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 2237 (1997): Prawns (Shrimps) - Frozen [FAD 12: Fish andFisheries Products]

IS 2237: 1987

n(~)-~-~

(ffRnr~" )

Indian Standard

PRAWNS (SHRIMPS) - FROZEN ­SPECIFICATION

( Third Revision)

ICS 67.120.30

OBIS 1997

BUREAU OF INDIAN STANDARDSMANAK BHAVAN, 9 BAHADUR SHAH ZAFAR MARO

NEW DEUII 110002

Ottobc:t' 1997 Prlee Group 7

Reaffirmed 2009

AMENDMENT NO. 1 JULY 2005TO

IS 1237: 1997 PRAWNS ( SHRIMPS) - FROZEN ­SPECIFICATION

( Tlalrtl RevLdon )

[Page 1, clause 2, para 2 ] - Substitute the following for the existingmatter against mevant IS Numbers:

SINo.

5887 (Part 7 ) : 1999

11427: 2001

Title

Methods for detection of bacteria responsible forfood poisoning: Part7 General guidance on methodsfor isolation and identification ofShigella

Fish aod fisheJy products - Sampling (first revision)

[ Page 3, Table 2, Sl No. (i), col 4 ] - Substitute 'soo000' /01' '1000 000'.

l Page 3, Table 2, SI No. (iii), col S and 6 ] - Substitute '100' lor '.Ab,.nt I.

[ Page 3, Table 2, Sl No. (vi), col 7 ] - Substitute 'IS 5817 (Part 7rfor 'IS ,a87 (Part 3 )'.

(FAD 12)

Repropaphy Unit,81S, New Delhi. Iadia

Fish and Fisheries Products Sectional Committee. FAD 12

FOREWORD

This Indian Standard (Third Revision) was adopted by the Bureau of Indian Standards, after the draftfinalized by the Fish and Fisheries Products Sectional Committee had been approved by the Food andAgriculture Division Council.

Fishis one of the most perishable of all foods and needs proper care fromthe time it is caughtuntil it isserved or processed. Lowering the temperatureof fISh by a prompt and efficient chilling procedure isfundamental for preservation of fish freshness. The quality of frozen fIShery products is influenced bymany different considerations. Among themostimportantarecompositionoffish, pre-freezing, handlingandtransport,methodoffreezing employed andtheenvironment towhich the frozen productissubjectedduring storage and handling. Of principal concern here is the temperature and humidity of the coldstoragearea and the protective packaging or glazing afforded the product.

Frozenprawns constitutean importantexportcommodity of India. Thisstandardwas first published in1962 and underwent first revision in 1971 andsecond revision in 1985. Thisrevision, incorporates amongother the following changes:

i) Existing microbiological requirements have been modified along with incorporation of newmicrobiological requirements alongwith their methods of tests;

if) Forsafeguarding the healthofconsumers. maximum permissible limi~lor heavy metals havebeenprescribed, alongwith their methods of tests;and

iii) Editorialchanges includes updating of referredstandards.

In the preparationof thisstandard,due consideration hasbeengiven to the provisions of the PreventionofFoodAdulterationAct, 1954 andStandards ofWeights andMeasures (Packaged Commodityv Rules, 1977.However, this standard is SUbject to the restrictions imposed under these,wherever applicable.

For the purposeof deciding whethera particularrequirement of this standardis complied with the finalvalue, observed or calculated, expressing the resultofa testor analysis, shallbe rounded off inaccordancewith IS 2 : 1960 'Rules for rounding offnumerical values (revised)'. The number of significant placesretained in the roundedoffvalue. shouldbe the sameas that of the specified valuein this standard.

IS 2237 : 1997

Indian Standard

PRAWNS (SHRIMPS) - FROZEN ­SPECIFICATION

( Third Revision )1 SCOPE

1.1 This standard prescribes the requirements andthe methods of sampling and test for frozen prawns(shrimps).

1.1.1 The frozen prawns (shrimps) shall be of anyedible species.

2 REFERENCES

The following Indian Standards listed containprovisions which through reference in this text,constitute provision of this standard. At the time ofpubltcation, the editions indicated were valid. Allstandards are subject to revision and parties toagreements based on this standard are encouragedto investigate the possibility of applying the mostrecent editions of the standards indicated:

Reagent grade water (third 3.1.14 Individually Quick Frozen (IQF) - Any ofrevision) the types mentioned above but quick frozen in­Code of hygienic condition dividually.for fish industry: Part 1 Pre-processing stage (first revision) 4 DESCRJJYrIONMethods for detection of bacteriaresponsible for food poisoning: The shell and flesh of frozen prawns shall havePart 3 Isolation and identification characteristic colour of the respective types andof Salmonella and Shigella tflrst shall be free from anyblack discoloration. The meatrevision) shall be firm and consistent.Methods for sampling for fishandfisheries products 5 REQUIREMENTS

[SHo.

1070: 1992

4303(Part 1) : 1975

5887(Part 3) : 1975

11427: 1985

Title

3.1.6 Peeled and Deveined - Head and shell anddorsal tract removed completely.

3.1.7 Cooked and Peeled - Peeled after cooking.

3.1.8 Peeled Deveined and Cooked - As in 3.1.6but cooked.

3.1.9 Whole, Cooked - As in 3.1.1 but cooked.

3.1.10 Peeled Undeveined - Head and shellremoved completely.

3.1.11 Headless Blanched - As in 3.1.2 butblanched.

3.1.12 Peeled and Deveined Blanched - As in 3.1.6but blanched.

3.1.13 Peeled Undeveined and Blanched - Asin 3.1.10 but blanched.

S.1 The material shall be prepared from clean,wholesome and fresh prawns, and shall not showany visible signs of spoilage. The material shall asfar as possible be free from any discoloration,off-odours, dehydration, black spots, whole insectsor its fragments, rodent droppings and any otherforeign matter. However, the defects for variousquality characteristics shall not exceed thetolerance limits given in Table 1.

3.1 Frozen prawns shall be of the following types.

3.1.1 Whole - Head and shell on.

3.1.2 Headless - Head removed, shell OD.

J.l.3 Fantail Round - Head and shell removedexcept on the last segment and tail.

3.1.4 Fantail Deveined - As in 3.1.3 but the dorsaltract removed. 5.2 The material shall be prepared and processed3.1.5 Fantail Butterfly - As in 3.1.3 in addition to as given in Annex ~ under hygienic conditions assplitting open. prescribed in IS 4303 (Part 1).

