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

IS 14988-2 (2002): Microbiology of Food and Animal Feeding

Stuffs - Horizontal Method for the Detection and
Enumeration of Listeria monocytogenes, Part 2: Enumeration
Method [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”
 IS 14988 ( Part 2 ) :2002
ISO 11290-2:1998
(Reaffirmed - 2012)
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Indian Standard
MICROBIOLOGY OF FOOD AND ANIMAL FEEDING
STUFFS — HORIZONTAL METHOD FOR THE
DETECTION AND ENUMERATION OF
Listeria monocytogenes
PART 2 ENUMERATION METHOD

Ics O7.1OQ.3O

@ BIS 2002

BUREAU OF INDIAN STANDARDS

MANAK BHAVAN, 9 BAHADUR SHAH ZAFAR MARG
NEW DELHI 110002

Aupust 2002 Price Group 8

.
Food Microbiology Sectional Committee, FAD 46

NATIONAL FOREWORD

This Indian Standard ( Part 2 ) which is identical with ISO 11290-2 : 1998 ‘Microbiology of food
and animal feeding stuffs — Horizontal method for the detection and enumeration of Lisferia
mortocytogenes — Part 2: Enumeration method’ 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.

IS 14988 consists of two parts, under the general title ‘Microbiology of food and animal feeding
stuffs — Horizontal method for the detection and enumeration of Listeria monocytogenes:

Part 1 Detection method, which is identical with ISO 11290-1 :1996

Part 2 Enumeration method, which is identical with ISO 11290-2:1998

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 6887-1 :1999 Microbiolo y of IS 10232:1982 Guidelines for the Equivalent

food and animal feeding stuf ?s — preparation of dilutions for micro-
Rules for the preparation of the test biological examination for food
sample, of initial suspension and of
decimal dilutions for microbiological
examination — Part 1 : General
rules for the preparation of initial
suspension and of decimal dilutions

ISO 11290-1 :1996 Microbiology of IS 14988 ( Part 1 ) :2001 Micro- Identical

food and animal feeding stuffs — biology of food and animal feeding
Horizontal method for the detection stuffs — Horizontal method for the
and enumeration of Listeria mono- detection and enumeration of
cytogenes — Part 1 : Detection Listeria rnonocytogenes : Part 1
method Detection method

The technical committee responsible for the preparation of this standard has reviewed the provisions
of ISO 7218:1996 ‘Microbiology of food and animal feeding stuffs — General rules for microbiological
examination’ and has decided that it is acceptable for use in conjunction with this standard.

In this adopted standard, the following additional information shall be treated as added:

Clauses B.1.2, B.2.1 .2, B.3.1 .2, For sterilization of culture media, a temperature of 121 ‘C for 15
B.4.2, B.5.2, B.6.1.2, B.7.1.2, min shall be maintained at a corresponding pressure of 103 kN/m2
B.8.2 and B.11.2 (15psi).

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 ‘Rules for
rounding off numerical values ( revised )’.

,
 A.

IS ‘14988 ( Patt 2 ) :2002

ISO 11290-2:1998

/nciian Standard
MICROBIOLOGY OF FOOD AND ANIMAL FEEDING
STUFFS — HORIZONTAL METHOD FOR THE
DETECTION AND ENUMERATION OF
Listeria monocytogenes
PART 2 ENUMERATION METHOD

WARNING — In order to safeguard the health of laboratory personnel, it is strongly recommended that
tests for detecting Listeria mor)ocytoger)es are undertaken in properly equipped laboratories, under the
control of a skilled microbiologist, and that great care is taken in the disposal of all contaminated materials.
In particular, it is strongly recommended that female laboratory staff are made aware of the particular risk
to the developing foetus presented by infection of the mother through exposure to L.isteria mono-
cytogenes. National legislation may involve more specific demands.

1 Scope
This part of ISO 11290 specifies a horizontal method for the enumeration of Listeria rnm?ocytogenes.

NOTE — The method also allows enumeration of other Listeria species which may be used as indicators of the hygienic .-2
quality of food or feed products.
,,
Subject to the limitations discussed in the introduction, this part of ISO 11290 is applicable to products intended for ~.
human consumption or animal foodstuffs.

In general (see note in 9.2.1), the lower limit of enumeration of this method is 10 L. n’ronocytogenes per millilitre of
sample for liquid products, or 100 L. rnonocytogenes per gram of sample for other products.

2 Normative references
The following standards contain provisions which, through reference to th’e text, constitute provisions of this
International Standard. At the time of publication, the editions indicated were valid. All Standards are subject to
revision, and parties to agreements based on this International Standard are encouraged to investigate the
possibility of applying the most recent editions of standards indicated below. Members of IEC and ISO maintain
registers of currently valid International Standards.

ISO 6887-1 :—l ), Microbiology of Iood and arrirna/ feeding stuffs — Preparation of test samples, initial suspension
and decimal dilutions for microbiological examination — Part 1: General rules for the preparation of the initial
suspension and of decimal dilutions. ,,,

ISO 7218:1996, Microbiology of food and anima/ feeding stuffs — General rules for microbiological examination.

