幽门螺旋菌在双相培养基中生长特性的研究

本文观察了幽门螺旋菌在固体及液体—固体双相培养基上生长的特性,37℃72小时培养后,定量实验表明,固体培养基上菌数为1.02×10~2cfu/ml,而双相培养基上则为1.14×10~4cfu/ml;用血液双相培养基,对CP进行了生长曲线的测定,从0小时加入1.02×10~2cfu/ml开始,按每天平皿活菌计数,共5天(24、48、72、96、120、144h)分别为2.5×10~2cfu/ml,4.97×10~2cfu/ml,1.14×10~4cfu/ml,1.24×10~5cfu/ml,2.87×10~5cfu/ml,9.75×10~4cfu/ml以后逐日下降;液相培养液24—144小时pH测定,pH由7.2下降至6.4—6.9。     

几丁质及其可溶性衍生物对霍乱弧菌在低温下生存的影响

<正> OI群霍乱弧菌C_5株式Mikasagawa株(由外环境分离的一株E1 Tor菌),悬浮于生理盐水或pH7.2缓冲液中,放置0℃保存,其活菌数迅速减少,培育第3天未能测出活菌;只有当细菌浓度低于10~6cfu/ml时才能测出这种在低温下的杀灭作用,而在细菌浓度较高时,此杀灭作用不明显。     

DNA重组技术：Ⅲ.重组质粒在大肠杆菌中的转化

氯化钙法是用质粒DNA转化细菌细胞,尤其是大肠杆菌细胞最常使用的方法。实验操作如下: (1) 将受体菌接种在平板培养基上划线,37℃过夜,进行活化。 (2) 挑选单菌落,接于5ml LB培养液,37℃震荡过夜。 (3) 从上述培养液中取0.5ml,转入50mlLB培养液,37期震荡培养2～4小时,到细菌密度大约为5×10~7/ml,OD_(575)约为0.8。     

绿色荧光蛋白标记的嗜水气单胞菌在越冬水体的饥饿存活及复苏

将绿色荧光蛋白标记的嗜水气单胞菌（Ah4332^GFP）置模拟越冬水体（8-10℃）内,进行饥饿存活试验,用三种计数方法检查水体中的细菌数量变化,在41d后平板计数法（PC）降至0。而细菌总数法（DC）和活菌直接计数法（DVC）结果相似,只是DVC计数结果低于细菌总数1-2个数量级,显示细菌已经变成活的非可培养（Viable but nonculturable,VBNC)状态,复苏试验表明,升高温度、添加鱼血清或新鲜培养的Ah4332^GFP细菌上清及通过兔肠管结扎,均使VBNC状态的Ah4332^GFP得到复苏,荧光显微镜和电镜观察处于可培养的VBNC状态的Ah4332^GFP,后者的细菌细胞比前者体积明显缩小,形态由杆状变成了球形,但细胞膜和细胞壁是完整的,不是细菌L型。     

降雨对秦皇岛西浴场细菌总数和可培养菌群组成的影响

【目的】研究降雨条件对浴场细菌总数和优势菌群组成的影响。【方法】2014年8月强降雨前后采集秦皇岛西浴场3个站位的海水样品,采用荧光显微镜计数法和平板计数法分别对细菌总数和可培养细菌总数进行计数;对群落结构组成进行分析,并对可培养细菌进行鉴定。【结果】雨前3个站位细菌总数和可培养细菌总数平均值分别为5.6×10~9 CFU/L和8.3×10~7 CFU/L,雨后分别为9.2×109 CFU/L和2.1×10~8 CFU/L。在可培养菌群中,变形菌门(Proteobacteria,雨前占80%,雨后占73%)是主要的微生物类群,其次为拟杆菌门(Bacteroides,雨前占12%,雨后占13%)、厚壁菌门(Firmicutes,雨前占7%,雨后占11%)等;肠杆菌属(Enterobacter spp.,21株)、海杆菌属(Marinobacter spp.,13株)、弓形菌属(Arcobacter spp.,13株)、假单胞菌属(Pseudomonas spp.,10株)、芽孢杆菌属(Bacillus spp.,10株)和弧菌属(Vibrio spp.,6株)为雨前可培养细菌优势属,而雨后可培养细菌优势属为肠杆菌属(22株)、海杆菌属(21株)、芽孢杆菌属(14株)、不动杆菌属(Acinetobacter spp.,11株)、假单胞菌属(9株)和弓形菌属(5株)等。【结论】降雨对细菌总数有显著的影响,同时降雨后浴场微生物群落结构发生了改变。     

