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 5887-2 (1976): Methods for detection of bacteriaresponsible for food poisoning, Part 2: Isolation,identification and enumeration of STAPHYLOCOCCUS AUREUS andfaecal streptococci [FAD 15: Food Hygiene, SafetyManagement and Other Systems]

IS : 5887 ( Part II ) - 1976

Indian Standard METHODS FOR

DETECTION OF BACTERIA RESPONSIBLE FOR FOOD POISONING

PART II ISOLATION, IDENTIFICATION AND ENUMERATION OF STAPHYLOCOCCUS AUREUS

AND FAECAL STREPTOCOCCI

( First Revision) Food Hygiene, Sampling and Analysis Sectional Committee, AFDC 36

Chairman Representing

DR RABNJIT SEN Serologist to the Government of India ( DGHS ), Calcutta

Members

AQRICULTIJRAL M A R K E T I N Q ADVISER TO THE GOVERNMENT

Directorate of Marketing & Inspection ( Ministry of

OF INDIA Agriculture & Irrigation ), Faridabad

SHRI T. V. MATHEW ( Alternate ) SHRI V. N. AMBLE Institute of Agricultural Research Statistics ( ICAR ),

New Delhi SHRI K. S. KRISHNAN ( Altcrnatc )

Da G. C. DAS Health Officer, Corporation of Calcutta DR P. K. DATTA All India Institute of Hygiene and Public Health,

Calcutta SHRI SUKUMAR DE

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

SHRI 0. P. DHAMIJA Export Inspection Council of India, New Delhi DIRECTOR Central Food Laboratory, Calcutta SHRI C. T. DWARKANATH Central Food Technological Research Institute

( CSIR ), Mysore DR M. A. KRISHNASWAMY (Alternate )

EXECUTIVE HEALTH OFFICER Municipal Corporation of Greater Bombay MUNICIPAL ANALYST ( Alternate )

HEALTH OBFICER Corporation of Madras COL KEWAL KRISHNA

DR A. D. KUMAR ( Alternat ) Health Department, Municipal Corporation of Delhi

( Continued on page 2 )

@ Copyright 1977 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 : 5887 ( Part II ) - 1976

( Continued from page 1 )

Members Repnscnting DR ( SMT ) S. KHOSLA

DR P. K. KYMAL

Department of Health h Family Planning, Govern- ment of Punjab, Chandigarh

Food & Nutrition Board ( Ministry of Agriculture & Irriaation ), New Delhi

DR 0. N. AQARWALA ( Alternate) - ‘. MAJV.A. NARAYANAN . Defence Food Research Laboratory ( Ministry of

Defence ), Mysore DR G. M. VERMA ( Alternate )

PUBLIC ANALYST (FOOD AND WATER )

Government of West Bengal, Calcutta

PUBLIC ANALYST ( BACTERIO- LOOY ) ( Alternate )

DR A. N. RAI CRAWDHURI MAJ-GIN D. C. SACHDEVA

National Institute of Communicable Diseases, Delhi Directorate General of Armed Forces Medical Services

SENIOR MEDICAL OFFICER

D!,%L::Nkx . .

SERI N. SRINIVASAN DR M. R. SUBBARAM

( Ministry of Defence ), New Delhi Northern Railway, New Delhi

Public Analyst, Government of Uttar Pradesh, Lucknow

Public Analyst, Government of Tamil Nadu, Madras Directorate of Sugar & Vanaspati ( Ministry of

Agriculture & Irrigation ) 1 SHRI I. A. SIDDIQI ( Alternate ,

DR T. A. V. SUBRAMANIAN Vallabhbhai Pate1 Chest Institute, Delhi DR M. C. SWAMINATHAN Directorate General of Health Services ( Ministry of

SHRI D. S. CHADHA ( Alternate ) Health & Family Planning ), New Delhi

COL R. N. TANEJA Quartermaster General’s Branch, Army Headquarters, New Delhi

LT-COL D. D. VOHRA ( Alternate ) SHRI P. c. VIN The Coca-Cola Export Corporation, New Delhi

