इंटरनेट मानक

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.

“जान1 का अ+धकार, जी1 का अ+धकार” “प0रा1 को छोड न' 5 तरफ”

Mazdoor Kisan Shakti Sangathan Jawaharlal Nehru
“The Right to Information, The Right to Live” “Step Out From the Old to the New”

IS 15187 (2002): Water Quality -- Detection of Salmonella

Species [FAD 14: Drinks and Carbonated Beverages]

“!ा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”
 IS 15187:2002
ISO 6340:1995

%’wfkTTm

@ a ymn - &pf)J dmmfaa%mm

Indian Standard
WATER QUALITY — DETECTION OF
Sahnone//a
SPECIES

Ics 07.100.20

@ BIS 2002
BUREAU OF INDIAN STANDARDS
MANAK BHAVAN, 9 BAHADUR SHAH ZAFAR MARG
NEW DELHI 110002

! Ju/y 2002
Price Group 6

/ ~
Drinks and Carbonated Beverages Sectional Committee, FAD 14

NATIONAL FOREWORD

This Indian Standard which is identical with ISO 6340:1995 ‘Water quality — Detection of Sa/mone//a
species’ issued by the International Organization for Standardization (ISO) was adopted by the
Bureau of Indian Standards on the recommendation of the Drinks and Carbonated Beverages 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’; and

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

International Standard Corresponding Indian Standard Degree of

Equivalence

ISO 3696:1987 Water for analytical IS 1070:1992 Reagent grade water Equivalent
laboratory use — Specification and (third revision)
test methods

ISO 6579:1993 Microbiology— IS 5887 (Part 3) : 1999 Methods Identical

General guidance on methods for for detection of bacteria responsible
the detection of Sa/mone//a for food poisoning : Part 3 General
guidance on methods for detection
of Salmonella

The technical committee responsible for the preparation of this standard has reviewed the provisions
of the following ISO Standards and has decided that they are acceptable for use in conjunction with
this standard:

ISO 5667-1:1980 Water quality — Sampling — Part 1: Guidance on the design of sampling
programmed

ISO 5667-2:1991 Water quality — Sampling — Part 2: Guidance on sampling techniques

ISO 5667-3:1994 Water quality — Sampling — Part 3: Guidance on the preservation and handling
of samples

In reporting the results 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 ‘Rules
for rounding off numerical values (revised)’.

I
 IS 15187:2002
ISO 6340:1995

Indian Standard
WATER QUALITY — DETECTION OF
Salmonella SPECIES

WARNING — In order to safeguard the health of laboratory personnel, it is essential that tests for
detecting Salmonella species are undertaken in properly equipped laboratories, under the control
of skilled microbiologists only, and that great care is taken in the disposal of all incubated
materials.

1 Scope ISO 6579:1993, Microbiology — General guidance on

methods for the detection of Salmonella.
This International Standard specifies a method for the
detection of Sa/rncme//a species in water samples for 3 Definitions
monitoring purposes. In special epidemiological situ-
ations, other media may also be required. For the purposes of this International Standard, the
following definitions apply.
The method can be applied to all kinds of water, ex-
-- cept raw sewage.
3.1 Salmonella species: Gram-negative, oxidase-
negative, facultatively anaerobic, non-sporeforming,
rod-shaped bacteria which generally form colonies of
2 Normative references 2 mm to 4 mm in diameter on solid selective media.
They form typical colonies on solid selective media
The following standards contain provisions which, and display the biochemical and serological charac-
through reference in this text, constitute provisions teristics described when tests are carried out in ac-
of this International Standard. At the time of publi- cordance with this International Standard.
cation, the editions indicated were valid. All standards
are subject to revision, and parties to agreements 3.2 detection of Salmonella organisms: Determi-
based on this International Standard are encouraged nation of the presence of these bacteria in a particular
to investigate the possibility of applying the most re- volume, when tests are carried out in accordance with
cent editions of the standards indicated below. this International S~andard.
Members of IEC and ISO maintain registers of cur-
rently valid International Standards.
4 Principle
ISO 3696:1987, Water for ana/ytica/ /aboratory use —
The detection of Sa/mone//a species requires four
Specification and test methods.
successive stages.

