Disclosure To Promote The Right To Information
Disclosure To Promote The Right To Information
Disclosure To Promote The Right To Information
( First Reoision )
UDC 613.2-099:664:576.851.31.078
6J Copyright 1977
Indian Standard
METHODS FOR
DETECTION OF BACTERIA RESPONSIBLE
FOR FOOD POISONIN
PART V ISOLATION, IDENTIFICATIQN AND
ENUMERATION OF VIBRIO CHOLERAE
AND VIBRIO PARAHAEMOLYTICUS
( First Reuision )
Food Hygiene, Sampling and Analysis Sectional Committee, AFDC 36
Chairman Representing
DR RANJIT SEN Serologist to the Government of India ( DGHS ),
Calcutta
Members
AGRICULTURAL MARKETING AD- Directorate of Marketing & Inspection .( Ministry of
VISOR TO THE GOVERNMENTOF Agriculture & Irrigation ), Faridabad
INDIA
SHRI T. V. MATHEW ( Alternate )
SHRI V. N. AMBLE Institute of Agricultural Research Statistics (ICAR),
New Delhi
SHRI K. S. KRI~HNAN( Alternate )
DR G. C. DAS Health Officer, Corporation of Calcutta
DR P. K. DA~TA All India Institute of Hygiene and Public Health,
Calcutta
SHRI SUKUMARDE National Dairy Research Institute ( ICAR ), Karnal
DR C. A. MULAY ( Alternate )
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. K. KRI~HNASWAMY( Alternate )
EXECUTIVEHEALTH OFFICER Municipal Corporation of Greater Bombay
MUNICIPAL ANALYST ( Alternate )
HEALTHOFFICER Corporation of Madras
COL KEWAL KRISHNA Health Department, Municipal Corporation of Delhi
DR A. D. KUMAR ( Alternate )
( Continued on page 2 )
@I Copyright 1977
BUREAU OF INDIAN STANDARDS
This publication is protected under the Zndiun 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 V ) - 1976
( Continuedfiom page 1 )
Members Representing
DR ( SMT) S. KHOSLA Department of Health & Family Planning, Govern-
ment of Punjab, Chandigarh
DR P. K. KYMAL Food & Nutrition Board ( Ministry of Agriculture &
Irrigation), New Delhi
DR 0. N. AGARWALA ( Alternate )
MAJ V. A. NARAYANAN Defence Food Research Laboratory ( Ministry of
Defence ), Mysore
DR G. M. VERMA ( Alternate )
PUBLIC ANALY~ ( FOOD AND Government of West Bengal, Calcutta
WATER )
PUBLIC ANALYST ( BACTERIO-
LOGY ) ( Alternate )
DR A. .N. RAI CHAWDHURI National Institute of Communicable Diseases, Delhi
MAI-GEN D. C. SACHDEVA Directorate General of Armed Forces Medical
Services ( Ministry of Defence ), New Delhi _
SENIOR MEDICAL 0 F F I c E R Northern Railway, New Delhi
( HEALTH )
DR S. B. SINGH Public Analyst, Government of Uttar Pradesh,
Lucknow
SHRI N. SRINIVASAN Public Analyst, Government of Tamil Nadq, Madras
DR M. R. SUBBARAM Directorate of Sugar & Vanaspati ( Mmistry of
Agriculture & Irrigation )
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
Health & Family Planning ), New Delhi
SHRI D. S. CHADHA ( AIternate )
COL R. N. TANEJA Quartermaster General’s Branch, Army Head-
quarters
LT-COL D. D. VOHRA ( Alternate )
SHRI P. C. VIN The Coca-Cola Export Corporation, New Delhi
SHRI J. D. CONTRACTOR( Alternate )
SHRI T. PURNANANDAM, Director General, ISI ( Ex-officio Member )
Deputy Director ( Agri & Food )
Secretary
SHRIs. K. SUD
Deputy Director ( Agri & Food ), ISI
Convener
DR RANJIT SEN Serologist to the Government of India ( DGHS ),
Calcutta t
i
Members
AGRICULTURAL MARKETING AD- Directorate of Marketing & Inspection ( Ministry of
VISER TO THE GOVERNMENTOF Agriculture & Irrigation ), Faridabad
INDIA
DIRECTOR OF LABORATORIES
( Alternate )
( Continued on page 15 )
2
IS : 5887 ( Part V ) - 1976
Indiun Standard
METHODS FOR
DETECTION OF BACTERIA RESPONSIBLE
FOR FOOD POISONING
PART V ISOLATION, IDENTIFICATION AND
ENUMERATION OF VIBRIO CHOLERAE
AND VIBRIO PARAHAEMOL YTICUS
( First Revision )
0. FOREWORD
0.1 This Indian Standard ( Part V ) ( 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.
3
IS : 5887 ( Part V ) - 1976
review and revision of the parts easier. The salient features of this
revision are:
a) besides detection, estimation procedure for various organisms,
where applicable, have been incorporated;
b) method of detection of Vibrio parahaemolyticus has been included;
and
c) 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:2-1960*.
