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“The Right to Information, The Right to Live” “Step Out From the Old to the New”

IS 14987 (2001): Microbiology - General Guidance for the

Detection of Vibrio Parahaemolyticus [FAD 15: Food Hygiene,
Safety Management and Other Systems]

“!ान $ एक न' भारत का +नम-ण”

Satyanarayan Gangaram Pitroda
“Invent a New India Using Knowledge”

“!ान एक ऐसा खजाना > जो कभी च0राया नहB जा सकता ह”

है”
ह
Bhartṛhari—Nītiśatakam
“Knowledge is such a treasure which cannot be stolen”
 + rayWFll=J R?T
Indian Standard
MICROBIOLOGY — GENERAL GUIDANCE FOR THE
DETECTION OF Vibrio parahaemolyticus

Ics 07.100.30

I
.,

0 BIS 2001

BUREAU OF IN DI AN STAN DARDS

MANAK BHAVAN, 9 BAHADUR SHAH ZAFAR MARG
NEW DELHI 110002

Ju/y 2001 Price Group 6

 . ...4
Food Microbiology Sectional Committee, FAD 46

r
,’

*..J

NATIONAL FOREWORD

This Indian Standard which is identical with ISO 8914:1990 ‘Microbiology — General guidance for /
the detection of Vibrio parahaen?o/yticus’ issued by the International Organization for Standardization
(ISO) was adopted by the Bureau of Indian Standards on the recommendation of the Food
Microbiology Sectional Committee and approval of the Food and Agriculture Division Council.

In the adopted standard certain terminology and conventions are not identical to those used in Indian
Standards. Attention is particularly drawn to the following:

a) Wherever the words ‘International Standard’ appear referring to this standard, they should be
read as ‘Indian Standard’.

b) Comma (,) has been used as a decimal marker while in Indian Standards, the current practice
is to use a point (.) as the decimal marker.

In the preparation of this standard, due consideration has been given to the Prevention of Food
Adu/feration Act, 1954, and the Rules framed thereunder and the Standards of Weights and Measures
(Packaged Commodities) R.des, 1977. The standard is subject to the restrictions imposed under
these Rules, wherever applicable.

CROSS REFERENCES

In this adopted standard, the following International Standards are referred to. Read in their respective
places the following:

International Corresponding Degree of

Standard Indian Standard Equivalence

ISO 6887:1983 Microbiology — IS 10232:1982 Guidelines for the Equivalent

General guidance for the pre- preparation of dilutions for micro-
paration of dilutions for microbio- biological examination for food
logical examination

ISO 7218 : 1996 Microbiology of IS 5404:1984 Code of practice for Related

food and animal feeding stuffs — handling of samples for microbio-
General rules for microbiological logical analysis (first revision)
examination

For the purpose of deciding whether a particular requirement of this standard is complied with the final
value, observed or calculated, expressing the result of a test or analysis, shall be rounded off in
accordance with IS 2:1960 ‘Rules for rounding of numerical values (revised)’. The number of
significant places retained in the rounded off value, should be the same as that of the specified value
in this standard.
 IS 14987:2001
ISO 8914:1990

Indian Standard
MICROBIOLOGY — GENERAL GUIDANCE FOR THE
DETECTION OF Vibrio parahaemo/yticus

1 Scope are carried out in accordance with this International

Standard.
This International Standard gives general guidance
for the detection of Vibrio parahaemolyticus present 3.2 detection of Vibrio parahaemo/yticus: Determi-
in products intended for human consumption or ani- nation of the presence or absence of these micro-
mal feeding stuffs. organisms in a particular mass of product when
tests are carried out in accordance with this inter-
The incubation temperature used may be 35 “C or national Standard.
37 ‘C, this temperature forming the subject of
agreement between the parties concerned.
4 Principle

