The use of R2A medium and the spread plate method for the enumeration of heterotrophic bacteria in drinking water

The pour plate method with yeast extract agar and a 3 d incubation period is the standard method in the UK for the enumeration of heterotrophic bacteria in drinking water. We have compared the standard method with other procedures using the spread plate technique, R2A medium and a longer incubation period. The R2A spread plate method with a 7 d incubation period gave an average estimate of bacterial numbers 520 times greater than for the standard method. This alternative method is recommended for obtaining a more accurate estimate of heterotrophic bacterial populations in drinking water.     

A novel agar medium to detect hydrogen peroxide-producing bacteria based on the prussian blue-forming reaction

The classic method for H(2)O(2) detection involving Prussian blue formation was adapted to formulate a novel agar medium that makes possible in situ detection of H(2)O(2) produced by bacteria. Using this medium, colonies of H(2)O(2)-producing species including Streptococcus pyogenes were easily identified by the appearance of blue halos. The utility of the medium was further illustrated by its successful application to the isolation of H(2)O(2)-producing mutants from a non-H(2)O(2)-producing stain of S. pyogenes.     

Comparison of agar plate and real-time PCR on enumeration of Lactobacillus, Clostridium perfringens and total anaerobic bacteria in dog faeces

AIMS: To compare agar plate and real-time PCR methods on enumeration of total anaerobic bacteria, Lactobacillus and Clostridium perfringens in dog faeces. METHODS AND RESULTS: Thirty-two faecal specimens from Labrador retriever dogs were used to compare agar plate and real-time PCR enumeration methods for Lactobacillus, C. perfringens and total anaerobic bacteria. Total anaerobic bacteria, C. perfringens and Lactobacillus of faeces were counted (as CFU g(-1) faeces) for 48-h incubation at 37 degrees C in an anaerobic gas chamber on genus-selective media. Total genomic DNA from samples was extracted by the QIAamp DNA stool mini kit. The quantification of DNA (as DNA copy per gram faeces) by real-time PCR was performed with a LightCycler system with the QuantiTect SYBR green PCR kit for PCR amplification. The results indicated that there was a significant correlation between CFU and DNA copy of Lactobacillus (R2 = 0.78, P < 0.01) and total anaerobic bacteria (R2 = 0.21, P < 0.05); but no correlation was found between CFU and DNA copy of C. perfringens. The regression equations for Lactobacillus and total anaerobic bacteria were log(DNA copy) = 0.83 x log(CFU) + 1.43 and log(DNA copy) = 1.62 x log(CFU) - 6.32 respectively. CONCLUSIONS: The real-time PCR method could be used to enumerate Lactobacillus within 2 days when compared with plating method which requires 5-6 days. SIGNIFICANCE AND IMPACT OF THE STUDY: The real-time PCR method and the primer set for Lactobacillus spp. harboured in the dog intestine can be used for rapid enumeration of lactobacilli and monitoring of the faecal Lactobacillus community.     

泥螺养殖滩涂异养菌群和弧菌的检测

1　引　　言泥螺 (Ｂｕｌｌａｃｔａｅｘａｒａｔａ)隶属腹足纲软体动物 ,肉质鲜嫩、营养丰富 ,是浙江沿海重要的滩涂养殖品种 .至 2 0 0 0年浙江省养殖面积已达 1.13× 10 4ｈｍ2 ,由于实行生态养殖 ,本轻利厚 ,广受养农的青睐 .随着泥螺养殖生产的发展 ,养殖业的自身污染和陆源排污水的大量入海 ,养殖生态环境越趋恶化 ,使生物病原大量滋生 ,自 1995年以来常诱发养殖泥螺的暴发性死亡 .有关海洋滩涂环境细菌的研究至今未见报道 .为探明泥螺养殖周期内暴发性死亡与滩涂环境的关系 ,对养殖滩涂的细菌菌群组成及其生态特性进行了检测分析 .2…     

杭州西湖水体中异养细菌生长的限制因子

通过添加不同营养盐类的纯种和自然培养试验,对异养细菌生长的限制因子进行了研究．结果表明,生物可利用的有机碳是主要的限制性营养因子,而氮源和磷源的影响相对较小;湖水的高pH、丰富的藻类和浮游动物生物量也制约了异养细菌的生长．此外还发现,在自然水体中添加0．5％葡萄糖后,一些自生固氮细菌得到富集;添加C+N+P后,有大量霉菌生长,添加0．01％牛肉膏后,假单胞菌属(PSeu-domonas)细菌由原来的30％提高到57％;湖水进行实验室培养时细菌的最大生长量可达10^5个·ml^-1。     

Statistical approach for comparison between methods of bacterial enumeration in drinking water