1. SCOPE
1.1 This standard ( Part V ) prescribes methods for isolation and identifica-
_-
tion of Vibrio cholerae and Vibrio parahaemolyticus, and their estimation in
foods, if required to be carried out.
4
IS : 5887 ( Part V ) - 1976
: .
t
IS:5887(PartV)-1976
4. MEDIUM
4.1 Alkaline Peptone Water - Dissolve 30 g peptooe ( see IS:6853-1973* )
and 5 g sodium chloride in water and make up to 1000 ml. Adjust pH to
8.2 and distribute into sterilized test tubes, and sterilize at 120°C for 15
minutes.
4.2 Thiosulphate-Citrate-Bile Salts-Sucrose Agar ( TCBS ) - Dissolve by
gentle heating 5 g yeast extract ( see IS: 7004-1973t ), 10 g peptone
(see IS: 6853- 1973* ), 20 g sucrose, 10 g sodium thiosulphate
(NaS,O,,SH,O), 10 g sodium citrate (Na,C,H,,2H,O), 3 g sodium
cholate, 5 g oxgall ( dehydrated bile ), 10 g sodium chloride, 1 g ferric
citrate, 0.04 g bromothymol blue, 0.04 g thymol blue and 15 to. 30 g
agar ( see IS: 6850-1973: ) in 1000 ~1 of water. Boil for 1 to 2 mmutes
only. This medium shall not be further sterilized by autoclaving. The
final pH of the medium shall be 8.6. Melt and pour into sterile petri
dishes and allow’ to cool.
NOTE I - Sodium cholate can be omitted when 8 g of oxgall is to be used.
6
IS:5887(PartV)-1976
4.4.1 Nutrient Agar for the Motility Test - The medium consists of 3 g
meat extract ( see IS: 6851-1973* ), IO g peptone ( see IS:6853-19737 ), 5 g
sodium chloride, and 4 to 5 g agar ( see aIS:6850-1973$ ) dissolved in
1000 ml of water. Adjust pH to 7.5 to 7.6. Put the dissolved medium-into
test tubes to fill a part of the tube and place into this a glass tube open
at both ends. One end of the glass tube shall project from above the
surface of the agar for not less than 1.5 cm. Sterilize the tubes with
the medium at 120°C for 15 minutes, and then cool. The consistency of
the agar should be soft but not liquid. This is achieved by altering the
amount of agar used, if necessary.
4.5 Medium for Hugh-Leifson’s Test - Dissolve by heating in 1000 ml
water, 2 g peptone ( see IS :6853-19737 ), 5 g sodium chloride, 0.3 g
dipotassium hydrogen phosphate, and 3 g agar (see 18:6850-1973: ).
Adjust to pH 7.1, filter and add 15 ml 0’2 percent alcoholic solution
of bromothymol blue. Sterilize at 115°C for 20 minutes. Add sterile
solution of glucose to give a final concentration of 1 percent. Mix
and distribute in tubes, 14x 100 mm, adding 3 to.4 ml per tube.
NOTE- In carrying out the test with V. parahaemolyticus. the medium as in
4.5 should contain an additional 2 to 3 percent sodium ‘chloride.
4.7 TSI Medium for H,S Test - Heat to dissolve in 1000 ml water, 3 g B
meat extract ( see IS :6851-1973* ), 3 g yeast extract ( IS :7004-19734 ), 20 g
peptone ( see IS:6853-19737 ), 1 g glucose, 10 g lactose, 10 g sucrose, 0.2 g
ferrous sulphate ( FeS0,,7H,O ), 5 g sodium chloride, 0.3 g sodium
thiosulphate ( Na,Sz0,,5H,0) and 15 to 30 g agar. Add 12 ml of
*Specification for meat extract, microbiqlogical grade.
tSpecification for peptone, microbiological grade.
SSpecification for agar, microbiological grade.
&Specification for yeast extract, microbiological grade.
7
IS : 5887 ( Part V) - 1976
O-2 percent phenol red solution, mix and tube. Sterilize by autoclaving
at 115°C for 20 minutes. Pour into sterile test tubes and cool to form a
slope with deep butts.