2 Normative references In general, the detection of Vibrio parahaemo/yticus

is carried out in three successive phases as follows.
The following standards contain provisions which,
through reference in this text, constitute provisions 4.1 Enrichment in selective media
of this International Standard. At the time of publi-
cation, the editions indicated were valid. All stan- Inoculation of the test portion into the following two
dards are subject to revision, and parties to selective enrichment media: .-
agreements based on this International Standard
are encouraged to investigate the possibility of ap- a) salt polymyxin B broth (SPB);
plying the most recent editions of the standards in-
dicated below. Members of IEC and ISO maintain b) alkaline saline peptone water or saline glucose
registers of currently valid International Standards. cullure medium with sodium dodecyl sulfate
(known previously as sodium Iauryl sulfate)
ISO 6887:1983, Microbiology – Genera} guidance for (GST).
the preparation of dilutions for microbiological ex-
amination, Incubation at 35 “C or 37 ‘C (as agreed) for 7 h to
8 h.l)
ISO 7218:1985, Microbiology — General guidance for
microbiological examinations.
4.2 Plating out and identification

From the cultures obtained in (4.1), inoculation of the

3 Definitions following two selective solid media:

For the purposes of this International Standard, the a) thiosulfate citrate bile sucrose agar (TCBS);
following definitions apply,
b) triphenyltetrazolium chloride soya tryptone agar
3.1 Vibrio parahaemolyticus: Halophilic micro- (TSAT),
organisms which form characteristic colonies on
solid selective media, and which display the Incubation at 35 ‘C or 37 “C, and then examination
biochemical characteristics described when tests after 18 h, 24 h or 48 h (depending on the medium)

1) It is recommended for deep-frozen products that two plating out operations as described in 4.2 be carried out, one after
incubation for 7 h to 8 h and the other after incubation for 18 h,

1
 .

IS 14987:2001
ISO 8914:1990

to check for the presence of colonies presumed to 5.4.2 Saline meat-yeast agar
be Vibrio parahaemolyticus by their visible charac-
teristics. The temperature forms the subject of See B.3.2.
agreement between the parties concerned and is
indicated in the test report. 5.4.3 Saiine medium for the detection of iysine
decarboxyiase

4.3 Confirmation
See B.3.3.
Testing of the presumptive Vibrio parahaemolyticus
coionies by confirmatory tests. 5.4.4 Medium and reagents for indoie detection

5.4.4.1 Saline tryptonehyptophane medium

5 Culture media and reagents
See B.3.4.1.

5.1 Generai
5.4.4,2 Kovacs reagent
For generai guidance, see ISO 7218.
See B.3.4.2.

5.2 Enrichment media 5.4.5 Reagent for oxidase detection

NOTE 1 Because of the large number of culture media

See B.3.5.
and reagents, it was considered preferable, for the ciarity
of the text, to give their composition and preparation in
annex B, which also includes details of dispensing, stor- 5.4.6 Reagent for fl-gaiactosidase detection
age, etc.
5.4,6.1 ONPG soiution
5.2.1 Sait poiymyxin B broth (SPB)
See B.3.6.I.
See B.1.l.
5,4.6.2 Buffer solution
5,2.2 Alkaline saline peptone water
See B.3.6.2.
See B.I.2.
5.4.6.3 Compiete reagent

5.2,3 Saline glucose cuiture medium with sodium

See B.3.6.3.
dodecyi suifate (GST)

See B.I.3. 5.5 Sodium chioride soiution

See clause B.4.

5.3 Piating-out media

5.6 Zinc powder

5,3.1 Thiosulfate citrate biie sucrose agar (TCBS)

See B.2. I. 6 Apparatus and giassware

NOTE 2 Disposable apparatus Is an acceptable alterna-

5.3.2 Trfphenyitetrszoiium chioride soya tryptone tive to reusable glassware if it has suitable specifications.
agar (TSAT) [11
Usuai microbiological laboratory apparatus and, in
See B.2,2. particular, the foliowing.

5.3.3 Saiine nutrient agar 6,1 Apparatus for dry sterilization (oven) or wet
sterilization (autoclave), see ISO 7218.
See B.2.3.

6.2 incubator, capable of operating at

5.4 identification media 35”C*1°C or37”C+l”C.

5,4.1 Tripie sugar saline iron agar (TSi saline agar) 6.3 Drying cabinet or oven, ventilated by con-
vection, capable of operating at between
See B.3.I. 37 °C+10Cand550C*10C.

