectified spirit( see IS : 323-1959* ).

d) Potassium chromate indicator solution - approximately 5 percent( wJv ).

11.2.2 Procedure-Take a suitable aliquot of the solution prepared asin 11.1.3. Ifit is acidic, neutralize it with the standard sodium hydroxidesolution using phenolphthalein as an indicator. Then titrate with thestandard silver nitrate solution using potassium chromate solution as anindicator.

11.2.3 Calculation - Sodium chloride, in the brine, percent by weightS·8 ( VN)

=-~V-

wherev = volume in ml of the standard silver nitrate solution used,N = normality of the standard solution, andW = weight in g of the brine in the aliquot.

12. DETERMINATION OF CALCIUM

12.1 Reagents12.1.1 Hydrochloric Acid - 0-1 N.

12.1.2 Sodium Acetate Solution - 20 percent.

12.1.3 Sodium Oxalate Solution - saturated.

12.1.4 Dilute Sulphuric Acid - one volume of concentrated sulphuric acid( sp gr 1·84 ) diluted with 4 volumes of water.

12.1.5 Standard Bromocresol Green Indicator Solution - prepared by dis­solving 0·1 g of bromocresol green in 100 ml of rectified spirit (see IS : 323·1959* ).

12.1.6 Standard Potassium Permanganau Solution - 0·05 N.

12.1.7 Acetic Acid

12.2 Procedure

12.2.1 Weigh accurately about 25 g of the composite sample (3.3.2) ina previously weighed 50 to 100 ml dish. Heat the dish at 100°C untilwater is expelled, and continue heating slowly until the swelling stops.Place the dish in a closed muffle furnace at 525 0 ± 20°0 and leave untilwhite ash is obtained. Treat the ash with 25 ml or more of 0·1 N

·Specification for rectified aplrit (,msld').

12

IS : 2860 • 1964

hydroChloric' acid and heat to incipient boiling on an asbestos plate till allthe soluble constituents have completely dissolved. Cool and filter through• WhatmaD No. 1 filter paper into a 250-ml graduated flask.

12.2.2 Take a 50 to 100 ml aliquot of the solution obtained in 12.2.1 ina 400-ml'beaker and add 8 to 10 drops of bromocresol green indicatorsolution and then the sodium acetate solution till the colour of the solution·iI distinctly blue. Adjust the pH of the solution at 4·4 to 4·6 adding aceticacid drop by drop until the colour changes to distinct green. Filter thesolution and bring it to boil. While still hot, add the saturated sodiumoxalate solution dropwise as long as any precipitate forms, and then addan excess, and keep on boiling water-bath for one hour. Allow to standfor 3 hours or longer, preferably overnight. Decant the clear solutionthrough a quantitative filter paper. Pour 15 to 20 ml of hot water on theprecipitate and again decant the clear solution. Dissolve any precipitateremaining on the filter paper by washing with hot dilute hydrochloric acid,into the original beaker; wash the filter paper thoroughly with hot water,Then reprecipitate, while boiling hot, by the addition of sodium acetateand a tittle of the saturated sodium oxalate solution. Allow to stand for3 houn or more as before; filter through the same filter, and wash with hotwater until it is chloride free.

12.2.3 Perforate the apex of the filter cone, wash the precipitate with50 m1 of'hot water into the beaker used for precipitation. Add roughly10 ml of dilute sulphuric acid, heat to 90°C and titrate with the standardpotassium permanganate solution.

12.3 Calca1adoD - Calculate as percent calcium. One millilitre of0·05 N potassium pennanganate solution is equivalent to 0'001 g ofcalcium.

13. DETERMINATION OF ARSENIC

13.1 Apparatu. (Gatzeit Bottle) - Assemble the apparatus shown inFig. 2 &I follows:

Take a 6O-ml wide mouth bottle. Equip it by means of a per­forated stopper with a glass tube A measuring 120 X 10 rom with aconstriction at a distance of 50 mm from the upper end. Place asmall wad of glass wool in the constricted bottom end of the tube andadd about 4 g of clean dry sand. Moisten the sand with lead acetatesolution ( lOlercent wlv) and remove the excess by light suction.Place dry lea acetate paper in the lower portion of the tube for theremoval of hydrogen sulphide. Connect the tube by means of arubber stopper with a narrow glass tube B, measuring 100 X 2·7 111mwith a constriction at a distance of 60 mm from the upper end. Placein this tube the strip of mercuric bromide paper ( 13.2.1 ).

13

IS 12860 .1964

B

II

MERCURICIRONto! PANR

2·'",,,, lOAI - ....U~I

10mm lORE - .........

SAND MOISTENED 10 AWt'l'H LEAD ACE- .~TAT! SOLUTIO~ ~~. \

GLASS WOOL~..

10",,,, lOA! - ~ JII~DRV LI!ADA~ I L 70

TATE PAPER .-JI'~: .j'Oml :.

IOTTL!__•

- - .. ._ .. - _. I

'--~~~':.

GRANULATED .:~.£;:~

ZINC~ {.:~~~~.~_.

1--41FIG. 2 APPAlU.TUI J'OB DBTBBKINATIOK O. AumfIO

13.2 Reagent.

13.2.1 Mercuric Bromide Pap'r - Prepare by cutting paper What.man No. 40, or its _equivalent filter paper, into strips 2'5 mm wideand 120 mm long and soaking the strips for one hour in a fresh 5 percentsolution of mercuric bromide in 95 percent ethyl alcohol. Dry and atorein a dark bottle covered outside with black paper. Use the strips U lOOn ·as possible.

13.2.2 C01J&lnlrQtld Nitric kid -sp gr 1'42.

13.2.3 ConemtrtJlld Sulphuric Acid- Ip gr 1-84.

1,3.2.4 ConemlrtJlld HJdroclalorie kid-Ip gr 1'16.

U.2.5 Potassium Todid, Solution - 15 percent (wiD)

14

IS I 2860 • 1964

13.2.6 Stannous Chloride Solution - Dissolve 40 g of arsenic-free stannouschloride (SnCI I , 2H.O) in hydrochloric acid and make up the volume 'to100 ml with hydrochloric acid.

