Comparison of plate count agar and R2A medium for enumeration of heterotrophic bacteria in natural mineral water

In order to recover as many viable bacteria as possible from natural mineral water, in this study we have compared the counts obtained with the standard method (pour plate procedure with Plate Count Agar (PCA)) and counts with alternative test methods (PCA/spread plates, R2A medium/pour plates and R2A medium/spread plates). The results showed that counts with R2A medium/spread plates at 22°C and after a 7-day incubation period were more than 343% higher than those obtained with PCA/pour plate method. At 37°C and after a 3-day incubation period, the R2A pour plate technique gave counts about 368% greater than for the standard method. Moreover, while Pseudomonas, Comamonas and Acinetobacter species were isolated both from PCA and R2A medium, Flavobacterium spp. and Arthrobacter spp. were isolated only from R2A medium. For its higher productivity, R2A medium should be recommended for heterotrophic plate counts in natural mineral water.     

An improved organ culture method for adult mammalian lung

Summary An improved method for maintaining adult rat lung in submerged organ culture is described in which the alveoli were inflated with agar and 200-μm-thick hand-cut sections were mounted in Rose chambers. The conventional single-compartmented Rose culture chamber was modified by adding a second chamber separated from the first by a gaspermeable membrane. One compartment functioned as an air reservoir and the other housed the explants submerged in nutrient medium. Visking dialysis membrane used underneath the explants prevented cell outgrowth and facilitated the exchange of nutrients and waste products at the glass-tissue interface. Because of the excellent optical properties of the Rose chamber and the thinness of the explants, individual cell types can be identified in the living tissue. The explants were studied with time-lapse cinematography, light microscopy, histology, and with erythrosine B for dye exclusion. With this modified system the functional life span of the explants was increased from 1 week to 1 month. This study was supported by NHLBI Grant No. HL15098-05.     

厌氧培养皿及厌氧培养管的临床应用——对传统的厌氧菌分离培养及鉴定方法的简化

利用我们研制的厌氧培养皿及管,共检验了39份牙髓炎及牙周炎标本,分离出厌氧菌59株,标本中厌氧菌分离的阳性率为100％。其中类杆菌28株,消化链球菌9株,消化球菌7株,韦荣氏球菌5株,真杆菌2株,梭形杆菌8株。在类杆菌中产黑素类杆菌21株,占类杆菌总数的75％。结果表明使用厌氧培养皿及管研究厌氧菌简便、有效、经济,适于基层单位应用。     

Agar culture of Piscirickettsia salmonis, a serious pathogen of farmed salmonid and marine fish

Piscirickettsia salmonis, a serious bacterial pathogen of farmed marine fish, previously considered culturable only in eukaryotic cell-culture systems, was grown for the first time on agar and broth containing enhanced levels of cysteine, thus greatly increasing the potential for isolation, in vitro culture and study of this organism. Virulence towards Atlantic salmon following passage on agar media was retained in a controlled laboratory trial. Of the studied temperatures, optimal growth on agar was observed at 22 degrees C.     

Guar gum: a cheap substitute for agar in microbial culture media

AIMS: To determine the possibility of using guar gum, a colloidal polysaccharide, as a cheap alternative to agar for gelling microbial culture media. METHODS AND RESULTS: As illustrative examples, 12 fungi and 11 bacteria were cultured on media solidified with either guar gum or agar. All fungi and bacteria exhibited normal growth and differentiation on the media gelled with guar gum. Microscopic examination of the fungi and bacteria grown on agar or guar gum gelled media did not reveal any structural differences. However, growth of most of the fungi was better on guar gum media than agar, and correspondingly, sporulation was also more advanced on the former. Bacterial enumeration studies carried out for Serratia sp. and Pseudomonas sp. by serial dilution and pour-plate method yielded similar counts on both agar and guar gum. Likewise, a selective medium, succinate medium used for growth of Pseudomonas sp. did not support growth of Bacillus sp. when inoculated along with Pseudomonas on both agar or guar gum supplemented medium. CONCLUSIONS: Guar gum, a galactomannan, which is 50 times cheaper than Difco-bacto agar, can be used as a gelling agent in place of agar in microbial culture media. SIGNIFICANCE AND IMPACT OF THE STUDY: As the media gelled with guar gum do not melt at temperature as high as 70 degrees C, these can be used for isolation and maintenance of thermophiles.     