A general statistical procedure based on the likelihood ratio test is presented for the purpose of comparing estimates of mean bacterial density derived from different sets of data. This approach is much more appropriate than the conventional ways of analyzing bacteriological results (e.g., analysis of variance) which usually require previous transformation of the data. An illustrative application of the method compares three distinct titration techniques for enumerating heterotrophic bacteria in drinking water at 20°C incubation temperature. It was shown that both the standard plate count (SPC) and the membrane filter (MF) procedures supplied substantially the same information, whereas the microplate technique using the most probable number (MPN) for total bacterial enumeration could yield considerably different estimates: MPN values were significantly lower in three cases and significantly higher in one case out of a total of five experiments. The results consistently indicate a strong interaction between the technique used and the sample analyzed. Three different media (nutrient agar, R-2A low nutrient agar and m-SPC agar) were then evaluated for enumerating heterotrophic bacteria, using the MF technique at 48, 72 and 96 h of incubation time at 20°C. Although the media recovered approximately the same numbers of bacteria after 96 h of incubation, statistically significant discrepancies occurred after intermediate periods of incubation, perhaps because the relative rates of bacterial growth differed among media.     

A new solid medium for the isolation and enumeration of Thiobacillus ferrooxidans and acidophilic heterotrophic bacteria

A solid medium (FeTSB) was developed for the simultaneous isolation and enumeration of the iron-oxidising bacterium Thiobacilluls ferrooxidans and acidophilic heterotrophic bacteria. The medium consisted of ferrous sulfate, tryptone soya broth and basal salts, solidified with agarose, although bacteriological agar could be substituted for some strains. The medium has been used to isolate bacteria from natural environments and also in laboratory studies of characterised strains. The factors which influence the success of colony growth on solid media are discussed.     

泥浸汁对太湖沉积物中的好氧可培养细菌多样性的影响

【目的】研究添加泥浸汁与否对太湖沉积物中可培养细菌的影响。【方法】采用R2A培养基和添加泥浸汁R2A培养基对沉积物中细菌进行分离培养,16S r RNA基因系统发育分析比较种群结构。【结果】培养基中添加泥浸汁,可使可培养细菌的种类数量增加到1.6倍。16S r RNA基因序列分析表明,培养的优势细菌类群存在明显差别。R2A培养基上生长的细菌主要为厚壁菌门(52%)、放线菌门(24%)、变形菌门(20%)和拟杆菌门(4%),其中大部分细菌与芽孢杆菌属、假单胞菌属、节杆菌属等关系密切;而添加泥浸汁的R2A培养基上生长的细菌则主要为变形菌门(40%)、放线菌门(35%)、厚壁菌门(22.5%)和拟杆菌门(2.5%),与鞘脂单胞菌属、芽孢杆菌属、副球菌属、节杆菌属等关系密切。【结论】添加泥浸汁原始营养因子可提高沉积物中可培养细菌的多样性,提高菌种可培养效率。     

A porous agar medium for improving the growth of plants under sterile conditions

Porous nutrient agar was prepared under sterile conditions by drawing molten 3.5% (w/v) nutrient agar into a plastic syringe, allowing it to set, extruding it into a test tube and giving the tube a firm flick. Simple colorimetric tests showed that gaseous diffusion was substantially faster through 3.5% (w/v) porous agar than through the 1% (w/v) non-porous agar frequently used for growing plants under sterile conditions. Root systems ofTrifolium subterraneum grew 80–90% larger in porous than in non-porous agar.     

Modified Pseudomonas agar: new differential medium for the detection/enumeration of Pseudomonas aeruginosa in mineral water.

Pseudomonas aeruginosa has been implicated as a foodborne and waterborne pathogen and is now considered a primary infectious agent. In the present study, the survival of P. aeruginosa inoculated in mineral water was evaluated by drop counts on Pseudomonas Agar Base (PAB), PAB with CN supplement X107, PAB with cetrimide, PAB with nalidixic acid, and these media with added FeSO(4). Initial counts, before starvation, were the same in all media tested. Following this period, P. aeruginosa became sensitive to PAB with added cetrimide. The addition of FeSO(4) did not improve the recovery of stressed P. aeruginosa but gave colonies a typical dark brown colour being easily differentiated from other species that can grow at 42 degrees C. The modified Pseudomonas agar medium was also tested with several P. aeruginosa strains, other species of Pseudomonas, and other genera. Only P. aeruginosa strains (pyocyanin positive) produced the typical colonies. Our results demonstrate that Pseudomonas agar with ferrous sulphate, used for the differentiation of P. aeruginosa colonies, and nalidixic acid, used as an inhibitor of Gram-positive bacteria, might be a useful medium for the detection of injured P. aeruginosa in mineral water.     