ISO 11290-1:1996, Microbiology of food and anima/ feeding stuffs — Horizontal method for the detection and
enumeration of Listeria monocy?ogenes — Part 1: Detection method.

1) TO be published. (Revision of ISO 6887:1993)

.,

1
IS 14988 ( Part 2.)-: 2002
ISO 11290-2:1998

3 Definitions

For the purposes of this part of ISO 11290, the following definitions apply.

3.1
Listeria monocytogenes
microorganisms which form typical colonies on the solid selective medium described and which display the
morphological, physiological and biochemical” characteristics described when the analysis is carried out in
accordance with this part of ISO 11290

3.2
enumeration of fJsteria monocytogenes
determination of the number of colony-forming units (CFU) of Listerki rnorrocytogenes (see 3.1), in a given quantity
of product, when the analysis is carried out in accordance with this part of ISO 11290

4 Principle

Within ,the limits of this part of ISO 11290, the enumeration of Listeria rnonocytogenes requires six successive steps
(see annex A for a flowchart). .
,.
4.1 Preparation of the initial suspension in one of the two diluents described, as necessary.
.

4.2 Resuscitation for 1 hat 20 “C.

4.3 Surface plating, on the solid selective culture medium contained in two Petri dishes, of a specified quantity of
the test sample for liquid products or the initial suspension for other products.

Preparation of other dishes, under the same conditions, using decimal dilutions of the test sample or initial
suspension.

4.4 Incubation of the dishes at 35 “C or 37 “C and examination after 24 h and 48 h.

4.5 Confirmation of presumptive colonies of Listeria rnonocytogenes with the tests described.

4.6 From the number of confirmed colonies, calculation of the number of Listeria rnonocytogenes per gram or per
millilitre of the test sample.

5 Culture media and reagents

For current laboratory practice, see ISO 7218.

NOTE — Because of the large number of culture media and reagents, it has been considered preferable, for clarity of the text,
to describe them in annex B.

6 Apparatus and glassware

Usual microbiological equipment (see ISO 7218) and, in particular, the following.

6.1 Apparatus for dry sterilization (oven) or wet sterilization (autoclave)

See ISO 7218.

2
 L.,

IS 14988 ( Part 2’ )“ :-”20.~2

ISO 11290-2: ~99g

6.2 Drying cabinet or incubator, capable of being maintained between 25 “C ~ 1 “C and 50 “C ~ 1’ ‘C: ‘“
,.
6.3 Incubators, for maintaining the inoculated media, plates and tubes within the following temperature ranges:’

a) 20 “C t 1 “C (optional);

b) 25 ‘C ~ 1 “C (optional); ,....

c) 35 °C*10Cor370C*10C. ,,

6.4 Water bath, capable of being maintained at 47 “C * 2 “C.

6.5 Loops and wires of platinumhidium or nickel/chromium, or Pasteur pipettes or single-use loops. “

6.6 Glass or plastic spreaders, sterile.

6.7 pH-meter, capable of being read to the nearest 0,01 ~H unit at 25 “C, enabling measurements to be made
which are accurate”to f 0,1 pH u~it.

6.8 Test tubes or flasks, of appropriate capacity, for sterilization and storage of culture media and incubation of
liquid media.

6.9 Total-delivery graduated pipettes, of nominal capacities 1 ml and 10 ml, graduated respectively in 0,1 ml
and 0,5 ml divisions.

6.10 Petri dishes, of diameter 90 mm and 140 mm.

6.11 Jars, suitable for microaerobic incubation (optional).

6.12 Gas mixture (optional), of specified composition for microaerobic incubation:

5 7. to 12 % C02, 5 0/~to 15 Y. 02, and N2 up to 100 Y..

6.13 Equipment for the Henry illumination test (optional)

See annex B. .. . . . .

6.14 Microscope, preferably with phase-contrast. ,---

7 Sampling ,.
Sampling is not part of the method specified in this part of ISO 11290. If there is no specific International Standard
dealing with sampling of the product concerned, it is recommended that the parties concerned come to an
agreement on this subject.

It is important that the laboratory receive a sample which is truly representative and has not been damaged or:,
changed during transport or storage (see ISO 7218).
.. . .

..’.

3“
IS 14988 ( Part 2 ) : 2002
ISO 11290-2:1998

8 Preparation of test sample

Prepare the test sample in accordance with the specific International Standard appropriate to the product
concerned. If there is no specific International Standard, it is recommended that the parties concerned come to an
agreement on this subject.

9 Procedure
WARNING — Whenever a choice is given between 35 ‘C or 37 ‘C for the incubation temperature, this
temperature shall be agreed between the parties concerned and recorded in the test report.

9.1 Test portion, initial suspension and dilutions

See ISO 6887-1 and any specific International Standard appropriate to the prociuct concerned.

Use as diluent for preparing the initial suspension either buffered peptone water (B.1 ), or half-Fraser broth base
medium (B.2).