Cultivation of syntrophic anaerobic bacteria in membrane-separated culture devices

A dialysis culture device was used for growth of syntrophic fatty acid-oxidizing and ethanol-oxidizing anaerobic bacteria. A pure culture of the fatty acid oxidizer Clostridium bryantii was grown inside dialysis tubing which was surrounded by a pure culture of Desulfovibrio vulgaris. The same apparatus was used for the syntrophic cultivation of Pelobacter acetylenicus and Acetobacterium woodii with ethanol as substrate. In both cases, substrate degradation and product formation were about half as fast as with the homogeneously mixed control cultures. In the compartment of the hydrogen producer, the concentration of free hydrogen during syntrophic ethanol degradation was about 10 times as high as in that of the hydrogen utilizer, whereas the homogeneously mixed culture exhibited an intermediate hydrogen partial pressure.     

Changes of α-Glycerophosphate Dehydrogenase Activity in Fatty Liver of Rats by Amino Acid Imbalance

An aminopeptidase was purified from an aqueous extract of mullet roe in the presence of 2-mercaptoethanol by fractionation with ammonium sulfate and column chromatography on DEAE-cellulose and Sephadex G-200. The molecular weight of the enzyme was 184,000 by gel filtration, and the enzyme appeared to consist of two homogenous subunits. The optimal pH and optimal temperature for activity were 7.4 and 45°C, respectively. Puromycin, p-chloromercuribenzoic acid, and o-phenanthroline inhibited the enzyme n on-competitively (their Ki = 1.34 μm, 0.113mm and 0.145 mm, respectively), while 2-mercaptoethylamine was competitive (Ki = 0.056 mm). The enzyme was also inhibited by l-amino acids, in particular glutamic acid. The enzyme could hydrolyze a variety of α-aminoacyl β-naphthylamides and was most active on l-alanyl-β-naphthylamide. Judging from these properties, the mullet roe aminopeptidase resembles soluble alanyl amino-peptidase [EC 3.4.11.14].     

Efficiency of colony formation and differentiation of human spermatogenic cells in two different culture systems

Optimization of in vitro culture system for the expansion and the maturation of male germ cells to post meiotic stages is a valuable tool for studies exploring spermatogenesis regulation and the management of male infertility. Several studies have reported promising results of mouse spermatogonial stem cells culture in three-dimensional (3D) culture systems and a subsequent production of sperm. In the present study, we investigated the capacity of a three-dimensional soft agar culture system (SACS) supplemented with Knockout Serum Replacement (KSR) in colony formation and inducing human germ cells to reach post-meiotic stages. Testicular cells from testes of brain -dead donors were first cultured for three weeks in proliferation medium. The cells were subsequently cultured in the upper layer of the SACS (3D group) in a medium supplemented with KSR and hormones, and the results were compared with that of a two-dimensional (2D) culture system. We found that the number and diameter of colonies and the levels of expression of Scp3 and Integrin α6 in the 3D culture group were significantly higher than in the 2D group. Our findings indicate that SACS can reconstruct a microenvironment capable of regulating both proliferation and differentiation of cell colonies.     

Use of cereals as basal medium for the formulation of alternative culture media for fungi

Summary The feasibility of developing alternative media to different culture media particularly potato dextrose agar was assessed using local cereal species as the basal media. Three cereal meal extracts – corn, sorghum and millet – were prepared, using them as substitute for the potato in potato dextrose agar. Potato dextrose agar (PDA) was the standard set up with which the performances of the formulated media were compared. Eight genera of fungi (Aspergillus niger, Fusarium moniliforme, Penicillium sp., Cercospora sp., Curvularia palescens, Botryodiplopodia sp., Rhizopus sp. and Rhodotorula rubra) were isolated and pure cultures of each species aseptically inoculated onto the three different formulated media including PDA and allowed to grow. Their growths were measured at 24, 48, 72, and 96 h after inoculation, using diameter of growth as an index. The set up was repeated thrice for each species on the three formulated media and the control (PDA). Growth of all the fungal species were observed to be about the same or sometimes better in the formulated media relative to those on the standard set up, except for Rhodotorula rubra. The radius of growth of F. moniliformehad an average of 15 + 0.58 mm on corn-dextrose agar relative to 12 mm on PDA at 96 h while Cercospora sp. measured 30 + 0.58 mm on millet-meal dextrose agar relative to 37 + 1.16 mm at 48 h. Botryodiplopodia sp. grew through the whole diameter of the plate (covering the total length of the radius of 45 mm) in both sorghum-meal and PDA at 96 h.     