13.2.7 Granulated Zinc - arsenic-free.

13.2.8 Standard Stock Solution of Arsenic - Dissolve 1·320 g of arsenictrioxide ( As,O.) in 25 ml of sodium hydroxide solution (20 percent wlv).Saturate the solution with carbon dioxide and dilute to 1 000 ml withrecently boiled water. One millilitre of this solution contains one milligramof arsenic.

13.2.9 Standard Dilut« Solution of Arsmic '- Dilute 40 ml of the stocksolution ( 13.2.8 ) to I 000 ml.. Mix thoroughly. Take 50 ml of the dilutedsolution and dilute it further to 1 000 mi. This solution contains 0'002 mgof arsenic per millilitre, Use this solution for the preparation of standardstains (St, 13.3.3 ).

13.3 Proceclllre

13.3.1 Pr,paration of tlu Solution - Weigh accurately 5u to 100 g of thecomposite sample (3.3.2 ), transfer to a 500-ml Kjeldahl digestion Bask andwet with 25 to 50 nil of concentrated nitric acid. Add 10 to -20 ml ()fconcentrated sulphuric acid and heat cautiously. Add concentrated nitricacid dropwise from time to time from a pipette to speed up the oxidationof the material. Note the total amount of concentrated sulphuric acid andnitric acid added. When 'the oxidation is complete and the solution iscolourless, add 20 ml of water and again boil to fuming. Coo) and dilutewith water to 50 ml in a graduated flask. Use a suitable aliquot for theestimation of arsenic and reserve the rest for the. determination of lead,copper t zinc and tin.

13.3.2 Place an aliquot of the prepared solution ( not exceeding SO ml )estimated to contain 0·01 to 0·03 mg of arsenic in the 60-ml bottle. Addsufficient concentrated' hydrochloric acid so as to have a total volume of5 ml of hydrcchloric 'acid in the aliquot. Cool, if necessary, and add 5 mlof potassium: iodide solution and 4 drops of stannous chloride solution.Add 2 to 5 g of granulated zinc, put in the strip of the mercuric bromidepaper in the narrow tube and immerse the whole apparatus in a water..bath· kept at 20° to 25°C within 2'5 em of the top of the narrow tube forIi hours, '

13.3.3 Remove the strip and average the length of the stains on bothsides in mllllmetres, Locate the length of the unknown on a standardgraph and" read the quantity of arsenic present from the abscissa. Thegraph ia prep.~, by running known quantities o.f the standard dilutesolution of arsenic ( 13.2.9 ) by the above method using the length of thestain as ordinate and milligrams of arsenic u abscissa. Carry out a blankdetermination in case of a positive result.

15

IS I 2860· 1964

13.4 Calculation - Calculate the quantity of arsenic present in thematerial as parts per million.

14. DETERMINATION OF LEAD

14.1 Reagents

14.1.1 Citric Acid - solid.

14.1.2 Ammonium Hydroxide Solution - sp gr 0·88 or diluted as required.

14.1.3 Potassium Cyanide Solution - 10 percent ( wlv).

14.1.4 Dithizone ( Diphenyl Thiocarbazone i Solution - 0'1 percent (wlv)in chloroform, freshly prepared.

14.1.5 Dilute Hydrochloric Acid - approximately 0'1 N.

14.1.6 Standard Lead Solution - two reference solutions of lead nitrate arerequired in this test as given under 14.1.6.1 and 14.1.6.2.

14.1.6.1 Standard strong lead solution - obtained by dissolving 0'160 0 gof lead nitrate [ Pb (N03)2] in 50 ml of dilute nitric acid and- making upthe volume in a IOO-ml graduated flask.

14.1.6.2 Standard dilute lead solution - freshly prepared before the testby diluting one millilitre of the standard strong lead solution to 100 mlwith water in a graduated flask.

14.1.7 Ammonium Acetate - solid.

14.1.8 Sodium Sulphide Solution - 10 percent ( wlt).14.2 Procedure

14.2.1 Take a suitable aliquot of the solution ( 13.3.1 ), add 2 g of 'citricacid and just neutralize with ammonia. Add one millilitre of the potas­sium cyanide solution and transfer the whole to a separating funnel.Extract the liquid with the dithizone solution. Carry out 3 extractions,using 10 rnl for the first extraction and 5 ml each for the subsequentextractions. If the last extraction gives any indication of a reddish tinge,extract again to ensure complete removal of lead.

14.2.2 Take 10 ml of water in another separating funnel and wash eachextract with this water. If suspended matter is.present in the chloroformextract, this shall be filtered before passing to the separating funnelcontaining the 10 ml of wash-water. Transfer the combined chloroformextracts to a separating funnel and extract lead by shaking successively with50 ml, 20 ml and 10 ml of dilute hydrochloric acid. Combine the. acidextracts iii a separating funnel) wash once or twice with 10 mJ of chloro­form and filter through a previously wetted filter' paper into a !QO.m!graduated flask. Make up the volume of the filtrate to 100 ml with dilutehydrochloric acid and use this as the test solution.

16

IS z 2860 • 1964

14.2.3 Estimate colorimetrically the lead present by comparison with thestandard dilute lead solution containing 0·000 01 g of lead per millilitre(using not more than 10 ml of the standard solution for matching) in thefollowing manner:

Transfer a suitable volume of the test solution to a Nesslercylinder. Add 2 g of ammonium acetate, followed by ammonia untiljust alkaline and then one millilitre of potassium cyanide solution.Dilute to 50 ml, add 2 drops of sodium sulphide solution and matchthe colour against a set of standards prepared in the same way.

14.2.4 A blank determination shall be run under the same conditions, onthe same reagents and by the same person but without using the material.

15. DETERMINATION OF COPPER

15.1 Apparatus

15.1.1 Spectrophotometer

15.2 Reagents15.2.1 Citric Acid - solid.15.2.2 Ammonium Hydroxide Solution - sp gr 0·90. .15.2.3 Sodium Diethyldithiocarbamate Solution - 0·1 percent ( w Iv), aqueous.15.2.4 Carbon Tetrachloride - redistilled.15.2.5 Sodium Sulphate - anhydrous.

15.2.6 Dilute Nitric Acid - concentrated nitric acid of sp gr 1'42 dilutedwith an equal volume of water.

15.2.7 Standard Copper Solution - Weigh accurately 0'1000 g of pure. copper turnings, carefully dissolve in the minimum amount of nitric acid,

cool and dilute to one litre in a graduated flask. Pipette 10 ml of thissolution into a 100-ml graduated flask and dilute ro the mark. Thissolution contains 10 micrograms of copper per millilitre.