ISO 5667-1:1980, Water quality — Sampling —

Part 1: Guidance on the design of sampling pro- 4.1 Pre-enrichment
grammed.
Pre-enrichment is necessary to enable injured cells to
ISO 5667-2:1991, Water quality — Sampling — grow. If necessary, samples can be concentrated us-
Part 2: Guidance on sampling techniques. ing membrane filtration. The membrane filter with
cells, or a known volume of sample or its dilution, is
ISO 5667-3:1994, Water quality — Sampling — transferred to non-selective broth (buffered peptone
Part 3: Guidance on the preservation and handling of water) for incubation at the optimal temperature for
samples. mesophilic bacteria.

/
 IS 15187:2002
!&w”wc. 1s0 6340:1995

ij=-----
——..
4.2 Enrichment in selective liquid medium 5 Culture media and confirmation media

h A selective enrichment step is necessa~ to increase

the proportion of Sa/rrtorre//a species in relation to
background flora. For this purpose, inoculum from
Use reagents of analytical quality for the preparation
of culture media, unless otherwise specified. Prepare
media using glass-distilled water, or water of equiv-
pre-enrichment broth is transferred to malachite alent quality, complying with grade 3 of ISO 3696.
green/magnesium chloride (modified Rappaport-
Vassiliadis) medium which is incubated at an elevated If commercially available dehydrated media are used,
temperature to increase its selectivity. prepare them according to the manufacturer’s in-
structions and add selective agents as supplements
NOTE 1 For the detection of Sa/mone//a typhi, which is to give the specified concentrations.
usually not important for water quality monitoring but may
be required under special circumstances, selenite cystine All pH values given in this International Standard are
medium (also available as dehydrated complete medium for media after sterilization; for pH correction, use
from different manufacturers) can be used for incubating sodium hydroxide or hydrochloric acid at concen-
the cultures at 36 ‘C + 2 ‘C for up to 24 h. In certain special trations of 1 mol/1 each.
epidemiological situations, addition of other media may be
necessary.
5.1 Culture media

5.1.1 Pre-enrichment medium: buffered peptone

4.3 Selection on agar media water

Solid selective media are used after the liquid

5.1.1.1 Composition
enrichment steps for the detection and isolation of
Salmonella species. In order to increase the prob- t
Single Double
ability of detecting Sa/monel/a organisms, at least two
strength strength
different media are inoculated from selective
enrichment cultures:
Peptone log 20 g
— brilliant green/phenol red lactose agar; Sodium chloride (NaCl) 5g log
Disodium hydrogen phosphate
— xylose Iysine deoxycholate agar; dodecahydrate
(Na2HP0412H20) 9g 18g
— bismuth sulfite agar (optional). Potassium dihydrogen phos-
phate (KH2P04) l,5g 3g

Water to 1 000 ml to 1 000 ml

4.4 Confirmation
5.1.1.2 Preparation
The occurrence of typical colonies of Salmonella spe-
cies on selective agar media is not sufficient evidence Dissolve all the constituents in water by heating gen-
for the presence of Salmonella species. Therefore, it tly, but do not boil the solution.
is necessary to subculture presumptive Salmonella
colonies on different media for biochemical and Adjust the pH to 7,2 A 0,1, with sodium hydroxide
serological confirmation (see table 1). solution or hydrochloric acid.

NOTE 2 Commercially available identification kits suitable Dispense the medium into culture bottles/tubes.
for the identification of Sa/mone//a species can be used in-
stead of the tests listed in table 1, provided that they are Sterilize the medium in the autoclave (6.1 .2) at
used according to the manufacturer’s instructions and on 121 “C*1 0Cfor15 min.
condition that they can be considered at least as reliable as
the tests listed in table 1. Store in a refrigerator for up to 3 months.

