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1、CARGILL JUICE BEVERAGE APPLICATIONS - AMSTERDAM NETHERLANDSL.2.28 ATSB MICROBIOLOGYDATE n.a.ISSUE 011. Method of detection of ATSB(ATSB = Acido-Thermoresistant Sporeforming Bacteria)Materials 材料- 250 ml bottle with heat resistant screwcap. 250 毫升带有抗热螺丝钉的瓶子- 100 ml bottle with aluminium screwcap. 100
2、 毫升带有率螺丝钉的瓶子- Demiwater - PDA agar merck PDA 培养基- Magnetic stirrer/heater 磁力加热器- 2000 ml erlenmeyer2000 毫升的 erlenmeyer- Dosagepipet of 1 milliliter 千分之一毫升的吸量管- Pipet 1 ml 1 毫升的吸量管- 9.5% tartaric acid solution.9.5%的酒石酸溶液- Temperature adjustable water-bath 可调节温度的水浴- Sterile sample spoon or sterile hyp
3、odermic syringe (10ml).消过毒的样品匙或者消过毒的注射器（10 毫升）- Petri dish 14cm diameter14 厘米直径的 Petri 盘- Incubator 培养器medium ATSB.ATSB 媒介- Fill 90ml demiwater in 250ml bottle with screwcap, sterilize in autoclave during 15 minutes at 121C. - 将 90 毫升的 demiwater 注入 250 毫升带有抗热螺丝钉的瓶子，放在高压锅中在 121C 条件下杀菌 15 分钟。- Weigh 1
4、17.0 g of PDA agar- 称量 117 克 PDA 培养基- Dissolve the agar into 2000 g of demi water while heating/steering.- 在加热搅拌过程中，将培养基溶解在 2000 克 demi water 中- Fill 100 gram of dissolved agar into 100 ml bottle with alucap and sterilize during 15 minutes at 121C.- 将 100 克溶解后的培养基注入 100 毫升带有率螺丝钉的瓶子，并在 121C 的条件下杀菌 15
5、分钟- Cool solution back to 60C and add 1.0 ml 9.5% tartaric solution - 将溶液冷却至 60C 并 加入 1 毫升 9.5%的酒石酸溶液- Cool further to 47.5C prior usage. - 在使用前进一步冷却至 47.5CProcedure- For single strength juice: pour 100 ml of sample in an empty sterile screw-cap bottle.- 单一浓度的果汁：将 100 毫升的样品注入杀过菌的带有螺丝的空瓶中。- For conce
6、ntrate add 10 grams of sample into a sterile screw-cap bottle containing 90 ml of sterile distilled water.CARGILL JUICE BEVERAGE APPLICATIONS - AMSTERDAM NETHERLANDSL.2.28 ATSB MICROBIOLOGYDATE n.a.ISSUE 01- 浓缩果汁：将 10 毫升样品加入含有 90 毫升灭菌蒸馏水的带有螺丝的瓶子中- Heat-shock the sample by placing the bottle in an 80
7、C water-bath for 10 minutes. - 在 80C 的水浴中加热样品 10 分钟。- Start timing when the sample temperature reaches 80C, and be sure that the water level in the bath is above the level of sample in the bottle.- 在样品温度达到 80C 时开始计时，并且保证水的液面高于瓶中样品的液面。- Quickly cool the sample by placing into ice or cold water.- 将样品至
8、于冰水或者冷水中快速冷却。- Pour 50 ml of the agar into the 100 ml sample.- 将 50 毫升的培养基注入 100 毫升的样品中。- Divide the 150 ml mix over 3 large petri dishes, and swirl to mix well- 将 150 毫升的混合物分在 3 个大的 petr 盘中，并充分搅匀。- Allow the plates to solidify- 允许盘子凝固- Incubate the plates for 5 days at 46C +/- 1 C- 在 46+/-1C 的温度下，培
9、养 5 天- Count the total of colonies on all three plates and record as:- 将 3 个盘子中的菌落群体计数并记录如下：- A) For single strength juice: Divide the total number of colonies on all three plates by 10 to obtain the count of Acido-Thermoresistant Sporeforming Bacteria in cfu/ 10 ml of juice. (Sensitivity = 0.01 cfu
10、/ml).- ）单一浓度的果汁：用除三个盘子中的菌落总数作为 10 克果汁中含有的 ATSB 数量（灵敏度= 0.01cfu/ml）- B) For concentrated juice: Record the total number of colonies on all three plates as is to obtain the amount of Acido-Thermoresistant Sporeforming Bacteria in cfu/10 gram of concentrate. - ）浓缩果汁：记录三个培养皿中的菌落总数作为 10 克浓缩果汁中含有的 ATSB 数量