低温寡营养条件下副溶血弧菌形成活的非可培养状态及其复苏研究

为研究在低温寡营养条件下副溶血弧菌(Vibrio parahaemolyticus)能否进入活的非可培养状态(VBNC),将浓度为1×1010CFU/mL的副溶血弧菌HW799在陈海水中4℃保存,每隔5天取样分别用吖啶橙染色荧光显微镜直接计数法(AODC)、活菌直接计数法(DVC)和涂布平板法(PC)测定细菌总数、活细菌数和可培养细菌数.在第30天时总细菌数基本不变,仍保持在109CFU/mL,活菌数为106CFU/mL,比总菌数低了约三个数量级,可培养细菌数为零,表明绝大部分副溶血弧菌HW799进入了VBNC状态;用扫描电镜、流式细胞仪对副溶血弧菌HW799活的非可培养状态、正常状态以及复苏后的细胞形态的研究表明进入VBNC状态后副溶血弧菌HW799形状变为球状,体积比正常状态明显变小,活细胞数也略有减少;采用在培养液中添加营养物质升温培养的方法,使VBNC状态的副溶血弧菌细胞在48h内复苏为可培养状态,复苏后的副溶血弧菌HW799与正常状态的细菌形态相似.     

生态制剂用于烧伤大鼠局部抗感染的初步观察

本实验使用皮肤常住菌分离株,筛选驯化后成为无毒菌株,经冷冻干燥后,制备成生态制备膏霜,涂于已被烧伤大鼠表面,2h后接种绿脓杆菌(10~9CFu/ml)或金黄色葡萄球菌(10~9CFu/ml),并以空白膏霜涂抹作为对照组,观察其抗感染作用,发现拮抗作用自48h始,痴上绿脓杆菌菌数由1.08×10~(11)CFu/g降至6.51×10~(10)CF/g,痂下由1.12×10~(11)至7.77×10~9CFu/g。至72 h拮抗作明显,痴上绿脓杆菌4.69×10~(10)至2.08×10~8,痂下为1.65×10~(10)至4.5×10~8。感染金黄色葡萄球菌从10~(10)CFu/g到10~8CFu/g,72h后便降至1.02—7.83×10~6CFu/g,并一定程度阻止了感染的细菌入血,而对肠道细菌迁移无作用。     

解淀粉芽孢杆菌TF28活菌定量PMA-qPCR技术

[目的]建立解淀粉芽孢杆菌TF28特异性活菌分子定量技术。[方法]基于TF28特有基因设计q PCR引物,建立菌株特异性q PCR技术;优化PMA处理条件,建立PMA-q PCR定量TF28活菌技术,对其特异性、灵敏性与可靠性进行检测。[结果]建立的q PCR技术对菌株TF28具有特异性。优化的PMA处理条件为:PMA终浓度150μmol/L、暗培养10 min、光照20 min,在此条件下,可封闭106cfu/m L以下死菌基因组DNA;建立的PMA-q PCR技术特异性强,灵敏度高,最低检测限102cfu/m L;线性关系好,R2=0.997;在菌浓度103cfu/m L～107cfu/m L范围内重复性好,CT值变异系数小于2%,与平板活菌计数比较,差异不显著。[结论]建立的PMA-q PCR技术对TF28具有特异性,能够对菌浓度103cfu/m L～107cfu/m L活菌进行定量。     

