- SCOPE适用范围
- This method is applicable to all materials, which contain a high sugar (> 40 o Brix ) content, where an estimate of the total Osmophilic yeast and mold count is desired.
1.1这种方法适用于所有高糖度的原料(> 40 o Brix ),可以评估嗜高渗压酵母菌和霉菌总数。
- APPARATUS设备仪器
- Sterile sampling devices 无菌取样装置
- Autoclavable 150 ml screw cap bottle 150 ml耐高温的带有螺帽的瓶子
- Autoclavable Erlenmeyer flasks 耐高温的锥形瓶
- Sterile Petri dishes (150mm) 灭菌培养皿 (150mm)
- Sterile 10 ml pipettes 灭菌10 ml移液管
- Bunsen burner 煤气灯
- Incubator set at 25 o C 25 o C培养箱
- Constant temperature water bath at 45-47 o C 45-47 o C 的恒温水浴锅
- Autoclave 高压灭菌器
- Balance: to 1000 grams capacity 可称量1000克的天平
- Stirrer/ Hot Plate, stir bar 搅拌器/轻便电炉,搅拌子
- MEDIA
- 40% Sucrose Malt Extract Agar 40%的蔗糖麦芽浸出物琼脂
- Prepare according to the following formula
- Malt Extract 麦芽膏30 grams
- Mycological Peptone 蛋白胨 5 grams
- Agar 琼脂粉15 grams
- Sucrose 蔗糖400 grams
- Distilled Water 蒸馏水 1 liter
- pH5.4
- Alternately, dissolve 400 grams of sucrose and 50 grams of Oxoid Malt Extract Agar in 1 liter of distilled water.
3.3 也可以选择溶解400克的蔗糖和50克的Oxoid公司生产的麦芽精琼脂1升蒸馏水中。
- Dispense into autoclaving autoclavable Erlenmeyer flask and sterilize by autoclaving at 121 o C for 10 minutes. Cool in water bath prior to using.
- 3.4分装到锥形瓶高压灭菌121 o C 10分钟在水浴中冷却待用。
DILUENT- Buffered Peptone Water Supplemented with Glucose用葡萄糖补充到缓冲蛋白胨水中
- Prepare according to the following formula:
- Peptone蛋白胨10 grams
- Sodium Chlori 氯化钠 5 grams
- Disodium Phosphate 磷酸氢二钠 3.5 grams
- Potassium Dihydrogen Phosphate磷酸二氢钾1.5 grams
- Glucose葡萄糖400 grams
- Distilled Water蒸馏水 1 liter
- Alternatively, dissolve 400 grams of glucose and 20 grams of Oxoid Buffered Peptone Water (CM509) in 1 liter of distilled water.
4.3 也可以选择溶解400克的葡萄糖和20克的Oxoid公司生产的缓冲蛋白胨水(CM509)在1升蒸馏水中。
- Dispense 94 mls into 150-ml screw cap bottles.
4.4 分装94ml到150-ml的带有螺帽的瓶子中。
- Cap and sterilize by autoclaving for 15 minutes at 121 o C.
4.5 121 o C灭菌15分钟。
- Cool to ambient temperature prior to use. After cooling, this yields 90 ml of Diluent in each bottle.
4.6在室温中冷却。冷却后每个瓶子里有90 ml稀释液。
- PREPARATION OF STANDARDS
- None.
- PREPARATION OF SAMPLES
- Use aseptic technique. Sample at least 20 grams of test material into a sterile container.
6.1 灭菌容器中最少有20克的样品。
INSTRUMENTAL CONDITIONS
- None
CALIBRATION- None
PROCEDURE- Aseptically pipette 10 grams if test material into 90 mls of Diluent
- 用灭菌移液管将样品10克加到稀释液中。
- Shake vigorously to mix.
8.2 剧烈振摇使其混合。
- Pipette 10 mls into each of the two 150-mm petri dishes.
8.3 吸取10 ml到150-mm的平皿中,并做平行样。
- Pour 35-40 mls of molten 40% Sucrose Malt Extract Agar into each petri dish and swirl to mix.
8.4 倾倒35-40 ml的40%的蔗糖麦芽精培养基到平皿中,旋转摇匀。
- After the agar has solidified, invert the petri dish and place in a 25 o C incubator for 5 days
8.5 等到琼脂凝固后,翻转平皿,放入25 o C培养箱中培养5天。
- Count the colonies on both dishes and calculate the average to yield the number of CFU's per gram of test material.
8.6 计数两个平皿上的菌落并且计算每克样品中所含有的菌落数。
- CALCULATIONS
- None
- SAMPLE CALCULATIONS
- None
- REPORTING RESULTS
- Record results and compare to specification.