Lipolytic micro organisms enumeration

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1Scope and field of application适用范围和领域

The method for the detection and enumeration of lipolytic micro organisms in foodstuffs and other materials. 本方法适用于食品和其他材料中分解脂肪微生物的检测和步骤。

2Principle of the method方法的原理

This method is based on Anderson (1939).这一方法建立在Anderson (1939)的基础上。

The culture medium contains tributyrin as a reactant; degradation of this compound gives rise to clear zones surrounding the lipolytic colonies in the otherwise turbid culture medium.
      培养基包含三丁酸甘油酯作为反应物，在浑浊的培养基中因分解这种混合物而清晰出现分解脂肪生物体菌圈。

3Definitions and abbreviations定义和缩写

4Reagents, materials, media试剂、原料、指示剂

Medium/reagenAbbreviation Advised maximal storage time¹

 方法/试剂                              缩写               最长建议保存时间                                             

Tributyrin Agar*), BaTBA1 month  4-10°C

三丁酸甘油酯                       TBA                         4-10°C   1个月

Glycerol tributyrate (Tributyrin)**) see manufacturers description

甘油三丁酸甘油酯                                                         视生产商说明

¹ The shelve life of the prepared media/solutions is given when stored in the dark. Media which can be kept longer than 1 month have to be stored in well closed (screw capped) bottles.

*…….e.g. Tributyrin Agar [Merck Darmstadt cat. No. 1.01957.0500]

**.......e.g. Glycerol tributyrate (Tributyrin) [Merck Darmstadt cat. No 1.01958.0100]

保存在黑暗处的制备好的溶液、指示剂的货架期：用瓶子密封贮存的指示剂能保存1个月以上

Use only media with a good quality, proven by suppliers quality certificate and/or ecometrical test results.使用合适指示剂的优良质量，通过供应商质量证书或检测结果予以证明。

Follow the manufacturer’s description on:制造商的描述为以下方面

1. Storage of the (powdered) media and reagents（粉末）指示剂和试剂的贮藏
2. Preparation of the media指示剂的制备
3. Storage of the prepared media制备好的指示剂的贮藏

5Equipment仪器

Usual microbiological equipment.通常微生物技术仪器

6Procedure 程序

6.1Enumeration 步骤

1. Prepare of a resuscitated sample, a suitable dilution series dependent on the presumed contamination level of the sample (see method M01_01ME). The number of colonies on a plate should not exceed 300 cfu. For a better suspension of oils add 1% Tween 80

样品复苏的制备：（参照方法M01）在估计样品污染水平的基础之上确定合适的稀释倍数， 一个平皿上的菌落数不超过300 cfu，在油中添加1%的土温80以制备好的悬浮液。

2. Pipette 1 ml of the appropriate dilution into a petri dish.

每一个碟中用刻度吸管加入1ml适当稀释的样品

3. Pour approximately 15 ml of the TBA (tempered at 45±2°C) into the petri dish, mix and allow to set.
   在有盖的培养皿中加入大约15ml TBA（温度在45±2°C），混合然后放置
4. Incubate the plates up side down for 72 ±3 h. at the 28°C *)
   平皿翻转倒放在28°C条件下培养72 ±3小时。

* To avoid drying out of the plates they should be packed in a well-closed plastic bag or box. Add some tissue paper to the plastic bag to absorb condensate water which helps to prevent spreading of the colonies.

放入密闭良好的塑料袋或盒子中避免烘干平皿；在塑料袋中加入一些薄的纸吸收冷凝水帮助防止菌体散布。

5. Lipolytic microorganisms produce colonies which are surrounded by clear zones in the otherwise turbid culture medium. Count all colonies with appearance as described before.

分解脂肪的微生物产生的菌体在混浊培养基里被明显的圈所环绕，按前面描述的外观计算所有的生物体。

6.2Confirmation证实

Not necessary 没有必要

6.3Internal control内部控制

Positive control: Pseudomonas aeruginosa ATCC 27853

阳性控制;

Staphylococcus aureusATCC 25923

                                                         葡萄球状菌

Bacillus subtilis                                    ATCC 6633

                                细菌    

Negative control: g Irradiated infant milk formula or a comparable g rradiated product

阴性控制: g辐照婴幼儿乳制品或相当于g辐照的产品。

7Expression of results, calculation结果的计算与表达

Calculate the number of cfu as described in M01_01AA. Express the results as cfu  per volume or gram of sample.

            按M01_01AA中的描述计算菌落数量，按每克或每体积样品表示结果

8Validation确认

outstanding

9References备注

The above mentioned methods is based on the following references but do not necessarily reflect the exact methodologies.上面提及的方法是基于以下参考但不能表现精确的方法理论。

1. ANDERSON, J.A.: The use of tributyrin agar in dairy bacteriology. - Ber. 3. Int. Mikrobiol. Kongress, 3; 726-728 (1939)
2. EL SADEK, G.M., a. RICHARDS, T.: Nile blue, aniline blue and neutral red as indicators of lipolysis. - J. Appl. Bact., 20; 137 (1959).
3. INNES, A.G.: Coagulase positive Staphylococci from bulk milk supplies low in solids-notfat. - J. Appl. Bact., 19; 39-45 (1956).
4. HAYES, P.R.: Studies on marine flavobacteria. - J. Gen. Microbiol., 30; 1-19 (1963).
   RAPP, M.: Elektive Nährmedien zum Nachweis von Lipolyten. - Milchwirtsch., 33; 493-496 (1978).
5. WILLIS, A.T.: The lipolytic activity of some clostridia. - J. Path. Bact., 80; 379-390 (1960).

10Enclosures, addenda

Not applicable