浓香型白酒窖泥中兼性厌氧细菌的分离鉴定

为探讨浓香型白酒窖泥中微生物区系的构成,采用传统微生物分类鉴定法,对泸州老窖不同窖龄窖底和窖壁泥样的可培养兼性厌氧细菌进行了分类计数,并初步鉴定到属。平板涂布法计数得出,老窖底样的菌落总数最多,达3.98×103cfu/g泥,新窖壁样菌落数最少,只有0.24×103cfu/g泥;经生理生化鉴定,参照伯杰氏细菌鉴定手册等将划线单离后的菌株归为8个属,其大部分属于芽孢杆菌属(Bacillus)和芽孢乳杆菌属(Sporolac-tobacillus),也存在假单胞菌属(Pseudomonas)、梭菌属(Clostridium)等。     

嗜酸乳杆菌固态发酵的初步研究

研究了嗜酸乳杆菌在 4种不同固态基质 (豆渣、麦麸、芋艿渣、纤维素 )中的生长情况 ,结果显示 :1)豆渣作为基质时嗜酸乳杆菌生长最佳 ,最高活菌数可达 1.6 45× 10 9cfu/g ,麦麸次之 ,达 3.2× 10 8cfu/g ,纤维素最差 ,仅为 2 .1× 10 7cfu/g ,并且豆渣固态培养基优于对照的 3种液体培养基 ;2 )豆渣的含水量以 75 %左右为宜 ;3)通过加入可中和所产酸量的CaCO3 ,并用缓冲液代替水配制培养基 ,结果发现用pH 6 .0的缓冲液配制的培养基 ,培养后活菌量可达 2 .12 4× 10 9cfu/g ,,比对照用自来水提高了 4.2倍 .     

Viable but nonculturable bacteria in drinking water.

Klebsiella pneumoniae, Enterobacter aerogenes, Agrobacterium tumefaciens, Streptococcus faecalis, Micrococcus flavus, Bacillus subtilis, and Pseudomonas strains L2 and 719 were tested for the ability to grow and maintain viability in drinking water. Microcosms were employed in the study to monitor growth and survival by plate counts, acridine orange direct counts (AODC), and direct viable counts (DVC). Plate counts dropped below the detection limit within 7 days for all strains except those of Bacillus and Pseudomonas. In all cases, the AODC did not change. The DVC also did not change except that the DVC, on average, were ca. 10-fold lower than the AODC.     

Viable but nonculturable bacteria in drinking water

Klebsiella pneumoniae, Enterobacter aerogenes, Agrobacterium tumefaciens, Streptococcus faecalis, Micrococcus flavus, Bacillus subtilis, and Pseudomonas strains L2 and 719 were tested for the ability to grow and maintain viability in drinking water. Microcosms were employed in the study to monitor growth and survival by plate counts, acridine orange direct counts (AODC), and direct viable counts (DVC). Plate counts dropped below the detection limit within 7 days for all strains except those of Bacillus and Pseudomonas. In all cases, the AODC did not change. The DVC also did not change except that the DVC, on average, were ca. 10-fold lower than the AODC.     

Long-term survival of Legionella pneumophila serogroup 1 under low-nutrient conditions and associated morphological changes

Abstract Extended survival of Legionella pneumophila , using both a clinical and an environmental isolate, was studied in drinking water, creek water, and estuarine water microcosms. Legionella populations were monitored by acridine orange direct counts (AODC) and viable count on buffered charcoal yeast extract agar amended with alpha-ketoglutarate (BCYEα). Initial colony counts of the clinical isolate in drinking and creek water microcosms were 2 × 10⁸ cfu/ml and, after incubation for 1.5 years, the plate counts decreased to 3 × 10⁶ cfu/ml. The AODC counts, however, did not change significantly. The clinical isolate in estuarine water decreased in plate counts to 10² (cfu/ml) over the same period. After incubation for 1.5 years at 15°C in the microcosms, Legionella plate counts of creek and drinking water decreased by two logs. Direct microscopic examination of aliquots removed from all microcosms revealed the presence of small bacilli, large bacilli and rare filamentous cells. The environmental isolate demonstrated only one colony morphology upon culture on BCYEα. Interestingly, after four months incubation in the microcosm, upon plating the clinical isolate on BCYEα, two distinct colony types were evident. Examination by immunofluorescent staining employing a monoclonal antibody against L. pneumophila revealed both bacillus and filamentous forms. The total cellular proteins of both morphotypes were examined by sodium dodecyl sulfate polyacrylyamide gel electrophoresis (SDS-PAGE), demonstrating identical protein patterns. Those Legionella cells remaining culturable during 1.5 years of incubation grew rapidly when transferred to BCYEα. Incubation was continued and it was found that some strains of L. pneumophila serogroup 1 can remain viable for longer than 2.4 years under low-nutrient conditions.     