2
 .,

IS 14987:2001
1s0 8914:1990

6.4 Water-bath, capabie of operating at 9.1.2 in generai, to prepare the first initiai suspen-
37 “c * 1 ‘c. sion, dispense a 25 g test portion in 225 ml of the
enrichment medium, sait polymyxin B broth (5.2.1).
if a test portion of 25 g is not available, use a smaller
6.5 Containers, especially test tubas of dimensions
amount and add the quantity of enrichment medium
16 mm x 180 mm and 9 mm x 180 mm, and.. fiasks
necessary to obtain a 10-1 dilution.
or boffles of appropriate capacity.

9.1.3 Prepare the second initiai suspension in a

6.6 Petrt dishes, made of glass or plastic, of 90 mm manner similar to that used in 9.1.2 but using the
to 100 mm diameter. second enrichment medium chosen, either the ai-
kaiine saline peptone water (5.2.2) or the GST broth
6.7 Total delivery pipettes (blow-out pipettes), with (5.2.3).
wide openings, having nominai capacities of 10 ml
and 1 ml, graduated in 0,1 ml divisions.
9.2 Enrichment

6.8 Rubber bulbs, or any other type of safety device Incubate the two initial suspensions in the incubator
suitabie for use with the pipettes (6.7). set at 35 ‘C or 37 ‘C (as agreed) for 7 h to 8 h. if the
dilution and incubation cannot be completed in one
working day, the diluted sample shall be stored
6.9 Loop, approximately 3 mm in diameter, and
overnight at between O ‘C and + 5 “C.
straight wire, both made of platinum-iridium or
nickel-chromium, andlor a glass rod.
NOTE 4 For deep-frozen products, it is recommended
that a second plating out be carried out after incubation
NOTE 3 A nickel-chromium loop or wire is not suitable
for 18 h.
for use in the oxidase test (see 9.4.3.1).

9.3 Plating out and identification

6.10 pH-meter, accurate to -10,1 pH unit at 25 ‘C.

9,3.1 After incubation, using a loop (6.9), inoculate

6.11 Microscope, with an immersion objective.
the culture obtained in the first enrichment medium
(9.1.2) on to the surface of a plate containing the first
selective plating out medium, TCBS (5.3.1), to ailow
7 Sampling
the development of clearly separated coionies.
Sampiing shall have been carried out in accordance
Proceed in the same way with the second selective
with the specific International Standard appropriate
plating out medium, TSAT (5.3.2).
to the product concerned.

If there is no specific International Standard, H is 9.3.2 Using the cuiture obtained in the second
recommended that agreement be reached on this enrichment medium (9.1.3), repeat the procedure
subject by the parties concerned. described in 9.3.1 with the two selective plating out
media.

8 Preparation of the test sample 9.3.3 Invert the plates (9.3.1 and 9.3,2) and place
them in an incubator ,(6.2) set at 35 ‘C or 37 “C (as
Prepare the test sample in accordance with the agreed).
specific International Standard appropriate to the
product concerned. Incubate for 18 h for the TCBS medium and for 20 h
to 24 h for the TSAT medium.
If there is no specific international Standard, it is
recommended that agreement be reached on this
9.3,4 Examine the plates to identify the presence
subject by the parties concerned.
of characteristic colonies of Vibrio paraf)aerno/yticus
as foilows:
9 Procedure
a) on TCBS medium, the characteristic coionies of
(See the diagram in annex A.) Vibrio parahaemolyticus are smooth, green
(sucrose negative) and 2 mm to 3 mm in diam-
ete~
9.1 Test portion and Initial suspensions
b) on TSAT medium, the characteristic colonies of
9.1.1 See ISO 6887 and the specific International Vibrio parahaemolyticus are smooth, flat, dark
Standard concerning the product to be tested. Use red (reduction of triphenyitetrazoiium chloride)
the enrichment medium as the diluent. and 2 mm to 3 mm in diameter.

