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食品水质等微生物菌落总数检测的所有相关问题
发布时间:2018-06-22   点击次数:957次

菌落总数主要是作为判定食品被细菌污染程度的标志,也可以应用这一方法观察食品中细菌的性质以及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科学依据。

菌落总数测定的几项说明

1.菌落总数的测定:是以检样中的细菌细胞和平板计数琼脂混合后,每个细菌细胞都能形成一个可见的单独菌落的假定为基础的。由于检验中采用36℃于有氧条件下培养(空气中含氧约20%),因而并不能测出每g或mL检样中实际的总活菌数,厌氧菌、微嗜氧菌和冷营菌在此条件下不生长,有特殊营养要求的一些细菌也受到了限制,因此所得结果,只包括一群能在普通平板计数琼脂中发育、嗜中温的、需氧和兼性厌氧的细菌菌落的总数。

 

2.鉴于食品检样中的细菌细胞是以单个,成双、链状、葡萄状或成堆的形式存在,因而在平板计数琼脂上出现的菌落可以来源于细胞块,也可以来源于单个细胞,因此平板上所得需氧和兼性厌氧菌菌落的数字不应报告活菌数,而应以单位重量、容量或表面积内的菌落数或菌落形成单位数(colony forming units,CFU)报告之。

 

3.每种细菌都有它一定的生理特性,培养时,应用不同的营养条件及其他生理条件(如温度、培养时间、pH、需氧性质等)去满足其要求,才能分别将各种细菌都培养出来。因此,要得到较全面的细菌菌落总数,应将检样接种到几种不同的非选择性培养基上,并培养在不同条件下,如温度,氧气供应等。但国家颁发的食品卫生标准对不同食品的菌落总数的规定,都是根据用平板计数琼脂进行需氧培养所得的结果确定的,因此在食品的一般卫生学评价中并不要用几种不同的非选择性培养基的培养。

 

 

菌落总数的测定

测定食品中菌落总数时,是将食品检样做成几个不同的10倍递增稀释液,然后从各个稀释液中分别取出一定量在平皿内与平板计数琼脂相混合,经培养后,按一定要求计算出皿内琼脂平板上所生成的细菌集落数,并再根据检样的稀释倍数,计算出每g或mL样品中所含细菌菌落的总数。

 

菌落总数测定中的一些要求和规定

WEILEZHENGQUEDIFANYINGSHIPINZHONGGEZHONGXUYANGHEJIANXINGYANYANGJUNCUNZAIDEQINGKUANG,JIANYANSHIBIXUZUNXUNYIXIAYIXIEYAOQIUHEGUIDING:

(一)所用器皿及稀释液:

1.JIANYANZHONGSUOYONGBOLIQIMIN,RUPEIYANGMIN,XIGUAN、SHIGUANDENGBIXUSHIWANQUANMIEJUNDE,BINGZAIMIEJUNQIANCHEDIXIDIGANJING,BUDECANLIUYOUYIJUNWUZHI。

球探体育2.YONGZUOYANGPINXISHIDEYETI,MEIPIDOUYAOYOUKONGBAIDUIZHAO。RUGUOZAIQIONGZHIDUIZHAOPINGBANSHANGCHUXIANJUNLUOSHI,YAOZHUIJIADUIZHAOPINGBAN,YIPANDINGSHIKONGBAIXISHIYE,YONGYUQINGZHUPINGMINDEPEIYANGJI,HAISHIPINGMINXIGUANHUOKONGQIKENENGCUNZAIDEWURAN。

3.JIANYANGDEXISHIYESUIKEYONGSHENGLIYANSHUIHUOLINSUANYANHUANCHONGYE,DANYIYONGLINSUANYANHUANCHONGYEWEIHESHI,YINWEILINSUANYANHUANCHONGYEDUIXIJUNXIBAOYOUGENGHAODEBAOHUZUOYONG,BUHUIYINWEIZAIXISHIGUOCHENGZHONGERSHISHIPINJIANYANGZHONGYUANYISHOUSUNSHANGDEXIJUNXIBAODAOZHISIWANG。RUGUODUIHANYANLIANGJIAOGAODESHIPIN(RUJIANGPINDENG)JINXINGXISHI,ZEYICAIYONGZHENGLIUSHUI。

 

(二)检样稀释:

球探体育1.JIANYANGXISHISHI,YINGYIWUJUNCAOZUOCHENGQU(HUOLIANGQU)YOUDAIBIAOXINGDEYANGPIN25g(HUOmL)JIANSUIFANGYUHANYOU225mlMIEJUNXISHIYEDEBOLIPINGNEI(PINGNEIYUZHISHIDANGSHULIANGDEBOLIZHU),JINGCHONGFENZHENYAOZUOCHENG1:10DEXISHIYE。RUROU、YUDENGGUTIYANGPIN,JIANXIYUMIEJUNRUBONEIYUXISHIYEYANYUN,HUOYUXISHIYETONGZHIYUMIEJUNJUNZHIQIBEIHUOPAIJISHIJUNZHIDAIZHONG,SHIZUOCHENGJUNYUNDE1:10XISHIYE。

 

球探体育2.GENJUSHIPINWEISHENGBIAOZHUNYAOQIUHUODUIBIAOBENWURANQINGKUANGDEGUJI,JIANGSHANGSHU1:10DEJIANYANGXISHIYEZAIZUOCHENGJIGESHIDANGDE10BEIDIZENGXISHIYE。JIQU1:10XISHIYE1mLYU9mLXISHIYEHUNHEZUOCHENG1:100DEXISHIYE,RANHOUYICIDIZENGXISHI,ZUOCHENG1:1000、1:10000DENGXISHIYE。ZHUYIMEIDIZENGXISHIYICI,BIXULINGHUAN1ZHIl mLMIEJUNXIGUAN,ZHEIYANGSUODEJIANYANGDEXISHIBEISHUFANGWEIZHUNQUE。

 

球探体育3.CONGXIGUANTONGNEIQUCHUMIEJUNXIGUANSHI,BUYAOJIANGXIGUANPENGZHEQITARENGLIUZAIRONGQINEIDEXIGUANDEWAILUBUFEN;ERQIEXIGUANZAIJINCHUZHUANGYOUXISHIYEDEBOLIPINGHESHIGUANSHI,YEBUYAOCHUJIPINGKOUJISHIGUANKOUDEWAICEBUFEN;YINWEIZHEIXIEBUFENDOUKENENGJIECHUGUOSHOUHUOQITAWURANWU。

 

4.ZAIZUO10BEIDIZENGXISHIZHONG,XIGUANCHARUJIANYANGXISHIYENEIBUNENGDIYUYEMIAN2.5cm;XIRUYETISHI,YINGXIANGAOYUXIGUANKEDU,RANHOUTIQIXIGUANLIKAIYEMIAN,JIANGTIEYUBOLIPINGHUOSHIGUANDENEIBISHIXIGUANNEIYETIDIAOZHISUOYAOQIUDEKEDU,ZHEIYANGQUYANGJIAOZHUNQUE,ERQIEZAIXIGUANCONGXISHIYENEIQUCHUSHIBUHUIYOUDUOYUDEYETIZHANFUYUGUANWAI。  

 

5.DANGYONGXIGUANJIANGJIANYANGXISHIYEJIAZHILINGYIZHUANGYOU9mLKONGBAIXISHIYEDESHIGUANNEISHI,YINGXIAOXINYANGUANBIJIARU,BUYAOCHUJIGUANNEIXISHIYE,YIFANGXIGUANWAICEBUFENZHANFUDEJIANYEYEHUNRUQIZHONG。

 

(三)平板接种与培养:

1.JIANGXISHIYEJIAZHIMIEJUNPINGMINNEISHI,YINGGENJUSHIPINWEISHENGBIAOZHUNYAOQIUHUODUIBIAOBENWURANQINGKUANGDEGUJI,XUANZE2~3GESHIYIXISHIDU,FENBIEZAIZUO10BEIDIZENGXISHIDETONGSHI,JIYIXIQUGAIXISHIDUDEXIGUANYIlmLXISHIYEJIARUMINNEI(CONGMINCEJIARU,BUYAOJIEQUMINGAI),HOUJIANGXIGUANZHILISHIYETILIUBI,BINGZAIMINDIGANZAOCHUZAICAYIXIAXIGUANJIANJIANGYUYEPAICHU,ERBUYAOCHUICHU。MEIGEXISHIDUYINGZUO2GEPINGMIN。

