Use of impedance test for testing effectiveness of drug preservatives

Pharmacopoeias contain preservation efficacy test for estimating antimicrobial activity of chemical compounds added to pharmaceutical preparations in multidose containers in order to inhibit bioburden growth. This method involves the treating of preserved products with bacteria and yeast cells and monitoring the survival of microorganisms through the specified time periods up to 28 days. The last stage of assay--incubation and colony counting--is very time consuming. Recent advance in technology enables faster and more convenient detection in comparison to traditional methods. Impedance method is based on the principle that conductance and capacitance of cultivation medium increases when bacteria grow and metabolize, Impedance time detection is inversely proportional to initial bacterial population. Six different products were utilised throughout the study. The calibration curves were calculated for each of the tested strains by comparison between standard plate count method and detection time measured in Bactometer system. In our study log reduction calculated in alternative method were similar to those obtained in plate count assay. All of the tested preparations, except one, exhibited acceptable activity against bacteria and fungi and meet the pharmacopoeal requirements. The studies indicated a positive correlation between standard plate count results and impedance reading. The procedure with the usage of Bactometer, provides a rapid and accurate system for the determination of bacterial content.     

Bacteria associated with granular activated carbon particles in drinking water

A sampling protocol was developed to examine particles released from granular activated carbon filter beds. A gauze filter/Swinnex procedure was used to collect carbon fines from 201 granular activated carbon-treated drinking water samples over 12 months. Application of a homogenization procedure (developed previously) indicated that 41.4% of the water samples had heterotrophic plate count bacteria attached to carbon particles. With the enumeration procedures described, heterotrophic plate count bacteria were recovered at an average rate of 8.6 times higher than by conventional analyses. Over 17% of the samples contained carbon particles colonized with coliform bacteria as enumerated with modified most-probable-number and membrane filter techniques. In some instances coliform recoveries were 122 to 1,194 times higher than by standard procedures. Nearly 28% of the coliforms attached to these particles in drinking water exhibited the fecal biotype. Scanning electron micrographs of carbon fines from treated drinking water showed microcolonies of bacteria on particle surfaces. These data indicate that bacteria attached to carbon fines may be an important mechanism by which microorganisms penetrate treatment barriers and enter potable water supplies.     

Bacteria associated with granular activated carbon particles in drinking water.

A sampling protocol was developed to examine particles released from granular activated carbon filter beds. A gauze filter/Swinnex procedure was used to collect carbon fines from 201 granular activated carbon-treated drinking water samples over 12 months. Application of a homogenization procedure (developed previously) indicated that 41.4% of the water samples had heterotrophic plate count bacteria attached to carbon particles. With the enumeration procedures described, heterotrophic plate count bacteria were recovered at an average rate of 8.6 times higher than by conventional analyses. Over 17% of the samples contained carbon particles colonized with coliform bacteria as enumerated with modified most-probable-number and membrane filter techniques. In some instances coliform recoveries were 122 to 1,194 times higher than by standard procedures. Nearly 28% of the coliforms attached to these particles in drinking water exhibited the fecal biotype. Scanning electron micrographs of carbon fines from treated drinking water showed microcolonies of bacteria on particle surfaces. These data indicate that bacteria attached to carbon fines may be an important mechanism by which microorganisms penetrate treatment barriers and enter potable water supplies.     

UV inactivation of pathogenic and indicator microorganisms

Survival was measured as a function of the dose of germicidal UV light for the bacteria Escherichia coli, Salmonella typhi, Shigella sonnei, Streptococcus faecalis, Staphylococcus aureus, and Bacillus subtilis spores, the enteric viruses poliovirus type 1 and simian rotavirus SA11, the cysts of the protozoan Acanthamoeba castellanii, as well as for total coliforms and standard plate count microorganisms from secondary effluent. The doses of UV light necessary for a 99.9% inactivation of the cultured vegetative bacteria, total coliforms, and standard plate count microorganisms were comparable. However, the viruses, the bacterial spores, and the amoebic cysts required about 3 to 4 times, 9 times, and 15 times, respectively, the dose required for E. coli. These ratios covered a narrower relative dose range than that previously reported for chlorine disinfection of E. coli, viruses, spores, and cysts.     

UV inactivation of pathogenic and indicator microorganisms.