3
 -.!&

IS 14987:2001 -4

ISO 8914:1990

9.3,5 If the development of colonies is slow, if the b) inocuiate a tube of the alkaiine saiine Dedone
coloration is weak, or in the absence of a charac- water (5.2.2). incubate in the” -incubato~ ~et at
teristic colony, continue the incubation for up to a 35 “C or 37 ‘C (as agreed) for 1 h to 6 h. Piace a
total of 24 h for the TCBS medium and for a total of drop of the culture on to a ciean microscope
48 h for the TSAT medium, and then re-examine slide, cover with a cover slip and examine the
them. micro-organisms microscopically for motility.
Note cuitures showing motiiity.

9.4 Confirmation
9.4.3.3 Seiection of coionies for biochemical tests

Retain for the biochemical confirmation colonies

9.4.1 Choice of colonies
which are oxidase positive, sucrose negative and
Gram negative, and which give a positive resuit for
Select at least five characteristic colonies from the
the motiiity test.
plates (9.3.4 or 9.3.5).

If there are fewer than five characteristic colonies

use them all. 9.4.4 Biochemical tests

Using a straight wire (6.9), inocuiate the identifica-

9.4.2 Preparation of pure cultures tion and reagent media (9.4.4.1 to 9.4.4.5) with each
of the pure cuitures prepared in 9.4.2 and seiected
Inoculate separately each of the selected character- in 9.4.3.3, incubate and carry out the biochemical
istic colonies on to the surface of a saline nutrient tests.
agar (5.3.3) to allow well-separated colonies to de-
velop. Incubate the inoculated plates in the
incubator set at 35 ‘C or 37 ‘C (as agreed) for 18 h 9.4,4.1 Saiine TSI agar (5.4.1)
to 24 h. Pick up a single well-isolated colony from
each of the plates and inoculate slants of saline nu- inocuiate the siant surface (siope) with a longitudi-
trient agar (5.3.3). Incubate in the incubator set at nal streak and stab the butt to the bottom of the
35 “C or 37 ‘C (as agreed) for 18 h to 24 h. Use these agar.
pure cultur~ for the microscopical examination and
incubate in the incubator set at 35 “C or 37 “C (as
biochemical tests.
agreed) for 24 h.

Interpret the changes in the medium as shown in

9.4.3 Preliminary tests
table 1

9.4.3.1 Oxidase test

Ti ,Ie 1

Using the platinum-iridium loop or straight wire, or ‘osition Coiour interpretation

a glass rod (6.9), take a portion of the pure culture
from the saline nutrient agar slant (9.4.2) and streak Yellow Glucose positive (fermen-
on to a filter paper moistened with the oxidase rea- tation of glucose)
gent (5.4.5) or use a commercially available test, Red or un- Giucose negative (no fer-
following the manufacturer’s instructions, A nickel- changed mentation of glucose)
chromium loop or wire shall not be used. Butt
Black Formation of hydrogen sul-
fide (H,S)
The test is positive if the colour changes to mauve,
violet or deep blue within 10 s. Bubbles or Gas formation from
cracks glucose

9.4.3.2 Microscopical examination Yellow Lactose andlor sucrose

positive (one or both
Take separate fractions of each of the pure cultures Siant sugars utilized)
(9.4.2), and test as in a) and b) below. surface
Red or un- Lactose and sucrose nega-
changed tive (neither sugar utiiized)
a) Place a droD of the sodium chioride soiution
(5.5) on to a ciean. microscope siide, add a frac-
tion of a pure cuiture and emuisify to give a thin Characteristic cuitures of Vibrio Darahaemo/vficus
suspension. Aiiow the siide to dry in air and fix show an alkaiine red siant (lact&e negativ~ and
by passing through a Bunsen burner flame. Carry sucrose negative) and an acid yeliow butt (glucose
out a Gram stain, and note the morphology and positive) without gas formation (gas negative) or
the Gram reaction of the micro-organisms. blackening (H2S negative).