Use of microcosms to determine the survival of the fish pathogen Tenacibaculum maritimum in seawater

The survival of the fish pathogen Tenacibaculum maritimum in different seawater microcosms was investigated during 160 days. The persistence of culturable cells was greater in sterile than in natural seawater. Standard plate counts showed that T. maritimum survived in sterile seawater for more than 5 months at concentration around 10(3) cfu ml(-1). However, T. maritimum proved to be very labile in non-sterile seawater, rendering culturable cells no longer than 5 days. These results were confirmed when DNA-based methods were applied. Regardless of the microcosms used, epifluorescence microscopy counts remained at about 10(6) cells ml(-1) throughout the experiment, even though we can not distinguish T. maritimum in the case of non-sterile microcosms. Resuscitation assays with addition of fresh medium to non-sterile microcosms did not favour the recovery of T. maritimum on solid media. Although morphological changes from filamentous to spheres were observed after 3 days in the non-sterile microcosms, in the case of the sterile microcosms this change was observed at the sixth day. The biochemical, physiological, serological and genetic characteristics were unaffected in the sterile microcosms. The overall results contribute to a better understanding of the behaviour of T. maritimum in natural seawater and suggest that the aquatic bacterial population play an important role in the survival of this fish pathogen.     

Characterization and resuscitation of viable but nonculturable <Emphasis Type="Italic">Vibrio alginolyticus</Emphasis> VIB283

The aim of this study was to investigate the viable but nonculturable (VBNC) state of the bacterium. Vibrio alginolyticus VIB283 was cultured in sterilized seawater microcosm at 4°C. Culturability of the cells in the microcosm was monitored by spread plate count (PC) on 2216E agar, PCs declined to undetectable levels (<0.1 CFU/ml) within 90 days. Total cell counts remained constant throughout the period as determined by acridine orange direct count (AODC). The direct viable counts, on the other hand, declined from 10¹⁰ to 10⁹ CFU/ml active cells and remained fairly constant at this level by direct viable count (DVC), which indicated that a large population of cells entered into the VBNC state. The VBNC cells could be resuscitated by temperature upshift with and without the presence of nutrition. The resuscitated time were 16 h and 8 days respectively. The resuscitation was not achieved in chick embryos. The morphology of the VBNC, normal and resuscitated cells was studied with scanning electron microscope and flow cytometry. The cells changed from rod or arc to coccoid and decreased in size when entered into the VBNC state. The resuscitated and the normal cells had almost no morphological differences.     

Lack of colonization of 1 day old chicks by viable, non-culturable Campylobacter jejuni.

Seven strains of Campylobacter jejuni, isolated from various sources [human (n = 2), chicken (n = 3), water (n = 2)], were studied under starvation conditions in filter-sterilized and pasteurized surface water by acridine orange direct count (AODC), viable count (DVC) and culture methods. Plate counts showed a rapid decline (2 log-units/day) for all strains under these conditions. Only one of the seven strains (14%) showed a (prolonged) viable, non-culturable 'state'. The ability of these viable, non-culturable cells to colonize the intestine was tested on day-old chicks. The infectious oral dose of freshly cultured cells of this model was 26-260 cfu; 1.8 x 10(5) viable, non-culturable C. jejuni were introduced to day-old chicks orally. Campylobacter jejuni was not isolated from the caeca of the chicks after incubation for 7 d. Also, passage through the allantoic fluid of embryonated eggs did not recover viable, non-culturable C. jejuni. These findings cast serious doubts on the significance of the viable, non-culturable 'state' in environmental transmission of C. jejuni.     