&RI J. D. CONTRACTOR ( Alternate) SHRI T. PURNANANDAM, Director General, ISI ( Ex-o#cio Member )

Deputy Director ( Agri & Food )

Secretary SHRI S. K. SUD

Deputy Director ( Agri & Food ), ISI

Food Microbiology Subcommittee, AFDC 36 : 7

Convener

Da RANJIT SEN r

Serologist to the Government of India ( DGHS ), 1 Calcutta ,

OF INDIA

DIRECTOR OF LABORATORIES ( Alternate ) MAJ G. S. BALI Defence Food Research Laboratory I Ministrv

Defence ), Mysore SHRI K. Cl. RAPON ( Alternak )

2 ( Continued on page

Members

AQRICULTURAL MARKETING Directorate of Marketing & Inspection ( Ministry of Anvrann TO THE GOVERNMENT Agriculture & Irrigation ), Faridabad

IS : 5887 ( Part II ) - 1976

Indians Standard METHODS FOR

DETECTION OF BACTERIA RESPONSIBLE FOR FOOD POISONING

PART II ISOLATION, IDENTIFICATION AND ENUMERATION OF STAPHYLOCOCCUS AUREUS

AND FAECAL STERPTOCOCCI

( First Revision )

0. FOREWORD

0.1 This Indian Standard ( Part II ) ( First Revision ) was adopted by the Indian Standards Institution on 13 December 1976, 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 Several micro-organisms contaminating food give rise to clinical symptoms. These are abdominal pain, nausea, vomitting, diarrhoea and sometimes pyrexia. A well-known exception is that of botulism where the symptoms are those of difficulty in swallowing, diplopia, aphonia and difficulty in respiration. Poisoning through food is characterized by the explosive nature with which the symptoms occur in otherwise healthy individuals. Often several persons after having consumed a particular item of food, develop symptoms that serve as important guide in suspect- ing food poisoning. Such explosive nature of food poisoning helps in differentiating conditions from those of out-breaks of food-borne infectious diseases which generally spread over a period of several days. The micro-organisms causing food poisoning belong to bacteria, protozoa and helminths, fungi and viruses. However, this standard covers the method for detection and estimation of important bacteria responsible for food poisoning food-borne diseases.

0.3 This standard was first published in 1970. It is being revised in_ parts covering methods of detection and estimation of various bacteria separately. This has been done with a view to making each part more comprehensive including various details of the methods. It is expected that publication of these methods in parts will facilitate better

3

F

IS : 5887 ( Part II ) - 1976

implementation and adoption of the standard by concerned orga- nizations. This will also make review and revision easier. The salient features of this revision are:

a) Besides detection, estimation procedures for various organisms where applicable have been incorporated; and

b) Methods of identification have been updated.

0.4 In reporting the result of a test or analysis made in accordance with this standard, if the final value, observed or calculated, is to be rounded off, it shall be done in accordance with IS : Z-1960*.

1. SCOPE

1.1 This standard ( Part II ) prescribes method for isolation, identi- fication and enumeration of Staflhylococcus aureus and faecal streptococci in foods.

2. SAMPLING AND QUALITY OF REAGENTS

2.1 Sampling -For microbiological examination the samples should be handled carefully. For this purpose, IS : 5404-1969t shall be followed.

2.2 Quality of Reagents shall be employed in tests

- Unless specified otherwise, pure chemicals and distilled water ( see IS : 1070-1960$ ) shall

be used where use of water as a reagent is intended. NOTE - ‘ Pure chemicals ’ h-J mean chemicals that do not contain impurities

which affect the results of analysis.

3. GENERAL CHARACTERISTICS

3.1 Staphylococcus aweus - Aerobic, Gram-positive cocci in clusters,

. usually, but not always producing a golden yellow coloured colonies on nutrient agar ( 4.2 ) and blood agar ( 4.3), and shiny black colonies with or without narrow grey-white margin when grown on Baird-Parker medium ( 4.5 >s Suspect colonies must show coagulase activity.

3.2 paecal Streptococci - Aerobic, Gram-positive cocci usually in pairs or short chains, producing small pink colonies on MacConkey agar ( 4.7 ) and colonies which are dark red or having red or pink centres when grown on ethyl violet azide dextrose agar ( 4.6). Growth is obtained also at 44°C.