15.3 Procedure15.3'.1 Transfer a lO-ml aliquot of the test solution ( 13.3.1 ) to a s~para­

ting funnel. Add one gram of nitric acid and dissolve it by shaking.Make the solution alkaline to litmus by adding ammonium hydroxidesolution in small quantities. Add 5 ml of the sodium diethyldithiocarba­mate solution, shake thoroughly and extract with 5-ml portions of carbontetrachloride until the final extract is colourless. Dry the combinedextracts by shaking thoroughly with anhydrous sodium sulphate. Filterthe dry extract and wash the filter paper with carbon tetrachloride. Makeup the volume of the filtrate to 25 ml with carbon tetrachloride andmeasure the absorption at 43 7 mum by means of the spectrophotometer.Simultaneously, carry out blank determinations on the water and thereagents.

17

IS I 2860 • 19M

15.3.2 Prepare a se'ries of'standardsby treating aliquots of the st&'ftdardeepper solution (15.2.7) in the same manner as the test solution. Fromthe absorption ot the standard solutions, prepare a standard curve plottiQgabsorption values against concentrations. From the curve, obtain theweight of copper present in the test solution.

16. DETERMINATION OP ZINC

16.1 Apparata.

16.1.1 S/JIctrophotomlUr

16.2 Reqeat.16.2.1 Mlthyl Red Indieator Solution - one percent (wID), aqueous.16.2.2 Copp" $ulphat, Solution - Dissolve 8 H ofcOJ?per sulphate ( CuSO.,

5H.O) in water and dilute to one litre. One nullilitre of this solutioncontains 2 mg of copper.

16.2.3 Ammonium Hydro_ Solution - redistilled, sp gr O,go.16.L4 Cone,ntrat,d Hydrochloric Acid- sp gr 1'16.

16.2.5 Hydrogen Sulphidl Gas - Passed through a wash-bottle containingwater.

16.2.6 Dilute Hydrochloric Acid - containing 5 percent by weight ofconcentrated hydrochloric acid.

16.2.7 Bromine Wattr'- saturated.16.2.8 Phenol Red Indicator Solution - prepared by dissolving 0'1 g of

phenol red in 100 ml of rectified spirit ( s" 323·1959· ).16.2.9 Hydrochloric Acid ( 1 : 1 ) - Dilute concentrated hydrochloric acid

with an equal volume of water.16.2.10 Ammonium Citrate Solution - Dissolve 225g of ammonium citrate

in water, make alkaline to phenol red with ammonium hydroxide and add75 ml in excess. Dilute to 2 litres. Extract this "solution immediatelybefore use as follows:

Add to the solution a slight excess of dithizone solution andextract with carbon tetrachloride until the solvent layer is clearbright-green. Remove the excess of dithizone by repeated extractionwith chloroform and finally extract once more with carbon tetrachlo­ride. (It is essential that excess dithizone be entirely removed, other­wise zinc will be lost during the elimination of cobalt and nickel. )

16.2.11 Di11Uthylg/yoxime Solution - Dissolve 2 g of dimethylglyoxime in10 ml of ammonium hydroxide solution and 200 to 300 ml of water, filterand dilute to one litre.

·Specification for rectified Inirit ("viJ,~).

18

18 I 2860 • 19M

16.2.12 s-Nitroso ~-Naphthol Solution - Dissolve 0'25 g of or.-nitroso ~.naphthol in chloroform and dilute to 500 m1 with chloroform.

16.2.13 ChkJroform - redistilled.

16.2.14 Dithu;o", (Diph,nylthiocarbal:,one) Solution - Dissolve 0'05 g of:dithizone in 2 ml of ammonium hydroxide solution and 100 ml of water

I and extract repeatedly with, carbon tetrachloride until the solvent layer isclear bright.~reen in colour. Discard the solvent layer and filter theaqueous portion through a washed ashless filter paper. ( This solution isbest prepared as needed since it is only moderately stable even when keptin the dark and under refrigeration. )

16.2.15 Carbon Tetrachloride - redistilled.

16.2.16 Hydrochloric Acid- approximately 0'04 N.

16.2.17 Stock Solution of Zinc - Dissolve exactly 0'500 g of pure granulatedzinc in slight excess of dilute hydrochloric acid and dilute to one litre in agraduated flask.

~ 16.2.18 Standard Solution ofjZinc - At the time of the experiment, dilute10 ml of the stock solution of zinc ( 16.2.17 ) to 1 000 ml with hydrochloricacid (0'04 N). This solution contains 5 micrograms of zinc per millilitre,

16.3 ProcedureI

116.3.1 Separation of Sulphid« Group - Take a suitable aliquot of the testsolution ( 13.3.1 ) to contain 25 to 100 micrograms of zinc. add 2 drops ofmethyl r-ed indicator solution, one rnillilitre of copper sulphate soluticn and'neutralize with ammonium hydroxide solution. Add sufficient quantity of'concentrated hydrochloric acid to make the solution about 0·15 N withrespect to this acid (about 0·75 ml excess in 50 ml of solution is satis­factory). The pH of the solution at this point should be 1'9 to 2'1 whenmeasured with a glass electrode. Pass a stream of hydrogen sulphide g.into the solution until precipitation is complete. Filter the contents of theflask through a fine textured filter paper ( Whatman No. 42 or equivalent)that has been previously fitted into the funnel and washed firstwith dilutehydrochloric acid and then with water, Collect the filtrate in a beakerand wash the flask and the filter with 3 to 4 small portions of water. Boilthe filtrate gently until the odour of hydrogen 'sulphide is no longerdetected; then add 5 ml of bromine water and continue boiling untilbromine has been expelled. Allow the solution to cool, neutralize withammonium hydroxide solution using l'henol red as indicator and, thenmake i; ilightly acidic with hydrochloric acid ( 1 : 1 ) by adding an excessof 0'2 ml, Dilute the resultant solution in a graduated flask to contain0'2 ml to 1'0 microgram of zinc per millilitre,

16.3.2 Elimination of Nickel andCobalt - Transfer a 20 ml aliquot of thesolution ( obtained under 16.3.1 ) to a separating funnel. Add to it 5 m1

19

U I 2860 • 19M

of ammonium citrate solution, 2 ml of dimethylglyoxime solution and 10 mlof «-nitroso ~-naphthol solution and shake the contents of the funnel fortwo minutes- Discard the lower layer and extract the aqueous layer with10ml of chloroform to' remove residual e-nitroso ~.naphthol_ Discard thechloroform layer.