3 lYPES

1

IS 2237 : 1997-

Table 1 ToleranceUmltl lor Deleetl lor VarioulCharacteristics

(Clause S.l)

5.3 The material shall conform torequirements prescribed in Table 2.

'.2.2.1 The use of the Standard mark is governedbythe provisions ofBureau ofIndianStandardsAct,1986 and the Rules and Regulations madethereunder.1b.edetails of conditions under whichthe licence for the use of Standard Mark may begranted to manufacturers or producers may beobtained from the Bureau of Indian Standards.

c) Batchor code number;

d) Count per kg (if required by the purchaser);e) Minimumnet massof the contents whichis

equivalent to the drained mass ascertainedafter complete thawing and excluding theglaze;

f) Name of the additives used, if any;8) Any other requirement as given under the

Standards of Weights and Measures(Packaged Commodities) Rules, 1977.

'.2.2 BIS CertificationMarking

The product may also be markedwith the StandardMark.

the

2

15

20

105

,OtherTypei

(percent)(4)

S

3

S2

10

Tolel'llDCe Umll"r

Headleu Shellon Type (percent)

(3)s

CbanacteriaUc

(1) (2)i) Deterioration with

spoiled piecel.ii) Discoloration ofabeD

and meatiii) Black spot on shell

and meativ) Broken,dama.ed

and 10ft sbelled piecesv) Lep, bits of veina,

100Ie ahelll, etcvi) Dehydration 15

SINo.

6.1 Paeklol

6 PACKING AND MARKING

1b.ematerial shall be packed in suitablecontainer 7 SAMPLINGas agreed between the purchaser and theprocessor" In the absence of ~ny SUCh, agreem~nt The methods ofdrawing representative samples ofthe mat~nal shall be packed In containers ~h~~ ...... material for test and criteria for conformitymay wlths,tand the stress and st~attr:or shallbe as prescribed in IS 11427.transportation and can prevent deterioration . . _during transportation and frozen storage. A layer NOTE - For determining the defects In cooked as well as

. raw frozen products, remove the wrapper of the frozenof polyethylene shall be used betweenthe material material and place it in a closed polythene bag. The polytheneand the containerwhenindividually frozen prawns bal II further immerled in potable water at roomare packed. temperature ina clean container wherein the potable water

is introduced from the bottom of the container at a flowrate6.2 Marking of2S IImin.The material shall not come in direct contactwith

WIler.After all the glazethat can be seen or felt is removed,6.2.1 Each container shall be marked legibly and and the pieces separate easily, transfer the products to theindelibly with the following information: preweiChedsieve. Drain for 2 minutes at the inclined position

and weigh. Calculate the net mall. Number of pieces ofa) Name and type of the material with brand shrimp may be determined in the above material bycounting

name, if any; the pieces in 1 kg.

b) Nameand addressof the processor;

2

IS 2237 : 1997

Table 2 Requirement For Prawns (Shrimps) - Frozen(Claust 5.3)

81 CbaracterisUc RequlnmeDt Method of Tesl,No. Ref to Annex, A

, and Other IndianStandard

Whole, Peeledand CookedType Blanched TypeHeadless Develned Type Includina Including Headlessand IQP Includinl Cooked-Peeled, Blanched, Peeled-

Type Butterfly, WholeCooked, Deveined Blanched,Fantail Peeled- and Peeled

Roundand Deveined, Undeveined BlanchedIOFType Cookedand IOF and IQFType

(1) (2) (3) (4) (5) (6) (7)

i) Total bacterial 500000 1000000 100000 200000 Bcount/g in thefinished product, Max

ii) Escherichia coli Count/g,Max 20 20 Absent Absent C

iii) Faecal Streptococci count/g,Max 100 100 Abient Absent 0

iv) Coagulase positiveStaphylococci/g, 100 100 Absent Absent EMax

v) SalmonellalArizona Absent Absent Absent Absent Fper2S g per25g per25a per 25 g

vi) Shigella Absent Absent Absent Absent IS5887 (Part 3)per2S g per25g per2S g per 25 g

vii) Vibrio cholera« Absent Absent Absent Absent Gper2S g per2S g per 25 g per 25 g

viii) ListeriD monocytogenes Abient Abient Absent Absent Hper2S g per2S I per25 I per2S g

ix) Formaldehyde mglkg, Max 10.0 10.0 10.0 10.0 J

x) Indole,mglk" Max 2.5 2.S 2.5 2.5 K

xi) HeavyMetal. :

a) Mercury, mg/kg,Max 0.5 0.5 0.:5 0.5 L-3

b) Copper, mglkg,Max 20.0 20.0 20.0 20.0 1.,·4

c) Zinc, mglkg,MQ 50.0 SO.O 50.0 50.0 L-4

d) Arsenic, mg/kl, Max 1.1 1.1 1.1 1.1 L-S

e) Lead, mglkg, Max 1.0 1.0 1.0 1.0 L-6

f) Tin, mglka, Max 250.0 250.0 250.0 250.0 L-7

3

IS 2237 : 1997

ANNEX A

(Clause 5.2)PROCESSING OF PRAWNS (SHRIMPS) - FROZEN

A-I Processing

A-l.I The prawns shall be iced and maintained ata temperature of 3°C till they reach the factory forfreezing.

A-l.2 The material along with crushed ice,shall bekept in clean stainless steel, aluminum or plasticcontainers of suitable size, It shall be processed,that is, converted into required product such asheadless (deheaded), peeled and deveined orpeeled and undeveincd, blanched, etc.

A-l.2.1 For blanched products, the material shallbe processed in stainless steel containers, heatedelectrically/or by steam by holding the product inwater with or without additives or exposing tosteam for required short period just to removeslimeand/or bring about desirablechange in colourof shell or meat but taking care that the product isnot cooked. Best results can be obtained by imme­diate cooling in crushed ice or chilled water sub­sequent to blanching.

A-l.3 The material shall be size graded, andwashedwith chlorinated water containing 10mg/kgavailable chlorine. It shall then be weighed andfilled in polythene bagsor waxed laminated cartonswith a lining of waterproof material. The materialafter washing and selecting may be arranged on aconveyor for freezing.

A-l.4 The material shall be ~uick frozen at atemperature not exceeding-40 C in the minimumpossible time. During freezing, the time taken forthe core of the material, 5 em thick, to reach atemperature ranging from -18 to -20°C, shall bepreferably llh, but shall not exceed 3 h under any

circumstances.