/
 IS 15187:2002
ISO 6340: 1995

5.1.2 Enrichment medium: malachite 5.1.3 Optional enrichment medium: selenite

green/magnesium chloride (modified cystine
Rappaport-Vassiliadis medium)
5.1.3.1 Composition

Casein-peptone 5g
5.1.2.1 Composition
L-Cystine 0,01 g

Lactose 4g

Dmodum hydrogen phosphate (Na2HPOJ log

Basic medium Sodium hydrogen selerme (NaHSeOJ 4g
Peptone, enzymatic djgest of arumal tissue 4g Water to 1 000 ml
Peptone, from soybeans Ig

Sodium chloride (NaCl) 8g

5.1.3.2 Preparation
Dipotasswm hydrogen phosphate tnhydrate
(K2HP043H20) 0,4 g
Dissolve all the constituents in water by heating gen-
Potassium dlhydrogen phosphate (KH2POJ 0,6 g
tly, but do not boil the solution.
Water to 1 000 ml

CAUTION — Do not sterilize the medium in the

autoclave, Apply sterile filtration instead, and do
Supplement 1 1)
not use the medium if red sediments appear.
Magnesium chlorlde hexahydrate (MgCl,6H,0) 31,7 g

Water to 100 ml Adjust the pH to 7,0 + 0,2.

1) As this salt IS very hydroscopic, it IS adwsable either to

WARNING — Inhalation of sodium hydrogen
store It r’ a desslcator or to dissolve the entire contents of a
newly opened container of magnesium chloride in such a way selenite dust and direct contact with the skin is
that the mass concentration of magneswm ch!oride very dangerous. The dust irritates the eyes, skin
hexahydrate IS 28,6 g/1 In the final medium. The magnesium and mucous membranes and can penetrate skin
-- chloride solutlon can be stored for a long time m a sealed con-
both as a powder and as a solution. It has long-
tainer.
term health effects and may be carcinogenic. Re-
action with acids liberates gaseous hydrogen
Supplement 2 selenide, which is very dangerous if inhaled and
Malachlte green oxalate 0,4 g irritate the eyes and mucous membranes. Sodium
Water to 100 ml
hydrogen selenite and its solution must be han-
dled under a hood using gloves and, if required, a
respirator mask should be used. Contact with ac-
ids must be avoided. Store in tightly closed con-
tainers in a well-ventilated area that is dry and
5,1.2.2 Preparation separated from acids.

5.1.4 Selective solid media

Dissolve all the constituents of the basic medium in
water by heating gently, but do not boil the solution. 5.1.4.1 Brilliant green/phenoi red lactose agar
(according to Edel and Kampelmacher)
Add the prepared magnesium chloride solution (sup-
plement 1) and 10 ml of the malachite green solution 5.1.4.1.1 Composition
(supplement 2) to the basic medium.

Basic medium
Adjust the pH to 5,2 + 0,1, with sodium hydroxide
Meat extract powder 5g
solution or hydrochloric acid.
Peptone, enzymatic digest of animal tissue 5g

Dispense about 10 ml of the medium into each cul- Disodium hydrogen phosphate (NazHPO.J lg
ture tube. Sodium dihydrogen phosphate (NaH2POJ 0,6 g

Agar about 15 g
Sterilize the medium in the autoclave (6.1 .2) at
Water to 900 ml
115 °C*10Cfor15 min.

/
 IS 15187:2002
ISO 6340: 1995
-

Supplement 1 Supplement

log Phenol red 0,4 g

Lactose
log Water to 100 ml
Sucrose
Phenol red 0,09 g

Water to 100 ml
\ 5.1.4.2.2 Preparation

Supplement 2 Dissolve all the constituents, including 20 ml of the

Brilliant green 0,5 g
phenol red solution (supplement), by heating to bring
Water to 100 ml to the boil.

Adjust the pH to 7,4 ~ 0,1.