16.3..1 Isolation and Estimation of Zinc

16.3.3.1 To the aqueous layer obtained after eliminating nickel andcobalt ( 16.3.2) which at this point has a pH of'B'O to 8'2, add 2·0 ml ofthe dithizone solution and 10 ml of carbon tetrachloride, and shake for twominutes. Allow the layers to separate and remove the aqueous layer ascompletely as possible, withdrawing it by means of a pipette attached tothe vacuum line. Wash down the sides of the separating funnel withabout 25 ml of water and draw off the aqueous layer without shaking.Add 25 m1 of hydrochloric acid (0·04 N ) to the carbon tetrachloride layerin the separating funnel and shake for one minute to transfer the zinc tothe acid-aqueous layer. Drain off and discard the carbon tetrachloridelayer taking care to dislodge and remove the drop that usually floats on thesurface, To the acid aqueous layer, add 5-0 ml of ammonium citratesolution and 10-0 ml of carbon tetrachloride (pH of the solution at thispoint is a·a to 9-0). Add the requisite volume of the dithizone solutlQllcalculated as follows: . '

Pipette 4-0 mJ of the standard solution of zinc into a separatingfunnel, add to it 21 ml of hydrochloric acid ( 0-04 N ) from a burette,5 ml of ammonium citrate solution, 10·0 ml of carbon tetrachlorideand then add the dithizone solution in 0·1 ml increments, shakingbriefly after each addition until a faint yellow colour in the aqueouslayer indicates a bare excess of the reagent. Note the total volumeof the dithizone solution added. Multiply this volume by 1-5.

After the addition of dithizone solution, shake the separating funnelfor two minutes, Pipette 5-0 ml of the carbon tetrachloride layer andtransfer it to the spectrophotometer cell. Dilute the solution with 10-0 mlof carbon tetrachloride, mix and determine its transmission at 540 mum,

NOT. - Dilution may be made in a clean dry test-tube if the design of the cell doesnot permit direct mixing,

16.3.3.2 Pipette into a series of separating funnels 0, 1, 2, 3 and 4 mlof the standard solution of zinc and add the necessary volume of hydro­chloric acid (0-04 N) to make 25 ml. Add into each separator 5-0 ml ofthe ammonium citrate solution and the calculated volume of the dithizonesolution. Shake the separating funnel, pipette out 5-0 ml of the carbontetrachloride layer and transfer it to the spectrophotometer cell. Dilutethe solution with 10'0 ml of carbon tetrachloride, mix and determine itstransmission at 540 mum, Proceed in the same manner with the solutionsconta\ned in other separating funnels.

20

IS z 2860 • 1964

16.3.3.3 Plot the transmittance for each of the series of separatingfunnels on logarithmic scale against concentration of zinc in microgramspresent in 25 ml of the diluted standard zinc solution in the particularseparating funnel and draw a smooth curve through the points, (Tnterceptof this curve may vary from day to day depending on the actual concentra­tion of dithizone used in the final extraction, but the slope of the curveshould remain essentially the same.) From this curve, obtain the weightof zinc in micrograms.

17. DETERMINATION OF TIN

17.1 Reagents

17.1.1 Ammonium Hydroxide Solution - sp gr o·go.17.1.2 Concentrated Hydrochloric Acid - sp gr 1·16.

17.1.3 Dilute Sulphuric Acid-I: 3 by volume.

17.1.4 Hydrogen Sulphide Gas - passed through a wash-bottle containingwater.

17.1.5 Wash Solution - Mix 100 mI of saturated ammonium acetatesolution with 50 ml of glacial acetic acid and 850 ml of water.

17.1.6 Ammonium Polysulphide Solution - Pass hydrogen sulphide gasthrough 200 ml of ammonium hydroxide solution contained in a bottle

.immersed in ice-cold water until no more gas is absorbed, add 200 ml ofthe ammonium hydroxide solution and dilute with water to make lOOO-mI.Add 25 g of flowers of sulphur to this solution and keep for several hoursto digest the sulphur and then filter. Use the filtrate.

17.1.7 Dilute Acetic Acid- Dilute one volume of glacial acetic acidwith 9 volumes of water.,17.2 Procedure

17.2.1 Take a suitable aliquot of the test solution (13.3.1), dilute to400 ml, cool and add ammonium hydroxide solution until the contents arealkaline. Add 20 ml of either concentrated hydrochloric acid or dilutesulphuric acid. Cover the beaker with a watch-glass. Heat the solutionto about 95°0 and pass a slow stream, of hydrogen sulphide gas through itfor one hour. Digest the mixture at 95°0 for one hour and 'allow it tostand for another 30 minutes. Filter the contents of the beaker through aquantitative filter paper and wash the residue of stannous sulphide on thefilter, alternatively with three portions each of wash solution and hot water.Transfer the residue along with the filter paper to a 50-m) beaker, add 10to 20 ml of the ammonium polysulphide solution, heat to boiling and filterby decantation. Add 10 ml of the ammonium polysulphide solution, boil

21

IS~I 2860 • 1964

~~Cl filter again. Repeat this once more and finally wash the filter. paperwith hot water. Acidify the combined filtrate and washings with dilutea~tic acid, gently boil for one hour and allow to stand overnight. Filter.the resulting mixture through a double l l-cm ashless filter paper. Washthe filter paper alternately with two portions each' of th« wash solution andhot water. Transfer the residue along with the filter paper to a taredporcelain crucible and dry it thoroughly in an air-oven. Carefully ignitethe filter paper, using a Bunsen flame and incinerate the contents to convertthe sulphide to oxide. Partly cover the crucible and heat strongly over aBunsen or Meker burner. (The sulphide shall be gently roasted to the

roxide which may then be heated ·to a high temperature without loss byvolatilization. ) (.

17.2.2 Cool the crucible in a desiccator and weigh as stannous oxide( SnOI ). Repeat the process of heating, cooling and weighing till thedifference between two successive weighings is Jess than one milligram.Note the lowest weight. Obtain the weight of metallic tin from the weightof stannous oxide by using the factor 0·787 7.

