ISO 15214 pdf download.Microbiology of food and animal feeding stuffs — Horizontal method for the emumeration of mesophilic lactic acid bacteria — Colony-count technique at 30 °C
1 Scope
This International Standard specifies a horizontal method for the enumeration of viable mesophilic lactic acid bacteria by counting the colonies growing in a solid medium after incubation at 30 °C for 3 days. NOTE In some food products, there exist psychotrophic or thermophilic lactic acid bacteria necessitating culture temperatures different from 30 °C. Moreover, not all lactic acid bacteria grow on MRS agar at pH 5,7 and some grow only weakly. Subject to the limitations discussed in the introduction and in the note above, this International Standard is applicable to products intended for human consumption or animal foodstuffs.
2 Normative references
The following standards contain provisions which, through reference in this text, constitute provisions of this International Standard. At the time of the publication, the editions indicated were valid. All standards are subject to revision, and parties to agreement based on the International Standard are encouraged to investigate the possibility of applying the most recent editions of the standards inidicated below. Members of IEC and ISO maintain registers of currently valid International Standards. ISO 6887-1 :— 1 ) , Microbiology of food and animal feeding stuffs — Preparation of test samples, initial suspension and decimal dilutions for microbiological examination — Part 1 : General rules for the preparation of the intial suspension and decimal dilutions. ISO 721 8:1 996, Microbiology of food and animal feeding stuffs — General rules for microbiological examinations.
3 Definitions
For the purposes of this International Standard, the following definition applies. 3.1 mesophilic lactic acid bacteria bacteria which form colonies 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 dishes using MRS agar at pH 5,7 contained in Petri dishes. Poured-plate or possibly surface 2) inoculation of the dishes 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 dishes, under the same conditions, using decimal dilutions of the test sample or of the initial suspension. Incubation of the dishes at 30 °C for 72 h. 4.3 Calculation of the number of mesophilic lactic acid bacteria (3.1 ) per gram or per millilitre of test sample from the number of colonies obtained in 4.2 in the dishes selected, and possibly confirmed 3) .
5 Diluent and culture medium
5.1 General For current laboratory practice, see ISO 721 8. 5.2 Diluent See ISO 6887-1 . NOTE The buffered peptone water does not always allow satisfactory resuscitation of lactic acid bacteria (see annex A, references [1 ], [2], [3]). 5.3 Culture medium: MRS medium (de Man, Rogosa and Sharpe) at pH 5,7 (see reference [4]) NOTE The use of commercially available ready-to-use media is acceptable. However, attention is drawn to the fact that variations in composition and pH may occur between products from different manufacturers and could therefore give results different from the ones obtained with the medium as specified in this International Standard.
5.3.2 Preparation 5.3.2.1 Dissolve the components or the dehydrated complete medium in the water by boiling. Using the pH-meter (6.7), adjust the pH so that after sterilization it is 5,7 ± 0,1 4) at 25 °C. Transfer the medium to bottles of appropriate capacity. Sterilize for 1 5 min in the autoclave (6.1 ) set at 1 21 °C. If the medium is to be used immediately, cool it before use to appoximately 47 °C in the water bath (6.5), or by any other technique giving equivalent results (see ISO 721 8). If not, in order to avoid any delay when pouring the medium before beginning the microbiological examination, completely melt the medium, in a boiling water bath (6.6), then cool it to approximately 47 °C in the water bath (6.5). 5.3.2.2 If there is a risk of extensive yeast contamination (e.g. in dried sausage), add sorbic acid to the MRS medium as follows. Dissolve 1 ,4 g of sorbic acid in about 1 0 ml of a 1 mol/l solution of sodium hydroxide. Sterilize by filtration. Add this solution to 1 000 ml of sterilized MRS agar, previously cooled to approximately 47 °C. The final pH of the medium shall be 5,7 ± 0,1 at 25 °C.