IS 14844 (2000): Meat and Meat Products - Enumeration of

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IS 14844 (2000): Meat and Meat Products - Enumeration ofLactic Acid Bacteria - Colony-Count Technique at 30C [FAD18: Slaughter House and Meat Industry]

IS 14844 : 2000 IS0 13721 : 1995

Indian Standard

MEAT AND MEAT PRODUCTS - ENUMERATION OF LACTIC ACID BACTERIA - COLONY-COUNT

TECHNIQUE AT 30°C

ICS 67.120.10

0 BIS 2000

BUREAU OF INDIAN STANDARDS MANAK BHAVAN, 9 BAHADUR SHAH ZAFAR MARG

NEW DELHI 110002

June 2000 Price Group 3

Slaughter House and Meat Industry Sectional Committee, FAD 56

NATIONAL FOREWORD

This Indian Standard which is identical with IS0 13721’ : 1995 ‘Meat and meat products - Enumeration of lactic acid bacteria - Colony-count technique at 30°C’ issued by the International Organization for Standardization (ISO) was adopted by the Bureau of Indian Standards on the recommendation of the Slaughter House and Meat Industry 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.

Technical Corrigendum 1 to the above International Standard has been incorporated.

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

In terna tional Standard Corresponding Indian Standard Degree of Equivalence

IS0 3190-1 :1991 Meat and meat products - Sampling and preparation of test samples - Part 1 : Sampling

IS0 3100-2 : 1988 Meat and meat products - Sampling and preparation of test samples - Part 2 : Preparation of test samples for microbiological examination

IS0 6887 : 1983 Microbiol-

ogy - General guidance for the preparation of dilutions for microbiological examination

IS0 7218 : 1996 Microbiol- ogy of food and animal fee- ding stuffs - General rules for microbiological examin- ations

IS 3100 ( Part 1) : 1996 Meat and meat products - Method of sampling

Identical

IS 5404 : 1984 Code of practice for handling of samples for microbiological analysis ( first revision )

Equivalent

IS 10232 : 1982 Guidelines for preparation of dilutions for microbiological examination for food

do

IS 5404 : 1984 Code of practice for handling of samples for microbiological analysis ( first revision )

Related

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 mumerical values ( revised )‘.

IS 14844 : 2000 IS0 13721 : 1995

Indian Standard

MEAT AND MEAT PRODUCTS - ENUMERATION -OF LACTIC ACID BACTERIA - COLONY-CO~UNT

TECHNIQUE AT 30°C

1 Scope

This International Standard specifies a method for the

enumeration of viable lactic acid bacteria in meat and meat products, including poultry, by counting the colonies growing in a solid medium after aerobic incubation at 30 “C for 3 days.

2 Normative references

The following standards contain provisions which, through reference in this 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 re- cent editions of the standards indicated below. Members of IEC and IS0 maintain registers of cur- rently valid International Standards.

IS0 3100-2:1988, Meat and meat products - Sam- pling and preparation of test samples - Part 2: Preparation of test samples for microbiological examination.

IS0 6887: 1983, Microbiology - General guidawe for the preparation of dilutions for microbiological examination.

IS0 7218:--“, Microbiology of food and animal feed- ing stuffs - General rules for microbiological exam- inations.

3.1 lactic acid bacteria: Bacteria which form colon-

ies at 30 “C in a solid selective medium (MRS at pH 5,7) under the test conditions specified in this International Standard.

4 Principle

4.1 Preparation of two poured plates, using MRS agar at pH 5,7 contained in Petri dishes. Inoculation of the plates with a specified quantity of the test sample if the initial product is liquid, or with a specified quantity of the initial suspension in the case of other products.

4.2 Inoculation of other pairs of poured plates, under the same conditions, using decimal dilutions of the test sample or of the initial suspension.

Incubation of the plates at 30 “C for 72 h.

4.3 Calculation of the number of lactic acid bacteria per gram or millilitre of test sample from the number of co!onies obtained in the dishes selected.

5 Dilution fluid and culture medium

5.1 General

For current laboratory practice, see IS0 7218.

5.2 Dilution fluid

See IS0 6887.

3 Definition

For the purposes of this International Standard, the following definition applies.

