饮用天然矿泉水中产气荚膜梭菌检测方法的改良

将饮用天然矿泉水中产气荚膜梭菌检测国标方法GB 8538-2016《食品安全国家标准饮用天然矿泉水检验方法》作一些改良,以使检测结果更加准确可靠。用产气荚膜梭菌CICC22949人工污染的水样通过孔径为0.22μm的无菌滤膜过滤,分别将胰胨-亚硫酸盐-环丝氨酸琼脂(tryptose-sulfite-cycloserine agar,TSC)培养基和亚硫酸盐-多黏菌素-磺胺嘧啶琼脂(sulfite-polymyxin-sulphadiazxine agarr,SPS)培养基采用双层培养基覆盖法进行试验。改良后的方法在滤膜上有黑色可疑菌落,而国标方法滤膜上的菌落无黑色。TSC培养基上目标菌计数值显著高于SPS培养基计数值(P<0.05)。用新鲜配制的TSC培养基经双层培养基覆盖改良后的方法检测产气荚膜梭菌,现象更明显,结果更可靠,更适合饮用天然矿泉水中产气荚膜梭菌的检验。     

矿泉水中产气荚膜梭菌检测能力验证经验

产气荚膜梭菌(C.perfringens)又称魏氏梭菌,厌氧芽孢杆菌属。广泛存在自然界的水源、土壤及人和动物肠道中。它是条件致病菌,主要引起人的食物中毒、气性坏疽和动物猝死症等疾病。产气荚膜梭菌的重点检测项目是活菌数的测定和证实试验。GB 8538-2016饮用天然矿泉水中产气荚膜的检测标准中说明,产气荚膜梭菌在SPS平板上36 ℃下厌氧培养24 h,菌落形态为黑色。但在实际检验中SPS平板上的菌落形态难以呈黑色,如果改用TSC平板可以长出较多黑色菌落。TSC培养基中柠檬酸铁铵和亚硫酸钠的含量比SPS要高,实验证明TSC培养基要比SPS培养基更能长出黑色菌落。     

水中产气荚膜梭菌在不同过滤培养体系中的表型特征的研究

目的比较水中的产气荚膜梭菌在2种不同孔径滤膜和2种培养基上的计数结果,并探讨其机理。方法用0.22μm和0.45μm 2种不同孔径的滤膜过滤添加产气荚膜梭菌的矿泉水后,将滤膜贴在亚硫酸盐-多粘菌素-磺胺嘧啶琼脂培养基(sulfite-polymyxin-sulfadiazine agar,SPS)和胰胨-亚硫酸盐-环丝氨酸琼脂培养基(tryptose sulfite cycloserine agar,TSC)上,36℃±1℃厌氧培养24 h,观察菌落形态并进行菌落计数。用扫描电镜观察0.22μm和0.45μm2种滤膜表面的开口大小。结果水样经过2种不同孔径的滤膜过滤后,产气荚膜梭菌在TSC和SPS上的计数结果无显著性差异(P0.05)。但与SPS相比,产气荚膜梭菌在TSC上更易形成特征性黑色菌落;与孔径为0.22μm滤膜相比,在孔径为0.45μm滤膜上生长的菌落更大。电镜扫描发现,0.22μm滤膜和0.45μm滤膜的平均表面开口分别为2 5μm、4 0μm,后者的表面开口更大。结论水样通过0.45μm孔径滤膜过滤,贴在TSC琼脂上培养,更适用于矿泉水中产气荚膜梭菌的检验。     

