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 6853 (1973): Peptone, Microbiological Grade [FAD 15:Food Hygiene, Safety Management and Other Systems]

X3:6853-1973

Indian Standard SPECIFICATION FOR

PEPTONE, MICROBIOLOGICAL GRADE

Food Hygiene, Samplin g and Analysis Sectional Committee, AFDC 36

Chairman

MAJ-GEN M. S. BOPARAI

Refiresenting

Directorate General of Armed Forces Medical Services, New Delhi

Members

AQRICULTURAL MARICE’~IN~ ADVISER Directorate of Marketing & Inspection, Faridabad TO THE GOVERNMENT OF INDIA

DR J. S. PRUTHI ( Alternate ) SHRI V. N. AMBLE Institute of Agricultural Research Statistics ( ICAR ),

New Delhi SHRI K. S. KRISHNAN ( Alterante )

SHRI K. BALASUBRAMANIAN Public Analyst, Government of Tamil Nadu, Madras SHRI D. S. CHADHA Central Committee for Food Standards, New Delhi DR G. C. DAS DR P. K. DATTA

Corporation of Calcutta All India Institute of Hygiene and Public Health,

Calcutta SHRI SUKUMAR DE

DR C. A. MULAY (Alternate) National Dairy Research Institute ( ICAR ), Karnal

DIRECTOR Central Food Laboratory, Calcutta EXECUTIVE HEALTH OFFICER Bombay Municipal Corporation

MUNICIPAL ANALYST ( Alternate ) HEALTH OFFICER Corporation of Madras DR ( SMT ) S. KHOSLA Department of Health & Family Planning,

DR P. K. KYMAL Government of Punjab, Cbandigarb

Food and Nutrition Board (Ministry of Agricul- ture ), New Delhi

I DR R. C. SINHA ( Alternate)

SHRI Y. N. LAKSHMAN Defence Food Research Laboratory ( Ministry of

DR G. M. VERMA ( Alternate) Defence ) (R & D ), Mysore

SHRI F. G. T. MENEZES Directorate of Sugar & Vanaspati (Ministry of Agriculture ), New Delhi

PUBLIC ANALYST ( FOOD AND Government of West Bengal, Calcutta WATER )

( Continacd on pugc 2 )

@ Co@right 1973

INDIAN STANDARDS INSTITUTION

This publication is protected under the Indian Copyright Act ( XIV of 1957) and reproduction in whole or in part by any means except with written permission of the publisher shall be deemed to be an infringement of copyright under the said Act.

IS : 6853 - 1973

( Continuedfrom page 1)

Members

DR T. N. RAMACHANDRA RAO

Representing

Central Food Technological Research Institute ( CSIR ), Mysore

SHRI C. T. DWARKANATH ( Alternate ) COL M. C. SANYAL Quarter Master General’s Branch, Army ._

Headquarters LT-COL 0. P. KAPUR ( Alternate )

DR RANJIT SEN Serologist to the Government of India, Calcutta SENIOR COMMERCIAL OFFICER, Chief Catering Officer, New Delhi

NORTHERN RAILWAY DR B. N. SINGH ISI, New Delhi DR S. B. SINGH Public Analyst, Government of Uttar Pradesh,

Lucknow LT.COL 0. N. TYACI HealtDh3epartment, Municipal Corporation of

DR A. G. AJWANI ( Alternate ) SHRI P. c. VIN The Coca-Cola Export Corporation, New Delhi DR HARI BKAGWAN, Director General, ISI ( Ex-o#cio Member)

Director ( Agri & Food )

Secretas

SHRI S. K. SUD

Assistant Director ( Agri & Food ), IS1

Media for Microbiological Tests Subcommittee, AFDC 36 : 7

Convener

DR RANJIT SEN Serologist to the Government of India, Calcutta

Members

DR A. N. BOSE Bengal Immunity Co Ltd, Calcutta DIRECTOR King Institute, Madras

DR D. V. MURTHY ( Alternate ) DR A. K. GHOSH Cholera Research Centre ( ICMR ), Calcutta HEAD, DIVISION OP BIOLOGICAL Indian Veterinary Research Institute ( ICAR ),