Survival was measured as a function of the dose of germicidal UV light for the bacteria Escherichia coli, Salmonella typhi, Shigella sonnei, Streptococcus faecalis, Staphylococcus aureus, and Bacillus subtilis spores, the enteric viruses poliovirus type 1 and simian rotavirus SA11, the cysts of the protozoan Acanthamoeba castellanii, as well as for total coliforms and standard plate count microorganisms from secondary effluent. The doses of UV light necessary for a 99.9% inactivation of the cultured vegetative bacteria, total coliforms, and standard plate count microorganisms were comparable. However, the viruses, the bacterial spores, and the amoebic cysts required about 3 to 4 times, 9 times, and 15 times, respectively, the dose required for E. coli. These ratios covered a narrower relative dose range than that previously reported for chlorine disinfection of E. coli, viruses, spores, and cysts.     

The pituitary gland mediates acute and chronic pain responsiveness in stressed and non-stressed rats.

The effect of hypophysectomy on the responsiveness of rats to acute (hot plate test) and chronic (formalin test) pain was studied. Hypophysectomy did not alter hot plate behavior but increased the duration of the paw withdrawal in the formalin test. Pre-exposure of rats to immobilization stress caused a transient yet significant increase in escape latencies from a hot plate as well as a significant decrease in the duration of paw withdrawal in the formalin test. Hypophysectomy blocked the effect of stress on these behavioral manifestations of acute and chronic pain. The effect of hypophysectomy was not reversed by adrenocorticotropin pretreatment. These results suggest that centrally acting pituitary hormones may have a requisite function in normal and adaptive pain control in mammals.     

Microflora associated with the digestive tract of the fairy shrimp Branchinella spinosa (H. Milne Edwards, 1840) (Crustacea, Branchiopoda)

An ectoperitrophic association of microorganisms with the fairy shrimp Branchinella spinosa was investigated by standard plate counts and scanning electron microscopy. Yeasts were found to form a monolayer structure attached to the inner surface of the peritrophic matrix suggesting a strong symbiotic association. The bacterial population consisted mostly of members of the genus Serratia, concentrated in the hindgut, and exhibiting chitinolytic activity.     

Measurement of hydrocarbon-degrading microbial populations by a 96-well plate most-probable-number procedure

A 96-well microtiter plate most-probable-number (MPN) procedure was developed to enumerate hydrocarbondegrading microorganisms. The performance of this method, which uses number 2 fuel oil (F2) as the selective growth substrate and reduction of iodonitrotetrazolium violet (INT) to detect positive wells, was evaluated by comparison with an established 24-well microtiter plate MPN procedure (the Sheen Screen), which uses weathered North Slope crude oil as the selective substrate and detects positive wells by emulsification or dispersion of the oil. Both procedures gave similar estimates of the hydrocarbon-degrader population densities in several oil-degrading enrichment cultures and sand samples from a variety of coastal sites. Although several oils were effective substrates for the 96-well procedure, the combination of F2 with INT was best, because the color change associated with INT reduction was more easily detected in the small wells than was disruption of the crude oil slick. The method's accuracy was evaluated by comparing hydrocarbon-degrader MPNs with heterotrophic plate counts for several pure and mixed cultures. For some organisms, it seems likely that a single cell cannot initiate sufficient growth to produce a positive result. Thus, this and other hydrocarbon-degrader MPN procedures might underestimate the hydrocarbon-degrading population, even for culturable organisms.     

How to optimize the drop plate method for enumerating bacteria

The drop plate (DP) method can be used to determine the number of viable suspended bacteria in a known beaker volume. The drop plate method has some advantages over the spread plate (SP) method. Less time and effort are required to dispense the drops onto an agar plate than to spread an equivalent total sample volume into the agar. By distributing the sample in drops, colony counting can be done faster and perhaps more accurately. Even though it has been present in the laboratory for many years, the drop plate method has not been standardized. Some technicians use 10-fold dilutions, others use twofold. Some technicians plate a total volume of 0.1 ml, others plate 0.2 ml. The optimal combination of such factors would be useful to know when performing the drop plate method.This investigation was conducted to determine (i) the standard deviation of the bacterial density estimate, (ii) the cost of performing the drop plate procedure, (iii) the optimal drop plate design, and (iv) the advantages of the drop plate method in comparison to the standard spread plate method. The optimal design is the combination of factor settings that achieves the smallest standard deviation for a fixed cost. Computer simulation techniques and regression analysis were used to express the standard deviation as a function of the beaker volume, dilution factor, and volume plated. The standard deviation expression is also applicable to the spread plate method.     