5.1.4.1.2 Preparation CAUTION — To avoid overheating, do not prepare

portions larger than 1 Iitre at a time. Do not ster-
Dissolve all the constituents of the basic medium in ilize the medium in the autoclave. After prep-
water, and sterilize in the autoclave (6.1 .2) at aration, transfer to a water bath at 50 “C and pour
121 “C*1 0Cfor15 min. into Petri dishes (6.7) as soon as the medium has
cooled.
Prepare supplement 1 by dissolving the constituents
in sterile water. Heat the solution in a water bath
(6.2) at 70 “C for 20 min. Cool it to 55 “C + 1 “C and 5.1.4.3 Bismuth sulfite agar (according to Wilson
use it immediately. and Blair)

Prepare supplement 2 by dissolving the brilliant green

in the water, Store the solution for at least 1 day in the
5.1.4.3.1 Composition
dark to allow autosterilization to occur.

Add the prepared sugar/phenol red solution (sup- Besic medium

plement 1) and 1 ml of the brilliant green solution Meat extract 5g
(supplement 2) to the agar before distribution into Peptone, enzymatic digest of animal tissue log
Petri dishes (6.7); ensure that the final pH is o(+) -Glucose 5g
7,0 f 0,1. Immediately before use, dry the agar plates 4g
Disodium hydrogen phosphate (NazHPOJ
until the surface of the agar is dry. Use freshly pre-
kon(ll) sulfate heptahydrate (FeSOd7H@) 0,3 g
pared plates.
Bismuth sulfite [BiJSOJJ 8g
Agar about 15 g

5.1.4.2 Xylose Iysine deoxycholate agar Water to 1 000 ml

Supplement
5.1.4.2.1 Composition
Brilliant green 0,5 g
Water to 100 ml
Besic medium

o(+)-Xylose 3,5 g

d+)-Lysine 5g

2,5 g 5.1.4.3.2 Preparation

Sodium deoxycholate

Yeast extract 3g
Dissolve all the constituents, including 5 ml of the
Saccharose 7,5 g
brilliant green solution (supplement), by heating. Do
Lactose 7,5 g
not sterilize the medium in the autoclave.
Sodium chloride (NaCl) 5g

Sodium thlosulfate (Na$*OJ 6,8 g Adjust the pH to 7,6 ~ 0,1 and pour about 20 ml of
Iron(lll) ammonium citrate 0,8 g the dissolved, but cloudy medium into each Petri dish
about 13 g
(6.7). Ensure that the agar is pale brownish or reddish
Agar
yellow (fallow) or greenish. If the colour is brown, do
Water to 1 000 ml
not use the medium.

/
 1S 15187:2002
ISO 6340: 1995

p———
—.. — 5.2 Confirmationmedia Sterilize the medium in the autoclave (6.1.2) at
121 “C+ 1 0Cfor15 min.

5.2.1 Nutrient agar Pour about 6 ml of the medium into each tube.

Allow the medium to set in a slopina-. Dosition to give

... 5.2.1.1 Composition a butt of length about 2,5 cm.
~,. .
Meat extract 3g

Peptone, enzymatic digest of animal tissue 5g

5.2.3 Urea agar (according to Christensen)
Agar about 15g

Water to 1 OOOml

Sodium chloride (NaCl) (optional) 5g 5.2.3.1 Composition

Baaic medium
5.2.1.2 Preparation
Peptone, enzymatic digest of animal tissue lg

o(+) -Glucose lg
Dissolve all the constituents by boiling.
Sodium chloride (NaCl) 5g
Adjust the pH to 7,0 + 0,1. Potasswm d!hydrogen phosphate (KH,pOJ 2g

Agar about 15 g
Sterilize the medium in the autoclave (6.1.2) at
Water to 1 000 ml
121 ‘C+ 1 “Cfor15 min.

Dispense into Petti dishes (6.7).

--
5.2.2

5.2.2.1
Iron/two-sugar agar (according to Kligler)

Composition
~1Supplement 2

Urea 40 g
Basic medium Water to 100 ml
Meat extract 3g

Yeast extract 3g

Peptone, enzymatic digest of animal tissue 20 g

5.2.3.2 Preparation
Lactose log

o(+)-Glucose lg
Dissolve all the constituents of the basic medium and
Iron(lll) citrate 0,5 g 3 ml of the phenol red solution (supplement 1), by
.
Sodium chloride (NaCl) 5g heating if necessaw.
Sodium thiosulfate (NaJ@J 0,3 g

Agar about 12 g Sterilize the medium in the autoclave (6.1 .2) at

121 “Ci 1 “Cfor15 min.
Water to 1 000 ml

Allow the agar to cool to about 50 “C.

