百事微生物检测方法.doc

百事：微生物检测方法1.目的 PURPOSE概述非碳酸饮料产品的耐酸细菌、酵母菌和霉菌的分析程序。To outline the procedure for analysis of NCB products for Aciduric Bacteria, Yeast and Mould.2.范围 SCOPE本程序适用于需要精确测定存在的耐酸细菌、酵母菌和霉菌的非碳酸饮料产品。这些有机物有可能导致饮料的腐败。采用浇灌平板法对这些参数进行分析，酵母菌和霉菌用的是PDA，耐酸细菌用的是OSA。The procedure is applicable to NCB products where an accurate enumeration of the Aciduric Bacteria, Yeast and Mould present is required. These organisms may potentially cause spoilage of beverages. Analysis for these parameters is conducted using a pour plate methods using PDA for Yeast and Mould and OSA for Aciduric Bacteria.3.程序 PROCEDURE3.1设备 Equipment-无菌移液管 10ml容量Sterile Pipettes 10ml Capacity-无菌的皮氏培养皿 150mmSterile Petri Dishes 150mm-设定为30C +/- 1C的恒温箱Incubator set at 30C +/- 1C-设定为25C +/- 1C的恒温箱Incubator set at 25C +/- 1C-恒温水浴，45 +/- 1CConstant Temperature Waterbath at 45 +/- 1C-高压蒸汽灭菌器Autoclave-微波Microwave-纸巾Tissues-0.2 m过滤器0.2 m Filter-注射器(5ml或10ml)Syringe (5ml or 10ml)3.2试剂 Reagents-橘汁琼脂（Oxoid或同类产品）Orange Serum Agar (Oxoid or Equivalent)-马铃薯右旋糖琼脂（Oxoid或同类产品）Potato Dextrose Agar (Oxoid or Equivalent)-去离子水Deionised Water-乙醇/异丙醇70%Ethyl/Iso-propyl Alcohol 70%3.3标准 Standards不适用N/A3.4试验的准备 Test Preparation3.4.1工作台的准备 Preparation of Workstation层流装置的准备（任选）Laminar Flow Preparation (Optional)清洁装置并喷上70%的异丙醇或普通酒精，并用纸巾擦干；或让其蒸发。保证载片是关闭的，同时打开紫外灯。务必保证下列条件：Clean the cabinet and spray with 70% isopropyl or ethyl alcohol and dry with tissues or allow to evaporate. Ensure the sliding glass is closed and turn on the UV lamp. Always ensure the following:-使用紫外线时滑动窗要保持关闭。Keep the sliding window closed when UV is in use.-打开滑动窗之前要关闭紫外灯。Turn off the UV light before open the sliding window.-样品、培养基或者你本人在任何时候都不得接触紫外线。Do not expose samples, media or yourself to UV light at any time.试验之前至少15分钟就要接通装置的鼓风机，以便清除装置中的空气并代之以经过过滤的空气。Turn ON the cabinet blower at least 15 minutes before testing to allow the cabinet purge the air inside the cabinet and replace with filtered air. 概述 General微生物学化验员洗手并喷上70%的异丙醇或乙醇。Microbiology analysts wash hands and spray with 70% isopropyl or ethyl alcohol.应当穿上清洁并且适合的专用实验室外套（即袖口和领口都系紧）。Clean suitable designated microbiology lab coats should be worn (i.e. tied cuffs and closed upto the neck).利用柔性的家庭用洗涤剂清洗分析区。Clean analysis area with a mild household detergent.利用70%的异丙醇或普通酒精擦拭分析区，用纸巾擦干或让其蒸发。Swab analysis area with 70% isopropyl or ethyl alcohol and dry with tissues or allow to evaporate.调整试验歧管并与真空烧瓶和真空泵相连（见图1.3）。Set up testing manifold and connect to vacuum flask and pump (see Fig. 1.3)真空泵必须能够形成20 27英寸汞柱或 0.2 巴的真空度。Pump must be capable of creating a vacuum of 20 - 27 inches Hg or 0.2 Bar.