NOTE- For carrying the test with Y. por&zemo/yricus, the medium as in 4.7
shall contain an additional 2 to 3 percent sodium chloride.
4.8 Tryptone Broth Medium - Tryptone broth medium, without added
‘sodium chloride, is prepared by dissolving 10 g tryptone ( see 13:7127-
1973* ) in 1000 ml water. Sodium chloride in requisite amount [see 3.2 (.m)
to (p) ] is to be added where indicated, for example, for Y. parahaemoly-
ticus. The medium is dispensed in 5 ml amounts into sterilized tubes and
autoclaved at 120°C for 15 minutes. t
4.9 Medium for Voges-Proskauer Reaction - Steam to dissolve in 1000 ml
water, 5 g peptone ( see IS:6853-1973t ) and 5 g dipotassium hydrogen
phosphate. Filter and adjust the pH to 7’5. Add 5 g glucose, mix
to dissolve; distribute into tubes. Sterilize at 115°C for 10 minutes.
NOTE - For carrying out the test with V. parahaemolyticus, the medium as
in 4.9 shall contain an additional 2 to 3 percent sodium chloride.
4.10 Medium for Dihydrolase and Decarboxylase Activity - Dissolve
with heat, 5 g peptone (see IS:6853-19737) and 5 g meat extract
(see IS:6851-1973: ), 5 mg pyridoxal, 0.5 g glucose in 1000 ml water.
Adjust the pH to 6’0 and add 5 ml of 0’2 percent solution of bromocresol
purple and 2.5 ml of 0’2 percent solution of cresol red. Sterilize in an
autoclave at 115°C for 20 minutes. Divide this base medium into four
equal volumes, and to each of three add the respective amino acid
( arginine, lysine and ornithine ) as a final concentration of 1 percent
if the i-form of the amino acid is used or as 2 percent if the dl-form of
the amino acid is used. The fourth aliquot of the base medium does
not contain amino acid and serves as control. Distribute the four
media in I- to 1’5-ml amounts into small sterilized tubes, 67x 10 mm,
and layer with sterile liquid paraffin to a height of about 5 mm. The
pH of the final medium is to be readjusted, if necessary, to 6’0. Sterilize
in an autoclave at 115°C for 10 minutes.
4.10.1 An alternative medium where the base medium contains 5 g
peptone ( see IS:6853-1973t ), 3 g yeast extract ( see IS : 7004-1973$ ), 1 g
glucose and 10 ml of 0’2 percent bromocresol purple in 1000 ml of
water may be used. Adjust the yH to 6.7. The remaining procedure
for preparation of the medium is as in 4.10.
NOTE - For carrying out the tests with V. poruhaemolyticus, the media as in 4.10
and 4.10.X shall contain an additional 2 to 3 percent sodium chloride.
__ ~~
*Specification for tryptone, microbiological grade.
TSpecification for pc!ptOiIe,microbio~ogicd~ grade.
SSpecification for meat extract, microbtological grade.
SSpecification for yeast extract, microbiological grade.
8
IS : 5887 ( Part V ) - 1976
9
IS : 5887 (Part V ) - 1976
not made on to the surface of the medium outside the g&s tube.
Incubate at 37°C for 18 to. 24 hours. Motile strains shall be found
to show growth on the surface of the medium outside the ‘inner glass tube’
having travelled through the entire medium inside this inner tube. If
negative on the first day, keep the inoculated tube at room temperature
for a further 4 to 6 days to see if evidence of motility is present.
6.3 Test for Catalase - Grow strain for 18 to 24 hours at 37°C on
nutrient agar ( 4.4 ) slope and pour 1 ml of hydrogen -peroxide over
the growth with the tube in a slanting position. ‘Release of oxygen,
shown as bubbles, from hydrogen peroxide indicates presence of catalase.
6.4 Test for Oxidase -To nutrient agar ( 4.4 ) slope of the culture,
freshly grown, is added a few drops of freshly mixed test reagents, that is,
1 percent solution of alpha-naphthol in 95 percent ethanol and equal
amount of 1 percent solution of p-aminnodimethylaniline hydrochloride
in water. A positive reaction is indicated by the appearance of a blue
colour within two minutes.
NOTE- For carrying out the test with V. parahaemolyticus~ nutrient agar as
in 4.4 shall contain an additional 2 to 3 percent sodium chloride.
6.5 Hugh-Leifson’s Test -The strain from fresh nutrient agar ( 4.4 )
growth is stabbed into two tubes of medium. ( 4.5 ), one of which .is
then layered over with a small amount of sterile liquid paraffin. Incubate
both tubes at 37°C and observe up to 4 days. Acid formation, shown by
yellow colour, in the tube without paraffin indicates oxidative utilization
of glucose. Acid in both tubes indicates fermentative reaction. Lack
of acid in either tube indicates the strain as not being able to utilize
glucose oxidatively or fermentatively.