A-I.! The quick frozen material shall be uniformlyglazed if frozen in containers other than waxedcartons and packed in suitable receptacles. Thepackedmaterial shall be immediatelyshifted to thecoldstorage and stored at a minimum temperatureof -lSoC.

A-I.S.I Individually quick frozen products shall beglazed, preferably hardened, prior to packing,weighingand stored at a minimum temperature of-22°C.

NOTE - Glazing shall be done with ice cold potable watercontaining S to 10 mglkg available chlorine. Glazed watercanalso be added to the material before freezing to produceuniform glaze. Citricacid,sugarand sodium chloride may beadded to the glazing water up to levels of 0.2, 0.5 and 0.5percent respectively. Permitted food colours and/or otherpermitted additives may be used in the case of cooked andpeeled; devened and cooked; whole cooked and blanched.

A-I.' Food colours and/or other food additivespermissible under the Prevention of Food Adul­teration Act and Rules, 1955 maybe used in the caseof cooked and peeled; deveined and cooked; wholecooked and blanched prawns.

ANNEX B

[Table 2,Sl No. (i)]DETERMINATION OF TOTAL BACTERIAL COUNT

3 g1 g5 g

15 g1 litre

B-1 MEDIUM

B-l.1 Tryptone glucose beef extract agar with thefollowing composition shall be used:

Beef extractDextroseTryptoneAgarDistilled water(see,IS 1070)

B-l.1.1 Dissolve the agar by steaming. Dissolvedthe other ingredients mentioned above (B-l.l)in the molten agar. AdjustpH to 7.2. Filter throughabsorbent cotton and distribute in suitable

4

quantities in glass containers. Sterilize in anautoclave at 121°C for 30 minutes.

B-l.2 Phosphate BulTer

B-l.2.1 Stock Solution

Dissolve34 g of potassium dihydrogen phosphatein500 mldistilledwater (see IS 1070), adjust thepHto 7.2 and make up the volume to 1 000 ml withdistilled water (see IS 1070). .

B-l.2.2 Solution for Use

Add 1.25 ml of the stock solution to 1 000 ml ofdistilled water (see IS 1070). Distribute in glasscontainers as required for serial dilution. Sterilizein an autoclave at 121°C for 30 minutes.

B-2 PROCEDURE

B-2.1 Collection 01Sample

Scrap off the surfaceglaze from the material with asterile scalpel. Usinga sterile core sampler,in thecase of blocks and sterile scissors, in the case ofindividual quick frozen materials, collect samplefrom the different parts of the frozen material tomakeup a total ofabout 10 g.

.B-2.2 Preparation of HomOlenate

Disintegrate the sample (B-2.1) with 90 ml ofphosphate buffer(B-l.2.2) in a sterile homogenizer

IS 2237 : 1997

of stomacher type. Prepare sterile dilutions up to0.000001using this homogenate.

B-2.3 Transferasepticallydilutionsbeginningwith1 m1 of 0.01 dilution and ending with 0.000 001dilutionIntosterile petri dishes.

B-2.4 Introduce nearly 10 ml of the melted andcooled (40°C) agar (B-t.l) into the petri dishes(B-2.3) and mixbyrotating gently.

B-2.5 Incubatethe petridishes (B-2.4) aftersettingat 31'C for 48 h.' Count the colonies in theappropriate dilutions and compute their numberpergram.

ANNEX C

[Table 2, Sl No. (ii)]DETERMINATION OF ESCHERICHIACOLI COUNT

e-l MEDIUM to 7.0 and filter throughabsorbentcotton.Add theC-l.l Sterile Tergitol Tergitol-7 and bromothymol blue in the filterate

andmix well. Distribute in appropriate quantities7 Agar with the following composition shall be and sterilize in an autoclave at 121°C for 15used: minutes.

Proteose peptone No.3 Difco 5 gYeast extract 3 gLactose 10 gAgar 15 gTh~W~ nl~

Bromothymol blue 0.025 gDistilled water 1 litre

C-l.l.l Dissolve the agar in three-fourths of thedistilled water by steaming. Dissolve the otheringredients mentioned above (C-I.i) exceptTergitol-7 and bromothymol blue in the restof thedistilled water by warming on water bath, ifrequired. Mix the two solutionswell, adjust thepH

C-l.2 To every 100 ml of molten agar,cooled to atemperature of 40°C, add 0.4 ml of 1.0 percentsolution.of 2, 3, S triphenyl tetrazolium chlorideprior to pouringinto sterile petri dishes.

C-I.3 Dry the dishes (C-l.2) at 4SoCfor one hour.

C-l.4 To the surtaceof the dried agar (e-l.3) add0.5 ml of 0.1 dilution of sample (see 8-2.2) andspread it using a sterile bent glass rod.

C-I.S Incubate the platesat 3,oC for 18to 24handcount the characteristic yellow coloured colonies.Computethe numberper gram.

ANNEXD

[Table 2, Sl No. (iii)]DETERMINATION OF FAECAL STREPTOCOCCI COUNT

D·l MEDIUM

10 g10 gS g

10 g20.0 g

1 g0.49 g

0.0636 g

D-I.l.1 Dissolve the agar in 750 ml of the distilledwater by steaming. All ingredients mentionedabove (D.I.I) except bromocresol purple andsodium carbonate are dissolved separately in2SD mlofdistilled water. Mix the two solutions well.Add sodiumcarbonate in small portions and thenfilter through absorbentcotton. Add Bromocresolpurple in the filterate and mixwen. Distribute in

D-l.l Sterile KF agar with the followingcomposition shall be used:

Proteose peptone No.3Yeast extractSodiumchloride ARSodium glycerophosphateMaltoseCPLactoseSodium azideSodium carbonateAR

5

Bromocresol purpleAgarDistilled water (see IS 1070)

0.015 g10 g1 litre

182237: 1991

approgriatequantitiesandsterilizein an autoclave D-l.3 Pour 1 ml each of the 0.1and 0.01dilutionat 121 C for 15 minutes. sample (B-2.2) to twoseparate sterilepetridishes.

NOTE -ID cue compounded apr medium is UIed,foU<M Add the cooled agar (nearly 10 ml). Mix it bymanufacturer'. instructions for Iterilizalion. rotating. Incubate the plates at 3,oC for 48 h.