食真菌线虫对热或铜胁迫下土壤生态功能稳定性的影响

在模拟胁迫条件下(施加 CuSO₄ 的持续胁迫或加热40 ℃的瞬时胁迫),以大麦叶粉短期分解过程代表土壤功能,采用室内培养试验研究了土壤食真菌线虫(Aphelenchus avenae)与微生物的相互作用对土壤生态功能稳定性(抗性和恢复力)的影响.结果表明：无论施加胁迫与否,食真菌线虫的活动都有促进土壤微生物活性的趋势,尤其是在施加铜胁迫后第8 天开始到培养期结束,接种食真菌线虫导致土壤基础呼吸显著增加(P<0.05),但加热胁迫后食真菌线虫对土壤基础呼吸的促进作用仅在第8天有显著差异(P<0.05),反映了食真菌线虫对土壤微生物活性的影响程度与胁迫类型有关.在两种胁迫条件下,接种食真菌线虫对土壤功能的抗性没有影响,但都能促进胁迫条件下土壤功能的恢复.培养后期,两种胁迫条件下接种食真菌线虫处理真菌生物量低于未接种线虫处理,表明胁迫条件下食真菌线虫对真菌的取食可能限制甚至抑制了真菌生长,导致真菌对细菌的竞争压力减少,从而使细菌获得更大的生长优势,间接促进了细菌的生长.     

平板混合培养木质纤维素降解菌研究进展

微生物的混合培养已广泛应用于木质纤维素类物质的转化与降解领域.不同木质纤维素降解菌在混合培养时的相互关系在很大程度上影响混合培养的效果.目前对这种相互关系的研究主要依托平板混合培养展开,所用到的平板主要有基础培养基平板和改进培养基平板两种.其中基础培养基平板法主要根据菌落形态、菌丝体颜色、胞外挥发性有机化合物成分和典型胞外酶活性等进行研究,而改进培养基平板则是将基础培养基平板中的碳源更换为天然木质纤维素类物质进行对比研究.本文综述了采用平板混合培养不同木质纤维素降解菌菌株的研究现状和进展,并对该领域研究应重点关注的问题进行了展望.     

Improved laboratory enrichment for enterohemorrhagic Escherichia coli by exposure to extremely acidic conditions

Analysis of food samples for E. coli O157:H7 using the standard U.S. Food and Drug Administration procedure is frequently complicated by overgrowth of nontarget microorganisms. A new procedure was developed for enrichment of enterohemorrhagic E. coli (EHEC) which utilizes exposure to pH 2.00 for 2 h. This procedure yielded larger populations of EHEC than the standard method by factors ranging from 2.7 to 7.7 and, when age-stressed cultures were used, by factors ranging from 2.7 to 11.5. Cultures of competing enterics were more effectively inhibited by the new enrichment protocol as well.     

Development of lyophilization procedure ensuring survival of bifidobacteria and preservation of their probiotic potential upon long-term storage

Survival of bifidobacteria and preservation of their morphological characteristics after 12-month storage of lyophilized cells was studied for the strains of Bifidobacterium bifidum and B. animalis isolated and maintained in the microbial collection of the Department of Microbiology, Moscow State University. A combined approach to pre-lyophilization treatment of microorganisms and subsequent storage was developed in order to improve cell survival. Compared to the standard cryoprotector concentrations, sucrose and glucose (5% and higher) in skim milk, as well as freezing at?70°C with subsequent storage at the same temperature resulted in improved survival of bifidobacteria. Under such conditions, the number of viable cells (CFU) after 12 months of storage was two to three orders of magnitude higher than in the case of the standard lyophilization procedure. Investigation of dynamics of resistance of reactivated clones to such gastrointestinal stress factors as gastric juice and bile acids revealed preservation of these properties at all storage modes. However, since the number of surviving cells decreased during storage according to the standard procedure, the number of stress-affected cells was correspondingly lower. Reactivated cultures exhibited high resistance to oxygen, with survival decreasing to 35% of the initial level.     