Half-Fraser broth base without the addition of selective agents may be used as a diluent for the food or feed
sample when both the detection method (see ISO 11290-1) and this enumeration method are carried out on the
same test sample. This procedure is to avoid the need to prepare two initial suspensions; the selective agents are
added to the suspension once the test portion for enumeration has been used. Use of this procedure should be
noted in the test report.

Let the initial suspension stand for 1 h ~ 5 min at 20 ‘C ~ 2 “C [by using, if necessary, the incubator 6.3 a)], in order
to resuscitate the stressed microorganisms.

If a dilution range is used, prepare it after resuscitation.

9.2 Inoculation and incubation

9.2.1 Transfer, by means of a sterile pipette (6.9), 0,1 ml of the initial suspension (9.1) to each of two dishes of
PALCAM agar (B.3), dried beforehand if necessary in the incubator (6.2).

Repeat the procedure using further decimal dilutions if necessary,

NOTE — When, for certain products, it is necessary to estimate low numbers of Listeria rnonocytogenes, the limit of
enumeration can be lowered by a factor of 10 by examining 1,0 ml of the initial suspension. Distribute the 1 ml of inoculum
either on the surface of the agar medium in a large Petri dish (140 mm) or over the surface of the agar medium in three small
dishes (90 mm) using a sterile spreader (6.6). In both cases, prepare duplicates by using two large dishes or six small dishes.

9.2.2 Carefully spread the inoculum as quickly as possible over the surface of the agar plate without touching the
sides of the dish with the spreader. Use a fresh sterile spreader for each plate.z) Leave the plates closed for about
15 min at ambient temperature for the inoculum to be absorbed into the agar.

9.2.3 Invert the dishes prepared in 9.2.2 and place them in an incubator [6.3 c)] set at 35 “C or 37 “C. Incubate
PALCAM agar dishes either microaerobically in a jar (6.11) containing the gas mixture (6.12) or aerobically.

9.3 Enumeration of characteristic colonies

9.3.1 After incubation for 24 h, and for an additional 18 h to 24 h if growth is slight or if no colonies are observed
after 24 h of incubation, examine the dishes (9.2.3) for the presence of colonies presumed to be Listeria spp.
(see 9.3.3).

2) It is possible to use the same spreader for a given sample, by beginning with the higher dilution.
?,

4
 IS 14988 ( Part 2 j :.2002
ISO 11290-2:1998 -.

9.3.2 For dishes incubated microaerobically, after incubation leave the PALCAM agar dishes (9.3.1) to air for 1 h to
allow the agar to regain its pink to purple colour.

9.3.3 After 24 h the characteristic colonies of L.isteria spp. grow as small or very small greyish green or olive green
colonies, sometimes with black centres, but always with black halos. After 48 h Listeria spp. appear in the form of
green colonies, about 1,5 mm to 2 mm in diameter, with a central depression and surrounded by a black halo.

9.3.4 Count all the colonies presumed to be Lkteria spp. (9.3.3) on each dish containing less than 150
characteristic or non-characteristic colonies.

9.4 Confirmation of Listeria spp.

9.4.1 Selection of colonies for confirmation

9.4.1.1 After the period of incubation (9.3.1), keep the dishes containing less than 150 presumptive Listeria spp.
colonies, at all dilutions and, if possible, at two successive dilutions.

Select five of the presumptive colonies on each plate retained. If there are fewer than five presumptive colonies on a
dish, select for confirmation all presumptive colonies.

9.4.1.2 Streak the selected colonies onto the surface of predried plates of tryptone soya yeast extract agar
(TSYEA) (B.4) in a manner which will allow well-separated colonies to develop.

Place the plates in the incubator [6.3 c)] set at 35 “C or 37 ‘C for 18 h to 24 h or until growth is satisfactory.

Typical colonies are 1 mm to 2 mm in diameter, convex, colorless and opaque with an entire edge. If the colonies
are not well separated, pick a typical Listeria spp. colony onto another TSYEA plate. Carry out the following tests
from colonies of a pure culture on the TSYEA.

NOTE — The Henry illumination test may be conducted if necessary (see annex C and note in B.4.2). The colonies then
appear bluish with a granular surface.

9.4.2 Catalase reaction

Take a colony separated in 9.4.1.2 and suspend it in a drop of hydrogen peroxide solution (B.1O) on a slide. The
immediate formation of gas bubbles indicates a positive reaction (see ISO 7218).

9.4.3 Gram staining

Perform the Gram stain on a colony separated in 9.4.1.2 (see ISO 7218). Lisferia spp. are revealed as
Gram-positive slim, shorl rods (of approximately 0,4pm to 0,5 pm diameter, and 1 pm to 2 pm length).

9.4.4 Motility test (if necessary)3J

Take a colony separated in 9.4.1.2 and suspend it in a tube containing TSYEB (B.5)

Incubate in the incubator [6.3 b)] set at 25 ‘C for 8 h to 24 h until a cloudy medium is observed.

Deposit a drop of the above culture using a loop (6.5) onto a clean glass microscope slide. Place a coverslip on top
and examine it with the microscope (6.14). Lisferia spp. appear as slim, short rods with tumbling motility.