4. MEDIA 4.1 Nutrient Broth - Mix and dissolve by heating 10 g peptone (St-e IS : 6853-1973$), 10 g meat extract ( see IS : 6851-197311 ), and 5 g sodium chloride in 1000 ml water. When cool, adjust pH to 7.5 to 7.6. Remove precipitate by filtration through filter paper. Sterilize by autoclaving at 120°C for 15 minutes.

*Rules for rounding Off numerical values ( revised ). TCode of practice for handling of food samples for microbiological analysis. :Specification for water, distilled quality ( revised ). @pecification for peptone, microbiological grade. l/Specification for meat extract, microbiological grade.

4

IS : 5887 ( Part II ) - 1976

4.2 Nutrient Agar - To the medium as in 4.1, add agar ( see IS:6850- 1973* ) in such a concentration as will solidify and produce a sufficiently firm surface when poured in sterile petri dishes. The concentration of agar to be added varies from batch to batch and should be adjusted accordingly. Usual concentrations required vary from 1.5 to 3 percent. Dissolve the agar in the nutrient broth and sterilize by autoclaving at 120°C for 15 minutes. Plates and slopes are prepared from sterile nutrient agar.

4.3 Blood Agar - Melt sterile nutrient agar as prepared in 4.2 and hold between 50 to 55°C in a water-bath. Add sterile blood free from preser- vatives to give a concentration of 10 percent. Mix well and pour plates. Horse blood is commonly used but when not available, that of sheep, human or rabbit may be used.

4.4 Salt Medium

4.4.1 Cooked Meat Medium - Mince 500 g fresh beef heart and place it in 500 ml of alkaline boiling water containing 1.5 ml of I N sodium hydroxide solution. Simmer for 20 minutes. Drain off the liquid through muslin filter while still hot and partially dry the meat in the cloth or on filter papers. To 500 ml of liquid filtered from the cooked meat add 2.5 g, peptone (see IS : 6853-1973t) and 1.25 g sodium chloride. Steam at 100°C for 20 minutes and add 1 ml concentrated hydrochloric acid and filter. Bring the reaction of the filtrate topH 8.2 and steam again at 100°C for 30 minutes; adjust PH to 7.8. Place meat in test-tubes or in about 30 ml screw-capped bottles to a depth of about 2’5 cm and cover with 10 ml of the broth obtained. Autoclave at 120°C for 20 minutes. A layer of sterile paraffin may be added to cover the surface.

4.4.1.1 For salt medium - Add further 10 percent sodium chloride to the broth ( 4.4.1)) adjust the PH to 7.8 and proceed as in 4.4.1.

4.5 Baird-Parker Medium - Dissolve by boiling in 950 ml of water, 10 g tryptone (see IS : 7127-1973$), 5 g meat extract (see IS : 68.51-1973§), 1 g yeast extract (see IS: 7004-1973/l), 12g sodium pyruvate, 12 g glycine, 5 g lithium chloride and 15 to 30 g agar (see IS : 6850-1973* ). Distribute in 95 ml amounts into sterilized flasks (or screw-capped bottles) and sterilize at 120°C for 15 minutes. The final PH should be 6.8 to 72.

*+,&fication for agar, micrqbiological grade. tSp&fication for pcptone, microbiological grade. *Specification for tryptone, mlcro~ological.grade. §Specification for meat extract, mlcrobiologlcal grade. llSpecification for yeast extract, microbiological grade.

5

IS : 5887 ( Part II ) - 1976

To 95 ml of the molten medium (4.5) kept at 45” to 50°C, add 5 ml of Bacto-E2’ tellurite enrichment which has been prewarmed to 45” to 50%. Mix well and pour on to sterilized petri dishes. The plates should be made freshly before use and should not be stored longer than 48 hours before use. The plates, before use, should be well-dried by keeping in an incubator at WC for about 30 minutes, with the lid removed and agar surface downward.

