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1、1. Scope and field of application 适用范围This LI describes routine methods for the detection of Aerobic Bacterial Count in foods.本操作描述了食品中菌落总数的检验方法Aerobic Bacterial Count is commonly used as microbiological indicator for contaminant evaluation in food. This method also can be used for evaluate the tren
2、d of bacteria increase in food and provide the evidence for hygiene evaluation of the test sample.菌落总数主要作为判定食品被污染程度的标志，也可以应用这一方法观察细菌在食品中繁殖的动态，以便对被检样品进行卫生学评价时提供依据。2. Definitions 定义Under appropriate condition (media, temperature, time, pH and aerobic culture etc.), the total plate count of forming col
3、onies in 1 ml (g) treated food sample. 食品检样经过处理，在一定条件下培养后（如培养基成分、培养温度和时间、pH、需氧性质等），所得1ml(g)检样中所含菌落的总数。3. Principle of the method 原理Aliquots of decimal dilutions of the sample are mixed with culture medium in Petri dishes. Colonies are counted after incubation at 361 OC for 482h and the number of mic
4、roorganisms per gram or ml of the sample is calculated.吸取样品稀释液，使之在平板内与培养基混合，361 OC培养482小时，然后计数。4. Reagent and media 培养基和试剂 Nutrition Agar 营养琼脂5. Procedure 步骤5.1 Preparation of samples 样品准备Weight 25g(ml) sample in 225ml sterilized buffer liquid, homogenized if necessary. 称25克（毫升）样品到225毫升缓冲液，如有必要将样品打匀
5、。5.2 Plating 接种和倒板Aliquots of 1ml of the dilutions to be analyzed are placed into Petri dishes. Add about 15ml of liquid NA (cooled to 461 OC in water bath) within 15 minutes after preparation of dilutions. Mix carefully inoculum and medium and allow the agar to cool and solidify. Test two parallel
6、plates for each dilution. 吸1ml样品稀释液到平板中，然后倒15毫升营养琼脂（在水浴锅内冷却至461 OC)进去, 从样品稀释至倒平板不应 超过15分钟, 小心混匀, 待其凝固. 每个稀释度做两个平皿。5.3 Incubation 培养Plates are inverted and incubated for 482h at 361 OC. Do not stack more than 6 plates.倒放于361 OC，培养482小时。把板垒成一叠时，一叠不要超过6块。5.4 Reading 读板After incubation colonies are coun
7、ted. Examine the plates carefully, if necessary with a lens, to avoid mistaking particle or precipitates for pinpoint colonies. Count the weighted mean of each dilution.板上所有菌落都数出来，如有必要可用放大镜。算出同稀释度的各平板平均菌落总数。* Note 注意Use counts from all plates containing between 30 and 300 colonies, calculate the wei
8、ghted mean of two plates for each dilution. The plate should not be counted which contains a big flake colony exceed half of dish, only the other be counted with no flake colony; if the flake colony is not meet half of dish and the colonies distribute equably in another half, the total plate count i
9、s two times of half dish number. If catenulate colonies (no clear border) presence, count each catenulate colony as one.选取菌落数在30-300之间的平板作为菌落总数测定标准。一个稀释度使用两个平板，应采用两个平板平均数。其中一个平板有较大片状菌落生长时，则不宜采用，而应以无片状的菌落生长的平板作为该稀释度的菌落数；若片状菌落不到平板的一半，而其余一半中菌落分布又很均匀，即可计算半个平板后乘2以代表全平皿菌落数。平板内如有链状菌落生长时（菌落至今无明显界限），若仅有一条链，可
10、视为一个菌落；如果有不同来源的几条链，则应将每条链作为一个菌落计。6. Calculation 计数6.1 General Case 一般情况Calculate the amount as the weighted mean from the successive dilutions, which contain between 30 and 300 colonies. (See example 1 in attached sheet 1). The calculate result is the weighted means of the successive dilution multip
11、ly by dilution factor.应选择平均菌落数在30-300之间的稀释度，乘以稀释倍数报告之（见表1中例1）。6.2 Special Case 特殊情况I. If all weighted means are between 30 and 300 of two different dilutions, the calculate result should be decided by the ratio of two weighted means. If the ratio less than or equal to 2, the calculate result is the
12、mean value of two weighted means; if the ratio more than 2, the calculate result is the smaller value of the two weighted means. (See example 2&3 in attached sheet 1)若有两个稀释度，其生长的菌落数均在30-300之间， 则视两者之间的比值如何来决定。若其比值小于或等于2，应报告其平均数；若大于2则报告其中较小的数字（见表1中例2及例3）。II. If all weighted means are more than 300, th
13、e calculate result is the weighted means of the highest dilution multiply by dilution factor. (See example 4 in attached sheet 1)若所有稀释度的平均菌落数均大于300，则应按稀释度最高的平均菌落数乘以稀释倍数报告之（见表1中例4）。III. If all weighted means are less than 30, the calculate result is the weighted means of the lowest dilution multiply
14、by dilution factor. (See example 5 in attached sheet 1)若所有稀释度的平均菌落数均小于30，则应按稀释度最低的平均菌落数乘以稀释倍数报告之（见表1中例5）。IV. If none of the Petri dishes contains any colonies, the result expressed as less than 1 multiply the lowest dilution factor. (See example 6 in attached sheet 1)若所有稀释度的均无菌落数生长，则以小于1乘以最低稀释倍数报告之（
15、见表1中例6）。V. If not all weighted means are between 30 and 300, some value more than 300 or less than 30, the calculate result is the weighted means which closed 30 or 300 multiply by dilution factor. (See example 7 in attached sheet 1)若所有稀释度的平均菌落数均不在30-300之间，其中一部分大于300或小于30时，则以最接近30或300的平均菌落数乘以稀释倍数报告之
16、（见表1中例7）。6.3 Result Reporting 结果报告If the calculate result less than 100, report the actual result; otherwise round off the results calculated to two significant figures. Also can expressed as a number between 1.0 and 9.9 times the appropriate power of 10. (See attached sheet 1)菌落数在100以内时，按其实有数报告；大于1
17、00时，采用两位有效数字，在两位有效数字后面的数值，以四舍五入方法计算。也可用10的指数法来表示（见表1）。表1 稀释度选择及菌落数报告方式例次 Example稀释液及菌落数Dilution and Colony Number两稀释液之比Number Ratio of Two Dilution菌落总数Calculate Result报告方式Report Result10-110-210-3cfu/g(ml)cfu/g(ml)1多不可计TNTC16420-16 40016 000 或 1.6 X 1042多不可计TNTC295461.637 75038 000 或 3.8 X 1043多不可计TNTC271602.227 10027 000 或 2.7 X 1044多不可计TNTC多不可计TNTC313-313 000310 000 或 3.1 X 105527115-270270 或 2.7 X 1026000-1 X 10107多不可计TNTC30512-30 50031 000 或 3.1 X 104