Cultures grown above 25 ‘C may fail to exhibit this motion. Always compare them to a known culture. Cocci, large
rods, or rods with rapid swimming motility are not Listeria spp.

3) This examination is not necessary in all cases if the analysis is carried out by a microbiologist who regularly works on tie
detection of L. monocytogenes.

5
IS 14988 ( Part 2 ) :2002
ISO 11290-2:1998

As an alternative test for motility, using an inoculating needle (6.5), stab the motility agar (B.8) with a typical colony
on TSYEA (9.4.1 .2). incubate it for 48 h in the incubator [6.3 b)] set at 25 “C.

Examine for growth around the stab. Lisferia spp. are motile, giving a typical umbrella-like growth pattern
immediately below the surface of the agar. If growth is not sufficient, incubate for up to an additional 5 days and
examine the culture during this time.

9.5 Confirmation of L. monocytogenes

9.5.1 Haemolysis test

9.5.1. 1 If the morDholo~ical and ~hvsiolo~ical characteristics and catalase reaction are indicative of Lisferia sPP.,
determine the haernolyti~ reaction ‘on-shee~ blood agar dishes (B.6).

Before use, thoroughly dry the blood agar surface, then mark the agar into squares. For each culture, take a colony
separated in 9.4.1.2 and stab one Iabelled square using a wire (6.5). Also stab positive (L. monocyfogenes) and
negative (L. irrrrocua) control cultures.

After incubation at 35 “C or 37 “C for 24 h ~ 2 h, examine the test strains and controls. L. monocytogenes show
narrow, clear, light zones of 13-haemolysis4J; L, innocua show no clear zone around the stab. L. see/igeri show a
weak zone of 13-haemolysis. L. ivanovii usually show wide, clearly delineated zones of B-haemolysis. Examine the
plates by transparency to compare test cultures with controls.

9.5.1.2 The haemolytic reaction may also be carried out by using the CAMP test (9.5.3), or by using red cells in
suspension as follows. Disperse thb colony in 150@ of TSYEB (B.5); incubate at 35 “C or 37 ‘C for 2 h. Add 150 VI
of sheep blood red cell suspension (B.12). Incubate at 35 ‘C or 37 “C for between 15 min and 60 rein, then
refrigerate at +3 “C t 2 ‘C for about 2 h. Examine for the presence or absence of B-haemolysis. If the reaction is not
definite, leave the culture at +3 “C t 2 “C for up to 24 h.

9.5.2 Carbohydrate utilization

Using a loop (6.5), inoculate each of the carbohydrate utilization broths (B.7) with the culture from TSYEB (9.4.4). .
Incubate at 35 “C or 37 ‘C for up to 5 days. Positive reactions [formation of acid(s)] are indicated by the evolution of
the colour from purple to yellow and occur mostly within 24 h to 48 h. .,.
“.

9.5.3 CAMP test

\
Streak each of the Staphylococcus aureus and F?hodococcus equi (B.9.4) cultures in single lines across the sheep
blood agar plate (B.6 or B.9.3) so that the two cultures are parallel and diametrically opposite (see figure 1). A thin.,
even inoculum is required. This can be obtained by using an inoculation loop or a wire (6.5) held at right angles to
the agar.

Streak the test strain separated in 9.4.1.2 in a similar fashion at right angles to these cultures so that the test culture
and S. aureus and f?. equi cultures do not touch but at their closest are about 1 mm to 2 mm apart. Several test
strains may be streaked on the same plate.

4) The zone of fl-haemolysis is more readily seen by removing any colony grown on the surface of agar around the inoculum
mark.
.

6
 IS 14988 (Part 2):2002
ISO 11290-2:1998

1. monocytogenes
I

1. innocua
Narrow band of P-haemotysis 1
1

L ivanovii
No haemolysis

/
Wide band of fi-haemolysis -

NOTES

1 Inoculate thin blood agar plates (B.6 or B.9.3) as shown in the diagram. Vertical lines represent streaks of S. aureus (S) and
R. equi (R). Horizontal lines represent streaks of the test cultures. Hatched areas indicate the locations of enhanced
haemolysis.
2 The dotted area indicates the zone of influence of the S. aureus culture.

Figure 1 — Inoculation and interpretation of CAMP test plates

Simultaneously, streak control cultures of L. rrronocytogenes, L. irmocua and L. ivar?ovii. If blood agar (B.6) is used,
incubate the plates at 35 ‘C or 37 “C for 18 h to 24 h. If double-layer plates (B.9.3) are used, incubate at 35 ‘C or
37 °Cfor12hto18h.

An enhanced zone of 13-haemolysis at the intersection of the test strain with the cultures of S. aureus and R. equi is -
considered to be a positive reaction.

The positive reaction with R. equi is seen as a wide (5 mm to 10 mm) “arrow-head of haemolysis. The reaction is ‘
considered as negative if a small zone of weak haemolysis extends only about 1 mm at the intersection of the test
strain with the diffusion zone of the R. equi culture.