PRODUCTS Izatnagar DR A. P. JOSHI Vallabhbhai Pate1 Chest Institute, Delhi-

DR ( SMT ) V. BAJAJ ( Alternate ) DR A. D. MUKHERJEE Bengal Chemical and Pharmaceutical Works Ltd,

Calcutta DR A. N. RAI CHOWDHURI DR T. N. RAMACHANDRA RAO

Central Research Institute,.Kasauli Central Food Technologrcal Research Institute

DR B. RANGANATHAN DR M. V. SALT DR N. S. SU~BA RAO

( CSIR 1, Mysore National Dairy Research Institute ( ICAR ), Karnal Haffkine Institute, Bombay Indian Agricultural Research Institute ( ICAR),

New Delhi

I!?+ : 6853 - 1973

Indian Standard SPECIFICATION FOR

PEPTONE, MICROBIOLOGICAL GRADE

0. FOREWORD

0.1 This Indian Standard was adopted by the Indian Standards Institution on 28 February 1973, after the draft finalized by the Food Hygiene, Sampling and Analysis Sectional Committee had been approved by the Agricultural and Food Products Division Council.

0.2 Unless the ingredients used in media for microbiological work are of uniform quality, the results obtained might be erroneous and might be unreliable. Since the media used in different laboratories often differ greatly in their quality, the results of microbiological work at different laboratories can not be compared. Therefore, with a view to unifying the practices of different laboratories dealing with microbiology and providing guidance to the indigenous manufacturers regarding the quality, it was decided to bring out a series of Indian Standard specifications for ingredients commonly used in media for microbiological work.

0.2.1 For specific purposes, any additional requirements shall be as agreed to between the purchaser and the supplier.

0.3 Peptone is the dried product of enzymatic hydrolysis of materials con- taining animal or plant proteins. Peptone contains a variable mixture of proteoses, peptides and amino acids. The composition of a peptcme varies with the raw material used and the method of manufacture. Therefore, each brand has its own characteristics; also, variations may occur between batches of the same brand. For most bacteriological work, it is necessary to use peptone manufactured from materials of animal origin. Peptones from soyabean can not be used for media to detect fermentation of carbohydrates.

0.4 For the purpose of deciding whether a particular requirement of this standard is complied with, the final value, observed or calculated, express- ing the result of a test, shall be rounded off in accordance with IS : 2-1960*. The number of significant places retained in the rounded off value should be the same as that of the specified value in this standard,

*Rules for rounding off numerical values ( revised).

3

IS : 6853 - 1973

1. SCOPE

1.1 This standard prescribes the requirements and the methods of test for peptone, microbiological grade.

2. REQUIREMENTS

2.1 It shall completely dissolve in water.

2.2 A 1.0 percent solution in distilled water after autoclaving at 121°C for 30 minutes shall have a pH between 5.0 and 7.0.

2.3 A 1.0 percent aqueous solution having PH adjusted to 7.0 and after V filtration and sterilization by autoclaving at 121°C for 30 minutes shall be clear.

i

2.4 It shall be able to support and shall not inhibit the growth of micro-organisms when it is incorporated in suitable medium as the sole growth supporting substance. The general guidelines for testing this characteristic are given in 19 of IS : 6854-1973*.

2.5 When sterilized at 121°C for 30 minutes, it shall not contain any viable micro-organisms when tested according to the method given in IS : 5402-19697.

2.6 It shall give a good positive indol reaction when tested in accordance with the method prescribed in Appendix A.

2.7 It shall give a good positive methyl red reaction when tested in accordance with the method prescribed in Appendix B.

2.8 The material shall also conform to the requirements given in Table 1.

3. PACKING, STORAGE AND MARKING

3.1 Packing - The material shall be securely packed in well filled wide mouth containers with tightly fitting lids. b

3.2 Storage - The material shall be stored in a cool and dry place, I

3.3 Marking-Each container shall be marked legibly to give the following information:

a) Name of the material including the words ‘ Microbiological Grade ‘,

b) Name and address of the manufacturer,

*Methods of sampling and test for ingredients used in media for microbiological work.