试验之前应当在试验台上摆放下列物品;The following items should be placed on the analysis bench prior to testing;-干净的空容器（废弃物用）clean, empty container (for waste)-开瓶器浸泡在70%的异丙醇或普通酒精中bottle opener dipped in 70% isopropyl or ethyl alcohol-70%异丙醇或普通酒精的喷雾瓶70% isopropyl or ethyl alcohol spray bottle-样品平板用的托盘tray for sample plates-纸巾 tissues试验台附近应当放两个空箱一个而用来处理打开样品后的瓶盖和纸巾，另一个用来处理在给样品进行过滤之后的一次性滤膜漏斗的圆柱部分。Two empty bins should be placed near testing station - one for disposal of caps and tissues after opening samples and the other for disposal of cylindrical section of monitors after filtering samples.3.4.3环境监测 Environmental Monitoring-空气平板PDA琼脂 Air Plates PDA Agar给两个预先浇灌的PDA平板贴上标签并记录在实验室空气监测记录中。Label 2 pre-poured PDA plates and record in Lab Air Monitoring record.将一个PDA平板放入样品分析区（靠近歧管），另一个放入样品制备区（打开样品的地方）。Place one of the PDA plates in sample analysis area (next to manifold) and the other in sample preparation area (where samples are opened).一旦开始样品分析，就要打开平板的盖子让其曝光30分钟。Once sample analysis begins, open lids of plates for 30 minutes exposure.盖上平板并将其在25 +/-1条件下倒置培养120小时（5天）+/- 3小时。Close and incubate plates inverted at 25C +/-1C for 120 hours (5 days)+/- 3hrs.或者 Or-空气平板m-Green肉汤 Air Plates m-Green Broth给两个0.45m的一次性滤膜漏斗贴上标签并记录在空气监测记录中。Label 2 0.45m monitors and record in Lab Air Monitoring record.分析开始前，将这些一次性滤膜漏斗放在歧管上，给每个滤膜漏斗加入2-3ml的YMB肉汤。如果使用安瓿，在从上面给每个滤膜漏斗加一份。Before analysis commences place these monitors on manifold and add 2-3ml YMB to each monitor. If using ampoules, add 1 to each monitor by the top.接通真空源而将肉汤吸入薄膜。短暂地接通真空源，让培养基渗入薄膜但不要完全渗透吸附垫。 薄膜表面不残留液体是很重要的，因为这会导致菌落扩散，从而不能得到精确的计数。不要将培养基吸引到吸附垫之外也是重要的，因为这会让薄膜变干并排干培养基，使其不利于有机物的生长。Turn on vacuum and pull broth into membrane. Care should be taken to apply the vacuum briefly to pull the media into the membrane but not all the way through the absorbent pad. It is important that no liquid remains on the surface of the membrane, as this will cause colonies to spread, making it impossible to get an accurate count. It is also important that the media not be drawn out of the absorbent pad as this will dry the membrane and will eliminate the media, making it inaccessible for organism growth.关闭真空源并取出一次性滤膜漏斗。Close the vacuum and remove monitors.将一个一次性滤膜漏斗放入样品分析区（靠近歧管），另一个放入样品制备区（打开样品的地方）。Place one of the monitors in sample analysis area (next to manifold) and the other in sample preparation area (where samples are opened).曝光30分钟后，解开一次性滤膜漏斗的圆柱段，将薄膜部分放到盖子上，盖住平板并在25C +/-1 C条件下培养120小时（5天）+/-3小时。After 30 minutes exposure, disassemble cylindrical section of monitors, place membrane portion onto lid, cap and incubate plates at 25C +/-1 C for 120 hours (5 days)+/- 3hrs.3.5样品分析 Sample Analysis开始分析之前，将OSA和 PDA 融化并在45 + 1的水浴中调整，直到做好使用准备。Prior to commencing analysis, melt OSA and PDA and temper in waterbath at 45 C + 1 C until ready to use.分析之前，制备要加入到PDA中而将其酸化至pH 3.5的酒石酸（10%）；Preparation of Tartaric Acid (10%) to be added to PDA to acidify it to pH 3.5 prior to analysis;称取10g的酒石酸并加入100 ml无菌的蒸馏水/去离子水。