A positive reaction with S. aureus appears as a small zone of enhanced haemolysis extending only about 3 mm to
4 mm from the test strain and within the weakly haemolytic zone due to growth of the S. aureus culture. Large
zones of ELhaemolysis do not occur in the proximity area between S. aureus and L. rnonocytogenes.

9.6 Interpretation of morphological and physiological properties and of the biochemical

reactions

All Listeria spp. are small, Gram-positive rods that demonstrate motility (see 9.4.4). If they are observed by Henry
illumination, they appear bluish with a granular surface. They are generally catalase positive.

f-. rnonocytogenes are distinguished from other species by the characteristics listed in table 1.

,
 -P, ,

‘&
-

1?3 14988 ( Part 2 ) :2002

ISO 11290-2: 1998

Table 1 — Reactions for the identification of Listerh spp.

Species Haemolysis Production of acid CAMP test

Rhamnose Xyiose S. aureus R. equi
L. monocytogenes + + +
L. innocua v —

L. ivanovii + — + — +

L. seeligeri (+) + (+) —

L. welshimeri v +
L. grayi — — — —

V: variabie reaction
(+): weak reaction
+: >90 “A of positive reactions
-: no reaction

NOTE — There exist rare strains of L. monocytogenes which do not show 13-haemoiysis or a positive
reaction to the CAMP test under the conditions described in this part of iSO 11290.
L

9.7 Definitive confirmation

Strains which are considered to be L. monocvtoaenes (9.6) mav be sent to a recognized Lkteria reference
laboratory for serological or, possibiy, iysogeni~ t~ping, o’r an alternative reiiabie moie~uiar typing method. The
dispatch shaii be accompanied by aii possibie information concerning the strain(s).

10 Expression of results (see ISO 721 8)

10.1 Counting of colonies of L. monocyfogenes
Caicuiate for each of the piates the number a of coionies of L. monocytogenes present, using the foiiowing formuia:

b
U.—XC
A

where

b ~ is the number of colonies conforming to the identification criteria (9.6);

A is the number of coionies plated out for confirmation (9.4.1.1);

C is the total number of characteristic colonies enumerated on the dish (9.3.4).

Round off a to a whole number.

10.2 Method of calculation

10.2.1 Plates containing less than 150 colonies of L. monocytogenes, one of which contains at least
15 L. monocytogenes

Caicuiate the number N of L. rnonocytogenes present in 1 mi or 1 g of product, using the following formula:

8
 IS 14988 ( Part 2 ) :2002
ISO 11290-2:1998

where

b is the sum of the colonies of L. monocytogenes calculated after confirmation, on all the dishes retained at
two consecutive dilutions, one of which at least contains at least 15 identified colonies;

V k the volume of the inoculum applied to each dish in millilitres;

nl is the number of dishes retained at the first dilution;

n2 is the number of dishes retained at the second dilution;

d k the dilution factor corresponding to the first dilution retained.

Round the results obtained to two significant figures (see ISO 7218).

Take as the result the number of L. rnonocytogenes per millilitre (liquid products) or per gram (other products),
expressed as a number between 1,0 and 9,9 multiplied by 10 to the appropriate power.

NOTE — See ISO 7218 for an example.

10.2.2 Estimation of small numbers

10.2.2.1 If the two dishes, at the level of the initial suspension, contain less than 15 colonies of L. rnonocytogenes,
calculate the number of confirmed colonies on each dish, using the formula given in 10.1. Calculate the arithmetical
mean y of the colonies counted on two dishes.

Express the result as follows:

— estimated number of L. rnofrocytogenes per gram or per millilitre NE = ~

dxV

where

d is the dilution factor of the initial suspension;

V is the volume of inoculum on each dish.

10.2.2.2 If the two dishes at the level of the initial suspension do not contain any colonies, express the result as
follows:

1
— less than — L. rnonocytogenes per gram or per millilitre
dxv

where

d is the dilution factor of the initial suspension;

V is the volume of inoculum on each dish.

11 Precision
See ISO 7218.
IS 14988 ( Part 2 ) :2002
ISO 11290-2:1998 :-

~!
$
12 Quality control of culture media .
,,.
Since there is yet no general standard on this topic, check the ability of the culture media to support the selective b
,’ ‘
growth of L. rnonocytogenes as follows. Introduce a dilution of the reference culture of recently isolated strains of ~
L. rncmocytogenes and negative control strains (e.g. Iactobacilli, Streptococcus) in a control flask of the selective
primary enrichment medium (see 9.2). Add 10 to 100 L. morrocytogenes cells or negative control strains per flask.
,, ,,,;,
Proceed with the control flasks as for the test cultures to demonstrate that the positive control culture is recovered. ,, ,~
1

13 Test report \

The test report shall specify the method used, the temperature of incubation and the results obtained. It shall also t
mention all operating details not specified in this part of ISO 11290, or regarded as optional, together with details of
any incidents which may have influenced the results.

The test report shall contain all information necessaty for the complete identification of the sample.