Supplement
Sterilize the urea solution (supplement 2) by filtration
Phenol red 0,4 g
and add 50 ml of this solution under aseptic con-
Water to 100 ml ditions to 950 ml of the agar (basic medium and sup-
plement 1).

5.2.2.2 Preparation Dispense about 6 ml of the urea agar medium into

each tube.
Dissolve all the constituents, including 6 ml of the
phenol red solution (supplement), by heating. Ensure that the final pH is 6,8 ~ 0,1.

Adjust the pH to a final value of 7,4 + 0,1. Allow to set in a sloping position.

i
 IS 15187 :2002
ISO 6340: 1995

5.2.4 L-Lysine decarboxylase medium (according ways required but often anti-Vi and anti-H sera are
to Falkow) used as well (see ISO 6579). Serological tests should
be carried out in laboratories with a sufficient internal
!5.2.4.1 Composition quality control and experience of serology, to enable
selection of relevant sera and controls.

Basic medium

L-Lysme monohydrochlorlde 5g 6 Apparatus

Yeast extract 3g
Usual microbiological laboratory equipment and the
Peptone from meat 5g
following.
o(+) -Glucose lg

Water to 1 000 ml
6.1 Apparatus for sterilization

6.1.1 Oven (dry sterilizer), for glassware, capable

of being maintained at 170 “C to 175 “C for 1 h.

~ 6.1.2 Autoclave (wet sterilizer), for equipment,

culture media and reagents, capable of being main-
5.2.4.2 Preparation tainedat 121 “C+ 1 OCand at 115°C~ 1 “C.

Dissolve all the constituents, including 3 ml of the

6.2 Water bath, capable of being maintained at
bromocresol purple solution (supplement), by boiling.
70 “c ~ 1 “c.
Adjust the pH to 6,8 + 0,1.
6.3 Incubator, capable of being maintained at
Dispense 5 ml of the medium into each tube. 36 “C & 2 “C.

Sterilize the medium in the autoclave (6.1 .2) at

121 ‘C* 1 OCforl Omin. 6.4 Water bath or incubator, capable of being
maintained at 42 ‘C ~ 0,5 “C.

5.3 Serological confirmation

6.5 pH-meter, having an accuracy of calibration of
0,1 pH unit at 25 “C.
5.3.1 Saline solution
6.6 Loops, of diameter about 3 mm.
5.3.1.1 Composition

6.7 Petri dishes, sterile, with a diameter of ether

Sodium chloride (NaCl) 0,85 g 90 mm or 100 mm.
Water to 100 ml

6.8 Membrane filter, sterile, usually about 47 mm

or 50 mm in diameter and with filtration character-
5.3.1.2 Preparation istics equivalent to a rated nominal pore diameter of
0,45 pm.
Dissolve the sodium chloride in the water.

Adjust the pH to 7,0 f 0,1. 7 Sampling

Sterilize in the autoclave (6.1.2) at 121 “C + 1 “C for
Take samples in accordance with ISO 5667-1,
15 min.
ISO 5667-2 and ISO 5667-3. Keep them cool at 2 “C
Dispense smaller volumes into tubes. to 5 “C and protect them from light. The maximum
suitable preservation time before analysis is 24 h.
Start the analysis as soon as possible within 24 h.
5.3.2 Sera
NOTE 3 The volume of the sample depends on the ex-
Agglutinating sera are available either commercially pected density of Sa/morre//a cells, on the character of the
or from specialized laboratories, Anti-O sera are al- water and on legal requirements for water quality standards.

/’ I
 IS 15187:2002
ISO 6340: 1995
.