18. TEST FOR M1CROBtOLOGICAL REQ,UIREMENTS

18.1 GeDeral- The test consists of the incubation of the cans at 37°Cand 55°C for not less than 7 days and their examination at the end of thisperiod for evidence of microbiological activity. To satisfy the requirementsof this test, the cans shall not show evidence of microbiological activity ofany type. The incubated cans may have undergone microbiologicalspoilage if any. can:

a) shows a positive pressure;b) bulges when being incubated and remains bulged after being

cooled, or remains bulged after incubation; and

c) leaks.

18.1.1 Whether the cans show a positive pressure or not, the evidence ofbacterial proliferation shall be judged by: '

a) a significant change in pH value,b) disintegration or digestion of the fruit or vegetable as compared

with the unincubated sanw1es,c) microscopic examination of direct smears, andd) culture methods for testing bacterial multiplication and presence

of pathogenic bacteria.

18.2 Apparatu.18.2.1 Glassiuar« - All the glass apparatus used in the microbiological

examination shall be sterile. Sterilization shall be performed for at leuttwo hOUR at 170°0 in.dry heat.

22

IS 12860 -1964

18.2.2 Accessories - Can-opener of the puncturing type, cork borers,sampling tubes, and cotton wool - all sterile,

18.3 Reageat.

18.3.1 Petroleum Ether.18.3.2 Mercuric Chloride Solution - 1'0 percent ( wlv).

18.3.3 Carbolic Acid Solution - 1'5 percent ( tu]» ).

18.3.4 Ethyl ALcohol Solutions - 97 percent, 95 percent and 98 percent(wlv),

18.3.5 Chloroform

18.3.6 Formaldehyd« ~ 40 percent ( to]» ).

18.3.7 Sterile Normal Saline or Ringers Solution18.3.7.1 Normal saline -- O,g percent ( tu]» ) solution ofsodium chloride

( analytical reagent ),

18.3.7.2 Ringers solution - It has the following composition:Sodium chloride 0'2 gPotassium chloride 0'42 gCalcium chloride 0'48 gSodium carbonate 0'2 g

Distilled water 1 000 ml ( glass distilled)

18.3.7.3 Ringers solution is diluted to quarter strength when actuallyin use. The sterilization of the normal saline and the Ringers solutionshall be performed at 7 kg pressure at 121°C for 20 minutes in anautoclave..

18.3.8 Sterile Vaselin, - Alternatively, sterile liquid paraffin) liquid agaror paraffin wax may be used.

18.4 MecUa

18.4.1 Dextrose- Tryptone-Broth - Mix 109 of tryptone, 5 g of dextrose,0-04 g of Iexomo-cresol-purple and one litre of water. Steam the mixtureuntil dissolved. Adjust the pH to 6'8 to 7-0. Filter and tube in lO·m1amounts and autoclave at 12100 for 30 minutes. This media is used fordetecting the growth of thermophilic anerobes not producing hydrogensulphide, putrefactive anerobes and other mesophilic anerobes,

18.4.2 Liwr Broth - Boil 500 g of' well-minced degutted beef liver in1 000 ml of distilled water for one hour. Adjust the pH of the mixture to7-0 and boil further for 10 minutes. Cool and separate all rat by straining

23

IS : 2860 - 1964

through several layers of muslin and make up the volume to one litre withwater. Add 109 of peptone, one gram of di potassium hydrogen phosphate(K2HP04 ) and again adjust the pH to 7-0, tube in l Ovml quantities, addto each tube 2 g of liver particles and autoclave at 121°C for 30 minutes.Before use, boil the medium for 10 to 15 minutes to remove dissolved air.Cool the tube before inoculation, "[he medium may further be improvedby sealing with paraffin wax or liquid paraffin.

18.4.3 Cooked Meat Medium - Mince 500 g of fresh bullock's heart, placein 500 ml of boiling distilled water to which has been added 1-5 ml of INsodium hydroxide solution and simmer for twenty minutes, by the end ofwhich the neutralization of lactic acid will be ensured. Drain ofTthe liquidthrough a muslin filter and, while still hot, press the minced meat in acloth and dry partially by spreading it on a cloth or filter paper. In thiscondition it may be introduced into bottles without boiling them. Placeabout 2-5 g of the minced meat in each bottle and cover with 10 ml ofpeptone infusion broth made as follows:

Add peptone 0-5 pe-rcent and sodium chloride 0-25 percent to theliquid filtered from the meat. Steam for 20 minutes, add 1-0 ml ofconcentrated hydrochloric acid and filter. Bring the reaction of thefiltrate to pH 7·7 to 7-8. Autoclave at 120°C for twenty minutes.After sterilization, the pH of the broth over the meat is 7·4 to 7·5.Introduce the inoculum towards the bottom of the tube in contactwith the meat. Cooked meat medium is used for detecting the pre­sence of spoilage bacteria in acid foods like tomato products.

18.4.4 Nutrient-Agar - prepared from the following ingredients:

Poly peptone ( peptone) 5 g

Beef extract 3 g

Agar 15 g

Water 1 000 mlpH 6·2 to 7·0

Sterilize the media at 121°C for 15 minutes. Nutrient-agar is usedfor the isolation of organisms.

18.4.5 Potato-Dextrase-Agar - prepared from the following ingredients:

Infusion from 200 g potato 1 000 mlGlucose 20 gAgar 15 g

Sterilize the media at 121°C for 15 minutes. Adjust the pH to3·5 ± 0-1 at the time of pouring into plates. Potato-dextrose-agar isused for the growth of yeasts and moulds) and their counts. -

24

IS 12860 ·19&4

18.4.6 Malt.Extrtzet-Agar - prepared from the following ingredients:Malt extract 45 gAgar 15 gWater 1 000 ml

Sterilize the media at 121°C for 15 minutes, Adjust the pH to3·5 ± 0·1 at the time of pouring into plates. This media is used for thegrowth of yeasts and moulds.

18.5 Proeeclure18.5.1 Preparation ofthe Can

18.5.1.1 Note down all the identification marks, brand marks, batchnumber, date and other information available on the label or embossed onthe tin. Remove the label and record all physical defects, such as rust,pin-holing, dents, signs or defective seaming of end-plates and of defectiveside seams. Note other points, if any, for examination after the can is~pened.