 

2.YONGYUQINGZHUPINGMINDEYINGYANGQIONGZHIYINGYUXIANJIARESHIRONGHUA,BINGBAOWENYU45±1℃HENGWENSHUIYUZHONGDAIYONG。QINGZHUPINGMINSHI,MEIMINNEIQINGRUYUE15~20mL。

 

球探体育3.WEILEFANGZHIXIJUNZENGZHIJICHANSHENGPIANZHUANGJUNLUO,ZAIJIANYEJIARUPINGMINHOU,YINGZAI20FENZHONGNEIXIANGMINNEIQINGRUPEIYANGJI,BINGLIJISHIQIYUPEIYANGJIHUNHEJUNYUN。

 

球探体育4.JIANYANGYUPEIYANGJIHUNHESHI,KEJIANGMINDIZAIPINGMIANSHANGXIANXIANGYIGEFANGXIANGXUANZHUAN,RANHOUZAIXIANGXIANGFANDEFANGXIANGXUANZHUAN,YISHICHONGFENHUNYUN。XUANZHUANZHONGYINGJIAXIAOXIN,BUYAOSHIHUNHEWUJIANDAOMINBIANDESHANGFANG。WEILEBAOZHENGHUNHEJUNYUN,ERBUJIANJIMINBIANDESHANGFANG,KESHIYONGZIDONGPINGMINXUANZHUANYI,ZHIJIEJIANGJIAYOUJIANYANGDEPINGMINFANGZAIXUANZHUANYISHANG(KETONGSHIFANG4ZHIPINGMIN),JIARUPEIYANGJIHOU,KAIDONGDIANNIU,ZAISHUSHIMIAOZHONGNEIJIKEZIDONGZUOYOUXUANZHUANERSHIMINNEIDEJIANYANGYUPEIYANGJIHUNHEJUNYUN。

 

5.MINNEIQIONGZHININGGUHOU,BUYAOZHANGJIUFANGZHI,RANHOUCAIFANZHUANPEIYANG,ERYINGYUQIONGZHININGGUHOU,ZAISHUFENZHONGNEIJIYINGJIANGPINGMINFANZHUANYUYIPEIYANG,ZHEIYANGKEBIMIANJUNLUOMANYANSHENGZHANG。

 

6.WEILEKONGZHIWURAN,ZAIQUYANGJINXINGJIANYANDETONGSHI,YUGONGZUOTAISHANGDAKAIYIKUAIQIONGZHIPINGBAN,QIBAOLUDESHIJIAN,YINGYUGAIJIANYANGCONGZHIBEI、XISHIDAOJIARUPINGMINSHISUOBAOLUDEZHANGDESHIJIANXIANGDANG,RANHOUYUJIAYOUJIANYANGDEPINGMINYIBINGZHIYUWENXIANGNEIPEIYANG,YILEJIEJIANYANGZAIJIANYANCAOZUOGUOCHENGZHONGYOUWUSHOUDAOLAIZIKONGQIDEWURAN。

 

7.PEIYANGWENDU:YINGGENJUSHIPINZHONGLEIERDING。ROU、RU、DANLEISHIPINYONG37℃PEIYANG,SHUICHANPINYONG30℃PEIYANG。PEIYANGSHIJIANWEI48±2XIAOSHI。PEIYANGWENDUHESHIJIANZHISUOYIYOUZHEIZHONGBUTONGDEQUFEN,NAISHIYINWEIZAIZHIDINGZHEIXIESHIPINWEISHENGBIAOZHUNZHONGGUANYUJUNLUOZONGSHUDEGUIDINGSHI,FENBIECAIYONGLEBUTONGDEWENDUHEPEIYANGSHIJIANSUOQUDEDESHUJUZHIGU。SHUICHANPINYINLAIZIDANSHUIHUOHAISHUI,SHUIDIWENDUJIAODI,YINERZHIDINGSHUICHANPINXIJUNFANGMIANDEWEISHENGBIAOZHUNSHI,YONG30℃ZUOWEIPEIYANGDEWENDU。

 

(四)对照试验:      