8 Procedure brilliant green/phenol red lactose agar (5.1.4.1);

xylose Iysine deoxychlolate agar (5.1 .4.2);

8.1 Pre-enrichment
and (options!)
For water samples exceeding 10 ml in volume, add
the sample to the same volume of buffered peptone bismuth sulfite agar (5.1 .4.3).
water (double strength) (5.1.1 .l)l] or filter through a
sterde membrane filter (6.8) and place the filter in Place in an incubator (6.3) at 36 “C + 2 “C fcr
50 ml of buffered peptone water (single strength) 24 h (48 h for bismuth sulfite agar).
(5.1.1 .1). Filtering aids can be used when needed.
8.4 Confirmation
For sample volumes of 10 ml or less, use a minimum
of 50 ml of buffered peptone water (single strength) For confirmation, take all (or at least five of) the dis-
or at least 10 times the volume of the sample. tinct typical Sahoneh colonies from each positive
agar medium (see 8.3):
Incubate at 36 “C + 2 “C for 16 h to 20 h, and pro-
ceed to 8.2. — colonies on brilliant green/phenol red lactose agar
(5.1 .4.1 ) which are red or slightly pink-white and
8.2 Enrichment in selective liquid media opaque with red surroundings;

Transfer 0,1 ml of the pre-enrichment culture (8.1) to — colonies on xylose Iysine deoxycholate agar
10 ml, or 1 ml to 100 ml, of malachite
(5.1 .4.2) which are colorless (but appear red),
green/magnesium chloride medium (5.1 .2) and incu- usually with a black centre;
bate [n a water bath (6.4) at 42 ‘C + 0,5 “C for 18 h
to 24 h. (If an incubator other than a water bath is — black colonies on bismuth sulfite agar (5.1 .4.3),
used, prewarm the media at this temperature before usually surrounded by a metallic sheen.
inoculation. ) The larger volume of inoculum might in-
crease the probability of detecting Sa/mone//a Plate out the selected colonies onto the surface of
organisms, In certain situations, the use of selenite predried nutrient agar plates (5.2.1) in a manner which
cystine medium (5.1 .3) in addition to malachite will allow well-isolated colonies to develop,
green/magnesium chloride medium is recommended
Place these plates in an incubator (6.3) at
(see note 1 to 4,2).
36 “C &2 °C for 18’hto 24h.
Proceed to 8.3 ~nd 8.4.
Use single isolated colonies only. If the plating fails to
NOTE 4 In order to detect slow-growing Sa/rnone//a spe- produce distinct colonies, repeat in a manner which
cies, It is recommended to reinoculate solid media (see ensures that single discrete colonies are produced.
8.3) after continued incubation of selective liquid media for
another 24 h. 8.4.1 Biochemical confirmation (basic
procedures)
8.3 Selection on agar media
Table 1 lists the biochemical reactions of typical
Plate a Ioopful (see 6.6) of enrichment medium (see Salmonella s~ecies. At least 90 ‘Y. of Salmonella
8.2) onto strains produce the indicated reaction.

Tabla 1 — Basic biochemical reactions

Markar Raaction Medium

Lactose — Iron/two-sugar agar

Glucose + iron/two-sugar agar
Hydrogen sulfide + Iron/two-sugar agar
Urea — Urea agar
Lysine decarboxylase + Lysine decarboxylase medium

1) Mineral waters with high salt contents and sea water should not be investigated by this Wpe of pre-enrichment due to their
high salt concentrations.

7
IS 15187:2002
ISO 6340: 1995

Use cells from single colonies on nutrient agar of the serological tests. It is better to select another colony
culture to be confirmed to inoculate the biochemical for serology, but, if desired, the aut~agglutinable col-
test media (see 8.4). Repeat for each culture to be ony can be investigated biochemically for confirmation
identified. as a Salmonella species.

8.4.1.1 Lactose/glucose fermentation and 8.4.2.2 Examination for O-antigens

hydrogen sulfide formation
Using one colony recognized as non-auto-agglutinable,
Streak a colony and stab the butt into iron/two-sugar proceed according to 8.4.2.1, using 1 drop of the
agar (5.2.2). anti-O serum (5.3.2) instead of the sallne solution.

Place in an incubator (6.3) at 36 “C ~ 2 “C for 24 h. Use the polyvalent and monovalent sera one after the
other.
Typical Salmonella cultures show red slants with gas
formation and yellows butts with blackening of the Regard agglutination with antiserum as a positive re-
agar. action.