D-l.2 Add 1 ml of 1.0 percent solution of 2, 3, 5 Count the red and pink colonies and compute theirtripbenyl tetrazolium chloride per every 100 ml of Dumber pergram.the melted and 000100 agar prior to use. ·

ANNEX E

[Table 2, Sl No. (iv)]DETERMINATION OF COAGUlASE POSITIVE STAPHYLOCOCCI

s-r MEDIUM

E-l.l Sterile Baired parker medium of thefollowing composition shall be used:

Tryptone 10 gBeef extract S gYeast extract 1 gLithium chloride 5 gAgar 15 gDistilled water (see IS 1070) 1 litre

E-l.l.l Dissolve the agar in 7S0 ml of the distilledwater (see IS 1070) bysteaming. Dissolve the otheringredients mentioned above (E-t.!) in 250 ml ofthe distilled water by warming on a water bath, ifrequired. Mixthe two solutions welland adjust thepH to 7.0 followed by fl1tering hot through cotton,Distribute the filterate into conical flasks inquantities of 90 ml and sterilize in an autoclave at121°C for 15 minutes.

NOTE- In cue compounded alar medium II used,(oUowmanufacturer'. instructionl (or slerilization.

E-l.l.2 To every 90 ml of melted and cooled(40°C) medium, add successively the followingsterile solutions:

Glycine, 20percent 7 mlPotassium tellurite, 1 percent 1 mlSodium pyruvate, 20 percent 5 ml

Eggyolk emulsion (yolk from one S-mlegg is aseptically removed andmixedwith 80 ml of sterilephosphate buffer)

Mixwell and pour in sterile petri dishes in 10ml to15 ml quantities. Allow to solidify and dry at 45°Cfor 1 hour.

E-1 PROCEDURE

E-2.1 To the surface of the dried agar, inoculate0.5 ml of 0.1 dilution of the sample and spread itusing a sterile bent glass rod. Incubate at 3r»C for24 h and count the black colonies surrounded by aclear zone of hydrolysis as coagulase positivestaphylococci and compute their number per gram.In doubtful cases further confirmation may be doneusing plasma coagulation test.

&-2.2 Plasma Coagulation Test

Take 0.2 ml of citrated rabbit or human bloodplasma in sterile t~ tube and dilute it with 0.6 mlof0.4 percent sodium chloride solution in distilledwater. Inoculate one loopful of a young culture ofthe organism under "test and incubate at 3t>C.Watch the tube at the interval of 30 minutes.Definite clotting of the plasma within 6 h indicatesthat the organism under test is coagulase positive.

The sample isenriched in a nutritious non-selectivemedium followed by enrichment in a growtbpromoting medium containing selectivelyinhibitory reagents. The orlanism is then identifiedby biochemicaland serologicalmethods.

F-t PRINCIPLE

ANNEX F

[Table 2, Sl No. (v)]DETECTION OF SALMONELLA! ARIZONA

F-2 APPARATUS

a) Sterile petri dishes.

b) Sterile pipettes 1 ml, 10 ml with 0.1 mlgraduation.

c) Inoculating needle and loop (3 mm),

d) Sterile culture tubes and tube racks.

6

IS 2237 : 1997

F-5 SEROLOGICAL IDENTIFICATION

Isolates that give characteristic biochemical testsfor SalmonellaIArizona are tested with Salmonellapolyvalent Somatic '0' anteserum in conjunctionwith a saline control. Isolates that agglutinate withSalmonella polyvalent '0' anteserum and also givetypical biochemical reactions are confirmed asSalmonella IArizona.

a) Acid butt (yellow) and alkaline slant (red),gas and H2S.

b) Acid butt, alkaline slant, no gas.

F-4.S If the reactions in F-4.4 are positive forSalmonella, perform the biochemical tests (seeF-4.6).

F-4.6 Biochemical Tests

BOA Light pink transparent colonies.Surrounding media red.

HEA Green-Blue colonies with blackcentre.

XLD Red colonies with black centre.BSA Black colonies with silvery white

metallic sheen.

F-4.4 Streak and stab all the typical Salmonellacolonies from the plates of F-4.3 (maximum 10) onTriple Sugar Iron Agar (TSIA) slants. Incubate for24h at 3't'Cand examine the tubes after incubationis complete. Salmonella, if present, will giveeither of the (ollowingreactions on TSI agar slantsmentioned below:

Yellowcolour

ReactionArizona

Acid andgas (late)

No acid,no gas

No change

Acid and gas

No colour No colourchange change

Purple Purplethroughout throughout

No change Bluecolour

Lysine Iron Agar

Onho NitrophenylBeta D. GalactoPyranoside Broth

Malonate Broth

Salicin Broth

Dulcitol Broth

Urea Broth

Media Incubation CharacteristicTune and Temp Salmonella

Lactose Broth 37°C I 24 h No acid,no gas

Sucrose Broth

F-4 ISOLATION OF SALMONELLA

F-4.1 Pre-enrichment

Transfer aseptically 25 g of homogenized sampleinto a 500 ml conical flask containing 225 ml sterilelactose broth. Incubate at 37°C for 24 h.

F-4.2 Selective Enrichment

Trausfer 1 ml each from F-4.1 to 9 ml oC selenitecystine broth and 9 ml Tetrathionate broth andincubate at 3~C for 24 h.

F-4.3 Streak loopful of culture from both the tubesof F-4.2 to separate sterile petri dishes of BrilliantGreen Agar (BOA), Hektoen Enteric Agar (HEA),Xylose Lysine Desoxycholate Agar (XLD) andBismuth Sulphite Agar (BSA). Incubate the petridishes for 24 h at 3r>C and observe forcharacteristic Salmonella colonies aftercompletion of incubation as given below:

F-3 MEDIA AND REAGENTS

a) Lactose Broth,

b) Selinit~ Cystine Broth,

c) Tetrathionate Broth,

d) Brilliant Green Agar (BOA),

e) Xylose Lysine Desoxycholate Agar (XLD),

t) Hektoen Enteric Agar (HEA),

g) Bismuth Sulphite Agar (BSA),

h) Triple Sugar Iron Agar rrsIA),

j) Tryptone Broth,

k) Urea Broth,

m) Malonate Broth,

n) Lysine Iron Agar (LIA),

p) Lactose, Sucrose, Salicin and Dulcitol inAndrades Fermentation Base,

q) Ortho Nitrophenyl Beta D. GalactoPyranoside (ONPG) Broth,

r) Gram Stain Reagents,

s) Physiological Saline,

t) Hydrochloric Acid (1 N),

u) Sodium Hydroxide (1 N),

v) Kovac's Reagent, and

w) Salmonella Polyvalent Somatic '0' An­tiserum.