Weigh 10g Tartaric Acid and add 100 ml sterile distilled/deionised water.搅拌直至完全溶解。Mix until fully dissolved.经过一个0.2微米的过滤器过滤而杀菌。Sterilise by filtering through a 0.2 m filter.采用无菌方式，向每100ml PDA中加入18 ml酒石酸，也就是向1升PDA中加入18ml酒石酸（调和至45）。Aseptically add 1.8ml Tartaric Acid per 100ml PDA i.e. 18 ml Tartaric Acid is added to 1 litre PDA (tempered to 45C).取出一部分培养基检查PDA的pH值，利用一个有刻度的pH计来检查pH值。仪表的示数应当为3.5 +/- 0.2。Check pH of PDA by removing an aliquot of media and checking pH with a calibrated pH meter. It should read 3.5 +/- 0.2.如果是在分析之前制备的，那么培养基就可以直接从高压蒸汽灭菌器转移到水浴中。Media may be transferred directly from autoclave to waterbath if prepared prior to analysis.在各个样品之前摆放150mm的皮氏培养皿。从样品上取下1个WinLIMS标签，并将其贴到皮氏培养皿的底部。Place 150mm petri dishes in front of each sample. Remove 1 WinLIMS labels from sample, and place on base of the petri dishes.轻轻地倒置饮料容器。Gently invert the beverage container.对于带皇冠盖的瓶子，要利用大约70%的异丙醇或普通酒精擦拭瓶顶的外侧。For bottles with crowns, wipe the outside of bottle top with 70% isopropyl or ethyl alcohol.保证酒精到达皇冠盖的下侧。Ensure that the alcohol reaches the under side of the crown.将开瓶器浸泡在大约70%的异丙醇或普通酒精中，然后开启皇冠盖。Dip opener in 70% isopropyl or ethyl alcohol and remove crown.如果存在着灰尘、锈迹或其它杂质的痕迹，则要用大约70%的异丙醇或普通酒精擦拭瓶子的边缘。Wipe lip of bottle with 70% isopropyl or ethyl alcohol if there is any evidence of dirt, rust, or other debris.对于易拉罐，要用大约70%的异丙醇或普通酒精擦拭可移动的拉环和邻近表面，并用无菌方式开启。For cans, wipe the removable tab and adjacent surface with 70% isopropyl or ethyl alcohol and open aseptically.注： Note:开启之后，要废弃 20ml的饮料来冲掉残留在容器开口附近的任何酒精。After opening, discard 20ml beverage to flush away any alcohol remaining around the container opening.以无菌方式，利用吸液泵或吸液球取10ml样品，再倒入皮氏培养皿中。Draw up 10ml sample aseptically using pipette pump or bulb and dispense into petri dish.利用无菌方式，向皮氏培养皿中倒入50-55ml调和至44 46C的桔汁琼脂。Aseptically pour 50-55ml of Orange Serum Agar, tempered to 44 46C, into petri dish.顺时针和逆时针轻轻摇动平板，让内含物混匀。Swirl plate gently clockwise and anti-clockwise to mix contents.向另一个预先贴有标签的皮氏培养皿中，采用无菌方式吸取10ml同样的样品。Aseptically pipette 10ml of the same sample into another pre-labelled petri dish.采用无菌方式，向皮氏培养皿中倒入50-55ml 调和到44-46C的酸化PDA。Aseptically pour 50-55ml of acidified PDA, tempered to 44 46 C, into petri dish.顺时针和逆时针轻轻摇动平板，让内含物混匀。Swirl plate gently clockwise and anti-clockwise to mix contents.对所有样品重复上述步骤。Repeat for all samples.让琼脂硬化并倒置平板。最多堆放6层高。Allow agar to solidify and invert plates. Stack at a maximum of 6 high.在将一次性滤膜漏斗或皮氏培养皿放入恒温箱时，每叠之间至少留有2.5 cm的距离，并且每叠限高六层或以下。When placing monitors or petri dishes in the incubator, allow a minimum space of 2.5 cm between each stack and place stacks restricting height to six or less.对于酵母菌和霉菌，将PDA平板在25C +/-1 C条件下培养120小时(5天) +/- 3小时。For yeast and mould, incubate PDA plates at 25C +/-1 C for 120 hours (5 days) +/- 3hrs.