Detection of bacterial contamination in cultures of eucaryotic cells by gas chromatography-mass spectrometry

The use of gas chromatography-mass spectrometry for early detection of bacterial contaminations in cultures of baker's yeast, Penicillium chrysogenum, and an animal cell line was evaluated; muramic acid and characteristic cellular fatty acids were used as analytes. By analyzing branched-chain and cyclopropane-substituted fatty acids as methyl esters, Staphylococcus epidermidis, Bacillus subtilis, Lactobacillus reuteri, Enterobacter cloacae, and Pseudomonas fluorescens were detected in a 500-fold excess (w/w) of baker's yeast; the amounts injected corresponded to 300 ng (dry mass) of the bacteria. Contamination with Bacillus was detected in cultures of Penicillium chrysogenum and animal cells by analyzing muramic acid, both as its alditol acetate derivative, using electron impact ionization, and its trifluoroacetyl methyl glycoside derivative, using negative ion-chemical ionization. The trifluoroacetylated derivative was detected in injected amounts corresponding to 1 x 10(3) bacterial cells in the contaminated animal cell line, whereas amounts corresponding to 1 x 10(5) bacteria were required for detection of the alditol acetate derivative; the amounts in the original samples were 5 x 10(5) and 5 x 10(6), respectively. However, the alditol acetate method exhibited lower chemical interferences than the trifluoroacetyl methyl glycoside procedure. The results show the potential of using gas chromatographic-mass spectrometric analysis of cellular constituents for the detection of bacterial contaminations in eucaryotic cultures as an alternative to conventional microbiological methods. (c) 1993 John Wiley & Sons, Inc.     

Detection of adeno- and lentiviral (HIV1) contaminations on laboratory surfaces as a tool for the surveillance of biosafety standards

Aims: As a biosafety laboratory, we survey the handling of adenovirus type 5 (Ad5) and HIV1‐derived lentivirus in contained‐use facilities in Switzerland to identify insufficiencies of the safety precautions taken by the laboratories. Methods and Results: In the past 9 years, we took 430 swab samples from various types of surfaces in research laboratories. Samples were examined for Ad5 contaminations by real‐time PCR and infectivity assay or for the presence of lentivirus (HIV1) nucleic acids by real‐time (RT) PCR. Samples collected from centrifuges did not only contain Ad5 DNA more frequently but also exhibited higher numbers of Ad5 and lentiviral (HIV1) DNA copies than swabs from any other area of sampling. Five of ten samples containing infectious Ad5 particles or lentivirus (HIV1) RNA were found in samples taken from centrifuges. Ad5 contamination rates were higher in the tube holder and lower on the inner wall of the rotor chamber in centrifuges that were fitted with aerosol tight covers compared to centrifuges without covers. Conclusions: Our results allowed the comparison of hygiene standards of different laboratories and lead to the identification of centrifuges as hotspots for contaminations. Significance and Impact of the Study: Based on our results, we propose to use the collected data as a tool for rating future swab results. Furthermore, the amount of Ad5 and HIV1‐derived lentivirus DNA could serve as an indicator of the level of good laboratory practice in contained‐use laboratories handling these viral vectors.     

Rapid detection of lactobacillus and yeast concentrations using a particle size distribution analyser

Aims:  To see the possibility of particle size distribution analyser (PSDA) in detecting concentration of lactobacillus contaminants in yeast fermentation.
Methods and Results:  A PSDA was used to rapidly determine the size and concentration of lactobacillus and Saccharomyces cerevisiae . Data showed that the aerodynamic diameters of Lactobacillus casei and S. cerevisiae cells were around 0·63 and 2·9 μm, respectively, with both cultures showing a linear relationship between cell density and particle count on a size distribution curve of PSDA. In addition, Lactobacillus fermentum showed high similarity in bacterial size distribution and particle count numbers with L. casei . The PSDA also rapidly detected (within 1 min) the cell concentrations of S. cerevisiae and L. casei in a mixed sample with different concentration ratios with 10⁷–10⁹ cells ml⁻¹ of detection range.
Conclusions:  PSDA was demonstrated to be useful for the rapid detection of lactobacillus and S. cerevisiae concentrations.
Significance and Impact of the Study:  This is the first report concerning PSDA to detect the concentration of bacteria and yeast. This method can be useful in the actual field during ethanol fermentation because of relatively easy handling and rapid detection.     