7

IS 2237 : 1997

ANNEX G[Table 2, Sl No. (vii)]

IDENTIFICATIONOF ~ CHOLBlUE

G·5 SEROLOGICAL IDENTIFICAll0N

G-5.1 Isolates that givecharacteristic biochemicaltests are tested with ~ cholerae polyvalent 01antiserum in conjunction with a saline control.

Cultures that agglutinate in 01 antiserum and alsogivetypicalbiochemical reactions are confirmed asVibrio cholerae 01.

Culturesthat do not agglutinate with 01 antiserumbut give typicalbiochemical reactions are groupedas Vibrio cholerae non 01 or uon-agglutinablevibrios.

surface growth on to TCSS Agar and incubate theplate at 3~C for 18 to 24 h.

G-4.3 Examine the TCBS plates from G-4.1 andG-4.2 for ~ cholerae colony characteristics. Ji:cholerae will appear as large, smooth yellowtransparent colonies with translucent peripheries.

G·4.4 Inoculate all the individual suspectedcolonies but to a maximumof 10 in to Kligler IronAgar (KIA) media by stabbing the butt andstreaking the surface of slant. Incubate theinoculated slants at 31'C for 18 to 24 h.

Examine the KIA slants. ~ cholera« will produceacid butt (yellowin colour) and alkaline slant (redin colour) and will not produce gas and hydrogensulphide.

G-4.5 Blocbemlcal Tests

G-4.5.1 With the suspicious colonies perform thefollowing biochemical reactions, with theundermentioned media:

Glucose Broth doSucrose Broth doMannitol Broth doInositol doArabinose Broth doLysineBroth 3t>C 1-4daysOrnithine Broth doArginine Broth doGram Stain do

Motile andfermentativeAcid, no gas

dodo

No acid, no gasdo

Purple colourdo

Yellow colourGram Negativecomma shape

Typical ReactionofV; Cholerae

IncubationTemperature

and Time

3-rC/24hHugh Liefson

Media

c-i PRINCIPLE

G-l.1 The isolation of Vibrio cholerae and otherbiochemicallyrelated vibrios isb~ on the rapidgrowth of these organisms Inan alkaline peptonewater in the presence of available oxygen. Undersuch conditions JI: cholerae and related vibrios willoutgrow most of the contaminating pathogenicmicro-organisms. They are then identified bybiochemicaland serological tests.

G·2 APPARATUS

a) Sterile petridishes;

b) Sterile pipettes - 1 ml, 5 ml, 10 ml with0.1 ml graduation;

c) Inoculating needle and loop (3 mm);

d) Sterile culture tubes and tube racks; and

e) pH meter.

G-3 MEDIAANDREAGENTS

a) Alkaline Peptone Water;

b) Thiosulpbate Citrate BileSalt SucroseAgar(TeBS); -

c) Kligler Iron Agar (KIA);

d) Hugh-Lierson Glucose Broth;

e) Glucose, Sucrose, Mannitol, Inositol andArabinose in Andrades Fermentation base;

t) Lysine-Ornithine Decarboxylase/Arginine·Dehydrolase Broths;

g) Gram Stain Reagents;

h) PhysiologicalSaline;

j) Hydrochloric Acid (1 N);

k) Sodium Hydroxide (1 N); and

m) II: cholerae Polyvalent 'Ol t Antisera.

G-4 ISOlATION OF JJ: CHOLERAE

G-4.1 Primary Enrichment

Transfer aseptically 2S gof homogenizedsampleinto a 500 ml conical flaskcontaining 225ml alkalinepeptone water. Incubate at 3-rC for 16 to 18 h.Streak one Joopfulof the inoculam from the surfacegrowth on to TeaS Agar and incubate the plate at37°C for 18 to 24 h.

G-4.2 Secondary Enrichment

Transfer 1 ml of inoculam from G-4.1 to 9 mlalkaline peptone water. Incubate at 37°Cfor 6 to8 h. Streak one loopful of the inoculam from the

8

IS 2237 : 1997

ANNEX B

[Table 2, Sl No. (viii)]IDENTIFICATION OF L. MONOCYTOGENES

With the suspiciouscolonies perform the followingbiochemical reactions, with the undermentionedmedia:

primary selective enrichment supplement.Incubate for 24 h at room temperature.

11-4.2 Selective Media

Streak one loopful from enrichment mediumafterincubation (see 11-4.1) to plates of Oxoid Listeriaselective agar base containing Oxoid Listeriaselective supplement and incubate at roomtemperature for 24 to 48 h.

11-4.2.1 ListeriaMonocytogenes

Listeriamonocytogenes will grow as purple browntransparent colonies changing the colour of thesurrounding media to purple brown.

H-4.3 Stab typical colonies to SIM media andincubate at room temperature for 48 h.L. monocytogenes will exhibit umbrella motility.

11-4.4 Biochemical Tests

do

do

do

do

No acid no gas

do

do

do

do

do

do

Incubation TYpical ReactionTemperature ofand Time L. Monocytogenes

Gram + rods2-3 days/30o C Tumbling motility

24 h - 48 wsr C Acid butt andslant no gas

Acid and no gasFermentation inDextrose broth

Fermentation inMaltose broth

Fermentation inEsculin broth

Fermentation inRhamnose broth

Fermentation inMannitol broth

Fermentation inXylosebroth

Gram StainMotilityTSIAgar

Media

B-1 PRINCIPLE

The sample is first enriched in a suitableenrichment medium followed by streaking onpresumptive media. Presumptive coloniesdeveloped after incubation at 300e for 24 to 48 hare confirmed by biochemical reactions.

B-2 APPARATUS

a) Binocular Research Microscope;

b) Stomacher Homogenizer;

c) Sterile Petridishes;

d) Conical Flasks;

e) 1 ml Graduated Pipettes-

f) Test Tubes;

g) Sugar Fermentation Tubes, Durham Tubes;

h) Platinum Wire with Holder;

j) Microscopic Slides; and

k) Cavity Slides.