IMethod for plate count of bacteria in foodstuffs.

IS : 6853 - 1973

c) Minimum net content, and ad) Batch or xode number.

3.3.1 The container may also be marked with the IS1 Certification Mark.

NOTE - The use of the IS1 Certification Mark is governed by the provisions of the Indian Standards Institution ( Certification Marks ) Act and the Rules and Regulations made thereunder. The IS1 Mark on products covered by an Indian Standard conveys the assurance that they have been produced to comply with the requirements of that standard under a well-defined system of inspection, testing and quality control which is devised and supervised by IS1 and operated by the producer. IS1 marked products are also continuously checked by IS1 for conformity to that standard as a further safeguard. Details of conditions under which a licence for the use of the IS1 Certification Mark may be granted to manufacturers or processors, may be obtained from the Indian Standards Institution.

SL No.

(1)

i)

ii)

iii)

iv)

I 4

i

vi)

vii)

viii)

ix)

x)

TABLE 1 REQUIREMENTS FOR PEPTONE

( Clause 2.8 )

CHARACTERISTIC ’ REQUIREMENT METHOD OF TEST, REF TO

Appendix of This

Standard

Cl No. of * IS : 6854~1973*

- (4)

Moisture, percent by mass, 5 - 4 Max

Ash, percent by weight, Max

5.5 - 6

Amino acid nitrogen, percent le.5 - 8 by mass, Min

To~tassni~cn, percent by 14 - 9 ,

Phosphorus, as PsO,, percent by mass, Max

1.5 C -

“02; czar&, percent by >

7.0 - 11

Iron ( as Fe), mg/kg, Max 75 - 13

Coye; ( as Cu 1, m&g, 10 - 15

Zinc ( as Zn ), mg/kg, Max

Fermentable carbohydrates

25 - 18

Nil - 20

*Methods of sampling and test for ingredients used in microbiological work.

5

IS : 6853 - 1973

4. SAMPLING

4.1 The representative samples of the material shall be drawn according to the method prescribed in 3 of IS : 6854-1973*.

5. TESTS

5.1 Tests shall be carried out by the methods prescribed in 2 and in co1 4 and 5 of Table 1.

5.2 Quality of Reagents - Unless specified otherwise, pure chemicals and distilled water ( see IS : 1070-1960t ) shall be employed in tests.

NOTE - ‘ Pure chemicals ’ shall mean chemicals that do not contain impurities which affect the experimental results.

APPENDIX A

( Clause 2.6 )

METHOD OF TEST FOR INDOL REACTION

A-l. REAGENTS

A-l.1 Peptoae Water Medium - Dissolve by gentle heating 10.0 g of peptone under test, 5.0 g of sodium chloride in 1 001) ml of distilled water. Adjust @H between 8.0 and 8.4 and boil for 10 minutes. Cool and filter through a filter paper and adjust the@H between 7.2 and 7.4. Place in tubes the suitable amounts and sterilize at 120°C for 15 minutes.

A-1.2 Kovac’s Reagent -Dissolve by gentle heating on water-bath at 50 to 55”C, 5.0 g of p-dimethylamino benzaldehyde in 75 ml of amyl or isoamyl alcohol. Cool and add slowly 25.0 ml of concentrated hydrochloric acid sp gr 1.18 Min. Keep the reagents either in amber coloured bottles or in bottles wrapped with black paper, to protect it from light. When not in use, keep at 4°C in a refrigerator. The colour of the reagent should be yellow to light brown.

A-2. PROCEDURE

A-2.1 Inoculate a tube of peptone water medium with a bacterial strain known to produce indol from peptone. A suitable strain is Escherichia coli. Jnoculate another tube of peptone water -medium with a bacterial strain known not to produce indol from peptone. Suitable strains are Enterobacter clodcue and Salmonella t@hi. Incubate the inoculated tubes at 37°C for 48 hours. Add to the ~growth 0.5 ml of Kovac’s reagent, shake well and examine after 1 minute. A red colour in the reagent layer indicates the presence of indol. ___~ --__I

*Methods of sampling and test for ingredients used in media for microbiological work.

tspecification for water, distilled quality ( revised).