植物离体培养中微生物污染的鉴定与控制(综述)

本文综述植物离体培养过程中微生物污染的鉴定与控制的研究进展,包括通过指示培养和菌种鉴别以鉴定污染菌;从保护条件下生长的植株上取材以及材料的预处理,以便有效地控制附生菌和应用抗生素控制内生菌.     

分子诊断实验室去除核酸污染的方法学研究

核酸检测因具有良好的灵敏度和特异性而被广泛应用于体外诊断、动植物商品检疫、法医鉴定等领域。然而操作过程中易受到核酸污染导致的假阳性结果,严重影响了检测准确性。因此寻找一种有效的防止和清除核酸污染的方案对于实验室正常运转及保障检测结果的可靠性具有重要意义。文中比较了几种不同清除核酸污染的方法,确认了84消毒液和PCRguard试剂可有效清除液体中及不同材质表面的核酸污染。另外,84消毒液和PCRguard试剂配合使用,可很好地解决核酸气溶胶污染。研究结果对于分子诊断实验室日常预防核酸污染及清除已发生的气溶胶污染提出了解决方案。     

Preparation of reference strains for validation and comparison of mycoplasma testing methods

Aims: To optimize growth conditions for preparation of stocks of mycoplasma reference strains to obtain highly viable and disperse samples with low ratios of genomic copy (GC) number to that of colony forming units (CFU). These stocks are required for assessment of relative limits of detection (LOD) of alternative nucleic acid testing (NAT)‐based methods in comparison to the conventional microbiological methods. Methods and Results: A kinetics study was used to assess the changes in ratios between the numbers of GC and CFU at different growth phases of six different mycoplasma cultures Acholeplasma laidlawii, Mycoplasma gallisepticum, Mycoplasma arginini, Mycoplasma fermentans, Mycoplasma orale and Mycoplasma pneumoniae. All tested mycoplasmas demonstrated low GC/CFU ratios (≤10) within the log and early stationary growth phases. A significant increase in GC/CFU ratios was observed at the very late stationary and death phases, when the titre of cultures has declined. Similar patterns of GC/CFU profiles were observed for A. laidlawii and Myc. gallisepticum co‐cultured with suspension of Chinese hamster ovary (CHO) cells. Conclusions: Tested mycoplasma strains harvested at the exponential‐early stationary phases of growth demonstrated the lowest GC/CFU ratios and low propensity to form filamentous structures or aggregates under proposed conditions and can be used for the preparation of a mycoplasma reference panel for methods comparability study. Significance and Impact of the Study: This study shows that the preparation and use of viable mycoplasma reference strains with low CG/CFU ratios is the most reliable way to adequately evaluate the LOD of alternative NAT‐based mycoplasma testing methods.     

Species-abundance-biomass responses by estuarine macrobenthos to sediment chemical contamination

Macrobenthic community responses can be measured through concerted changes in univariate metrics,including species richness, total abundance, and totalbiomass. The classic model of pollution effects onmarine macrobenthic communities recognizes thatspecies/abundance/biomass (SAB) curves varydistinctively in a nonlinear manner with the magnitudeof organic enrichment. For example, at moderatelevels of organic enrichment, small-bodiedopportunistic species boost the abundance curve, whilespecies richness falls. Ratios among the metrics formuseful indicators of how the community changes withorganic enrichment. However, the classic SAB model isbased on organic enrichment effects over small spatialand temporal scales, and the applicability of the SABmodel to sediment chemical contamination and acrossbroad natural estuarine gradients is largely unknown. Here, SAB responses were examined with respect toprimary gradients in metals and organic chemicalsbased on an extensive dataset comprising 319 estuarinesites from throughout the northern Gulf of Mexico. Each SAB metric was first adjusted with respect to thethree primary natural estuarine gradients, salinity,depth, and sediment silt/clay content. Adjusted SABrelationships varied in their details with respect todifferent classes of sediment contamination, but alltypes of SAB stress responses appear to exhibitsimilar basic characteristics. As in the SAB model,all three SAB metrics were notably low at the highestconcentrations of both metal and organic-chemicalcontaminants. Moreover, rapid decreases in the B/Aratio with increasing contamination supported theconcept that relatively long-lived, large-bodied,equilibrium taxa decline markedly at highconcentrations of toxicants.     