1.JIARUPINGMINNEIDEJIANYANGXISHIYE(TEBIESHI1:10DEXISHIYE),YOUZHOUDAIYOUSHIPINKELI,ZAIZHEIZHONGQINGKUANGXIA,WEILEBIMIANYUXIJUNJUNLUOFASHENGHUNXIAO,KEZUOYIJIANYANGXISHIYEYUPEIYANGJIHUNHEDEPINGMIN,BUJINGPEIYANG,ERYU4℃HUANJINGZHONGFANGZHI,YIBIANZAIJISHUJIANYANGJUNLUOSHIYONGZUODUIZHAO。

 

2.WEILEFANGZHIJIANYANGZHONGSHIPINKELIYUJUNLUOHUNXIAO,YEKEZAIYIRONGHUAERBAOWENYU45℃SHUIYUNEIDEPEIYANGJIZHONG,ANMEI100mlJIAlmL,0.5%LVHUASANBENSIDANZUO(TTC)SHUIRONGYEZHILIANGJIARUSHILIANGDETTC。PEIYANGHOU,RUSHISHIPINKELI,BUJIANBIANHUA,RUWEIXIJUN,ZESHENGCHENGHONGSEJUNLUO,PEIHAODETTCRONGYE,YINGXIANYONGLAIYUBUJIATTCDEZUODUIZHAO,YIGUANCHAQIDUIJISHUSHIFOUYOUBULIDEZUOYONG(TTCZAIYIDINGNONGDUXIADUIGELANSHIYANGXINGJUNYOUYIZHIZUOYONG)。TTCRONGYEYAOFANGLENGANCHUBAOCUN,YIFANGSHOUREYUGUANGZHAOERFASHENGFENJIE。WUSEDETTCSHIZUOWEISHOUQINGTIJIARUPEIYANGJIZHONG,RUGUOYOUXIJUNCUNZAI,PEIYANGHOU,ZAIXIJUNTUOQINGMEIDEZUOYONGXIA,TTCBIANJIESHOUQINGERCHENGWEIHONGSEDESANBENJIJIASHOU(formazan),SHIJUNLUOCHENGXIANHONGSE。

 

(五)菌落计数:

1.CONGWENXIANGNEIQUCHUPINGMINJINXINGJUNLUOJISHUSHI,YINGXIANFENBIEGUANCHATONGYIXISHIDUDELIANGGEPINGMINHEBUTONGXISHIDUDEJIGEPINGMINNEIPINGBANSHANGJUNLUOSHENGZHANGQINGKUANG。PINGXINGSHIYANDE2GEPINGBANYUJUNLUOSHUYINGGAIJIEJIN,BUTONGXISHIDUDEJIGEPINGBANSHANGJUNLUOSHUZEYINGYUJIANYANGXISHIBEISHUCHENGFANBI,JIJIANYANGXISHIBEISHUYUEDA,JUNLUOSHUYUEDI,XISHIBEISHUYUEXIAO,JUNLUOSHUYUEGAO。

 

球探体育2.JISHUJUNLUOSHI,YINGXUANQUJUNLUOSHUZAI30~300CFUZHIJIANDEPINGBANZUOWEIJUNLUOZONGSHUCEDINGDEBIAOZHUN。1GEXISHIDUSHIYONGLIANGGEPINGBAN,YINGCAIYONGLIANGGEPINGBANPINGJUNSHU;RUQIZHONGYIGEPINGBANYOUJIAODAPIANZHUANGJUNLUOSHENGZHANGSHI,ZEBUYICAIYONG,ERYINGYIWUPIANZHUANGJUNLUOSHENGZHANGDEPINGBANZUOWEIGAIXISHIDUDEJUNLUOSHU,RUOPIANZHUANGJUNLUOBUDAOPINGBANDEYIBAN,ERQIYUYIBANZHONGJUNLUOFENBUYOUHENJUNYUN,JIKEJISUANBANGEPINGBANHOUCHENG2YIDAIBIAOQUANMINJUNLUOSHU。RUZAIYIGEXISHIDUDELIANGGEPINGBANZHONG,YIGEPINGBANDEJUNLUOSHUZAI30~300ZHIJIAN,LINGYIGEDAYU300HUOXIAOYU30SHI,ZEYIJUNLUOSHUZAI30~300JIANDEPINGBANZUOWEIJISHUDEBIAOZHUN。

 

3.ZHUYISHIXIANG:

(1)RUGUOXISHIDUDADEPINGBANSHANGJUNLUOSHUFANBIXISHIDUXIAODEPINGBANSHANGJUNLUOSHUGAO,ZEJIANYANGONGZUOZHONGFASHENGDECHACUO,SHUSHIYANSHISHIGU。CIWAI,YEKENENGYINYIJUNJIHUNRUYANGPINZHONGSUOZHI,JUNBUKEYONGZUOJIANYANGJISHUBAOGAODEYIJU。

 

(2)RUGUOPINGBANSHANGCHUXIANLIANZHUANGJUNLUO,JUNLUOZHIJIANMEIYOUMINGXIANDEJIEXIAN,ZHEISHIZAIQIONGZHIYUJIANYANGHUNHESHI,YIGEXIJUNKUAIBEIFENSANSUOZAOCHENG。YITIAOLIANZUOWEIYIGEJUNLUOJI,RUYOULAIYUANBUTONGDEJITIAOLIAN,MEITIAOLIANZUOWEIYIGEJUNLUOJI,BUYAOBALIANSHANGSHENGZHANGDEGEGEJUNLUOFENKAILAISHU。

 

(3)RUGUOSUOYOUPINGBANSHANGDOUYOUJUNLUOMIBU,BUYAOYONGDUOBUKEJIZUOBAOGAO,ERYINGZAIXISHIDUDADEPINGBANSHANG,RENYISHUQIZHONG2cm2GE,CHU2QIUCHUMEIcm2NEIPINGJUNJUNLUOSHUCHENGYIMINDIMIANJI63.6cm2SHU,ZAICHENGQIXISHIBEISHUZUOBAOGAO。LIRU10-1~10-3XISHIDUDESUOYOUPINGBANSHANGJUNJUNLUOMIBU,ERZAI10-3XISHIDEPINGBANSHANGRENSHU2GEcm2NEIDEJUNLUOSHUSHI60GE,MINDIZHIJINGWEI9cm,ZEGAIJIANYANGMEIg(HUOm1)ZHONG“GUJI”JUNLUOSHUWEI:60/2×63.6×1000=1908000HUO1.9×106。

63.6cm2SHUXIANMINDIZHIJINGWEI9cmSHIJISUANERDE,JI:(9/2)2×3.14=63.6;RUSUOYONGPINGMINDEMINDIZHIJINGBUSHI9cm,ZEKEANQIZHIJINGDESHIJIcmSHUDAIRENYUANMIANJIGONGSHIQIUCHU。

(4)JUNLUOJISHUZHONG,YEKEYIXUANYONGJUNLUOJISHUQIZEBIJIAOFANGBIAN,DENGGUANGYOUCEMIANSHEXIANGPINGBAN,JUNLUOYIYUGUANCHA。YOUYUYIQIDAIYOUDIANZIYINXIANGXUNHAO,YONGTEZHIJINSHUTANBIDIANSHUZHONG,ZAIFACHUYINXIANGZHIXIASHIXIANSHISHUZIZENGJIA,BUHUIFASHENGCHACUO。QIEDENGGUANGYUPINGBANZHIJIANJIAYOUDUOYONGFANGGEBOZHIGUIBAN(FANGGEMIANJIWEI1cm2),YEYOUZHUYUDUIJUNLUOMIBUDEPINGBANJINXINGJISHU。

 

球探体育(5)JIANYANGRUSHIWEISHENGWULEIZHIJI(RUSUANNIURU、JIAOMUZHISUANXINGYINLIAO),ZEPINGBANJISHUZHONGYINGXIANGYINGDIJIANGYOUGUANWEISHENGWU(RUSUANGANJUN、JIAOMUJUN)PAICHU,BUKEBINGRUJIANYANGDEJUNLUOZONGSHUNEIZUOBAOGAO。YIBANZAIXIAOZHENGJIANYANGDEpH7.6HOU,ZAIJINXINGXISHIHEPEIYANG,CILEISHISUANXINGWEISHENGWUWANGWANGJIBUYISHENGZHANG。BINGKEYONGGELANSHIFARANSEJIANBIE。RANSEJIANBIESHI,YAOYONGBUXIAOZHENGpHDEJIANYANGZUOCHENGXIANGTONGXISHIDUDEXISHIYEPEIYANGSUOSHENGCHENGDEJUNLUOTUPIANRANSEZUODUIZHAO,YIZIBIANBIE。JIAOMUJUNLUANYUANXING,YUANBIXIJUNDA,DAXIAOWEI2~5×5~30μm,GELANSHIYANGXINGZHESE。RUSUANGANJUNZAI24XIAOSHINEI,YUPUTONGYINGYANGQIONGZHIPINGBANSHANGZAIYOUYANGTIAOJIANXIAPEIYANG,TONGCHANGSHIBUSHENGZHANGDE。