A note on a selective agar medium for the enumeration of Flavobacterium species in water

A selective nutrient agar medium containing kanamycin at 50 micrograms/ml was developed for the isolation and enumeration of yellow-pigmented colonies from the River Sowe, Coventry. Such organisms were shown to be members of the heterogeneous genus Flavobacterium. Typically, yellow pigmented colonies constituted less than 10% of the colonies on nutrient agar alone but up to 70% on nutrient agar plus kanamycin. This medium is a useful addition to the range of media available for the isolation and further ecological study of particular species of this important group of micro-organisms.     

A note on a selective agar medium for the enumeration of Flavobacterium species in water

A selective nutrient agar medium containing kanamycin at 50 μg/ml was developed for the isolation and enumeration of yellow-pigmented colonies from the River Sowe, Coventry. Such organisms were shown to be members of the heterogeneous genus Flavobacterium . Typically, yellow pigmented colonies constituted less than 10% of the colonies on nutrient agar alone but up to 70% on nutrient agar plus kanamycin. This medium is a useful addition to the range of media available for the isolation and further ecological study of particular species of this important group of micro-organisms.     

A new medium for the enumeration and subculture of bacteria from potable water

Plate count agar is presently the recommended medium for the standard bacterial plate count (35 degrees C, 48-h incubation) of water and wastewater. However, plate count agar does not permit the growth of many bacteria that may be present in treated potable water supplies. A new medium was developed for use in heterotrophic plate count analyses and for subculture of bacteria isolated from potable water samples. The new medium, designated R2A, contains 0.5 g of yeast extract, 0.5 g of Difco Proteose Peptone no. 3 (Difco Laboratories), 0.5 g of Casamino Acids (Difco), 0.5 g of glucose, 0.5 g of soluble starch, 0.3 g of K2HPO4, 0.05 g of MgSO4 X 7H2O, 0.3 g of sodium pyruvate, and 15 g of agar per liter of laboratory quality water. Adjust the pH to 7.2 with crystalline K2HPO4 or KH2PO4 and sterilize at 121 degrees C for 15 min. Results from parallel studies with spread, membrane filter, and pour plate procedures showed that R2A medium yielded significantly higher bacterial counts than did plate count agar. Studies of the effect of incubation temperature showed that the magnitude of the count was inversely proportional to the incubation temperature. Longer incubation time, up to 14 days, yielded higher counts and increased detection of pigmented bacteria. Maximal bacterial counts were obtained after incubation at 20 degrees C for 14 days. As a tool to monitor heterotrophic bacterial populations in water treatment processes and in treated distribution water, R2A spread or membrane filter plates incubated at 28 degrees C for 5 to 7 days is recommended.(ABSTRACT TRUNCATED AT 250 WORDS)     

A new medium for the enumeration and subculture of bacteria from potable water.

Plate count agar is presently the recommended medium for the standard bacterial plate count (35 degrees C, 48-h incubation) of water and wastewater. However, plate count agar does not permit the growth of many bacteria that may be present in treated potable water supplies. A new medium was developed for use in heterotrophic plate count analyses and for subculture of bacteria isolated from potable water samples. The new medium, designated R2A, contains 0.5 g of yeast extract, 0.5 g of Difco Proteose Peptone no. 3 (Difco Laboratories), 0.5 g of Casamino Acids (Difco), 0.5 g of glucose, 0.5 g of soluble starch, 0.3 g of K2HPO4, 0.05 g of MgSO4 X 7H2O, 0.3 g of sodium pyruvate, and 15 g of agar per liter of laboratory quality water. Adjust the pH to 7.2 with crystalline K2HPO4 or KH2PO4 and sterilize at 121 degrees C for 15 min. Results from parallel studies with spread, membrane filter, and pour plate procedures showed that R2A medium yielded significantly higher bacterial counts than did plate count agar. Studies of the effect of incubation temperature showed that the magnitude of the count was inversely proportional to the incubation temperature. Longer incubation time, up to 14 days, yielded higher counts and increased detection of pigmented bacteria. Maximal bacterial counts were obtained after incubation at 20 degrees C for 14 days. As a tool to monitor heterotrophic bacterial populations in water treatment processes and in treated distribution water, R2A spread or membrane filter plates incubated at 28 degrees C for 5 to 7 days is recommended.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evaluation of water corrosion,scaling extent and heterotrophic plate count bacteria in asbestos and polyethylene pipes in drinking water distribution system