18.5.1.2 Scrub the can with soap and water. If necessary, applypetroleum ether and clean. After washing with soap, immerse for at leastone hour in a tall container filled with mercuric chloride solution or car­holic acid solution.

18.5.1.3 After removing from the antiseptic bath, clean the exteriorof the can with sterile cotton wool. When dry. scrub well with sterilecotton wool dipped in ethyl alcohol (97 percent). Cover the top of the.~ with sterile petri dish.

18.5.2 Prtparation of th, Sampl« for Bacuriological Tests- Puncture the canand enlarge the puncture under aseptic conditions with a thoroughly steri­lized can-opener to give an opening of 2,75 em diameter. If a cuttingtype of instrument is used, cut a triangle with a base of 2-5 em and side.5 em long. Cover the enlarged opening at once with sterile petri dish.

18.5.3 Rmaoval oftill Inoculum (Sampl,)18.5.3.1 RtmolJtJl of liquid inoeulum - The inoculum is preferably

removed by means of a sterile pipette graduated in millilitres, It 11 essentialthat the rear end of the pipette be plugged for a length not less than 1·5 emwith sterile cotton wool. Deliver 2 ml of the inoculum to each of thebroth tubes and one millilitre to each of the melted agar tubes. Mix wellto ensure uniform distribution and pour into the petri dishes swirling 10 asto obtain uniform spread. Alternatively, pour 2 ml of the inoculum intothe petri dish and then 'add the melted agar medium and mix by swirling.

18.5.3.2 RmunJal of solid inoculum - For removing the samples of solidmateria), use sterile cork borers, Remove cores of the pack from the centreand side regions of the can. Remove about 15 g of the sample and transferit to a sterile flask containing 50 ml of sterile water or sterile normal saline

25

IS: 2860 • 1964

or sterile Ringers solution. The flask should contain at least a dozensterile glass beads to facilitate mixing. Mix by. swirling for at least 5minutes. Take out 2-ml portions of the inoculum and add them 'to boththe broths and the agars.

18.5.4 Preparation of th« Anarobic Culture - The dextrose-tryptone-brothand the liver-broth media are rendered aneerobic by sealing the access toair by placing a layer of sterile vaseline, liquid paraffin, liquid agar orparaffin wax on the top of the medium, This layer should be not Jess than1-5 em in thickness. Just before using the culture tubes, exhaust the mediaby packing the tubes for at least 10 minutes in a water-bath and maintainthe water-bath at 70°C. Cool the media to 37°C and inoculate the mediumbelow the paraffin agar layer with as little disturbance as possible and coolthe medium after inoculation in a cold water-bath so that the top seal isset.

18.5.5 Intubation

18.5.5.1 Incubate the inoculated tubes and petri dishes as follows:a) D,xtros,-tryptonl-broth - two tubes each, cerobically and aneerobi­

cally, at 37°0 for 3 to 5 days and similarly another set at 55°C for3 to 5 days.

b) LiVIT broth - two tubes anerobically at 37°0 and two tubes at55°C for 5 days.

c) Malt-extraet-broth·- two tubes rrobically, one at 25°0 and theother at 37°C for 5 days.

d) Cooked meal medium - one tube anaerobically at 37°0 for 5 days.

e) PolakJ-duIrOl,-agar or mtllt-ut'tI&t-agar- two dishes erobically,one at 25°0 and the other at 37°0 for 5 days.

18.5.'.2 In the case of a blown can, run a separate series of culturetubes parallel to the tubes from the sound can. The incubation period itthe same as given in 18.5.5.1.

18.5.6 ExamiMtitm 0/Cultur, Afln Incubation18.5.6.1 In case there is growth in the nutrient-broth tubes, streak

them on agar plates and incubate. Examine the contents or each culturetube for the organisms of the following types:

a) Spoilage tyPes,b) Potential spoilage types, andc)" Pathogenic types.

18.5.6.2 In case there is a growth of cocci, establish their nature by.treaking them on blood agar and ..tudy ability for hleDlOlysia and nature

" by <a) coagulase tests, and (b) by microscopic examination.

26

IS I 2860 • 1964

18.5.7 Examination of the Cans After the Remoual of the Inoculum from th«Cans - Carry out the following tests:

a) Prepare a direct smear of the contents. Stain by Gram's methodor methyl violet and examine the preparations microscopically;

b) Determine the pH value;c) Examine contents for deterioration, discolouration, disintegration

and other signs of spoilage;d) The tinplate should be examined for nature of etching, staining

or corrosion disturbance of lacquer and rust; ande) The seams should be tested for leaks and soundness of fabrication.

18.5.7.1 Correlate the above findings with the incidence and natureof microBora and conclude about the cause of the spoilage, if any.

APPENDIX A

( Clause 9.3.2.1)

DEGREES BRIX, SPECIFIC GRAVITY AND DEGREESBAUME OF SUGAR SOLUTIONS

DZGBBEI BBIX O. SPJCOII10 ORAVITY SPECInO GRAVITY DEona BAtUJI.PBRCENT BY AT 20o/20°C AT 20o/4°C ( MODlJ'LU8 145)W.IOBT OJ'

SUCBO••

0-0 1-000 00 0-998 234- 0°000'2 1-000 78 0'999010 0-110-4 1-001 55 0~999 786 0·220-6 1-002 33 1-000563 00S40'8 1-003 11 1°001 342 0-45

1-0 1-00389 1-002 120 0-561°2 1-00467 1-002897 0-671-4 1°00545 1°003675 0-191°6 1-006 23 1-004453 0-901°8 1-00701 1-005234 1°01

2-0 1-00179 1-006cis 1-122-2 1-008 58 1-006796 1-252-4 1°009 36 1-007580 1-34-2-6 1-010 15 1-008965 1·462-8 1°01093 1°009 148 I-57

3-0 r-on 72 1'0099,. 1-683'2 1'01251 1-010721 1-793-. 1-01330 I-OIl ~10 1-90

. 3-6 1-01409 1-012298 21»2S-8 11)1488 1-013089 2'13

(~)

27

IS I 2860 • 1964

DEnKEEI Barx OR SPJtClrlC GuVITY SP.CIJ'IC GilAVITY D&ORBB. B.olI.PBRCENT BY .AT 20o/20°C

AT 20o/4°C( MODULUS J.5 )

W.nOHT orSUCBOI.