4
 IS 14987:2001
ISO 8914:1990

9.4.4.2 Saline meat-yeast agar(5.4.2) Replace thetube in the water-bath (6.4) set at 37°C
for 24 h and examine at intervals during the 24 h.
Using a loop, inoculate the molten regenerated agar
A positive reaction (presence of ~-galactosidase) is
cooled to 45 “C throughout its depth without intro-
ducing air bubbles. Immediately immerse the tubes indicated by a yellow colour that usually appears in
in cold water to solidify the medium. less than 30 min. The absence of coloration after
24 h indicates a negative reaction.
Incubate in the incubator set at 35 “C or 37 ‘C (as
agreed) for 24 h. 9.5 Interpretation of results

Examine the bacterial growth and determine the

Vibrio parahaernolyficus generally shows the follow-
breathing type.
ing reactions:

9,4.4.3 Saline for the detection of Iysine Sucrose (9,4.4,1) —

decarboxylase (5.4.3) Oxidase (9,4.3.1) +
Motility (9.4.3.2) +
Inoculate just below the surface of the liquid me- Glucose (9.4.4.1) +
dium. Gas formation from glucose (9.4.4.1)
Lactose (9,4.4.1) .-
Incubate in the incubator set at 35 ‘C or 37 ‘C (as H2S (9.4.4.1) —
agreed) for 24 h. Aerobic and anaerobic growth (9.4.4.2) +
Lysine decarboxylation (9.4.4.3) +
A purple colour and turbidity afler incubation indi-
Indole detection (9.4.4.4) +
cates a positive reaction (bacterial growth and —
~-galactosidase detection (9.4.4.5)
Iysine decarboxylation).
Conclude that Vibrio parahaemolyticus is present if
A yellow colour indicates a negative reaction
at least one colonv is confirmed as Vibrio ~ara-
haemolyticus. -
9.4.4.4 Saline medium for indoie detection (5.4.4)
Identification kits currently available commercially,
Inocuiate a tube containing tryptone/tryptophane and permitting the identification of Vibrio para-
mecium (5,4.4.1). haemolyticus, may be used. In this case, the
emulsion of the culture shall be prepared with the
Incubate in the incubator set at 35 “C or 37 ‘C (as
saline solution recommended by the manufacturer.
agreed) for 24 h.

After incubation, add 1 mi of Kovacs reagent 10 Expression of results

(5.4.4.2).
in accordance with the results of the interpretation,
The formation of a red ring indicates a positive re-
indicate the presence or absenc& of Vibrio para-
action (indole formation).
haemo/yticus in a test portion of x g of product.
A yellow-brown ring indicates a negative reaction.
/ 11 Test report
9.4.4.5 Reagent for /?-gaiactosidasedetection
(5.4.6) The test report shail specify the method used, the
incubation temperature used and the result ob-
Suspend a Ioopful of the pure culture in a tube con- tained. It shail also mention ail operating details not
taining 0,25 ml of the saline solution (5.5). specified in this international Standard, or regarded
as optional, together with details of any incidents
Add one drop oftoiuene and shake the tube. which may have influenced the result.

Put the tube in a water-bath (6.4) set at 37 ‘C for a The test report shall include ali information necess-
few minutes. ary for the complete identification of the sample.

Add 0,25 ml of the &galactosidase reagent (5.4.6.3)

and mix, or use a commercially available test and
foiiow the manufacturer’s instructions,

5
IS 14987:2001
ISO 8914:1990

Annex A
(normative)

Diagram of procedure

Tsst partlon, x g

UE!EE-1
‘0-7
Satt polymyxln B
Y Alkallns sallns peptme water
broth (5.2.1) (5.2.2) or GST broth (5.2.3)
Incubation tor 7 h to 8 h (possibly far 18 h to 24 h
for deep-frozen products) at 35 “Cor 37”[ (OS agreed)

&
/1
TCBSagor (5.3.1) TSAT agar (5.3.2)
Incubation for 16 h (24 h If nscessary) Incubation far 20 h to 24 h (46 h If
nt 35 “Cor 37 ●C (as agreed) necessary) at 35 ●[ or 37 “C (as agreed)

5 characteristic cohmles
(from eachelate)
“t
1====1
i
9 Inoculation on saLln* nutrlmt agor (5.3.3)
Incubotlan tor 18 h ta 24 h at 35 “Cm 37 “C(a~ agreed)

i
1 colony (from each plate)

i
Inoculation on slants of satlne nutrient agar (S.3.3)
Inctbattm for W h to 24 h at 3S”[ or 37 “C (as agreed)

t
Prellmtnary tests (9.4.3)
loxldase test, mlcroecoplcal sxamlnatlon)

t
Blochemlcal tests (9.4.4)

t
Interpretation of resdts (9.5)

I
 -J-L
.