The use of Alcalase 2·5L in the acridine orange direct count technique for the rapid enumeration of bacteria in beef mince

The acridine orange direct count (AODC) technique was used to count bacterial numbers in beef mince. The ability of Alcalase 2·5L to degrade beef mince was compared with the previously used Alcalase 0·6L. Both methods were evaluated against the standard plate count. The results showed that Alcalase 2·5L can be used in the AODC technique for the rapid counting of bacterial numbers in beef mince. Prediction equations obtained for the relationship between the AODC and standard plate counts were validated with commercial beef mince samples.     

Lack of colonization of 1 day old chicks by viable, non-culturable Campylobacter jejuni

G.J. MEDEMA, F.M. SCHETS, A.W. VAN DE GIESSEN AND A.H. HAVELAAR. 1992. Seven strains of Campylobacter jejuni , isolated from various sources [human ( n = 2), chicken ( n = 3), water ( n = 2)], were studied under starvation conditions in filter-sterilized and pasteurized surface water by acridine orange direct count (AODC), viable count (DVC) and culture methods. Plate counts showed a rapid decline (2 log-units/day) for all strains under these conditions. Only one of the seven strains (14%) showed a (prolonged) viable, non-culturable 'state'. The ability of these viable, non-culturable cells to colonize the intestine was tested on day-old chicks. The infectious oral dose of freshly cultured cells of this model was 26–260 cfu; 1.8×10⁵ viable, non-culturable C. jejuni were introduced to day-old chicks orally. Campylobacter jejuni was not isolated from the caeca of the chicks after incubation for 7 d. Also, passage through the allantoic fluid of embryonated eggs did not recover viable, nonculturable C. jejuni. These findings cast serious doubts on the significance of the viable, non-culturable 'state' in environmental transmission of C. jejuni.     

Viable but nonrecoverable stage of Salmonella enteritidis in aquatic systems

An environmental isolate (13- 1BB ) of Salmonella enteritidis serogroup C1 was inoculated into sterile Potomac River water microcosms to observe survival and culturability of the organism by employing acridine orange direct count, fluorescent antibody direct count, direct viable count, plate count on veal infusion agar and xylose lysine decarboxylase agar, and indirect enumeration by the most-probable-number method (MPN), using media selective for Salmonella. Loss of culturability on laboratory media was observed within 48 h. However, cultures could be "resuscitated" and cultured on solid media, following addition of nutrients to the microcosms . Cells, resuscitated 4 days after apparent "die-off" (0 colony-forming units (cfu)/mL) using plate count techniques, yielded numbers of cfu in the same order of magnitude as had been observed before the onset of nutrient limitation. Microscopic techniques for direct viable counting indicated that viability is maintained for as long as 60 days after depletion of nutrients, although attempts to culture these cells, by addition of nutrient, after 21 days yielded apparently sterile plates. Thus, longer periods of "dormancy" appear to require conditions other than simple nutrient addition for resumption of cell growth and division.     

Restoration of culturability of starvation-stressed and low-temperature-stressed Escherichia coli O157 cells by using H2O2-degrading compounds

Late-exponential-phase cells of Escherichia coli O157:H- strain E32511/HSC became nonculturable in sterilized distilled water microcosms at 4 °C. Plate counts declined from 3 × 10⁶ to less than 0.1 CFU/ml in about 21 days. However, when samples of microcosms at 21 days were inoculated onto an agar medium amended with catalase or nonenzyme peroxide-degrading compounds such as sodium pyruvate or α-ketoglutaric acid, plate counts increased to 10⁴–10⁵ CFU/ml within 48 h. The proposed mode of action of the catalase or pyruvate is via the degradation of the metabolic by-product H₂O₂, rather than through supplementation of a required nutrient in the recovery of nonculturable cells. Our studies were based on the assumption that E32511/HSC strain responds to starvation and a low temperature by entering a nonculturable state and that the correction of oxidative stress upon the inoculation of bacteria on agar plates promotes recovery of nonculturable cells. Received: 15 January 1999 / Accepted: 8 April 1999