8.4.1.2 Urea degradation 8.4.2.3 Definitive confirmation

Inoculate a colony into a tube of urea agar (see Send strains which are considered to be Sa/mone//a
5.2,3). species, or which may be Salmonella species, to a
reference laboratory for definitive typing.
Place in an incubator (6.3) at 36 “C + 2 “C for 24 h

Typical Sa/mone//a cultures show a negative reaction 9 Expression of results

(no rose-pink colour followed by deep cerise).
In accordance with this procedure, indicate the pres-
8.4.1.3 L-Lysine decarboxylase medium ence or absence of Sa/mone//a in the test portion
examined, the results for biochemical reactions listed
Inoculate a colony just below the surface of the liquid in table 1, serological test results and the procedures
L-!ysine decarboxylase medium (5.2.4). Overlay the in 8.4.2.
medium afterwards with sterile liquid paraffih or oil.

Place in an incubator (6.3) at 36 “C I 2 ‘C for 24 h 10 Test report

Typical Sa/mone//a cultures show a purple colour. The test report shall include the following information:

8.4.2 Serological confirmation a) a reference to this International Standard;

Use slide agglutination with the appropriate antisera b) all details necessary for complete identification of
(see ISO 6579) to detect Sa/mone//a antigens from the sample;
discrete single colonies on nutrient agar after auto-
agglutinable strains have been eliminated. c) the result, expressed as the presence or absence
of Salmonella in a test portion of x ml of water;
8.4.2.1 Elimination of auto-agglutinable strains
d) the number of strains isolated from selectlve solid
media (see 8.4), test results of biochemical con-
Place one drop of the saline solution (5.3.1) onto a
firmation (8.4.1 ) and serological confirmation
thoroughly cleaned glass slide. Disperse this drop in
the smooth colony to be tested, to obtain a uniform (8.4.2) indicating the species/serotypes observed.
suspension. If the colony is not smooth, proceed as
for auto-agglutinable strains. 11 Quality assurance
Rock the slide gently for 30 s to 60 s.
Performance characteristics for the detection of dif-
Observe the result against a dark background, prefer- ferent Sa/mone//a species and subspecies cannot be
ably with the aid of a magnifying glass. given because of variations between species and
subspecies. However, in the selective enrichment
If the suspension forms large clumps, regard the step (8,2) the magnesium chloride concentration and
strain as auto-agglutinable. Do not use it for further incubation temperature have been optimized to yield

8
 IS 15187:2002
ISO 6340:1995

good recovery without losing selectivity (see [2] in flasks of the pre-enrichment medium (5.1.1) and pro-
annex B). teed with these control flasks according to 8.1 to 8.4.
For example, about ten Sa/mone//a cells in the
To check the ability of the laboratory to detect inoculum with the background of 108 cells of
Sa/mone//a, introduce reference samples into control Escherichia coli shall be detected.
IS 15187:2002
ISO 6340:1995

Annex A
(inbrrnative)

Additional microbiological information relevant to examination of water

samples for Salmonella organisms

For routine water examination purposes, the Sa/mone//a organisms may be described generally in microbiological,
although not in taxonomic terms as follows.

The Salmone/ia genus belongs to the family Enterobacteriaceae. Sa/mone//a organisms are Gram-negative, non-
scoring, oxidase-negative, rod-shaped bacteria, which are capable of aerobic and facultatively anaerobic growth.
They are able to produce hydrogen sulfide, L-lysine decarboxylase and to ferment glucose, but usually not lactose.
They are not able to produce urease.

10
ma
r
IS 15187:2002
ISO 6340: 1995

Annex B
(iriformative)

Bibliography

[1] ISO 8199:1988, Water quality — General guide to the enumeration of micro-organisms by culture.

[2] PETERZ, M., WIBERG, C. and NORBERG, P. The effect of incubation temperature and magnesium chloride
concentration on growth Sa/mone//a in home-made and in commercially
of available dehydrated Rappaport-
Vassiliadis broths, J. App/, Bact. 66 (1989), PP. 523-528.

11
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