. .*
IS 14987:2001
ISO 8914:1990

Annex B
(normative)

Composition and preparation of culture media and reagents

Distribute aseptically the medium in bottles or

6.1 Enrichment media
flasks of appropriate capacity (see 9.1,2).

B.1.l Salt polymyxin B broth (SPB) By preference use the same day. Otherwise,
store the broth overnight at O ‘C to + 5 “C.
B.1.1.1 Base
B.I.2 Alkaline saline peptone water
Composition

Peptone 10,0 g Composition

Yeast extract 3,0 g Peptone 20,0 g

Sodium chloride 20,0 g Sodium chloride 30,0 g

1000mI Water 1000 ml

Water

Preparation Preparation

Dissolve all the components or the dehydrated Dissolve the components in the water, by heating
base in the water, by heating if necessary. if necessary.

AdJust the PH. if necessary, so that after Adjust the PH, if necessary, so that after
sterilization it is 7,4 at 25 ‘C. sterilization it is 8,6 at 25 ‘C.

Sterilize in an autoclave set at 121 “C for 15 min. Distribute the medium in bottles or flasks of ap-
propriate capacity (see 9,1.3).
B.1.I.2 Polymyxin B solution
Sterilize in an autoclave set at 121 ‘C for 15 min
Composition
6.1.3 Saline glucose culture medium with
Polymyxin B 1000oo Iu sodium dodecyl sulfate (GST)
Water 100 ml
Composition
Preparation 10,0 g
Peptone

Dissolve the polymyxin B in the water. Meat extract 3,0 g

Sterilize by filtration. Sodium chloride 30,0 g

Glucose 5,0 g
9,1.1.3 Complete medium

Methyl violet 0,002 g

Composition
Sodium dodecyl sulfate 1,36 g
Base (B.1.l.1) 900 ml
Water - 1000mt
Polymyxin B solution (B.1.l .2) 100 ml
Preparation
Preparation
Dissolve the Components or the dehydrated
Add aseptically the freshly prepared pdymyxin
complete medium in the water, by heating gently
B solution to the previously cooled base.
if necessaty.

7
IS 14987:2001
ISO 8914:1990

Adjust the pH, if necessary, so that after than 4 h at laboratory temperature or more than
sterilization it is 8,6 at 25 “C. 1 day between O “C and + 5 “C.

Distribute the medium in bottles or flasks of ap- B.2.2 Triphenyltetrazolium chloride soya
propriate capacity (see 9.1.3).
tryptone agar (TSAT) [11
Sterilize in an autoclave set at 121 “C for 15 min.
B.2.2.1 Basic medium

B.2 Plating-out media Composition

Tryptone 15,0 g
B.2.1 Thiosulfate citrate bile sucrose agar
(TCBS) Soya peptone 5,0 g

Composition Sodium chloride 30,0 g

Peptone 10,0 g Sucrose 20,0 g

Yeast extract 5,0 g Bile salt 0,5 g

Sodium citrate 10,0 g Agar 8,0 g to 18,0 gz)

Sodium thiosulfate 10,0 g Water 1000 ml

Iron(lll) citrate I,og Preparation

Sodium chloride 10,0 g Dissolve the basic components or the basic de-
hydrated medium in the water, by heating if
Ox bile 8,0 g
necessary.

Sucrose 20,0 g
Adjust the PH, if necessary, so that after
Bromothymol blue 0,04 g sterilization it is 7,1 at 25 ‘C,

Thymol blue 0,04 g Distribute the medium in bottles or flasks of ap-

propriate capacity.
Agar 8,0 g to 18,0 gz)
Sterilize in an autoclave set at 121 ‘C for 15 min.
Water 1000ml

Preparation B.2.2.2 1 % triphenyltetrazolium chloride solution .