对于耐酸细菌，将OSA平板在30C +/-1 C 条件下培养48小时(2天) +/- 3小时。For aciduric bacteria, incubate OSA plates at 30C +/-1 C for 48 hours (2 days) +/- 3hrs.此时应当记录培养的时间和日期，以及样品类型和使用的恒温箱。Time and date of incubation plus sample type and incubator used should be logged at this time.为了避免交叉污染，在样品培养之后，应当实施培养基控制措施。在主动控制校验之前，应当实施无菌性控制措施。To avoid cross contamination, media controls should be carried out following incubation of samples. Sterility controls should be carried out prior to positive control checks.3.5.1日常无菌性控制校验 Daily Sterility Control Checks在分析结束时，要对每瓶培养基进行无菌性检测，方法是：Carry out sterility tests on each bottle of media used at the end of analysis by;采用无菌方式，向一个未接种的皮氏培养皿中倒入大约5055ml的OSA，向另一个未接种的皮氏培养皿中倒入大约50-55ml的PDA。让其硬化。Aseptically pouring 5055ml of OSA into an uninoculated petri dish and 50-55ml of PDA into a second uninoculated petri dish. Allow to solidify.3.5.2日常主动控制校验 Daily Positive Control Checks分析结束时，对用于每批样品的每个培养基瓶，进行定性生长支持实验：Carry out qualitative growth support tests on each bottle of media used for each batch of samples at the end of analysis:采用无菌方式，向一个未接种的皮氏培养皿中导入大约50-55ml 的OSA，让其硬化。Aseptically pour 50-55ml OSA into an uninoculated petri dish and allow to solidify.给皮氏培养皿迅速地加上一整圈耐酸细菌参比培养基。Streak a loopful of an aciduric bacteria reference culture onto the petri dish.采用无菌方式，向一个未接种的皮氏培养皿中导入大约50-55ml 的PDA，让其硬化。Aseptically pour 50-55ml PDA into an uninoculated petri dish and allow to solidify.给皮氏培养皿迅速地加上一整圈酵母菌参比培养基。Streak a loopful of a yeast reference culture onto the petri dish.培养期过后，从恒温箱中取出全部皮氏培养皿。Following incubation period, remove all petri dishes from the incubator.3.6计算 Calculations培养之后要立即清点薄膜表面上的所有菌落。如果培养之后不能立即清点，则要将平板或一次性滤膜漏斗在大约4的条件下存放不超过24小时的时间。After incubation, promptly count all colonies on the membrane surface. If it is impossible to count immediately after incubation, store plates or monitors at approximately 4 for a period of not more than 24 hours.不要将任何培养基或样品颗粒或者薄膜表面上的沉淀物混淆成微小的菌落。Avoid mistaking any media or sample particles, or precipitating matter that may be on the membrane surface for pinpoint colonies.清点各个平板上的各个菌落。Count individual colonies on each plate.利用形态学来区分酵母菌、霉菌和细菌。如果需要进一步检查，则要用湿法制备菌落，并在显微镜下观察细胞结构。Differentiate between yeast, mold and bacteria by morphology. If further examination is required, prepare a wet mount of the colony and examine the cell structure under the microscope.注： Note:对于其他耐酸微生物这也是重要的，特别是橘汁琼脂中可能也会出现酵母菌。This is significant as other acid tolerant microorganisms; especially yeast may be picked up on Orange Serum Agar.如果平板上存在的菌落数超过大约200个，则要将平板分成合适的扇面（如2、4、8个），清点一个或多个扇面上的菌落数。将各个扇面上的总数乘以适当的系数，就能获得每种样品菌落总数的估算值。If there are more than 200 colonies approximately on the plate, divide the plate into convenient radial sections (e.g. 2,4,8) and count all the colonies in one or more sections. Multiply the tota