H-3 MEDWREAGENTS NEEDED

a) Oxoid Listeria Enrichment Broth BaseContaining Oxoid Listeria PrimarySelective Enrichment Supplement.

b) Oxoid Listeria Selective Agar BaseContaining.Listeria Selective Supplement.

c) SIMMedia

d) Triple Sugar Iron Agar (TSIA)

e) Urea Agar Slants

1) Dextrose, Maltose, Esculin, Mannitol,Rhamnose Xylose Broths

g) Crystal Violet, Grams Iodine, Ethyl Al­cohol, Saffranine, etc, for Gram's Reaction

h) Nutrient Agar Slants

8-4 PROCEDURE

H-4.1 Enrichment

Inoculate 2S g sample in 225 ml of Oxoid Listeriaenrichment broth base containing Oxoid Listeria

9

IS 2237 : 1997

ANNEX J[Table 2,Sl No. (ix)]

DETERMINATION OF FORMALDEHYDE

Place Sml trichloroaceticacid extractof the tissuein a SO ml beaker. Add 10 ml of distilled water (seeIS 1070) and make the solution alkaline with a fewdropsof30 percent NaOH and adjust thepH to 6.0with S percent aceticacid. Make up the solution to2Smlwithwaterandmix5mlofitwith5mlofacetylacetone - ammonium acetate reagent. Keep themixture still for 50 minutes at 3~C and read thecolour at a wave length of 410'nm',

J-4 PROCEDURE

trichloroacetic add for 2 minutes using a mortarand pestle and then filter through a filter paper(Whatman No. 42) aDd make up the volume to100ml in a standard volumetric flask.

For a blank, take distilled water in duplicate in theplaceof sample and for standard, prepare a seriesof tubes using 100 PI formaldehyde standard

J·3 EXTRACI' solution, concentration rangingfrom o-sopg.

Mincethe fISh muscletissue usinga homogenizer. J.' CALCUlATIONSMacerate 10 g of the fish mince in 10 percent

F Id b de ( t) Absorbanceof sample)( Concentration in standard x 2S x 100 x 100orma e., mg,percen - Absorbance of standard )( S x S )( J0 x 1000

J-t PRINCIPLEQuantitative determination of formaldehydeinvolves extraction of the fish muscle tisslle withtrichloroacetic acidfor the removal ofproteinsandtreatment with acetyl acetone - ammoniumacetate reagent to form a coloured formaldehydederivative. The absorbance is measured byspectrophotometer.J-2 REAGENTS

a) Trichloroacetic acid (10 percent solution);b) Sodium hydroxide (30 percent solution);c) Aceticacid (S percent solution); and

d) Acetyl Acetone - ammonium acetatereagent. To prepare 1 litre of the reagent,dissolve ISO gofammonium acetate.3mlofglacialaceticacidand 2 m1 ofacetylacetonein distilledwater (see IS 1070) and makeupvolume to lUtre.

ANNEX K

[Table 2, Sl No. (x)]DmmRM~ATIONOF~DOLE

x-i PRINCIPLE

Indole is extracted with ligbt petroleum fromtrichloroaceticacid- precipitated shrimpmuscle.The extractedindole, soluble in light petroleum, isreacted and re-estracted with Ehrlich's reagent.Indole in the form ofa rose indole complex can bedetermined spectrophotometricaUy.

K-2 APPARATUS

a) Spectrophotometer,and

b) Refrigerated centrifuge.

K·3 REAGENTS

a) Trichloroacetic Acid(TCA) - Dissolve6 gTCA in 100 ml distilled water.

b) Ught petroleum (BoDingpoint 4O-6CfC).

c) Ehrlich's Reagent - Dissolve 9 gparadimethylaminobenzaldcbyde (pABA)

in 45 ml concentrated Hel in 250 mlvolumetric flask and dilute to volumewithethanol.

d) Standard Indole Solutions - Accuratelyprepare stock solution of 10mg indoleIn 100 mllight petruleum.Use 1:10dilutionas working solution. Refrigerate indolesolutions.

K·4 PROCEDURE

K-4.1 Homogenize 40gshrimpwith80mlice-coldtrichloroacetic acid solution (TeA) in a waringblender for 1 minute. Add 80 ml ice-cold lightpetroleum and blend fot 1 minute. Transferhomogenate to 250 till centrifuge bottle andcentrifuge for 10 minutesat 10000 rev/min. Filtersupernate through Whatman No. 1 paper undersuction. Transfer filtrate to 250 ml separatoryfunnel. After the two layers haveseparated, tansfer

10

acid layer (lower) to second 2SO ml separatoryfunnel.

K-4.2 Wash TCA

Denatured protein precipitate (separated bycentrifugation) with 40 mllight petroleum and fil­ter as described above. Transfer filtrate to second250 ml separatory funnel already oontaining TeAlayer from first extraction. Shake for 1 minute andlet two layers separate. Transfer lower acid layer tothird separatory funnel and extract for third timewith 40 mllight petroleum.K-4.3 Combine all light petroleum extracts into 1separatory funnel. Extract indole with exactlySmlfreshly prepared Ehrlich's reagent by viaorouslyshaking for 1 minute. The rose indole oomplexformed is quantitatively transferred to Ehrlich'sreagent layer. When layers have separated, transfer

IS 2237 : 1997

lower layer to 1 em path ceU and read at 570 nmapinst reagent black solution.

K-4.4 Prepare standard curve as follows: Ac­cuntely measure volumes from 0.5 to 4 ml stockindole solution (working solution) and add into80 ml TCA in a separatory funnel. Extract indole byprocedures described abov~and construct standardcurve. Rose indole complex from indole standardand from TCA-extraeted shrimp is stable up to4h.

K-5 CALCULATION

With the help of the standard curve the amount ofindole present in 40 I shrimp can be determined.Indole oontent is usually expressed as the amountof indole in microgram per 100 g shrimp muscle.

ANNEX L

[Table 2, Sl No. (xi)]DETERMINATION OF HEAVY METAlS

1.,.1 PRINCIPLE

The sample is digested with nitric acid andsulphuric acid (mineralisation) in a closed system.The heavy metals are then determined by AtomicAbsorption Spectrophotometer. Mercury isdetermined using cold vapour technique andarsenic after generation of metal hydrides.

1.,.2 PREPARATION OF MATERIAL

Preparation ofsample of fISh involves inclusion ofskin and exclusion of bones, but subject to overallrule ofedibility. TIlis material is thoroughly mincedto get a homogeneous sample. This material is usedfor the determination of trace metals.

1.,.3 DETERMINATION OF MERCURY IN nSH

1.,.3.1 Prlndple

Mercury present in fish muscle is convened intoionic mercury bywet oxidation using concentratednitric acid and concentrated sulphuric add in theratio 4:1. The solution containing Hi+ ions arereduced to elemental mercury using stannouschloride. The liberated mercury vapour ismeasured using a Mercury Analyser, in the coldstate.