Dissolve the components or the dehydrated Composition

complete medium in the water by boiling.
Triphenyltetrazolium chloride 0,1 g
Adjust the pH, if necessary, so that it is 8,6 at
Water 10 ml
25 “C.
Preparation
Do not sterilize.
Dissolve the component in the water
Preparation of plates

Sterilize by filtration.
Pour 15 ml to 20 ml of the complete medium thus
prepared, and cooled to about 45 “C, into Petri
B.2.2,3 Complete medium
dishes (6.6) and allow to set.

Composition
Just before use, thoroughly dry the agar plates
(preferably after having removed the covers and Basic medium (B.2.2.1) 1000 ml
turned the plates upside down) in an oven (6.3)
until the agar surface is dry. 1 VO triphenyltetrazolium chloride

If they have been prepared in advance, the un- solution (B.2.2.2) 3 ml

dried agar plates shall not be stored for more

2) According to the gel strength of the agar.

8
 4
.-

1S 14987:2001
ISO 8914:1990

Preparation Preparation of slants of saline nutrient agar

Add aseptically to the basic medium, previously Distribute about 10 ml of the medium, cooled to
cooled to 45 “C, the sterile triphenyltetrazolium about 45 “C, into tubes of appropriate capacity.
chloride solution and mix.
Allow to set in a sloping position
Preparation of plates

B.3 Identification media

Distribute the complete medium, in quantities of
15 ml to 20 ml in Petri dishes (6.6) and allow to
solidify. B.3.1 Triple sugar saline iron agar (TSI saline
agar)
Just before use, thoroughly dry the agar plates
(preferably after having removed the covers and Composition
turned the.plates upside down) in an oven (6.3),
Peptone 20,0 g
until the agar surface is dry.

Meat extract 3,0 g

Prior to drying, the plates may be stored for no
longer than 2 weeks at between O “C and Yeast extract 3,0 g
+ 5 “c.
Sodium chloride 30,0 g
B.2.3 Saline nutrient agar 10,0 g
Lactose

Composition Sucrose 10,0 g

Peptone 5,0 g Glucose I,og

Meat extract 3,0 g Iron(lll) citrate 0,3 g

Sodium chloride 30,0 g Phenol red 0,024 g

Agar 8,0 g to 18,0 g3) Sodium thiosulfate 0,3 g

Water 1000 ml Agar 8,0 g to 18,0 gs)

Preparation Water 1000 ml

Dissolve the components or the dehydrated Preparation

complete medium in the water, by heating if
necessary. Dissolve the components or the dehydrated
complete medium in the water, by hbating if
Adjust the PH, if necessary. so that after necessary.
sterilization it is 8,5 at 25 ‘C,
Adjust the PH, if necessary, so that after
Transfer the medium to containers of appropriate sterilization it is 7,4 at 25 ‘C.
capacity.
Transfer the medium in quantities of 10 ml to test
Sterilize in an autoclave set at 121 ‘C for 15 min. tubes (6.5) of appropriate capacity.

Preparation of plates Sterilize in an autoclave set at 121 ‘C for 10 min.

Transfer 15 ml to 20 ml of the medium, cooled to Allow to set in a sloping position to give a butt
about 45 “C, to Petri dishes (6.6) and allow to set. of depth about 2,5 cm.

Immediately before use, thoroughly dty the agar If the medium is used more than 8 days after its
plates (preferably after having removed the cov- preparation, regenerate it by melting on a boiling
ers and turned the plates upside down) until the water-bath or in flowing steam for 10 min.
agar surface is dry. Resolidify as described above.