A microassay-based procedure for measuring low levels of toxic organophosphorus compounds through acetylcholinesterase inhibition

Using a microtiter plate spectrophotometric system, an assay procedure was developed for the following toxic organophosphorus compounds: 1,2,2-trimethylpropyl ester of methylphosphonofluoridic acid (1, soman); ethyl N,N-dimethylphosphoramidocyanidate (3, tabun); O-ethyl S-[2-[bis(1-methylethyl)amino]ethyl]- methylphosphonothiolate (4, VX); the diethyl 4-nitrophenyl ester of phosphoric acid (5, paraoxon); and bis(1-methylethyl) phosphorofluoridate (6, DFP). The procedure, based on the Ellman assay method, uses inhibition of eel acetylcholinesterase (0.01 unit per well) to carry out the determination of inhibitor concentrations for both a standard curve and the unknown samples on a single 96-well microtiter plate. On a typical plate, samples of both unknowns and standards (a minimum of six concentrations were used per standard curve) were assayed five times per sample, with three control (uninhibited) enzyme activity points included for each sample. The time required for carrying out a single plate was approx 30 min. Sensitivity for the most potent acetylcholinesterase inhibitor tested was 0.4 nM under the conditions used for a typical assay. It should be noted, however, that no attempt was made to optimize the assay procedure for sensitivity.     

重金属胁迫下土壤微生物和微生物过程研究进展

通过对重金属胁迫下土壤微生物和微生物过程研究的进程和研究进展的归纳综述,分析了该研究尚存在的问题,并阐述了其可能原因．认为土壤微生物和微生物学过程的重金属胁迫研究存在如下问题：一是从实验室、田间试验和实地监测得到的结果间无法进行比较,从而使实验室和田间试验的研究丧失了其科学指导意义,并且在实地监测研究中缺乏相应的“精确”对照;二是在重金属的胁迫下土壤微生物不但数量有消长,而且区系结构上也发生了变化,但是用于校园微生物区系结构变化的手段(PLFA、BI-OLOG和DNA等方法)尚处在探索阶段并需要昂贵的设备,难以普及,需发展一些可广泛普及的新方法来代替传统的平板分离法分析土壤微生物结构;三是重金属对土壤微生物和微生物过程产生胁迫的形态、离子效应和根际效应尚未得到有效的研究和探讨;四是土壤微生物和微生物过程重金属胁迫的表征体系尚未建立．     

Effect of air drying on bacterial viability: A multiparameter viability assessment

The effect of desiccation on the viability of microorganisms is a question of great interest for a variety of public health questions and industrial applications. Although viability is traditionally assessed by plate counts, cultivation-independent methods are increasingly applied with the aim to gain more insight into why cells might not form colonies and to optimize production processes. To evaluate their usefulness, we applied in this study a multiparameter viability assay to selected bacteria (Escherichia coli, Pseudomonas aeruginosa, Enterococcus hirae, and Staphylococcus aureus) subjected to air-drying in the absence or presence of supplements. Tests included growth on solid culture medium and the measurement of membrane integrity, membrane potential, esterase and respiratory activities using fluorescent dyes. All measured parameters were responsive to desiccation stress. Results suggested that extending plate count analysis with cultivation-independent methods can greatly enhance resolution especially for moderate stress conditions, which do not get reflected in plate counts due to cellular recovery. Whereas plate counts reflect the final effect on viability, immediate measurement of cellular functions provides a snapshot picture of the fitness status at a specific point in time. Special emphasis was given to MgCl(2) which in concentrations≥50mM dramatically increased the bacterial susceptibility to desiccation in the case of the gram-negative bacteria and to a lesser extent also for the gram-positive bacteria. The study in addition confirmed a good agreement of results obtained with the recently developed real-time viability (RTV) assay and the BacLight LIVE/DEAD method in combination with a fluorescence plate reader.     