Corrosion and scaling is one of the most important factors influencing drinking water quality that cause health disorders and economic problems. The aim of this study was to evaluate these phenomena in two sources of surface (Makou city) and ground water (Khoy city) in water networks. Corrosion and scaling potential was surveyed by Langelier, Ryzener, aggressiveness, Larson and Puckorius Indices and with measuring water physical, chemical, and microbial parameters. Statistical paired samples t-test displayed significant difference in means value of Langelier, Ryzener, Puckorius indices between cold and warm seasons of the year in Khoy samples and significant difference in means value of Ryzener, Puckorius and aggressiveness indices between cold and warm seasons of the year in Makou samples (p-value <0.001). Heterotrophic plate count water samples investigated in two cold and hot seasons in Khoy were respectively 14 ± 16 cfu ml^?1 and 41 ± 26 cfu ml^?1 and in the town of Makou were 11 ± 7 cfu ml^?1 and 61 ± 29 cfu ml^?1, respectively. In terms of health impacts, corrosion in different mains is important, then providing proper measures for balancing water quality before entering to the network and substituting of mains to prevent economic and health problems are necessary.     

PPP2R2A在乳腺癌细胞中结合GFPT2并导致其去磷酸化

PPP2R2A是PP2A磷酸酶的调控亚基之一,以往的研究报道显示,PPP2R2A可促进肿瘤细胞生存和生长。本研究通过串联亲和纯化联合HPLC-Chip-ESI/MS/MS筛选PPP2R2A的相互作用蛋白质,分析结果显示,L-谷氨酰胺-D-果糖-6-磷酸转氨酶1(Glutamine-fructose-6-phosphate transaminase 1,GFPT1)和L-谷氨酰胺-D-果糖-6-磷酸转氨酶2(Glutamine-fructose-6-phosphate transaminase 2,GFPT2)是PPP2R2A可能的结合蛋白。通过免疫荧光共定位、GST Pull-down和免疫共沉淀等方法,进一步确认了PPP2R2A和GFPT1及GFPT2的相互结合。通过shRNA下调PPP2R2A后,GFPT2的磷酸化水平显著增加,但GFPT1的磷酸化水平改变不明显。GFPT2是O-GlcNAC糖基化修饰通路中的一个限速酶,在乳腺癌细胞MDA-MB-231中下调PPP2R2A后,蛋白质O-GlcNAC糖基化修饰水平增加。这些结果表明,PPP2R2A可直接结合GFPT2,并导致其去磷酸化,进而影响细胞内O-GlcNAC糖基化修饰。     

3M Petrifilm快速菌落总数测试片法与食品中菌落总数检测国标方法（GB 4789.2‒2010）的比较

目的 比较3MTM PetrifilmTM快速菌落总数测试片（RAC）法与食品中菌落总数检测国标方法（GB 4789.2‒2010）检测熟肉样品、人工污染熟肉样品中的菌落总数结果的一致性。方法 分别用两种方法对129份熟肉样品和166份人工污染熟肉样品进行菌落总数项目的检测,并对3MTM PetrifilmTM快速菌落测试片法与国标方法的实验结果进行配对资料t检验、线性回归分析以及对数值差值绝对值（|dlog|）汇总分析。结果 第一部分：两种方法检测熟肉样品、人工污染熟肉样品的菌落总数检测结果t=1.5704、P=0.1188,差异无统计学意义;相关系数R2值分别为0.897、0.964;|dlog|≤0.500所占百分比分别为97.7％、100.0％。第二部分：两种方法检测295份样品,t=1.1336,P=0.2586;相关系数R2=0.992;|dlog|≤0.500的结果百分率为99.0％。结论 在检测熟肉样品、人工污染熟肉样品时,3MTM PetrifilmTM快速菌落总数测试片法与国标方法检测结果的一致性较好。     

Oligotrophic bacteria in ultra-pure water systems: Media selection and process component evaluations

Summary Presently, tryptic soy agar (TSA) medium is used in the semiconductor industry to determine the concentration of viable oligotrophic bacteria in ultra-pure water systems. Deionized water from an ultra-pure water pilot plant was evaluated for bacterial growth at specific locations, using a non-selective medium (R2A) designed to detect injured heterotrophic as well as oligotrophic bacteria. Results were compared to those obtained using Tryptic Soy Agar. Statistically greater numbers of bacteria were observed when R2A was used as the growth medium. Total viable bacterial numbers were compared both before and after each treatment step of the recirculating loop to determine their effectiveness in removing bacteria. The reduction in bacterial numbers for the reverse osmosis unit, the ion exchange bed, and the ultraviolet sterilizer were 97.4%, 31.3%, and 72.8%, respectively, using TSA medium, and 98.4%, 78.4%, and 35.8% using R2A medium. The number of viable bacteria increased by 60.7% based on TSA medium and 15.7% based on R2A medium after passage of the water through an in-line 0.2-m pore size nylon filter, probably because of the growth of bacteria on the filter. Our results suggest that R2A medium may give a better representation of the microbial water quality in ultra-pure water systems and therefore a better idea of the effectiveness of the various treatment processes in the control of bacteria.