,*.,-
,:,

10
 IS 14988 ( Part 2 ) :2002
ISO 11290-2:1998

Annex A
(normative)

Diagram of procedure

Test portion (x g or x ml)

+
Diluent for the preparation of the initial suspension
[buffered peptone water [8.1) or base medium for hatf-Fraser (B.2.1)1
(9x g or 9X ml)

Resuscitation for 1 h at 20 “C (9.1)

I
1

I Surface inoculation of PALCAM agar (B.3) 1

I
Incubation at 35 “[ or 3? “C for 2f4 h to 48 h
I

I Identification and enumeration of Listeria spp. (9.3) I

Confirmation of Listeria SPP. genus (9.4):

- inoculation on TSYEA and incubation
at 35 ‘C or 37 “C (9.4.1):
- catalase reaction (9.k.2): >---
- Gram staining (9.4.3);
- motility test (9,4.4) (if necessary).
1

F Confirmation

- carbohydrate
of L. rnortocytogenes (9.6):

utilization (9.5.2);

Y1
IS 14988 ( Part 2 ) :2002
ISO 11290-2:1998

AnnexB
(normative)

Composition and prepat’ation of media and reagents

B.1 Buffered peptone water

B.1.1 Composition

Enzymatic digest of animal tissues 10,0 g

Sodium chloride (NaCl) 5,0 g

9,0 g
Disodium hydrogen phosphate dodecahydrate (Na2HP041 2H20)
Potassium dihydrogen phosphate (KH2P04) l,5g

Water 1000 ml

B.1.2 Preparation

Dissolve the components in the water, by heating if necessary.

Adjust the pH [measured with the pH-meter (6.7)], if necessary, so that after sterilization it is 7,0 f 0,2 at 25 ‘C.

Dispense the medium in flasks (6.8) of suitable capacity to obtain portions appropriate for the test (see 9.1).

Sterilize for 15 min in the autoclave (6.1) set at 121 “C.

,----

B.2 Base medium for half-Fraser broth with ammonium iron(lll) citrate (see ISO 11290-1) ,.
B.2.1 Base medium for half-Fraser broth
B.2.1.1 Composition

Enzymatic digest of animal tissues 5,0 g

Enzymatic digest of casein 5,0 g
Meat extract 5,0 g
Yeast extract 5,0 g
Sodium chloride (NaCl) 20,0 g
Disodium hydrogen phosphate dihydrate (Na2HP04.2H20) 12,0 g
Potassium dihydrogen phosphate (KH2P04) 1,35 g
Aesculin l,og
Water 1000 ml

B.2.1.2 Preparation

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

Adjust the pH, if necessary, so that after sterilization it is 7,2* 0,2 at 25 “C.

Dispense the base in flasks (6.8) of suitable capacity to obtain portions appropriate for the test (see 9.1).
.

12
 IS 14988 ( Part 2 ) :2002
ISO 11290-2:1998

Sterilize for 15 min in the autoclave (6.1) set at 121 “C.

B.2.2 Ammonium iron(lll) citrate solution

B.2.2.1 Composition

Ammonium iron(lll) citrate 5,0 g

Water 100 ml

B.2.2.2 Preparation

Dissolve the ammonium iron(lll) citrate in the water.

Sterilize by filtration.

B.2.3 Preparation of the medium

Immediately before use, add 1,0 ml of the ammonium iron(lll) citrate solution (B.2.2) to each 100 ml portion of the
base medi~m for half-Fraser broth (B.2.1 ).

9.3 Selective plating-out medium: PALCAM agars)

B.3.1 Agar base
B.3.1.1 Composition

Peptones 23,0 g
Starch l,og
Sodium chloride (NaCl) 5,0 g
Yeast extract 3,0 g
Agar 9gto18gl)
D-Glucose 0,5 g
D-Mannitol 10,0 g
Aesculin 0,8 g
Ammonium iron(lll) citrate 0,5 g
Phenol red 0,08 g
Lithium chloride (LiCl) 15,0 g
Water 960 ml
1) Depending on the gel strength of the agar.

5) Details are given in: VAN NETTEN P., PERALES L, VAN DE MOOSDIJK A., CURTIS G.D.W. and MOSSEL D.A.A. Liquid
and solid selective differential media for the detection and enumeration of L. morrocytogenes and other Listeria spp. ht. J.
Food Microbial., 8, 1989,pp. 299-316. .

13
IS 14988 (Part 2):2002
ISO 11290-2:1998

B.3.1.2 Preparation

Dissolve the components or the dehydrated complete base in the water, by boiling.
..’
Adjust the pH, if necessafy, so that after sterilization it is 7,2 * 0,2 at 25 ‘C.

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

B.3.2 Polymyxin B sulfate solution

B.3.2.1 Composition

Polymyxin B sulfate (100 000 IU) 0,1 g

Water 100 ml

B.3.2.2 Preparation

Dissolve the polymyxin B sulfate in the water.

Sterilize by filtration.