Development and application of an enzymatic and cell flotation treatment for the recovery of viable microbial cells from environmental matrices such as anaerobic sludge

Efficient dissociation of microorganisms from their aggregate matrix is required to study the microorganisms without interaction with their native environment (e.g., biofilms, flocs, granules, etc.) and to assess their community composition through the application of molecular or microscopy techniques. To this end, we combined enzymatic treatments and a cell extraction by density gradient to efficiently recover anaerobic microorganisms from urban wastewater treatment plant sludge. The enzymes employed (amylase, cellulase, DNase, and pectinase) as a pretreatment softly disintegrated the extrapolymeric substances (EPS) interlocked with the microorganisms. The potential damaging effects of the applied procedure on bacterial and archaeal communities were assessed by studying the variations in density (using quantitative PCR), diversity (using capillary electrophoresis single-strand conformation polymorphism fingerprinting [CE-SSCP]), and activity (using a standard anaerobic activity test) of the extracted microorganisms. The protocol preserved the general capacity of the microbial community to produce methane under anaerobic conditions and its diversity; particularly the archaeal community was not affected in terms of either density or structure. This cell extraction procedure from the matrix materials offers interesting perspectives for metabolic, microscopic, and molecular assays of microbial communities present in complex matrices constituted by bioaggregates or biofilms.     

Effect of an activated sludge wastewater treatment plant on ambient air densities of aerosols containing bacteria and viruses

Bacteria- and virus-containing aerosols were studied during the late summer and fall seasons in a midwestern suburb of the United States before and during the start-up and operation of an unenclosed activated sludge wastewater treatment plant. The study showed that the air in this suburban area contained low-level densities of indicator microorganisms. After the plant began operating, the densities of total aerobic bacteria-containing particles, standard plate count bacteria, total coliforms, fecal coliforms, fecal streptococci, and coliphages increased significantly in the air within the perimeter of the plant. Before plant operations, bacteria were detected from five genera, Klebsiella, Enterobacter, Serratia, Salmonella, and Aeromonas. During plant operations, the number of genera identified increased to 11. In addition to those genera found before plant operations, Escherichia, Providencia, Citrobacter, Acinetobacter, Pasteurella, and Proteus, were also identified. Enteric viruses were detected in low densities from the air emissions of this plant. Only standard plate count bacteria remained at significantly higher than base-line densities beyond 250 m downwind from the center of the aeration tanks. Fecal streptococci and coliphages appeared to be more stable in aerosols than the other indicator microorganisms studied. In general, the densities of microorganism-containing aerosols were higher at night than during the day. The techniques used in this study may be employed to establish microorganism-containing aerosol exposure during epidemiological investigations.     

AN IMPROVED METHOD OF CELL ENUMERATION FOR FILAMENTOUS ALGAE AND BACTERIA1

A new simple method for estimating the number of individual cells per milliliter in suspensions of filamentous microorganisms is described. This 2-part procedure utilizes a standard microscopic counting chamber and is independent of filament length or individual cell size. A statistical analysis of the method is also presented.     

Review: Microbial endocrinology: intersection of microbiology and neurobiology matters to swine health from infection to behavior

From birth to slaughter, pigs are in constant interaction with microorganisms. Exposure of the skin, gastrointestinal and respiratory tracts, and other systems allows microorganisms to affect the developmental trajectory and function of porcine physiology as well as impact behavior. These routes of communication are bi-directional, allowing the swine host to likewise influence microbial survival, function and community composition. Microbial endocrinology is the study of the bi-directional dialogue between host and microbe. Indeed, the landmark discovery of host neuroendocrine systems as hubs of host–microbe communication revealed neurochemicals act as an inter-kingdom evolutionary-based language between microorganism and host. Several such neurochemicals are stress catecholamines, which have been shown to drastically increase host susceptibility to infection and augment virulence of important swine pathogens, including Clostridium perfringens. Catecholamines, the production of which increase in response to stress, reach the epithelium of multiple tissues, including the gastrointestinal tract and lung, where they initiate diverse responses by members of the microbiome as well as transient microorganisms, including pathogens and opportunistic pathogens. Multiple laboratories have confirmed the evolutionary role of microbial endocrinology in infectious disease pathogenesis extending from animals to even plants. More recent investigations have now shown that microbial endocrinology also plays a role in animal behavior through the microbiota–gut–brain axis. As stress and disease are ever-present, intersecting concerns during each stage of swine production, novel strategies utilizing a microbial endocrinology-based approach will likely prove invaluable to the swine industry.