 

(六)报告方式

球探体育1.DANGJIANYANGDEJUNLUOSHUWEI1~100SHI,ANSHIYOUSHUBAOGAO;DAYU100SHI,ZHIJILUZUOMIANTOULIANGWEISHUZI(YONGLIANGWEIYOUXIAOSHUZIZUOBAOGAO,KEBIMIANCHANSHENGXUJIADEGAINIAN),ZUOMIANDISANWEISHUZIZEYONGSISHEWURUFAJISUAN,CONGDIERWEISHUZIZHIHOUDOUJIWEI0,WEILESUODUANSHUZIHOUMIANDE0SHU,YEKEYONG10DEZHISHULAIBIAOSHI,LIRUJUNLUOSHUWEI37750SHI,JIKEXIECHENG3.8×105。

 

2.JIANYANGDEJUNLUOSHURUXICONGJUNLUOMIBUDEPINGBANSHANGANBILIJISUANERQIUDE,BAOGAOJIEGUOSHI,YINGZAIJUNLUOSHUQIANJIASHANG“GUJI”ERZI。

 

3.JIANYANGWEIGUTI,YONGZHONGLIANGFAQUYANGJIANYANSHI,YIgWEIDANWEIBAOGAOQIJUNLUOSHU;JIANYANGWEIYETI,YONGRONGLIANGFAQUYANGJIANYANSHI,YImLWEIDANWEIBAOGAOQIJUNLUOSHU;RUJIANYANGWEIYANGPINBIAOMIANDETUCAYE,ZEYINGYIcm2WEIDANWEIBAOGAOQIJUNLUOSHU。

 

4.RUJIANYANGWEIYETI,ZAILIANGGEPINGMINNEISUOJIAlmLWEIJINGXISHIDEJIANYANG(YUANYE)PEIYANGZHONG,JUNWUXIJUNJILUOSHENGCHENG,ZEBAOGAOWEIlmLJIANYANGNEIWEIYOUJUNLUOSHENGZHANG,HUO1mLJIANYANGNEIJUNLUOSHU<1。

 

 

特殊的方法

(一)平板表面涂布法

将营养琼脂制成平板,经50℃,1-2小时或35℃,18-20小时干燥后,于其上滴加检样稀释液0.2mL,用L棒涂布于整个平板的表面,放置片刻(约10分钟),将平板翻转,移至36±1℃恒温箱内培养48±2h(水产品用30±1℃培养48±2h),取出,按前述方式进行菌落计数,然后乘以5(由0.2mL换算为1mL),再乘以样品稀释的倍数,即得每g或mL检样所含菌落数。

此法较上述倾注法为优,因菌落生长在表面,便于识别和检查其形态,虽检样中带有食品颗粒也不会发生混淆,同时还可使细菌不必遭受融化琼脂的热力,不致因此而使菌细胞受到损伤而不生长,从而可避免由于检验操作中的不良因素而使检样中细菌菌落数降低。但是本法取样量较倾注法为少(仅倾注法的五分之一),代表性将受到一定的影响。同时由于不是国标规定方法,因此选用需要谨慎

 

(二)平板表面点滴法

与涂布法相似,所不同者,只是用标定好的微量吸管或注射器针头按滴(使每滴相当于0.025ml)将检样稀释液滴加于琼脂平板上固定的区域(预先在平板背面用标记笔划成四个区域),每个区域滴1滴,每个稀释度滴两个区域,作为平行试验。滴加后,将平板放平片刻(约5~10分钟),然后翻转平板,如前移入温箱内培养后进行计数,将所得菌落数乘以40(由0.025mL换算为1mL),再乘以样品稀释的倍数,即得每g或mL检样所含菌落数。与平板表面涂布法一样,由于也不是国标规定的检测方法,需谨慎选用。

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