6

IS : 6853 = 1973

APPENDIX B

( Clause 2.7 )

METHOD OF TEST FOR METHYI., RED REACTION

B-l. REAGENTS

B-l;1 Glucose Phosphate Medium - Steam to dissolve in 1 OOO*O ml of ~distilled water, 5.0 g of peptone under test and 5.0 g of dipotassium

/

1

hydrogen phosphate ( K,HPQ, ). Cool, filter and adjust to pH 7.5. Add 5.0 g of glucose, mix to dissolve, and distribute into tubes. Sterilize at 115°C for 10 minutes. *

B-l.2 Methyl Red Reagent-Dissolve 0.04 g of methyl red powder in 40.0 ml of absolute ethanol. Dilute with distilled water to a total volume of 100.0 ml.

B-2. PROCEDURE

B-2.1 Inoculate a tube of the medium with a bacterial strain known to give positive methyl red reaction. A suitable strain is Escherichia cola’. Inoculate another tube of the medium with a bacterial strain known to give negative methyl red reaction. A suitable strain is Enterobacter cloacae. Incubate preferably at 30°C for 5 days or alternatively at 37°C for 2 days. Add to the growth 2 drops of methyl red reagent, shake and examine, red colour indicates positive reaction and yellow colour indicates negative reaction.

APPENDIX C

[ Table 1, Item (v) ]

DETERMINATION OF PHOSPHORUS

C-l. REAGENTS

C-l.1 Hydrochloric Acid - 25 ml of concentrated hydrochloric acid diluted to 100 ml.

C-1.2 Concentrated Nitric Acid - sp gr 1’42 ( see IS : 264-1950* ) .

C-1.3 Nitric Acid ( 1: 1) - a mixture of equal volumes of concentrated nitric acid and water.

C-l.4 Ammonium Molybdate Stock Solution -Take 200 g of powdered ammonium molybdate in a stoppered graduated cylinder

*Specification for nitric acid.

7

IS : 6853 - 1973

of 1 000 ml capacity, add to it 800 ml of water and shake well for 25 minutes to dissolve the ammonium molybdate. Add gradually 25 percent ( m/v) ammonium hydroxide solution till the solution is clear ( about 100 to 140 ml of ammonium hydroxide may be required ). Avoid adding -excess of ammonia. Make up the volume to one litre. If necessary, filter the solution through a fluted filter paper and stock this solution.

C-l.5 Dilute Nitric Acid Solution - 2 percent ( m/v ).

C-l.6 Potassium Nitrate Solution - 3 percent ( m/v ),

C-1.7 Standard Sodium Hydroxide Solution,- 0.1 N.

C-l.8 Standard Nitric Acid Solution - 0.1 N.

C-l .9 Phenolphthalein Indicator Solution - Dissolve 0.1 g of phenolphthalein in 100 ml of 60 perpent ( m/v ) rectified spirit ( see IS : 323- 1959*). -

C-2. PROCEDURE

~-2.1 Preparation of the Solution - Weigh accurately about one gram of the material in a silica crucible and ignite at 600 f 20°C in a muffle furnace to carbon-free ash. Boil the ash in 40 ml of hydrochloric acid and a few drops of nitric acid (see C-I.2 ). Transfer to a 250-ml graduated flask. Cool, dilute to the mark and mix thoroughly.

C-2.2 Precipitation -Take a IO-ml aliquot of the prepared solution ( see C-2.1 ) in a 150-ml beaker. In a dry beaker, prepare ammonium molybdate solution by pouring into it, quickly and simultaneously 10 ml of the ammonium molybdate stock solution (see C-1.4) and 10 ml of con- 1 centrated nitric acid; or take 10 ml of concentrated nitric acid; first in the beaker and into ~this pour quickly 10 ml of the ammonium molybdate stock solution, swirling the beaker during addition. Pour this freshly prepared, clear liquid quickly into the beaker containing the aliquot and stir.