4种方法测定矿泉水中产气荚膜梭菌测量审核结果的比较分析

目的比较分析4种方法对矿泉水中产气荚膜梭菌测量审核结果的影响。方法以测量审核作业指导书、GB 8538—2016《食品安全国家标准饮用天然矿泉水检验方法》为依据,GB 4789.13-2012《食品安全国家标准食品微生物学检验产气荚膜梭菌检验》为参考,分别采用两标准中的亚硫酸盐-多粘菌素-磺胺嘧啶(sulfite polymyxin sulphadiazine,SPS)琼脂培养基和胰(?)-亚硫酸盐-环丝氨酸(tryptose sulfite cycloserine,TSC)琼脂培养基,再以以滤膜法倾注上层培养基与不倾注上层培养基的方法同时检测测量审核样品和模拟水样。比较2种培养基的产气荚膜梭菌回收率,运用方差分析测量审核样品、运用配对t检验模拟水样,进而评价4种方法对矿泉水中产气荚膜梭菌计数结果一致性的影响。结果 4种方法检测结果无明显差异,但是经不倾注上层培养基培养后目标菌难显黑色,经倾注上层培养基培养后目标菌显黑色。3个厂家的SPS培养基、TSC培养基产气荚膜梭菌的平均回收率分别为:89%、98%。采用TSC培养基和滤膜法倾注上层培养基这一方法的标准差最小,最为稳定。测量审核样品|Z|2、空白样品为0 CFU/50 mL,结果满意。结论采用滤膜法倾注上层培养基的方法更适用于矿泉水中产气荚膜梭菌的检验,并建议优先采用TSC培养基,以确保检测结果的准确性和质量。     

包装饮用水中铜绿假单胞菌检验方法相关问题的探讨

为了研究包装饮用水中铜绿假单胞菌项目的检测,确保铜绿假单胞菌检测的准确性,采用GB8538—2016 《食品安全国家标准饮用天然矿泉水检验方法》中滤膜法测定,探讨了试验过程中水样稀释、滤膜放置、绿脓菌素试验等关键环节对试验结果的影响。结果表明,按照GB 4789.2—2016 《食品安全国家标准食品微生物学检验菌落总数测定》中"样品的稀释"对水样进行递增稀释,能够保证滤膜上生长的菌落数量在适宜范围内,提高计数准确性;其次,滤膜的正确使用以及绿脓菌素试验注意事项,能够有效避免漏检和假阴性结果。     

郑州市熟肉制品中产气荚膜梭菌污染状况调查

目的了解郑州市熟肉制品中产气荚膜梭菌(Clostridium perfringens)的污染状况,为熟肉制品储存过程中产气荚膜梭菌的安全控制提供技术支撑。方法按照GB 4789.13—2012《食品安全国家标准食品微生物学检验产气荚膜梭菌检验》中的方法分别对11种不同样品进行检测分析,结合VITEK鉴定法和分子生物学鉴定法进一步验证。结果 259份样品中,产气荚膜梭菌检出份数为38份,总检出率14.7%,不同样品的检出率范围为0.0%~33.3%,其中盐焗鸡样品检出率最高为33.3%(7/21),卤半片鸭和卤鸡肝样品未检出。烧鸡、烤鸡和卤鸡腿样品的产气荚膜梭菌菌落总数均超过5.0log10CFU/g,其他样品的菌落总数均在4log_(10)~5log_(10) CFU/g之间。结论郑州市熟肉制品中的产气荚膜梭菌污染现象较严重,烧鸡、烤鸡和卤鸡腿的菌落总数较高,有引起食物中毒的可能。     

饮用天然矿泉水中疑似粪链球菌的鉴定与分析

目的 检测某品牌饮用天然矿泉水样品中是否含有粪链球菌,并对出现的疑似菌落进行分析。方法 按照GB 8538-2016《食品安全国家标准 饮用天然矿泉水检验方法》中规定的检测方法对样品进行检测,对疑似菌落进行确证性试验,并通过VITEK 2 COMPACT全自动微生物鉴定系统对疑似菌进行鉴定。结果 滤膜上出现典型红色菌落,经确证性试验确认为非粪链球菌;经VITEK 2 COMPACT鉴定,该菌为粘质沙雷氏菌。结论 样品中的疑似菌落非粪链球菌而是粘质沙雷菌,该菌作为干扰菌对滤膜法检测粪链球菌具有参考意义。     

膜过滤在微生物检测中的应用

过滤的目的无非是得到滤液或截留的成分。过滤的类型有多种．如膜过滤、筛网过滤、离心过滤等。其中膜过滤的应用有很多种,如针对微生物检测,膜过滤是通过使用一定孔径的滤膜从液体中截留微生物,使该微生物在膜表面生长成为菌落．然后对其进行培养,计数．鉴定等。     