~·O 1'01567 1-013881 2-24.'2 1·01647 1·0J4673 2'354'4 1'0172(; 1-015467 2'464'6 1'01806 1'016261 2'574'S l'OH~ 86 1'017058 2-68

5'0 1'01965 1-017854- 2·795'2 1·02045 1'018652 2-915'4 1'02125 1-019451 3-025'6 1-02206 1'020251 3'135'S I"02~ 86 1'021053 3-246"0 1°02366 1-021 855 3-356°2 1"02447 1-02:l659 3'466°4 1"02527 1'023463 3°576"6 1"02608 1°024270 3°696"S 1'02689 1'025077 3°80

7°0 1'02770 1'025885 3'917"2 1'02851 1'026694 4"027"'- 1"02932 1-027 504- 4'137"6 1"030,13 1°028316 4°24-7"S 1°030;95 1'029 128 4'35

8'0 1°031 76 1'029~2 4"468·2 1'03258 1°030757 4-588°'- 1°03340 1°031 573 4°698'6 1'03422 1'032391 4°80S'S 1°03504 1"033209 4°91

g·O 1-03586 1"QM029 5"029"2 1°036 68 10034MO 5°159'4 1-037 50 11)35671 5·24-9·6 r-osa !3 1'036494- 5"5g08 1-03915 1'037'18 5-46

10'0 I-G!9f1 1003S143 5'5710"2 J0()4.() 1 lo()!8 970 5"6810'4 1"04164 1'039797 S08010°6 1'01247 1"040626 S.g1100S 1°0+330 I"OfI4.56 6°02

11-0 I·Off 13 1°042288 6°1311°2 1-()f4g7 11K! 121 6°24-11°'- 1°04580 1-04! 9M 6°3511-6 1-04664 100M 711 6°4611·8 liN747 10045625 6·S7

1200 1-04851 1-046462 6°6812°2 1·049 I-S I·Of7300 6°7912-'- 1-04999 I1M8I40 6°9012-6 1-«»084 ·1-048 980 7-021211 11»51 68 le0f9322 7-13

(OM"')

28

IS : 2860 • 19M

DBOBEE8 BBIX oa SPECIJ'rO GRAVITY SPECII'IC GIlA VITY DEGREES BAt1I1BPERCENT BY .AT 20°/20°C AT 200/4°C

( ~IODULU8 145 )WEIGHT 01'

SUCBOSB

13·0 1'05252 1'050665 7'24-13'2 ) '05337 1'051510 7'3513'4- )'05422 )'052 356 7'46J3'6 1'05506 1'0:;3202 7·5713'8 1'05591 I·O~.O~ 7-68

14-0 1'05677 1'054900 7·7914'2 1'05762 1'055751 7'9014'4 1'05847 1'056602 8'0114'6 1'05933 1-057455 8'1214'8 1'060 18 1·058310 8'23

15-0 1·061 04 1'059165 8'M15·1 1'06J 90 1'060022 8-4515-'" 1'062 76 1·060880 8'5615'6 1'06362 1'061 738 8-6715-8 1'06448 1'062598 S·78

16'0 1'06534 1'063460 8-8916'2 1·06621 1-064324- g-OO16'4- 1-06707 1'065 188 g.1116·6 1·06794- 1-066OM 9-2216·8 1-068 81 1'066921 9'33

17'0 1'06968 1·067789 9'4.517'2 1'07055 1'0686~8 9-5611'4 1'0'11 42 1-069.529 9'6717'6 1-07229 1'070400 9'7817'8 1'073 17 1'071275 9-89

18-0 1'07404- 1-072 147 10-0018·2 1'07492 1'073025 10'11IS·4 1'07580 1'073900 1~22

18'6 1'07668 1'074777 1'~"18'& 1'07756 1'015657 10·...

19'0 1'078" 1'076557 Ig'5519'2 1'07932 1'077419 1 -6619'4- 1'08021 1'078502 1~77

19'6 1'081 10 1'079187 1&8819'& 1'08198 1'080072 l&gg

20'0 1'08287 1-080959 11'10.20'2 1'08376 1-081 848 11'2120'4- 1'08465 1'082737 11-3220'6 1'08554 1'083628 11-4320'8 1'08644- 1·084520 II-M

21·0 1-OS7 35 1'085414- 11-6521'2 1'08823 1'086309 11-7621-4 1'08913 1-087205 11-8721'6 1·09005 10Q88 101 11-1821'8 1'09093 1'089000 12-01

(C..II I)

29

IS: 2860 • 1964

DEGREES BRIX OR SP:F.CIFIC r; It.\ '-lTV S,'[o:('I ...II' (;I:,-\\'Y'fY I)EonEEs BAtiME

PERCK~'rDY AT '20.)/,20 C ,,\T .!U / I C ( ~lo1)L'Lu~ 14~)

W}';IOJlT OJ'SUCROSE

22-0 I-OQI B3 l'OB9900 12'2022'2 1'u~t! 7~J I'()~JO Hg'2 12'3122'4 1'093 h-t- l'Oql 7) ~ 12'4222'6 1'094-54 l'O~~2 (i07 12'5222'S 1'095-15 l'OV3513 1:l'G3

23'0 1'09636 1,O~)~ 420 12·7·l23'2 J '097 27 J '095 :t!H )2'8523'4 1·098 18 1'09G 236 12'9ti23'6 1'09909 )'0971-l7 13'0723'8 1'10000 1'098058 13-18

24'0' 1.100 92 " 1'098971 13'2924'2 1'101 B~~ 1'099 a86 13'4024'4 1'101 75 1'100802 13'5124'6 1'10367 1'101718 13'6224'8 1'10459 1'102637 13'73

25'0 1'10551 1-103557 13'8425'2 1'10643 1'104478 13'9525'4 1'10736 I-lOS 400 14'0625'6 1'10828 1'106324 14'1725'8 1'10921 1'107248 14'28

26'0 1'11014 1'108175 14'3926'2 1'111 06 1'109103 14'4926-4 1'11200 1'110033 14'6026'6 1'11293 1"110963 14"7126"8 1-11386 1"111895 14"82

27'0 I'] ]480 )'112828 14"9327-2 1"11573 1'113763 15-0427'4 1"11667 1'114697 15"1527'6 1-11761 1-115635 15"2627-8 1'11855 1-116572 15-37