Prevalence and genetic diversity of waterborne pathogenic viruses in surface waters of tropical urban catchments

Aims: To study the virological quality of surface water from highly urbanized tropical water catchment areas and to determine predominant enteric viral genotypes in surface water. Methods and Results: A wide range of human pathogenic viruses in urban surface waters was screened by nested PCR assays after concentration by ultrafiltration. Among the 84 water samples collected, at least one virus was detected in 70 (83·3%) of these samples. Noroviruses were determined to be the most prevalent enteric viruses detected in urban surface water samples, followed by astroviruses, enteroviruses, adenoviruses and hepatitis A viruses. The molecular characterization of environmental viral isolates suggested co‐circulation of multiple genotypes of both noroviruses GI and GII, astroviruses and enteroviruses in urban surface waters. Conclusions: Human enteric viruses with great genetic diversity were detected in surface waters, indicating the presence of human origin of faecal contamination in highly urbanized water catchment areas. Significance and Impact of the Study: The present study identifies and characterizes potential viral hazards of source waters for drinking water supply and recreational activities. This will enable scientific decisions to be made regarding the selection and prioritization of human pathogenic viruses to be included in the future risk assessment and treatment evaluation for water and wastewater.     

The rare occurrence of plant tissue culture contamination by Methylobacterium mesophilicum

A pink-pigmented, facultative methylotrophic (PPFM) bacterium, Methylobacterium mesophilicum, which is found on the leaf surface of most plants, has been reported to be a covert contaminant of tissue cultures initiated from Glycine max (soybean) leaves and seeds by Holland and Polacco (1992). The bacteria can be detected as pink colonies when leaves are pressed or tissue culture homogenates are plated on a medium with methanol as the sole carbon source. Since the presence of contaminating bacteria can confound any biochemical results obtained with such cultures (Holland and Polacco 1992), we wanted to determine the extent of the contamination of our tissue cultures of soybean and other species. No PPFMs were detected in any soybean culture we have, and previous results describing the biochemical characteristics of ureide utilization by one of our soybean suspension cultures (27C) also indicates that PPFM bacteria were not present. Analysis of about 200 other strains of 11 different species maintained in this lab showed that only three of about 160 callus cultures, recently initiated from Datura innoxia leaves, contained PPFMs. The D. innoxia leaves did have PPFMs on their surface but in most cases they did not survive the surface disinfestation and culture regimes. Thus PPFM bacterial contamination should not be a serious problem in most plant tissue cultures.Abbreviations AMS ammonium mineral salts medium - PPFM pink-pigmented facultative methylotrophic bacteria     

Arbuscular Mycorrhizal Fungi and Arsenate Uptake by Brachiaria Grass (Brachiaria decumbens)

ABSTRACT Using a compartmentalized treatment technique, the role of arbuscular mycorrhizal fungi (AMF; Acaulospora scrobiculata) on arsenic (As) uptake and translocation in Brachiaria decumbens. Treatments consisted of a factorial arrangement of three As doses (0, 50, and 100 mg kg^?1) and the presence/absence of AMF inoculates. In the absence of AMF, B. decumbens did not show As accumulation, indicating the probable presence of tolerance mechanism via As exclusion by the roots. B. decumbens plants showed high AMF colonization levels, especially in the arsenic treatments, with AMF improving shoot and root growth independent of As concentrations. Arsenic accumulation occurred only with AMF inoculation. Phosphorous uptake was reduced in B. decumbens roots in the presence of arsenic with and without inoculation of AMF. Results suggest that B. decumbens can be used in phytoremediation procedures when inoculated with A. scrobiculata, although pasture formation should be strictly avoided in contaminated sites.     

微生物处理地下水石油污染的应用研究

探讨中国淄博地区地下水源石油污染的微生物治理技术 .从污染地分离筛选出1 0株除油菌 ,经测定单菌株的降油率在 2 0～ 50 % ,混合菌群MZ940 2的降油率可达71 .4% .在含油地下水模拟反应器中 ,当投菌浓度保持在 1 0 6 个·ml- 1 时 ,混合菌群MZ940 2的降油率可达 53.1 % .在地下水石油污染的现场治理时 ,MZ940 2的投菌量为1 0L·d- 1 ( 1 0 9个·ml- 1 ) ,1 1d后在地下水流段面形成较稳定的微生物带 ,降油率保持在35%左右 .经初步鉴定 ,所投加的MZ940 2菌群未发生变异 .     