降胆固醇干酪乳杆菌WB生长培养基的筛选

降胆固醇干酪乳杆菌WB在不同种类选择性培养基上生长培养后,比较活菌菌落数、菌落及溶钙圈大小。结果显示:MRS培养基最适合降胆固醇乳酸菌WB的培养,活菌数最多、菌落及溶钙圈最大。厌氧培养后效果更佳。     

一起疑似产气荚膜梭菌腹泻暴发事件实验室检测分析

目的 分析一起疑似由产气荚膜梭菌导致腹泻暴发事件的实验室检测结果，为产气荚膜梭菌食物中毒实验室检测策略的改进奠定基础。方法 采集暴发事件中4例病例肛拭子样本，应用荧光PCR方法检测肛拭子及其增菌液中cpa基因与cpe基因。对肛拭子样本进行产气荚膜梭菌分离培养，对部分分离单菌落进行产气荚膜梭菌毒力基因检测和脉冲场凝胶电泳（PFGE）分子分型。结果 4例病例样本中均检测到cpa基因和cpe基因。从病例1样本中挑取并鉴定为产气荚膜梭菌的18个单菌落中获得1个cpe+菌落，构成比为5.56%（1/18）；从病例2样本中挑取并鉴定为产气荚膜梭菌的6个单菌落中获得1个cpe+菌落，构成比为16.7%（1/6）；病例3未分离到cpe+菌落，病例4未分离到产气荚膜梭菌。11株产气荚膜梭菌菌株包括A型和C型两种，包含5种PFGE带型，分离自病例1和病例2的cpe+阳性菌株PFGE带型一致。结论 本次暴发事件可能由产气荚膜梭菌导致，综合使用各种实验室检测方法可在产气荚膜梭菌诊断标准滞后的情况下协助暴发事件分析。     

优化生活饮用水中微生物检测的处理方法

目的优化生活饮用水微生物检测中总大肠菌群、耐热大肠菌群、大肠埃希氏菌滤膜法的稀释及过滤方法,并验证方法可行性。方法待测水样稀释1倍,旋转加入稀释液过滤,再依据国家标准GB/T5750.12-2006《生活饮用水标准检验方法微生物指标》滤膜法的条件,培养、验证;同时按照该方法处理能力验证待测水样,检验并上报结果。结果总大肠菌、耐热大肠菌、大肠埃希氏菌菌落在滤膜上生长均匀,可计数,能力验证结果|Z|均小于2。结论通过能力验证证实本实验稀释方法合理,过滤手段有效,适用于复杂水样总大肠菌群、耐热大肠菌群、大肠埃希氏菌的检测。     

Comparison of five anaerobic incubation methods for enumeration of Clostridium perfringens from foods.

This study compared the cost, speed, convenience, and sensitivity of five anaerobic systems. Fung's double-tube (FDT) method, the minitube method (MT), the sandwiched microtiter plate (SMP) method, and the Mitsubishi AnaeroPack System were compared with the Brewer anaerobic jar for total anaerobic bacterial counts in foods. Incubation was at 37 degrees C for 4, 8, 12, and 24 h. The results indicated that FDT, MT, SMP, and the Mitsubishi AnaeroPack System were as sensitive as the Brewer anaerobic jar for the detection and enumeration of Clostridium perfringens from food products. The FDT, MT, and SMP methods recovered higher numbers of C. perfringens compared to the Brewer anaerobic jar (P < 0.05) after 12 and 24 h incubation. The Brewer anaerobic jar was the most expensive method.     

The Detection and Enumeration of Clostridium perfrinqens in Foods

SUMMARY— A method that uses SPS agar and incorporates an improved egg yolk agar and nitrate motility medium has been developed for the enumeration and confirmation of vegetative cells and/or spores of Clostridium perfringens in foods. The method is based upon several diagnostic criteria and can be completed within 48 hr.     