L-3.2 Reagents Required

1,3.2.1 Stannous ChlorilU (SnCI2) Solutioll, (20percent m/V) - Dissolve 20 g stannous chloride(SnCI2)in 10 ml distilled hydrochloric add (Ha)and boil it for about a minute; cool and dilute to

100 mi. Add 1 to 2 g of tin metal after thepreparation of the solution.1,3.2.2 Pottuliwn Dkhromate (K2Cn07) Solution(1 percent mJv ill Wate)

L-3.2.3 PotlUsium PmntJngtDUJ~ (KMn04) Solu­tion ( S percent mJv in Water) - KMn04 solutiontends to absorb mercury from ambient atmosphereif kept exposed. Hence the bottle must be keptstoppered.

1,3.2.4 Nitrk .Acid (HN03) (10 percent v/v) ­Prepare in distilled water (see IS 1070).

1,3.3.5 Sodium HytJroxiIU (NaOH) Solution (20percent mJv) - Dissolve SO g of sodium hydroxidepellets in distilled water (see IS 1070) and dilute to250 mi.

1,3.2.6 Sulplaurk Acid (H2S04) (1:1) - Prepareby diluting with distilled water (see IS 1070).

1,3.%.7 Standtud Mercury Solution

1,3.2.7.1 Stock solution - Dissolve 0.135 4 g ofmercuric chloride (Hg02) in 2S mI of S percentnitric acid (HN03). Add 1 ml potassiumdichromate (K2Cf207) and make up the volume to100 mIwith Spercent nitric acid (HN03).

1.-3.2.7.2 WorlciIIgstillldm'd - 2SJlg, SOP g, 100 Jlg,150 P& and 200 PI per S ml or 10 ml solution bysuccessivedDution ofthe above standard. MaintainS percent nitric add (HN03) and 0.01 percentpotassium dichromate (K2Cn07) in the solution.

11

IS 1137: 1997

1,3.3 Digestion Procedure lor the Determlnadonof Mercury

Weigh about S to 10 g of wet material(homogenized) and transfer to the oxidation tlaskfollowed by the addition or a cold mixture orconcentrated nitric acid (HN03) and concentratedsulphuric acid (H2S04) in the ratio of4:1 (v/v). It isheated cautiously at first, collecting the distillate inthe reservoir. When the mixture starts to darken,add a little of the distillate into the flask. Theprocedure is continued, maintaining a slight excessof nitric acid (HN03) in the oxidation Oask, untilthe solution ceases to darken and fumes ofS03 areevolved. The solution is cooled and make up to aknown volume using distilled water (see IS 1070).

L-3.4 Determination of Mercury Using MercuryAnalyser

A suitable aliquot of the sample (S to 10 ml) ispipetted into the reaction vessel of the apparatusand the required amount of 10 percent nitric acid(HN03) solution is added in order to maintain totalvolume of 10 ml or 20 ml. Add 2 ml of stannouschloride (SnCI2) solution and stopper immediately.Stir the contents vigorously for 5 minutes. Theabsorbance is noted on the meter as early aspossible. The sample procedure is adopted with aseries of standards and reagent blank. The reactionvessel is thoroughly cleaned after eachmeasurement. Standard curve is prepared usingmercuric chloride (HgCI2).

1,.4 DETERMINAnON OF COPPERAND ZINC

L-4.1 Apparatus

L-4.1.1 Atomic Absorption Spectrophotometerwith hollow cathode lamps.

L-4.1.2 Air-Acetylene Fame

L-4.2 Reagents

L-4.2.1 Concentrated Nitric Acid (HN03)­redistilled.

L-4.2.2 Perchloric Acid (HCI04) - 70 percentdouble vacuum distilled.

L-4.2.3 Concentrated Sulphuric Acid (H2S04).

L-4.3 Copper Standard Solution

1..-4.3.1 Stock Solution (1 mglml) - Place 0.200 gcopper wire or foil into a 125 ml Erlenmeyer flask.Add 15 ml nitric acid (1:4), cover flask with watchglass and let copper dissolve, warming to completesolution. Boil to expel fumes, cool and dilute to200 ml with distilled water (see IS 1070).

L-4.3.2 Working Standard (Range 2 to 8 pglml) ­Prepare required concentrations daily, usingsulphuric acid (2N H2S04).

L-4.4 Zinc Standard Solution

L-4.4.1 Stock Solution ( 1 mglml) - Dissolve 1 gZinc powder in 20 ml hydrochloric acid (1:1) in 1litre volumetric Oast and dilute to volume withdistilled water (see IS 1070).

1..-4.4.2 Working Standard - (0.2,uglml, 0.5 Jlglml,1.0pg/mland 1.S,uglml). Pipette 1 mlstoeksolutioninto tOO ml volumetric Dask and make up thevolume with distilled water. From this solutionsuitably dilute to get the required concentration.

L-4.5 Digestion of the Sample (Wet OxIdation)

L-4.5.1 Weigh 2S g of sample into a suitable sizekjeldahl flask and add 10 to 15 ml concentratednitric acid (HN03) and keep overnight forpre-digestion. Heat cautiously until first vigorousreaction subsides somewhat; then add 5 mlconcentrated sulphuric acid (H2S04). Continueheating, adding more concentrated nitric acid(HN03) in small portions as needed to preventcharring, until fumes of S03 evolve and solutionremains clear and almost colourless. Add 0.5 mlpe.rchloric acid (HCI04) and continue heating untilIt IS almost completely removed. Excess nitric acidis destroyed by adding saturated ammoniumoxalate solution to the cooled, digested solution. Itis evaporated again to get 503 fumes. The solutionis cooled and diluted to a known volume usingdistilled water (see IS 1070).

1..-4.5.2 The metals are determined directly byaspirating the solution over the flame in an atomicabsorption spectrophotometer and thecorresponding readings are noted. calibration ofthe instrument is carried out using standardsolutions ofcopper and zinc. Air acetylene flame isused for atomisation.

L·5 DETERMINATION OF ARSENIC BYIIYDRIDE GENERATION METHOD

L-5.1 Principle

The vapour generation method works by reactingthe analyte element in an acidified solution withsodium borohydride. The resulting metal hydridesare relatively unstable and decompose at lowtemperature.

L-5.2 Apparatus

1,.5.2.1 AtomicAbsorption Spectrophotometer

1,.5.2.2 Vapour Generation Assembly

L-5.3 Reagents

L-5.3.1 Acids for Digestion (see L-4.2.1 to L-4.2.3)

L-5.3.2 Sodium Borohydride Pellets

12

L-5.3.3 PotassiumIodideSolution - Dissolve 20 gKI in 100 ml distilled water (see IS 1070).