28-0 1'11949 1'117512 15'4828'2 1'12043 1-118453 15'5928-4 1'121 38 1-119395 15"692&-6 1-12232 1'120339 15'802&'& 1-12327 1'121284 15'91

29'0 1'12422 1-122231 16'0229-2 1'125 17 1-123 179 16'1529'4 1-126 12 1'124128 16'2429-6 1-12707 1-125079 16-3529'S 1-12802 1-126030 16'46

SO-O 1-12898 1'1269&1- 16·5730-2 1-129 93 1-121939 16-6730-4 1·13089 1-128896 16-78!O'6 1-13185 1-129853 16-89!CHI 1-13281 1-130812 17'00

(c..,....J

30

IS I 2860 ·1964

DEORER8 BRIX OR SPECU'IC r;n.A\·ITY Sl~ECIVH' t(; Il,\ \'ITY l)1';Onl';}~S HAUIII:

PEltc'F.ST BY AT ·20o/:ll)C'C AT ~( o/4"C ( :\IODULl;& 145 )\VJo:IOllT OP

St:CR08B

31'0 1'1337K 1'131 773 17'11~1'2 1'13474 1'132 735 17'2231'4 1'115 iO 1'133 (i9H 17'3331'6 1'13667 1'13·l.iG:J 17'4331:a 1'137 G-t l'I:~5 (i28 17'54

32'0 1'13B til 1'136596 17'6532'2 1'13~} 58 1'137 SG5 17'7632'4 )'1-1055 t'138-534 17'87:\2·f) 1'14152 1'13950G 17'9632'8 1·1....2 50 l'I<lO479 18'08

33'0 1'14347 1'141 -1-53 18'1933'2 1'14445 1'142429 18'3033'4 1'14543 1'143405 18'413:i'6 1'14641 1'144 384- 18'5233'8 1'14739 1-1-4-5363 1&63

301-0 1'14837 1-146345 18-7334'2 1'14936 1'147 328 1&8434'4 1'15034 1'148313 18-9534'6 1'15133 1-149298 19'0634-8 ('15232 1'150286 19'17

35'0 1'15331 1-151275 19-283)'2 1-15430 1'152265 19'3835'4 1-15530 1'153256 19-493)'6 1'15629 1'1542·19 19'6035'8 1'15729 1'155242 19'71

36-0 1-15828 1'156238 19'8136-2 1'15928 1'157235 19'9236-4- 1-16028 "15A 233 20-0336'6 1-16128 1'159233 20-14-36'8 1'162 28 1'160233 20'25

37'0 1'16329 1'161236 20'3597'2 1-16430 "162240 20-4637-4 1'16530 1'163 245 20-5737'6 1'16631 1'164252 20'6837'8 1'16732 1'165259 20'78

38'0 1-16833 1'166 269 20-8938'2 1°16934 1·161281 21'00SS-4- 1-17036 \'-168 293 21-1138-6 1'17138 1-169 307 21-21saa 1'17239 1-170322 21-32

39-0 1'17341 1'171 SfO 21-"39'2 1'17443 1-172359 21'5439-4 1-17545 1'173379 21-6439-6 1-17648 .'174400 21-7;39-8 1'177 50 1-17542' 21-86·

<c.tiIftIM )

31

IS I 2860 ·1964

DBoB••8 BBIX OR SPECfWllC GBAVl1'Y SPltOll'lC GRAVITY DJ:O.B~ BAU••PEROENT BY AT 200j20°C

AT 200j4°C ( ?\10DUL1TII 145)WBIOBT O.

Sucaoa.

40'0 1'17853 1'176447 21'974002' 1'17956 1°177473 22~740°. 1°180 58 1°178501 22-1840°6 1°181 62 loJ79527 22'2940-8 1°18265 1°180 560 22°39

41°0 1-18368 1-181 592 22'5041°2 1°18472 1-182625 22-6141°4 1-185 75 1"183660 22"7241°6 1°18679 1-184696 22"8241-S 1"18783 1"185734 22°93

42-0 1°18887 1°186 773 23-0442°2 1'18992 1°187814 23"1442-4 1°19096 1°188 856 23"2542'6 1°192 01 1°189 901 23"3642°8 1°19305 1°190 946 23°46

4'-0 1°19410 1"191 993 23'5743°2 1°195 15 1"193041 23'684S'4 1°196 20 1"194090 23'7845-6 1°19726 1°195 141 2S'8943°8 1°198 31 1°196 193 24"00

44-0 1'19956 1"197247 24'1044°2 1°200 42 1°198 303 24°2144'4 1°201 48 1'199360 24°3244'6 1°202 54 1°200 420 24-42440S 1°20360 1°201 480 24-53

4500 1°204 67 1'202540 24-6345'2 1"20~ 73 1°203603 24'7445-4 1°20680 102CK668 24'U45'6 1'20787 1'205733 241546'8 1°20894 1°206801 25-06

46"0 1'21001 1'207870 25'1746~ 1'21108 1°208940 25'2746-4 1'21215 1'210013 25'.46-6 1'21323 1'211086 25'4846'8 1'21431 1'212 162 25'59

47-0 J-21538 1"213258 25'1047-2 1-21646 J0214 SI7 25'8047'. 1"21755 1'215395 .25°91

'47'6 1'218es 1-216.76 261)147'8 1'21971 1'21759 26-12.-0 1'220 80 1-218643 26'U= "22119 1-219729 28-SS

1-22298 1'220815 26-44.. 1-22406 1°221 904 ..,... 1-22516 1'222.5 21~(c.IIt••, I ")

32

IS 12860·1964

DEGREES Bsrx OR SPECIFIC Ga"vITY SPKCrrJC GRAVITY DEGREE. &UM.PERCENT BY AT 20o/20°C

AT 20o/4°C ( MODULUS 145)WJUOHT 6~

SUCROSE

49-0 1-22625 1-224086' 26-7549-2 1-22735 1-225180 26-8649'4 1'22844 1-226274 26-9649-6 1'22954 )'227371 27-0749'8 1'23064- 1-228469 ~7'18

SO'O 1-231 74- 1-229567 27'2850'2 1'23284 1-230 668 2J"3950-4- 1-23395 1'231 770 27'4950'6 1'23506 1-232874 27-60SO'8 1-23616 l'2l3979 27'70

••

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