超滤-改良活性污泥法处理桉木CTMP制浆废液可行性研究

按四种不同工艺条件，实验室模拟制浆过程提取出四种桉木CTMP制浆废液。采用超滤法对废液进行处理，找出最佳处理的膜孔径，并用该孔径的膜去处理四种不同工艺条件的废液。结果表明：经超滤处理，使废液的污染负荷大大降低；清洗后膜的通量可恢复99％；滤过液的生物可处理性明显提高，对经过最佳膜孔径的超滤膜处理后的废液进行改良活性污泥法处理后，出水水质达到GB 3544-92二级排放标准。     

Lessons from the organization of a proficiency testing program in food microbiology by interlaboratory comparison: analytical methods in use, impact of methods on bacterial counts and measurement uncertainty of bacterial counts

The proficiency testing program in food microbiology RAEMA (Réseau d'Analyses et d'Echanges en Microbiologie des Aliments), created in 1988, currently includes 450 participating laboratories. This interlaboratory comparison establishes proficiency in detection of Salmonella and Listeria monocytogenes, as well as enumeration of aerobic micro-organisms, Enterobacteriaceae, coliforms, beta-glucuronidase-positive Escherichia coli, anaerobic sulfito-reducing bacteria, Clostridium perfringens, coagulase-positive staphylococci, and L. monocytogenes. Twice a year, five units samples are sent to participants to assess their precision and trueness for enumeration and detection of micro-organisms. Most of participating laboratories use standard or validated alternative methods, they were 50-70% in 1994 and, for 5 years, they are 95%. An increasing use of alternative methods was also observed. This phenomenon is all the more significant as standard methods are laborious and time consuming; thus, 50% of the laboratories use alternative methods for the detection of Salmonella and L. monocytogenes. More and more laboratories use ready-to-use media and although the percentage is variable according to the microflora, we can consider that, today, 50-60% of the laboratories participating to the proficiency program only use ready-to-use media. The internal quality assurance programs lead also to an increasing use of media quality controls. The impact of analytical methods on bacterial counts was assessed by grouping together the results obtained by participating laboratories during the 10 last testing schemes from 1999 to 2003. The identified significant factors influencing enumeration results are variable from one microflora to another. Some of them significantly influence many microflora: the plating method (spiral plating or not) is influential for aerobic micro-organisms, Enterobacteriaceae, coliforms, and staphylococci, the type of culture medium and the medium manufacturer is influential for aerobic micro-organisms, Enterobacteriaceae, coliforms, E. coli, anaerobic sulfito-reducing bacteria, staphylococci, and L. monocytogenes. Others are specific of some micro-organisms: the resuscitation broth for L. monocytogenes, the mode of medium preparation for staphylococci and the incubation temperature for C. perfringens. These effects lead generally to small differences of about 0.1 log10 cfu g(-1), except for the enumeration of anaerobic sulfito-reducing bacteria, where the difference reaches 0.7 log10 cfu g(-1). These results, although difficult to extrapolate to all actual situations, which associate numerous food constituents and physiological states of bacteria to detect or numerate, allow nevertheless the quantification of interlaboratory variations linked to the methods in use. The analysis of bacterial counts obtained by the laboratories participating to the RAEMA proficiency testing program allowed also to validate a formula to calculate the repeatability of bacterial counts and to estimate the between-laboratory uncertainties for the majority of micro-organisms enumerated in food microbiology. The repeatability uncertainty is only indirectly affected by the method in use but depends essentially on the number of counted colonies. On the other hand, the between-laboratory uncertainty varies with the enumeration method in use, this variability is relatively small for the enumerations calling for methods without colony confirmation, i.e. for the enumeration of aerobic micro-organisms, Enterobacteriaceae, 'total' and thermotolerant coliforms, beta-glucuronidase-positive E. coli and coagulase-positive staphylococci with the technique using the rabbit-plasma fibrinogen agar. For these methods, the average between-laboratory standard deviation is 0.17 log10 cfu g(-1). The between-laboratory uncertainty is, on the contrary, larger for more complex techniques. For the enumeration of coagulase-positive staphylococci with the Baird-Parker agar, the between-laboratory standard deviation is equal to 0.23 log10 cfu g(-1), it is equal to 0.28 log10 cfu g(-1) for the enumeration of L. monocytogenes, to 0.34 log10 cfu g(-1) for the enumeration of C. perfringens, and to 0.47 log10 cfu g(-1) for the enumeration of anaerobic sulfito-reducing bacteria.     