The effects of culture medium rigidity on adventitious bud production and tissue vitrification in needle cultures of Sitka spruce [Picea sitchensis (Bong.) Carr.]

Adventitious bud formation on Sitka spruce [ Picca sitchensis (Bong.) Carr.] needle explants was strongly dependent upon the rigidity of the culture medium. In general, of organogenesis was greatest on weak gels and poorest on more rigid gels resulting from increased medium pH or agar strength. There was a significant interaction between agar strength and pH, with the optimum pH for organogenesis declining with increasing agar strength. Poor organogenesis at high agar concentrations was not due to toxic impurities since increased adventitious bud production could be stimulated by decreasing the medium pH whilst maintaining a high agar strength and an agar washing treatment had no significant effect. Although high levels of organogenesis could be sustained on weak gels the resultant adventitious shoots often showed severe vitrification. The frequency of shoots showing vitrification could be reduced by growing the tissues on harder media but this resulted in reduced shoot elongation. Vitrification of needle tissues did not stimulate the formation of adventitious buds in the absence of cytokinins.     

滩涂贝类养殖环境的细菌分布

从贝类养殖滩涂的底泥中共分离到2976株细菌,经鉴定可归于18个属与肠杆菌科的部分属,其中梭状芽孢杆菌属(Clostridium)、芽孢杆菌属(Bacillus)、棒状杆菌属(Corynebacterium)、肠杆菌科(Enterobacteriaceae)的部分属、发光杆菌属(Photobacterium)、黄杆菌属(Flavobacterium)和假单胞菌属(Pseudomonas)等为优势菌属.统计结果表明,在养殖滩涂的沉积物环境中,异养细菌的菌群组成具有明显的陆源性特点,而且在时空分布上有着较大的差异.在数量分布上,全年异养细菌、反硝化细菌、氨化细菌和硫酸还原菌的数量分别波动在1.62×103-2.00×105cfu/g、1.50×104-5.00×107个/g、9.00×104-9.0×107个/g和9.00×103-4.00×106个/g之间,此外,反硝化细菌、氨化细菌均与异养细菌在数量上呈正相关,而且氨化细菌与异养细菌之间的相关性极其显著,而硫酸还原菌与异养细菌在数量上呈负相关.     

连续培养技术在微生态学研究中的应用

对连续培养技术在微生态学研究中的应用及进展作了综述。连续培养技术可用来建立体外模型以模拟正常菌群的生态系统,如人或动物的肠道菌群、口腔菌群等;用来研究正常菌群与病原菌之间的相互作用,菌群的生理生化特性及代谢产物。并可用于微生态制剂的研制和生产。     

环凯食(饮)具大肠菌群检验纸片检测能力测试研究

研究以营养琼脂培养基的计数结果为基准,采用幅度卫生部食检所监制的两种不同厂家生产的食（饮）具大肠菌群检验纸片作为对照,对环凯食（饮）具大肠菌群检验纸片进行了检测能力测试研究,在测试中采用10cells/mL,50cells/mL和100cells/mL3个不同菌液浓度,环凯纸片的检测结果和营养琼脂平板计数结果基本一致。与其它两种纸片的检测结果无明显差异。完全可以用于食（饮）具大肠菌群的监督检验。     

细弱绒泡菌的培养及个体发育初探

通过对细弱绒泡菌(Physarum tenerumRex)基物培养和有饲培养的研究,得到了细弱绒泡菌的子实体,观察到细弱绒泡菌个体发育各形态的变化过程,比较了基物培养和有饲培养所得到的子实体的不同。     

Mixed culture of nitrifying bacteria and denitrifying bacteria for simultaneous nitrification and denitrification

A mixed culture containing nitrifying bacteria and denitrifying bacteria was investigated for aerobic simultaneous nitrification and denitrification. A mixture of NaHCO₃ and CH₃COONa was selected as the appropriate carbon source for cell growth and nitrogen removal, the concentrations of carbon and nitrogen sources were also examined. Ammonia could be oxidized aerobically to nitrite by the mixed culture, and the intermediate nitrite was then reduced to dinitrogen gas. No nitrite was detected during the process. 0.212 g of ammonia/l could be removed in 30 h and nitrate could not be utilized aerobically by the mixed culture. Nitrite could be degraded aerobically as well as anaerobically. Very little ammonia was degraded anaerobically, but the ability to degrade ammonia could be recovered even after oxygen had been supplied for 42 h.     