4.6 Ethyl Violet Azide Dextrose Broth - Dissolve in 1 000 ml water 20 g tryptose, 5 g dextrose, 2’7 g dipotassium phosphate, 2.7 g monopotassium phosphate, 5 g sodium chloride, 0.4 g sodium azide and O*OOO 83 g ethyl violet. Distribute into tubes. Sterilize at 120°C for 15 minutes. The final PH should be 7’0.

4,6.1 Ethyl Violet Azide Dextrose Agar-To medium as in 4.6, add 15 to 30 g agar (see IS : 6850-1973* ), and dissolve by heating. Sterilize at 120°C for 15 minutes, and pour on to sterilized petri dishes.

4.7 MacConkey Agar Medium - Mix 5 g sodium taurocholate or bile salts (see IS : 6852-19737 ), 20 g peptone (see IS : 6853-1973: ), 5 g sodium chloride and 15 to 30 g agar (see IS : 6850-1973” ), with 1000 ml water. Steam until the solids are dissolved. Cool to about 5o”C, and at this temperature adjust reaction to pH 7.6 to 7.8. Auto- clave at 120°C for 15 minutes and filter while hot through a good grade of filter paper, or a plug of cotton wrapped in gauze and placed in the funnel. Adjust reaction of the filtrate to PH 7.3 at 50°C or PH 7.5 at room temperature. Add 100 ml of 10 percent aqueous solution of lactose (or 10 g lactose ) and 3.5 ml of 2 percent solution of neutral red in 50 percent ethanol. Mix thoroughly, distribute into flasks and sterilize in the autoclave at 120°C for 15 minutes. For use, melt in the steamer, pour into sterile petri dishes and allow to set.

5. PROCEDURE FOR ISOLATION

5.1 Staphylococcus aureus - Inoculate sample on blood agar (4.3 ) and in salt medium ( 4.4 ) and on Baird-Parker medium ( 4.5 ), if available.

5.1.1 Incubate the blood agar and salt medium at 37°C overnight and the inoculum in Baird-Parker medium at 37°C for at least 30 hours. From the salt medium, make subcultures on the one or both solid media mentioned for overnight incubation at 37°C in case of blood agar, and for 30 hours in case of Baird-Parker medium.

*Specification for agar, microbiological grade. tspecification for bile salts, microbiological grade. *Specification for peptone, microbiological grade.

6

IS : 5887 ( Part II ) - 1976

5.1.2 Examine not less than 5 of the suspect colonies of Staphylococcus on the solid media and mark out as many suspect colonies as possible to investigate. A portion of each colony picked with a straight nichrome wire may be used for preliminary coagulase test using the slide technique. The remainder of the colony is streaked out on blood agar medium for incubation at 37°C to check purity and to proceed with identification. If preliminary slide testing is avoided, the suspect colony is checked for purity as just described. Pure colonies are stained by Gram’s method and tested for coagulase by the slide and tube methods.

5.2 Faecal Streptococci - To estimate the number of faecal strepto- cocci in the sample, the procedures given in 8.2 shall be followed.

6. TESTS FOR IDENTIFICATION

6.1 Staphylococcus aureus

6.1.1 Gram’s Stain -The stain consists of: (a) 0.5 percent methyl violet or crystal violet in water; (b) iodine solution ( 1 percent iodine and 2 percent potassium iodide in water ); and (c) counterstain ( 0.1 g neutral red, O-2 ml of 1 percent acetic acid and 100 ml water ).

On a clean grease-free slide, very light and thin smear covering a small area is made directly from liquid culture and in clean tap water if from solid media. and cooled.

The smear is fixed by passing to and fro over a flame

the stain Cover the smear with the stain (a) for 30 seconds, pour off

and wash with (b) and then cover with (b) and allow to remain for 30 seconds. Wash off with ethanol until the dye ceases to stream out. Wash in running tap water and apply (c) for about one minute. Wash in tap water and dry for examination.

6.1.2 Colonial Character -By growth on nutrient agar ( 4.2 ), blood agar (4.3) and/or on Baird-Parker medium ( 4.5 ), as described in 3.1.