NOTE -The temperature developed in the molybdate solution is suffkient to precipitate all the phosphorus present in the aliquot. Under no circumstances the phosphomolybdate precipitate should be heated either on a water-bath or directly over a burner so as to avoid precipitation of molybdic anhydride.

C-2.3 Filtration and Washing - Allow the precipitate to stand over- night and then filter through a disc of Whatman filter paper ( No. 42 or equivalant ) in a Gooch crucible by suction or through a g-cm Whatman filter paper ( No. 42 or equivalent ) over a funnel. As far as possible, only ~the supernatant liquid is passed through the filter papey, retaining the precipitate in the beaker. When the supernatant liquid IS decanted off,

*Specification for rectified spirit (revised).

IS : -6853 - 1973

the precipitate is washed twice with dilute nitric acid and then with potassium nitrate solution until the washings are free from acid. If ordinary funnel and filter paper are used, freedom from acidity may be tested by collecting sufficient filtrate in a test-tube to which a few drops of phenolphthalein indicator solution and one drop of the standard sodium hydroxide solutionare added. If the pink colour appears with one drop of the standard alkali, the precipitate is free from acid.

C-2.4 Titration - Transfer the precipitate with the filter paper back to the beaker in which precipitation was carried out. When Gooch crucible is used for filtration, transfer the whole crucible along with the filter paper to the beaker in which precipitation was carried out, Add standard sodium hydroxide solution from a burette just sufficient to dissolve the precipitate and then add 5 ml in excess. See* that no yellow precipitate sticks to the filter paper. Note the total volume of the standard sodium hydroxide solution added. Add about 10 drops of phenolphthalein indicator solution and titrate the excess of alkali with the standard nitric acid,

C-3. CALCULATION

C-3.1 Phosphorus ( as P,O,), percent by mass

0.772 ( A - B ) =

M

where

A = volume in ml of the standard sodium hydroxide solution used ( see C-2.4 ),

B = volume in ml of the standard nitric acid used to neutralize the excess alkali ( see C-2.4 ), and

M = mass in g of the material taken for the test ( see C-2.1 ).

!NDIPN S’kANDAkDS

ON

FOOD HYGIENE, SAMPLING AND ANAL%%&

IS:

2491-1972 Code for hygiene conditions for food processing units (first reuis&z )

5059-1969 Code for hygienic conditions for large scale biscuit manufacturing units atld bakery units

5126 ( Part I)-1969 Glossary of general terms for sensory evaluation of foods : Part I

5126 (Part

5398-1969

5399--1969

5400-1969

5401-1969

5402-1969

5403-1969

5404-1969

5835-1970

5837-1970

5838-1970

5839-1970

5886-1970

5887-1970

6510-1972

6541-1972

6542-1972

6850-1973

6851-1973

6852-1973

6853-1973

6854-1973

Methodology

II )-1969 Glossary of general terms for sensory evaluation of foods : Part II Quality characteristics

Methods of estimation of thiamine ( Vitamin B, ) in foodstuffs

M-thods of estimation of reboflavin ( vitamin B, ) in foodstuffs

Methods for estimation of nicotinic acid ( niacin ) in foodstuffs

Methods for detection and estimation of coliform bacteria in foodstuffs

Method for plate count of bacteria in foodstuffs

Method for yeast and mould count of foodstuffs

Code of practice for handling of food samples for microbiologica Ianalysis

Methods for estimstion of Vitamin D in foodstuffs

Code for hygienic conditions for soft drinks manufacturing units

Methods for estimation of Vitamin~C in foodstuffs

Code for hygienic conditions for manufacture, storage and sale of ice-creams

Methods for estimation of carotenes and vitamin A ( retinol ) in foodstuffs

Methods for detection of bacteria responsible for food poisoning and food- borne diseases

Code for hygienic conditions for manufacture and handling of ice for human consumption

Code for hygienic conditions for establishment and maintenance of mid-day school meal programme

Code for hygienic conditions for fruit and vegetable canning units

Agar, microbiological grade

Meat extract, microbiological grade

Bile salts, microbiological grade

Peptone, microbiological grade

Methods of sampling and test for ingredients used in media for microbiologi- cal work