Application of cell culture toxicity tests to the development of implantable biosensors

Cell culture toxicity testing methods were modified and applied to the development of implantable glucose microsensors, and positive and negative control materials suitable for the microsensor assessment were established. The location, source and degree of the toxic effect in a multi-component biosensor was spatially visualized with cell monolayers. A freshly prepared sensor showed moderate toxicity, mainly as a result of the presence of glutaraldehyde and the residual solvents in the polymer layers. However, it was possible to reduce the toxicity by removing the leachable toxic substances through extraction in phosphate, buffer, and a non-toxic sensor was readily obtained.     

不同增菌和培养条件下沙门氏菌检出效果的比较

通过在鸡精样本中添加低含量的鼠伤寒沙门氏菌和干扰菌(弗氏柠檬酸杆菌和奇异变形杆菌),参考国标和美国药典中沙门氏菌的定性方法,研究比较了3种选择性增菌液、4个培养时间段以及4种选择性平板对于沙门氏菌的不同检出效果。结果表明,在RV肉汤及四硫磺酸钠煌绿增菌液(TTB)中培养18 h～20 h,使用沙门显色培养基及亚硫酸铋琼脂(BS平板)进行选择性培养沙门氏菌的检出效果最好。     

不同病区血培养病原菌分布及耐药性分析

目的 了解各病区血培养分离病原菌的分布特点及其耐药性,指导临床医生合理有效地使用抗生素.方法 对宁波市泌尿肾病医院·鄞州第二医院2008年8月至2011年7月临床送检的8409例血液标本经Bact/A-lert3D全自动血培养仪培养,分离所得菌用美国德灵公司Walkaway 40系统进行鉴定和药敏.对检测结果进行统计学分析.结果 共检出病原菌853株,其中革兰阴性杆菌422株,革兰阳性球菌396株,真菌35株.血浆凝固酶阴性葡萄球菌占首位,其次分别为大肠埃希菌及肺炎克雷伯菌.检出菌数量居前三位的病区是重症监护病房、消化内科及肾内科.各病区的病原菌分布不尽相同.丁胺卡那、亚胺培南对大肠埃希菌、肺炎克雷伯菌显示出较高的敏感性,未发现耐利奈唑胺及万古霉素菌株.结论 该院血流感染病原菌以革兰阴性杆菌为主,各主要病区病原菌分布不尽相同,临床医生应跟据药敏结果合理有效地使用抗生素.     

A tissue culture assay for direct detection of sodium channel blocking toxins in bacterial culture supernates

A quantitative assay for sodium channel blocking toxins such as tetrodotoxin and saxitoxin has been developed for use with a microtitre plate reader. Mouse neuroblastoma cells, which die rapidly in the presence of ouabain and veratridine, were protected by tetrodotoxin; surviving cells were detected by their uptake of the vital dye Neutral red which was quantified with a microtitre plate reader at 540 nm. A sigmoidal dose response curve was obtained and tetrodotoxin concentrations were readily measured over the range 10 nM to 500 nM (3.2-160 ng/ml). With this method, sodium channel blocking toxins were detected directly, without processing or concentration, in culture supernates of several marine bacteria, including Shewanella alga, Alteromonas tetraodonis, Listonella (Vibrio) pelagia, V. alginolyticus, V. anguillarum and V. tubiashi. Culture supernates of Shewanella alga contained up to 510 ng/ml of sodium channel blocking toxin (using tetrodotoxin as a standard).     

A model for tumorigenicity and metastatic potential: Growth in 1.0% agar cultures

Summary We developed a method to determine the amount of work performed by cells through cell division in 1.0% agar cultures. There was no correlation between the cloning efficiencies of 1.0 and 0.3% agar cultures. Growth in 1.0% agar cultures correlated well with such malignant properties as tumorigenicity and the invasive and metastatic potentials. Our method revealed that metastatic MC and F cell lines possess different means of taking advantage of energy to proliferate against an environmental pressure from those possesed by nontumorigenic (ME and T-C3H) cell strain/line or nonmetastatic but tumorigenic (L, MR, and magc₁) cell lines.