6.1.3 Coagulase Test - methods.

This test may be carried out by the following The tube method shall be preferred:

a)

b)

Slide method - Emulsify a portion of the suspect colony in normal saline or water. Mix this with a straight wire dipped in human or rabbit plasma. Coagulase-positive staphylococci produce visible clumping immediately.

Tube method - Emulsify a single suspect colony from a 24 hour growth of blood agar medium ( 4.3 ) in 1 ml titrated rabbit plasma diluted 1 in 5 in 0.85 percent saline. The test is usually carried out in narrow tubes. Place in an incubator or

7

IS : 5887 ( Part II ) - 1976

preferably in water-bath at 37°C. Observe every hour to note clotting of plasma. Reading should be carried out for as long as possible, preferably avoiding overnight incubation. Positive control with a known coagulase-positive strain of Staphylococcus and a control of the diluted plasma without inoculum should be included in the test.

NOTE 1 - If the slide method gives a negative result, the tube method shall be carried out.

NOTE 2 -False positive results may occur on the slide test. A small number of strains give a positive slide test with a negative tube test due to the production of ‘ bound ’ coagulase alone.

6.2 Faecal Streptococci

6.2.1 Gram’s Stain - See 6.1.1.

6.2.2 Colonial Character - By growth on MacConkey agar ( 4.7 ) at 44°C and ethyl violet azide dextrose agar ( 4.6.1 ) at 37”C, as described in 3.2.

7. PHAGE TYPING

7.1 A single colony of coagulase-positive strain of Sta@hylococcus tested by the tube method may be maintained on nutrient agar ( see 4.2 ) slopes, and sent for phage typing.

NOTE - Presently phage typing facilities are available at the Stophylocnccur Phage TypinP; Centre, Department of Microbiology, Maulana Azad Medical College, Bahadur Shah Zafar Marg, New Delhi 110002.

8. ENUMERATION

8.1 Staphylococcus aureus - Since the presence of small numbers of coagulase-positive staphylococci does not necessarily indicate association of the isolate with food poisoning, estimation of approximate number of organisms per gram of suspect food form more valid determination if the suspect strains are to indicate food poisoning. While a rough estimate may be made on direct smear of the material stained by Gram’s method ( 6.I.I), quantitative estimation shall always be made by the procedure given in 8.1.1.

8.1.1 Twentyfive to fifty grams of the sample is taken in a sterile i blender jar and to this is added diluting fluid to have dilution of 10-r. The diluting fluid shall be peptone (8.~ IS : 6853-1973* ) 0.1 percent in water sterilized at 120°C for 20 minutes, final PH 6.8 f O,l or 3.4 percent potassium dihydrogen phosphate ( KHeI’O,) in water, pH adjusted to 7-2 and sterilized at 120°C for 20 minutes. Blend at 8000 to 10000 rev/min for 2 minutes. Alternatively, macerate the sample with diluting

*Specification for peptone, microbiological grade.

8

IS : 5887 ( Part II ) - 1976

fluid in a sterile mortar with sterile sand. Make serial ten-fold dilutions with the diluting fluid in duplicate series up to 10m6. Streak 0.1 ml from each tube evenly on to blood agar ( 4.3 ) and/or Baird-Parker medium ( 4.5 ). Incubate the blood agar plates at 37°C overnight and the Baird-Parker plates at 37°C for at least 30 hours. Enumerate the colonies which are as described in 3.1. These colonies are to be confirmed as being S. aureus by the coagulase test described in 6.1.3. The number of viable colonies per gram of sample is determined by multiplying the dilution factor( s ) and dividing by the mass of the sample.

8.2 Faecal Streptococci

8.2.1 Plate Count-Take 25 to 50 g of the sample in a blender jar and add diluting fluid (8.1.1 ) to have dilution of 10-l. Blend at 8 000 to 10 000 rev/min for 2 minutes. Alternatively, macerate with diluting fluid ( 8.1.1 ) in a sterile mortar with sterile sand. Make serial ten-fold dilutions with the diluting fluid in duplicate series up to 10-s. Streak 0.1 ml from each tube evenly on to the ethyl violet azide dextrose agar ‘( 4.6.1) and incubate at 37% for 48 hours. Enumerate the colonies which are as described in 3.2 and confirm these by Gram’s stain (6.2.1) and by growth at 44°C in MacConkey agar ( 4.7 ) for typical small pink colonies. The number of viable colonies per gram of sample is determined by multiplying by the dilution factor(s) and dividing by the mass of the sample.