1) To be published. (Revision of IS0 7218:1985)

5.3 Culture medium (MRS medium) at pH 5,7 (see reference Cl])

IS 14844 : 2000 IS0 13721 : 1995

NOTE 1 The use of commercially available media is acceptable, however attention is drawn to the fact that vari- atrons in composition may occur between products from drfferent manufacturers

5.3.1 Composition

2eptone 10,o 9 Meat extract 8.0 g Yeast extract 500 g Triammonium citrate [(NH,),C,H,O,] 20 g Sodium acetate (CH,COONa) 58 g Magnesium sulfate heptahydrate (MgS0,.7H,O) 0.2 g Manganese sulfate tetrahydrate (MnSO,.4H,O) 0,05 g Dipotassium hydrogen phosphate (K,HPO,) 20 g Glucose (C,H,,O,) 20,o g Sorbitan monooleate (Tween 30) 1.0 g Agar 12gtol8g’)

Water 1 000 ml

1) Depending on the gel strength of the agar.

5.3.2 Preparation

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

Adjust the pH (6.71 using hydrochloric acid (approx. 1 mot/l solution) so that after sterilization it is 5,7 at 25 “C.

Transfer the medium to bottles of capacity not more

than 300 ml.

Sterilize for 15 min in the autoclave (6.1) set at 121 “C. If the medium is to be used immediately, cool it to 47 “C in the water bath 16.5) before use.

If not, before beginning the microbiological exam-

ination, in order to avoid any delay when pouring the medium, completely melt the medium in a boiling

water bath (6.6). then cool it to 47 “C in the water bath (6.5).

5.3.2.2 If there is a risk of extensive yeast contami- nation (e.g. in dried sausage), add sorbic acid to the MRS medium as follows.

Dissolve 1,4 g of sorbic acid in about 10 ml of a 1 mol/l solution of sodium hydroxide. Adjust the pH to 5,8 and sterilize by filtration. Add this solution to 1 000 ml of sterilized MRS agar. The final pH of the medium should be 5.7.

6 Apparatus and glassware

NOTE 2 Disposable apparatus is an acceptable alterna- tive to reusable glassware if it has suitable specifications.

Usual microbiological laboratory apparatus and, in particular, the following.

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

See IS0 7218.

6.2 Incubator, capable of operating at 30 oc

* 1 "C.

6.3 Petri dishes, made af glass or plastic, of diam- eter 90 mm to 100 mm.

6.4 Total-delivery (Mow-out) pipettes, having wide openings and having a nominal capacity of 1 ml, graduated in 0,l ml divisions.

6.5 Water bath, or similar apparatus, capable of operating at 47 “C * 2 “C.

6.6 Boiling water bath

6.7 pH-meter, accurate to within f 0,l pH unit at 25 “C.

7 Sampling

It is important that the laboratory receive a sample which is truly representative and has not been dam- aged or changed during transport or storage.

Sampling is not part of the method specified in this International Standard. A recommended method is given in IS0 3100-Ill.

Store the sample, if necessary, in such a deterioration and change in composition

vented.

8 Preparation of test sample

sampling

way that are pre-

Prepare the test sample in accordance with IS0 3100-2.

2

9 Procedure

9.1 Test portion, initial suspension and dilutions

Prepare the intial suspension and dilutions in accordance with IS0 6887.

9.2 Inoculation and incubation

NOTE 3 Surface plating in combination with incubation under anaerobic or microaerobic conditions can be applied instead of the pour-plating procedure described. Candle jars may be used to obtain appropriate conditions.

9.2.1 Take two sterile Petri dishes (6.3). Using a sterile pipette (6.4), transfer to each dish 1 ml of the test sample if the product is liquid, or 1 ml of the in-

itial suspension in the case of other products.

Take two other sterile Petri dishes. Using a fresh sterile pipette, transfer to each dish 1 ml of the first decimal dilution (IO- ‘1 of the test sample if the product is liquid, or 1 ml of the first decimal dilution of the initial suspension (1 O-*) in the case of other products.

Repeat the procedure described with the further dilutions, using a fresh sterile pipette for each decimal dilution.

NOTE 4 If high numbers of lactic acid bacteria are ex- pected, plate out only those dilutions necessary to achieve the correct colony range (9.3).

9.2.2 Pour into each Petri dish approximately 15 ml

of the MRS medium (5.3) which has been prepared then cooled to approximately 47 “C in the water bath !6.5). The time elapsing between the end of the preparation of the initial suspension (or of the IO-’ dilution if the product is liquid) and the moment when the medium (5.3) is poured into the dishes shall not exceed 15 min.