大豆粗脂肪酸值检测方法的改进

以大豆粗脂肪酸值检测方法为研究对象,对GB/T 14488.1-2008《植物油料含油量测定》与GB/T 5530-2005的标准测定方法进行改进,减少称量步骤、缩短抽提时间,以提高检测效率。试验结果表明:改进后的大豆粗脂肪酸值检测方法在保证检测结果准确性的前提下,可大幅缩短检测时间,提升检测效率。     

Feasibility of using food-grade additives to control the growth of Clostridium perfringens

Previously, it was demonstrated that the combination of sucrose laurate (SL) ethylenediaminetetraacetate (E) and butylated hydroxyl anisole (B) (SLEB) was an effective antimicrobial agent against both gram-negative (aerobes) and gram-positive (facultative anaerobes) foodborne bacteria. This investigation examines the sensitivity of Clostridium perfringens to SLEB relative to: (1) the minimum inhibitory concentration (MIC) of SLEB required to inhibit the growth of C. perfringens and (2) the antibacterial effectiveness of different combination ratios of SLEB in fluid thioglycollate medium (FTM). Results indicated that the MIC of SLEB (1:1:1, v/v/v) against C. perfringens on tryptose sulfite cycloserine (TSC) agar was > 150 ppm at 37 degrees C. However, in FTM, a SLEB (1:1:1, v/v/v) concentration of > 100 ppm inhibited C. perfringens during an incubation (anaerobic) period of 196 h at 37 degrees C. The sensitivity of C. perfringens to different combination ratios was also investigated in FTM. The results showed that, when the concentrations of SL and E were held at 75 ppm in the SLEB combination, and the concentration of B increased from 0 to 75 ppm, C. perfringens growth increased initially during the first 24 h of incubation (37 degrees C) but remained constant during the next 48 h. Similarly, when concentrations of SL and E were held constant at 150 ppm in the SLEB combination and the B ratio increased from 50 to 150 ppm in FTM, C. perfringens viability decreased in all of the treated samples during 72-h incubation at 37 degrees C. The results indicated that SLEB was an effective inhibitor of C. perfringens growth activities, and the ratios of the components of SLEB can be adjusted to meet specific preservation needs.     

矿泉水中铜绿假单胞菌及溴酸盐污染情况分析

了解国标《饮用天然矿泉水》(GB 8537-2008)实施前后矿泉水中铜绿假单胞菌(Pseudomonas aeruginosa)及溴酸盐污染情况,分析其污染原因,采用国标GB/T 8538-2008/54滤膜法和GB/T 5750.10-2006离子色谱法对样品中PA和溴酸盐进行分别检测。结果发现,在2008年1月至2009年9月期间,水源水和成品水中PA超标率分别为11.43%和35.42%,成品水中溴酸盐超标率为42.12%。在2009年10月至2010年9月期间,水源水和成品水PA超标率分别为30.30%和7.65%,成品水中溴酸盐超标率为18.10%。国标实施前后成品水PA污染主要原因分别在于加工过程的污染和水源水的污染。因此国标实施后矿泉水中仍存在PA和溴酸盐超标现象。     

RapidChek检测食品中单增李斯特菌的方法评价

目的评价RapidChek法检测食品中单增李斯特菌的检测效果并验证。方法采用t检验,比较RapidChek方法与GB 4789.30-2016方法的培养基增菌效果。依据ISO 16140:2003《食品和动物饲料微生物学-可替代方法的验证方案》,比较RapidChek检测方法与GB 4789.30-2016检测方法的检测效果,涉及的性能指标有相对准确性、相对特异性和相对灵敏度、包含性和排他性。结果 RapidChek检测方法增菌培养基效果优于GB 4789.30-2016方法增菌培养基LB_1。根据RapidChek检测方法、GB 4789.30-2016培养基+RapidChek试纸条方法、RapidChek培养基+GB 4789.30分离鉴定方法的统计检验得出,3种方法与参照方法在相对准确性、相对特异性和相对灵敏度方面无显著性差异。在包含性和排他性方面,RapidChek检测方法与GB 4789.30-2016检测方法的检测结果均一致。结论在所验证的指标上,RapidChek检测方法(包括与GB4789.30-2016组合的方法)与GB 4789.30-2016方法无差异。