B.3.3 Acriflavine hydrochloride solution

B.3.3.1 Composition

Acriflavine hydrochloride 0,05 g

Water 100 ml

B.3.3.2 Preparation

Dissolve the acriflavine hydrochloride in the water.

Sterilize by filtration.

B.3.4 Sodium ceftazidime pentahydrate solution

B.3.4.1 Composition

I
0,116g

I
Sodium ceftazidime pentahydrate
Water 100ml

B.3.4.2 Preparation

Dissolve the sodium ceftazidime in the water.

Sterilize by filtration.

14
 IS 14988 ( Part 2 ) :2002
ISO 11290-2:1998

B.3.5 Complete medium

B.3.5.1 Composition

Agar base (B.3.1) 960 ml

Polymyxin B sulfate solution (B.3.2) 10ml
Acriflavine hydrochloride solution (B.3.3) 10ml
Sodium ceftazidime Dentahvdrate solution (B.3.4) 20 ml

B.3.5.2 Preparation

Add solutions B.3.2, B.3.3 and B.3.4 to the molten base (B.3.1 ) at 47 ‘C, mixing thoroughly between each addition.

The pH of the complete medium shall be 7,2* 0,2 at 25 ‘C.

I
B.3.6 Preparation of the agar plates
Place in each of an appropriate number of Petri dishes (6.1 O) appropriate quantities of the freshly prepared
complete medium (B.3.5). Allow to solidify.

Store the medium away from light.

B.4 Solid culture medium: Tryptone soya yeast extract agar (TSYEA)
B.4.1 Composition

Enzymatic digest of casein 17,0 g

Enzymatic digest of soya meal 3,0 g
Sodium chloride (NaCl) 5,0 g
Dipotassium hydrogen phosphate (K2HP04) 2,5 g
D-Glucose 2,5 g
Yeast extract 6g
Agar 9gto18glJ
Water 1000 ml
1) Depending on the gel strength of the agar.

B.4.2 Preparation

Dissolve the components or the dehydrated complete medium in the water by boiling.

Adjust the pH, if necessary, so that after sterilization it is 7,3 t 0,2 at 25 ‘C.

Dispense the medium into tubes of .$uitable capacity to obtain portions appropriate for the test.

Sterilize for 15 min in the autoclave set at 121 ‘C.

Allow to set in a sloping position.

For the preparation of agar plates, dispense the medium into sterile Petri dishes in portions appropriate for the Wt.
Allow to solidify.

NOTE — If the Henry illumination test is conducted, it is important that the agar medium layer is thin (approximately 12 ml per
dish of 90 mm diameter).

.,

15
 —

IS 14988 ( Part 2 ) :2002

ISO 11290-2:1998

B.5 Liquid culture medium: Tryptone soya yeast extract broth (TSYEB)
6.5.1 Composition

Enzymatic digest of casein 17,0 g

Enzymatic digest of soya meal 3,0 g

Sodium chloride (NaCl) 5,0 g

Dipotassium hydrogen pho.sphate(K2HP04) 2,5 g

D-Glucose 2,5 g

Yeast extract 6g

Water 1000 ml

6.5.2 Prepamtion
Dissolve the components or the dehydrated complete medium in the water, by heating if necessafy.

Adjust the pH, if necessary, so that after sterilization it is 7,3 f 0,2 at 25 “C.

Dispense the medium into flasks or tubes of suitable capacity to obtain portions appropriate for the test.

Sterilize for 15 min in the autoclave set at 121 ‘C.

. .
B.6 Sheep blood agar
6.6.1 Base
B.6.1.1 Composition

15g --- .,
Enzymatic digest of animal tissues
Enzymatic liver digest 2,5 g

Yeast extract 5g

Sodium chloride (NaCl) 5g

Agar 9gto18glJ
Water 1000 ml
I 1) Depending on the gel strength of the agar.

B.6.1.2 Preparation

Dissolve the components or the dehydrated complete medium in the water by boiling.

Adjust the pH, if necessary, so that after sterilization it is 7,2 ~ 0,2 at 25 “C. ,.J

Dispense the medium into flasks of suitable capacity to obtain portions appropriate for the test.

Sterilize for 15 min in the autoclave set at 121 ‘C.

16
 A*

IS 14988 (Pmt2 ):2002

ISO 11290-2:1998

B.6.2 Complete base

B.6.2.1 Composition

Base (B.6.1 ) 100 ml

Defibrinated ShW?D blood 5mlto7ml

B.6.2.2 Preparation

Add the blood to the base previously cooled to about 47 ‘C. Mix well. ,,

Dispense the medium into sterile Petri dishes in portions appropriate for the test. Allow to solidify.”
,.,

B.7 Carbohydrate utilization broth (rhamnose and xylose)

B.7.1 Base

B.7.1.1 Composition

Enzymatic digest of animal tissues log

Meat extract Ig . .
Sodium chloride (NaCl) 5g
Bromocresol purple 0,02 g
Water 1000 mi

B.7.1.2 Preparation

Dissolve the components in the water, by heating if necessary.