1,5.3.4 Arsenic Standard Solution

L-5.3.4.1 Stock solution - Dissolve 1.320 g As203in minimum volume of 20 percent sodiumhydroxide (NaOH) in 1 litre volumetric flask,acidify with hydrochloric acid (1:1) and dilute tomake up volume to 1 litre with distilled water (seeIS 1070).

L-S.3.4.2 Working standard - (1 Jlglml, 2 ,uglml,3,uglml, 4 Jlglml and 5 pglml). Pipette 10 ml stocksolution into 100 ml volumetric flask and dilute tovolume with water. Pipette 1 ml, 2 ml, 3 ml, 4 mland 5 ml ofabove diluted solution into separate 100ml volumetric flasks and make up the volume withdistilled water (see IS 1070).

L-S.4 Digestion of Sample (Wet Oxidation)

Weigh 25 g of sample into a suitable size kjcldahlflask and add 10 to 15 ml concentrated nitric acid(HN03) and keep overnight for pre-digestion.Heat cautiously until first vigorous reactionsubsides somewhat; then add 5 ml concentratedsulphuric acid (H2S04). Continue heating, addingmore concentrated nitric acid (HN03) in smallportions asneeded to prevent charring, until fumesof S03 evolve and solution remains clear andalmost colourless. Add 0.5 ml perchloric acid(HCI04) and continue heating until it is almostcompletely removed. Excess nitric acid is destroyedby adding saturated ammonium oxalate solution tothe cooled, digested solution. It is evaporated againto get S03 fumes. The solution is cooled and dilutedto a known volume using distilled water (see IS1070).

L-S.S Determination

I~S.S.l Add 5 ml of hydrochloric acid (Hel) and 5ml distilled water (see IS 1070) to 10 ml of workingsolution to make up 20 ml ofsolution. Arsenic mustbe reduced to Arsenic (III), by the addition of 1 mlof20 percent m/vpotassium iodide (KI) solution toeach aliquot ofsample. Reduction takes 1 Itat roomtemperature or 5 minutes at srrc, Cool thesolution to ambient temperature before analysis.

L-5.S.2 The sample is placed in the reaction vessel.Light the flame and adjust the fuel for a blue flame.Turn the stirrer on and wait for 20 seconds. Dis­pense 5 ml sodium borohydride via the septum.Allow the reaction to complete. The hydrideformed is carried from the reaction vessel by a flowof inert gas (N2) and thermally decomposed in a

. quartz tube heated by an acetylene flame.

IS 2237 : 1997

1.,.6 DETERMINATION OF LEAD

L-6.1 Apparatus (see L-S.2)

1,6.2 Reagents (see L-S.3)

1,6.3 Lead Standard Solution

L-6.3.1 Stock Solution (1 mglml) --Dissolve 1 glead powder in 20 ml nitric acid (1:1) in 1 litrevolumetric flask, and dilute to volume with distilledwater (see IS 1070).

L-6.3.2 Working Standard (S,uglm), 10,uglml, 15,uglml to 20 Jlg/mI). Pipette 1 ml, 2ml, 3ml and 4 mlstock solutions into 200 ml volumetric flasksrespectively and make up the volume with distilledwater (see IS 1070).

1..-6.4 Digestion Procedure

Dry the homogenized sample and make it into apowder. Take 1 to 2 g material in a silica crucibleand place in a furnace at 250°C. The temperature isslowly raised to 350°C with holding at thistemperature till smoking ceases. The temperatureis then increased to 500°C and the sample is ashcdat this temperature for 16 h. The carbon free ash iscooled and dissolved in 5 ml of nitric acid (1 NHN03). It is then warmed on a hot plate to aiddissolution and is made up to 50 ml in a standardflask using distilled water (see IS 1070).

The solution is aspirated over the flame of AtomicAbsorption Spectrophotometer and theconcentration is measured,

L-7 DETEIlMINATION OF TIN

L-7.1 Prlnclple

Samples are digested with nitric acid and thenhydrochloric acid and are diluted with distilledwater. Aqueous potassium chloride is added tosamples and standards. Tin is determined by atomicabsorption spectrophotometer at 235.5 nm withoxidizing acetylene flame.

L-7.2 Apparatus

L-7.2.1 Atomic Absorption Spectrophotometer(AAS)

L-7.3 Reagents

L·7.3.1 NitricAcid (HN03)

Test purityoClot bydiluting with distilled water (1:4v/v) and aspirating into atomic absorptionspectrophotometer. Absence of tin signal indicatessuitability for analysis.

L-7.J.2 Potassium Chloride Solution

Dissolve 1.91 g KCl and dilute to 100 ml withdistilled water (see IS 1070).

13

IS 2237 : 1997

L-7.3.3 Tin (Sn) Standard Solution

1.,.7.3.3.1 Stock solution - (1 mg Iml). Dissolve 1 gof tin (reagent grade) metal in200 ml concentratedhydrochloric acid (Ha). Add 200 ml of distilledwater.cool and make up the volume to t lure,

I~·7.3.3.Z Working standard - (O,ug/ml, SO ,ug/ml,100 Jlg/rnl, 150,ug/ml and 200,ug/ml). Pipette 5 ml,10 ml, 15 ml or 20 ml ofstock solution into 100 mlvolumetric flasks respectively and add 10 mlconcentrated hydrochloric acid (Hel) and 1.0 m1(potassium chloride) KCI solution. Make up thevolume with distilled water (see IS 1070).

1~7.4 Digestion orSample

Oask and within 15 minutes heat gently to initiatedigestion (avoid excessive frothing). Boil gently tillsamples just begin to dry at the bottom (avoidcharring). Add 2S ml concentrated hydrochloricacid (Hel) and heat gently for 15 minutes untilevolution of chlorine gas stops. Boil until solutionreducesto 10 to 15 mi. Add 40 ml water, swirl, andpour into 100 ml volumetric flask. Conduct tworeagent blanks.

I

L-7.4.2 Pipette 1.0 ml potassium chloride (KCI)solution into above 100 ml volumetric flask. Coolto ambient temperature and make up the volumewith distilled water (see IS 1070). Mix well and filterthrough mediumporositypaper.

1~.7.4.1 Weigh accurately about 25 g or sample in L-7.4.3 Aspirate water before and after eachan Erlenmeyer flask and dry in oven at 12(fC. Add sample. Calibrate the instrument using tin standard30 ml concentrated nitric acid (HN03) into the and read the concentration of the sample.

14

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