NOTE- For V. ptirahaemolyticus, the medium given in 4.5 should contain
additional sodium chloride.
6.6 Tests for Fermentation of Carbohydrates
6.6.1 V. cholerae -Inoculate each of peptone water medium (4.6 )
containing respectively, mannitol, inositol and glucose and incubate
at 37°C for 18 to 48 hours.
6.6.2 V. parahaemolyticus - Inoculate each of peptone water medium
with added sodium chloricje containing respectively, mannitol and !,
sucrose. Incubate at 37°C for 4 to 5 days. i(
r,
6.7 Test for H.,S Prodaction -Inoculate TSI medium ( 4.7) by
stabbing the strain into the butt and streaking the slope. Incubate
at 37°C and observe daily for up to 7 days. The presence or absence
of blackening in the butt of the medium is to be recorded.
NOTE- For V. parahaetilyticus. the medium given in 4.7 should contain
additional sodium chloride.
11
IS:5887(PartV)-1976
8. ENUMERATION
8.1 Pre-enrichment and Blending - Take 50 g of the sample in a sterile
blender jar and to this add 450 ml of a sterile solution containing @1
percent peptone ( see IS:6853-1973* ) and 0.8 percent sodium chloride at
pH 7.8 to 8-O for enumerating V. cholerue. For V. parahaemolyticus,
450 ml of sterile 3 percent sodium chloride solution only is added. Keep
for about 15 minutes at room temperature. Blend at 8000 to 10000
rev/min for 2 minutes. If the temperature of the contents of the mixture
increases by more than 5”C, the procedure of blending is to be carried
out by keeping the container in an ice-bath. After blending, allow the
solid particles, if any, to settle down. If a blender is not available,
macerate in a sterile mortar with sterile sand.
8.2 Dilutions-The procedure as in 8.1 provides a dilution of 10-l and
is then serially diluted ten-fold with the same solutions as in 8.1, for
V. cholerae and V. parahaemolyticus.
8.3 Enumeration
8.3.1 V. cholerue - Ten-fold dilutions of the sample up to 10s4 as
in 8.2, are taken and from each dilution and 10 ml aliquots are inoculated
into 10 ml of medium consisting of 2 percent peptone ( see IS :6853-1973* )
and 2 percent sodium chloride at pH 7.8 to 8*0. Incubate at 37°C for
18 hours, and streak a measured loopful (3 mm) from each tube on to
bile salt agar medium as in 4.3. Incubate at 37°C for 18 hours.
Count the colonies of V. cholerae, whose characters are as described
in 5.1. Confirm the suspect cplonies as being V. cholerae by the
characters as in 3.1 and by the tests which have been described in 6.
Calculate the number of viable colonies of V. cholerae per gram of
sample by multiplying by the dilution factor(s) and dividing by the
mass of the sample.
*Specification for peptooe, microbiological grades.
13
IS : 5887 ( Part V ) - 1976
Members Representing
MAI G. S. BALI Defence Food Research Laboratory ( Ministry of
Defence ), Mysore
SHRI K. C. RAPON ( Alternafe )
DR A. N. BOSE The Bengal Immunity Co Ltd, Calcutta
DR SUBRATACHAKRAVORTY Bengal Chemical and Pharmaceutical Works Ltd,
Calcutta
DIRECTOR King Institute, Madras
DR A. K. GHO~H Cholera Research Centre ( Indian Council of
Medical Research ), Calcutta
HEAD, DIVISION 01: BIOLOGICAL Indian Veterinary Research Institute ( ICAR ),
PRODUCTS Izatnagar
DR.A. P. JOSHI Vallabhbhai Pate1 Chest Institute, Delhi
DR (SM.T) V. BAJAJ ( Ahernafe )
DR M. A. KRISHNASWAMY Central Food Technological Research Institute
( CSIR ), Mysore
SHR~C. T. DWARAKANATH( Alternate )
SHRI K. R. NARASIMHAN The Metal Box Company of India Ltd, Calcutta
DR S. C. CHAKRAVORTY ( AIternare )
DR A. N. RAI CHOWDHURY Central Research Institute, Kasauli
DR B. RANGANATHAN 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 Agricultural Research Institute ( ICAR 1,
New Delhi
COL.R. N. TANEJA Food Inspection Organization, Quartermaster
General’s Branch&my Headquarters
LT-COL D. D. VOHRA( Alternate )
15
F