8.2.2 Enterococci Index- Obtain serial dilutions of the sample as in 8.2.1. Transfer, with a fresh sterile pipette, a measured volume of 1 ml of the homogenized mixture and of the five following serial dilutions of both dilution series in triplicate to the tubes of 10 ml of ethyl violet azide dextrose broth ( 4.6 ). Start with the highest dilution and proceed to the, lowest, filling and emptying the pipette three times before transferring the 1 ml portions to the tubes of medium ( see 4.6 ). When the number of enterococci is assumed to be very small, start by transferring, using a sterile 10 ml pipette, 10 ml of the homogenized mixture in triplicate to 10 ml of double strength broth which contains twice the amounts of the ingredient as in 4.6 in 1000 ml of water. Incubate at 37°C for 48 hours. Record as positive the tubes which have t developed turbidity ( growth ) and the growth having been confirmed as being faecal streptococci as described in 6.2. Using Table 1, obtain the most probable number ( MPN) of faecal streptococci per gram of the sample. Use for the calculation the results from three dilutions, select- ing the highest dilution showing three positive tubes below which no sets with a smaller number of positive tubes occur, and the two following higher dilutions. The number obtained from Table 1 has to be multiplied by the lowest dilution factor, that is, that of the first set of tubes, to obtain the most probable number of faecal streptococci per gram of the sample. For example, when dilution 100 ( = 10 ml of

9

IS: 5887 (Part II)-1976

macerate ), 10-I and lo-* are found to give the following numbers of positive tubes: 2, 2, 1, the MPN is 2’8 bacteria per gram, and when the dilutions 100, 10-l, 10m2, lo-*, lo-‘ and 10-s are found to give the following numbers of positive tubes: 3, 3, 3, 2, 0, 0, the MPN is 9.3 ( 3, 2, 0 ), multiplied by the dilution factor lo*, that is, 9.3 x IO2 bacteria per gram. The MPN is reported as the average of the results obtained from each of the duplicate dilution series.

TABLE1

NUMBER OF POSITIVE TUBES PER DILUTION

r---__*-_-_‘~ ioa

(1)

0 0 0 0 0 0 0 0 0

x 0

0

0

0

0

1

1

1 1 1

10-l

(2)

0 0 0 0 1 1 1 1

2

2' 2 3 3 3 3 0 0 0 0 1

lo-”

(3)

0 1 2 3 0 1 2 3 0

,: 3 0 1 2 3 0 1 2 3 0

MOST PROBABLE NUMBER(MPN) OF FAECAL STREPTOCOCCI

( Claure 8.2.2 )

MPN

(4)

0.30 0.30 0’60 0’90 0’30 0’61 0’92

1’2 0’62 0’93 1’2 1’6 0’94 1’3 1’6 1’9 0’36 0’72 1’1 1’5 0’73

NUMBER OF POSITIVE TUBES PER DILVMON

-___A _____ _ .10*

(1)

1 1 1 1 1 1 1 1 1

:

2 2 2 2 2 2 2

2 2

2’

IO-’

(2)

1 1 1 2 2 2 2 3 3

3” 0 0 0 0 1 1 1 1 2

;

IO-"

(3)

1

2 3 0 1 2 3 0 1

3’ 0 1 2 3 0 1 2 3 0 1 2

MPN

(4)

1’1 1.5 1.9

. 1’1 1.5 2’0 2’4 1’6 2’0

;:;

0’91 1’4

2’0 2’6 1’5 2.0 2’7 3’4 2-l 2-8 3’5

NUMBER OF MPN POSITIVE TUBES PER DILUTION

‘--“_A--__~ .lO”

(1)

2 2 2 2 2 3 3 3 3

3” 3 3 3 3 3 3 3 3 3 3

10-l

(2)

2 3 3 3 3 0 0 0 0 1 1 1 1

2 2 2 2 3 3 3 3

10-1

(3)