.. --
Adjust the pH, if necessary, so that after sterilization it is 6,8 * 0,2 at 25 “C. ,,
3 4

Dispense the medium into tubes of suitable capacity to obtain portions appropriate for the test.
“i

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

B.7.2 Carbohydrate solutions

B.7.2.1 Composition

I Carbohydratef) 5gl
Water 100ml
1) L-Rhamnose or D-xylose.

B.7.2.2 Preparation

Dissolve separately each carbohydrate in 100 ml of water.

Sterilize by filtration.

B.7.3 Complete media

For each carbohydrate, add aseptically x ml of solution B.7.2 to 9X ml of the base (B.7.I ).

17
IS 14988 ( Part 2 ) :2002
ISO 11290-2:1998

B.8 Motility agar

6.8.1 Composition

Enzymatic digest of casein 20,0 g

Enzymatic digest of animal tissues 6,1 g
Agar 3,5 g
Water 1000 ml

B.8.2 Preparation
Dissolve the components in the water by boiling.

Adjust the pH, if necessary, so that after sterilization it is 7,3 * 0,2 at 25 “C.

Dispense the medium into tubes in quantities of about 5 ml.

Sterilize for 15 min in the autoclave set at 121 ‘C.

B.9 CAMP (Christie, Atkins, Munch-Paterson) medium and test strains

Sheep blood agar plates (B.6) maybe used for this test, but it is preferable to use double-layered agar plates with a
very thin layer of sheep blood medium (see B.9.3).

B.9.1 Base
See 3.6.1.

6.9.2 Sheep blood medium

See B.6.2.

6.9.3 Complete medium

Dispense the base (B.9.1 ) into sterile Petri dishes, in quantities of about 12 ml for Petri dishes of 90 mm of diameter,
and allow to solidify. Pour evenly a very thin layer of the sheep blood medium (B.9.2) using amounts not greater
than 3 ml per plate. Allow to solidify.

If the blood medium is added to dishes containing the base which have been prepared in advance, it may be
necessary to warm the dishes for 20 min by placing them in an incubator set at 35 ‘C or 37 ‘C before pouring the
blood medium layer.

B.9.4 CAMP reaction strains

A f3-haemolytic strain of S. aureus (e.g. NCTC 1803 or ATCC 25923), a strain of R. equi (e.g. NCTC 1621 or
ATCC 6939) and a strain of L. monocylogenes (e.g. NCTC 11994) are required to undertake the CAMP test. Not all
strains of S. aureus and L. monocytogenes are suitable for the CAMP test..

Maintain stock cultures of S. aureus, R. equi, L. monocytogenes, L. innooua and L. ivanovii by inoculating the
TSYEA slopes (B.4), incubating them at 35 ‘C or 37 ‘C for 24 h to 28 h, or until growth has occurred, and storing in
the refrigerator at +3 ‘C f 2 ‘C. Subculture at least once per month, and confirm their purity by streahng on TSYEA,
plates (B.4).
 IS 14988 ( Part 2 ) :2002
ISO 11290-2:1998

B.1O Hydrogen peroxide solution

Use a 3 ‘XO(rnhn), i.e. 10 volume, solution.

I
1 6.11 Phosphate-buffered saline (PBS) (optional)
6.11.1 Composition

Disodium hydrogen phosphate dihydrate (Na2HP04.2H20) 8,98 g

Sodium dihydrogen phosphate (Na2HP04) 2,71 g

Sodium chloride (NaCl) 8,5 g
Water 1000 ml

)
B.11.2 Preparation
Dissolve the components in the water.

Adjust the pH, if necessaty, so that after sterilization it is 7,2 k 0,2 at 25 ‘C.

Sterilize in the autoclave for 15 min at 121 ‘C.

.,
6.12 Sheep blood red cell suspension (optional)
Keep the sheep blood cells at +3 ‘C ~ 2 “C before use.

Examine before use for signs of haemolysis (reddening) in the upper serum layer.

If no haemolysis is present, take 2 ml of the lower red cell layer into 98 ml of PBS buffer (B.1 1).

If some haemolysis has occurred, suspend about 4 ml of the red cell layer in 10 ml of PBS buffer and mix gently,
then centrifuge. If the supernatent is obviously reddened by significant haemolysis, do not use and discard the stock
suspension. Otherwise decant the supernatant and add 2 ml of this cells suspension to 98 ml of PBS buffer.

Store the suspension for up to 5 days at +3 “C 12 ‘C. Discard if any haemolysis occurs.

19
IS 14988 ( Part 2 ) :2002
ISO 11290-2:1998

Annex C
.-
.
(informative)

Henry illumination test

Examine the plates using a source of beamed white light, powerful enough to illuminate well the plates, striking the ‘
bottom of the plate at a 45° angle (see figure C.1 ). When examined under this obliquely transmitted light (Henry
illumination) from directly above the plate, colonies of Lkteria spp. exhibit a bluish colour and a granular surface.

Observed by looking
straight down

k
Beamed white light source
1

~“a’e
\

Tripod

~
>.-

a
1n 1 m
I

Figure C.1 — Examination of platesfor suspectcolonies

.,

20
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