3) According to the gel strength of the agar,

9
1S 14987:2001
ISO 8914:1990

.A
n
B.3.2 Saline meat-yeast agar Transfer the medium to 9 mm x 180 mm test
tubes (6.5) in quantities of approximately 2 ml 1
Composition per tube. L. A
h.
Peptone 10,0 g Sterilize in an autoclave set at 121 ‘C for 10 min.
i
Meat extract 2,0 g
B.3.4 Reagents for indoie detection
Yeast extract 6,0 g
B,3.4.I Saiine tryptone/tryptophane medium
Sodium chloride 30,0 g
Composition
Cysteine hydrochloride 0,3 g
Tryptone 10,0 g
Glucose 2,0 g
DL4@OphatW I,og
Agar 5,0 g to 8,0 g’}
Sodium chloride 30,0g
Water 1000ml
Water 1000ml
Preparation
Preparation
Dissolve the components or the dehydrated
complete medium in the water, by heating if Dissolve the components in the water, by heating
necessaty. if necessary, and filter.

Adjust the PH, if necessary, so that after Adjust the PH, if necessary, so that after
sterilization it is 7,5 at 25 ‘C. sterilization it is 7,5 at 25 ‘C.

Transfer the medium in quantities of 4 ml to Transfer the medium, in quantities of 5 ml, to test
9 mm x 180 mm test tubes (6.5). tubes (6.5) of appropriate capacity.

Sterilize in an autoclave set at 121 “C for 15 min. Sterilize in an autoclave set at 121 “C for 15 min.

Just before use, heat the test tubes on a boiling B.3,4.2 Kovacs reagent
water-bath or in flowing steam for 10 rein, and
then cool rapidly to about 45 ‘C. Composition

(A) 4-dimethylaminobenzaldehyde 5,0 g

B.3.3 Saline medium for the detection of
Iysine decarboxylase (B) hydrochloric acid (p 1,18 glml to

Composition 1,19 g/ml) 25 ml

L-lysine monohydrochloride 5,0 g (C) 2-methylbutan-2-ol 75 ml

Yeast extract 3,0 g Preparation

Glucose l,og
Dissolve A in C, then slowly add B.
Bromocresol purple 0,015 g
Store the reagent in the dark.
Sodium chloride 30,0 g
B.3.5 Reagent for oxidase detection
Water 1000 ml

Composition
Preparation
N, N, N, N’-tetramethyl-p-
Dissolve the components in the water, by heating
if necessary. phenylene diamine dihydrochloride l,og

Adjust the PH, if necessary, so that after Water 100 ml

sterilization it is 6,8 at 25 ‘C.

4) According to the gel strength of the agar.

10
 .s-.

Is 14987: 2W1
ISO 8914:1990

Preparation Adjust the pH to 7,0 with 0,1 moljl sodium hy-

droxide solution.
Dissolve the component in the water (cold) im-
mediately before use. Add water to a final volume of 50 ml

Store at between O “C and + 5 “C.

B.3.6 Reagent for fl-galactosidase detection

9.3.6.1 ONPG solution B.3.6.3 Complete reagent

Composition
Composition
ONPG solution (B.3.6.1) 15 ml
2-orthonitropheny l-~-D-galactopy ranoside

(ONPG) 0,08 g Buffer solution (6.3.6.2) 5 ml

Water 15 ml Preparation

Preparation Add the buffer solution to the ONPG solution.

Dissolve the ONPG in the water at approximately

B.4 Sodium chloride solution
50 ‘c.
Composition
Cool the solution
Sodium chloride 30,0 g
B.3.6.2 Buffer solution
Water 1000 ml
Composition
Preparation
Sodium dihydrogenorthophosphate
Dissolve the component in the water, by heating
(NaH2P0,) 6,9 g if necessary.

Water to a final volume of 50 ml Adjust the PH, if necessary, so that after

sterilization it is 7,5 at 25 ‘C.
Preparation

Transfer the solution to bottles or flasks (6.5) of

Dissolve the component in approximately 45 ml
appropriate capacity.
of water.

Sterilize in an autoclave set at 121 “C for 20 min.

11
 t.,
——

IS 14987:2001
ISO 8914:1990

Annex C
(informative)

Bibliography

[1] KouRANY MEDluM, App/ied andenvironmenta/ microbiology, Januaw1983, Vol. 45, No. l.

‘t2
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