3 0 1

2 3 0 1 2 3 0 1 2 3 0 1 2 3 0 1 2 3

(4)

4.2 2’9 3’6 44 5.3 2’3 3’9 6’4 9’5 4’3 7’5

12’0 16.0 9’3

15’0 21’0 29’0 ” 240

46’0 110’0 i

110’0

P

IS t 5887 ( Part II ) - 1976

( Continued from /page 2 )

Members DR A. N. BOSE DR SUBRATA CHAKRAVORTY

DIRECTOR DR A. K. GHOSR

Representing The Bengal Immunity Co Ltd, Calcutta Bengal Chemical and Pharmaceutical Works Ltd,

Calcutta King Institute, Madras Cholera Research Centre ( Indian Council of Medical

Research ), Calcutta HEAD, DIVISION OF BIOLOQICAL Indian Veterinary Research Institute ( ICAR ).

PRODUCTS Izatnagar . ,-

DR A. P. JOSHI Vallabhbhai Pate1 Chest Institute, Delhi DR ( SXT ) V. BAJAJ ( Alternate)

DR M. A. KRISRNASWAMY Central Food Technological Research Institute ( CSIR ), Mysore

SRRI C. T. DWAREANATH ( Alternate ) SHRI K. R. NARASIMHAN The Metal Box Company of India Ltd, Calcutta

DR S. C. CHAKRAVORTY ( Alternate) DR A. N. RAI CHOWDHURY Central Research Institute, Kasauli DR B. RANUANATRAN National Dairy Research Institute ( ICAR ), Karnal DR M. V. SANT Haffkine Institute, Bombay DR SHRINIWAS All India Institute of Medical Sciences, New Delhi DR N. S. SUBBA RAO Indian Aericultural Research Institute ( ICAR 1.

New Gelhi \ ,.

COL R. N. TANEJA Food Inspection Organization, Quartermaster

LT-COL D. D. VOHRA ( Alternate ) General’s Branch, Army Headquarters

11

INDIAN STANDARDS

ON

FOOD MICROBIOLOGY

IS:

5401-1969 Methods for detection and estimation of coliform bacteria in foodstuffs

5402-1969 Method for standard plate co&t of bacteria in foodstuffs

5403-1969 Method for yeast and mould count of foodstuffs

5404-1969 Code of practice for handling of samples for microbiological analysis

5887 ( Part I )-1976 Methods for detection of bacteria responsible for food poisoning: Part I Isolation, identification and enumeration of Escherichia coli (first revision )

5887 ( Part II)-1976 Methods for detection of bacteria responsible for food poisoning: Part II Isolation, identification and enumeration of Staphylococcus oweus and Faeeal streptococci (jut revision )

5887 ( Part III )-1976 Methods for detection of bacteria responsible for food poisoning: Part III Isolation and identification of Salmonella and Shigella ( jirst revision )

5887 ( Part IV )-1976 Methods for detection of bacteria responsible for food poisoning: Part IV Isolation and identification of Clostridium welchii, Clostridium botulinum and bacillus cereus and enumeration of Clostridium welchii and Bacillus cereus (first revision )

5887 ( Part V )-I976 Methods for detection of bacteria responsible for food poisoning: Part V Isolation, identification and enumeration of Vibrio cholerae and Vibrio parahaemolyticus (Jirst revision )

f&F,l)_1973 Agar, microbiological grade

6851-1973 Meat extract, microbiological grade

6852-1973 Bile salts, microbiological grade

6853-1973 Peptone, microbiological grade

6854-1973 Methods of sampling and test for ingredients used in media for microbio- logical work

7004-1973 Yeast extract, microbiological grade

7127-1973 Tryptone, microbiological grade

7128-1973 Proteose peptone, microbiological grade

7203-1973 Casein hydrolysate ( acid digested ), microbiological grade

7535-1975 Liver extract, microbiological grade

7536-1975 Soluble starch, microbiological grade

7590-1975 Gelatin, microbiological grade

7591-1975 Malt extract, microbiological grade

7801-1975 Trypsine, microbiological grade