蚯蚓-秸秆及其交互作用对黑麦草修复Cu污染土壤的影响

以高沙土为供试土壤,加入Cu^2＋以模拟成：0,100,200,400mg／kgCu^2＋的Cu污染土壤,设置接种蚯蚓（E）、表施秸秆（M）,同时加入蚯蚓和秸秆（ME）及不加蚯蚓和秸秆的对照（CK）4个处理,并种植黑麦草。研究蚯蚓、秸秆相互作用对黑麦草吸收、富集铜的影响。结果表明：加入秸秆显著提高了蚯蚓的生物量,一定程度上缓解了重金属对蚯蚓的毒害,同时蚯蚓显著提高了秸秆的分解率,较无蚯蚓对照提高了58．11％～77．32％。接种蚯蚓（E,ME）还提高了土壤有效态重金属（DTPA-Cu）含量,秸秆处理（M）则降低了土壤有效态重金属含量。研究还发现,E处理促进了黑麦草地上部生长,而M和ME处理均显著提高了黑麦草地下部的生物量。E和ME处理同时提高了植物地上部和地下部的Cu浓度及Cu吸收量,M处理则只对植物的地下部Cu浓度和Cu吸收量有显著促进作用。总体来看,E处理、M处理及ME处理分别使黑麦草地上部Cu富集系数提高了31．22％～121．07％．2．12％～61．28％和25．56％～132．64％。     

Reduced expression of distinct T-cell CD molecules by collagenase/DNase treatment

DNase/collagenase treatments are widely used to obtain single-cell suspensions of tumour cells and tumour-infiltrating T lymphocytes (TIL) from solid tumours. Since the functional integrity of such cells has been questioned, we have studied whether treatments with commonly used preparations of these enzymes could affect the expression of lymphocyte surface molecules and lymphocyte proliferative responsiveness. With peripheral-blood-derived T cells as a model, flow-cytometric analysis revealed strongly reduced expression of distinct CD molecules for each enzyme, notably CD2, CD4, CD8 and CD44 for DNase, and CD4, CD14, CD16, and CD56 for collagenase. The effects were found to be due to protease contaminations present in all but the purest enzyme preparations tested. Addition of serum or trypsin inhibitor abolished the effects. Since serum-free media are widely used to expand tumour-infiltrating T cells for clinical therapeutic use, data from early phenotypic analyses can be strongly misleading. Even after an 18-h rest period following the enzyme treatments, re-expression of the affected membrane markers was still far from complete. On the other hand, despite strongly reduced expression of CD2 molecules on the lymphocyte membrane, anti-CD2-induced proliferation was not affected, showing the redundancy of this signal molecule. Since other important T cell activation molecules (TCR, CD3, CD28) were not affected by enzymatic treatment, the use of expensive, highly purified collagenase/DNase preparations does not seem to be mandatory in clinical studies with expanded TIL.     

Studies on Cadmium Accumulation by Some Selected Floating Macrophytes

The results of investigation of the process of cadmium accumulation by floating plants of Eichhornia crassipes and Pistia stratiotes are discussed. The main specialty of this study is that it puts more emphasis on the mechanism of penetration of pollutant within the plant and its fate during accumulation act. As a result it was shown that at the first stage of cadmium uptake the sorption of the metal on the surface of the roots due to the presence of carboxylic groups takes place. At the root of the plant cadmium mainly localized in the cortex and rhizodermis, then the pollutant penetrates into the tissues of the stem according to its translocation factor. It has been also assumed that flavonoids perform an intermediate role in the accumulation of cadmium by the plant, taking part in the transport and combat an oxidative stress.     

Mycoplasma contamination of cell cultures: Incidence, sources, effects, detection, elimination, prevention

The contamination of cell cultures by mycoplasmas remains a major problem in cell culture. Mycoplasmas can produce a virtually unlimited variety of effects in the cultures they infect. These organisms are resistant to most antibiotics commonly employed in cell cultures. Here we provide a concise overview of the current knowledge on: (1) the incidence and sources of mycoplasma contamination in cell cultures, the mycoplasma species most commonly detected in cell cultures, and the effects of mycoplasmas on the function and activities of infected cell cultures; (2) the various techniques available for the detection of mycoplasmas with particular emphasis on the most reliable detection methods; (3) the various methods available for the elimination of mycoplasmas highlighting antibiotic treatment; and (4) the recommended procedures and working protocols for the detection, elimination and prevention of mycoplasma contamination. The availability of accurate, sensitive and reliable detection methods and the application of robust and successful elimination methods provide powerful means for overcoming the problem of mycoplasma contamination in cell cultures. This revised version was published online in August 2006 with corrections to the Cover Date.