Carefully mix the inoculum with the medium by horizontal movements and allow the mixture to solidify, with the Petri dishes standing on a cool horizontal surface.

9.2.3 Invert the prepared dishes and incubate them in the incubator (6.2) set at 30 “c for 72 h -C 3 h.

9.3 Counting and selection of colonies

After the specified period of time (see 9.2.3). count the colonies in each dish. Retain dishes containing fewer than 300 colonies at two successive dilutions.

10 Expression of results and calculation

IS 14844 : 2000 IS0 13721 : 1995

10.1 Calculate the number N of lactic acid bacteria present in the test sample, as the weighted mean from two successive dilutions, using the equation:

c a N=

V(n, + 0,l n,)d

where

Ca

V

n1

"2

d

is the sum of the colonies counted on all the dishes from the two successive dilutions which contain at least 15 colonies;

is the volume of inoculum applied to each dish, in millilitres;

is the number of dishes retained at the first dilution;

is the number of dishes retained at the second dilution;

is the dilution factor corresponding to the first dilution retained.

Round off the results calculated to two significant figures. For this, if the last figure is below 5, the preceding figure is not modified; if the last figure is 5 or more, the preceding figure is increased by one unit. Proceed stepwise until two significant figures are ob-

tained.

Take as the result the number of lactic acid bacteria per millilitre (liquid product) or per gram (other product), expressed as a number between I,0 and 9,9 multiplied by the appropriate power of 10.

EXAMPLE

c a N= = 168+215+14+25 =

V(n, + O,l%)d I(2 + 0,l x 2)10-*

422 =-= 19 182 0,022

Round off to two significant figures, namely 19 000

or I,9 x 1 O4 lactic acid bacteria per gram of product.

10.2 If the two dishes, at the level of the test sample (liquid product) or of the initial suspension (other product), contain less than 15 colonies, calculate the arithmetical mean y of the colonies covnted on two dishes.

3

IS 14844:2000 Iso 13721:lQQS

Express the result as follows:

for liquid products: estimated number of lactic acid

bacteria per millilitre NE = y

for the other products: estimated number of lactic acid bacteria per gram NE = y x f

where d is the dilution factor of the initial suspension.

10.3 If the two dishes at the level of the test sample (liquid product) or of the initial suspension (other product) do not contain any colonies, express the result as follows:

less than 1 lactic acid bacterium per millilitre

(liquid product)

less than 1 x f lactic acid bacterium per gram (other product)

where d is the dilution factor of the initial suspension.

11 Confidence limits

12 Test report

The test report shall specify:

- the method in accordance with which sampling was carried out, if known;

- the method used;

- the test result(s) obtained; and

- if the repeatability has been checked, the final quoted result obtained.

It shall also mention all operating details not specified in this International Standard, or regarded as optional, together with details of any incidents which may have influenced the test result(s).

The test report shall include all information necessary for the complete identification of the sample.

For calculation of the confidence intervals, see IS0 7218.

4

IS 14844 : 2000 IS0 13721 : 1995

Annex A (informative)

Bibliography

[I] Pharmacopoeia of Culture Media for Food Microbiology: De Man, Rogosa and Sharpe agar with sorbic acid (MRS-S agar). Int. J. Food Microbial., 5, 1987, pp. 230-232.

[2] IS0 3100-l :I 991, Meat and meat products - Sampling and preparation of rest samples - Part 1: Sampling.

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BIS is a statutory institution established under the Bureau oflndian StandardsAct, 1986 to promote harmonious development of the activities of standardization, marking and quality certification of goods and attending to connected matters in the country.

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Review of Indian Standards

Amendments are issued to standards as the need arises on the basis of comments. Standards are also reviewed periodically; a standard along with amendments is reaffirmed when such review indicates that no changes are needed; if the review indicates that changes are needed, it is taken up for revision. Users of Indian Standards should ascertain that they are in possession of the latest amendments or edition by referring to the latest issue of ‘EIS Handbook’ and ‘Standards : Monthly Additions’.

This Indian Standard has been developed from Dot : No FAD 56 ( 1024 ),

Amend No.

Amendments Issued Since Puhlicatipn

Date of Issue -

Text Affected

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IS 14844 (2000): Meat and Meat Products - Enumeration of