Influence of Temperature and Method of Reconstitution on the Standard Plate Count of Casein and Caseinates

Sample solubility, in addition to method and temperature of reconstitution was found to affect the standard plate count of casein. The best combination for recovery of bacteria from casein and caseinates was pre-warming the buffer to 45 °C and the 'Stomacher'used for reconstitution.     

Multiregional evaluation of the SimPlate heterotrophic plate count method compared to the standard plate count agar pour plate method in water

A new SimPlate heterotrophic plate count (HPC) method (IDEXX Laboratories, Westbrook, Maine) was compared with the pour plate method at 35 degrees C for 48 h. Six laboratories tested a total of 632 water samples. The SimPlate HPC method was found to be equivalent to the pour plate method by regression analysis (r = 0. 95; y = 0.99X + 0.06).     

Agar Plate Method for Detection and Enumeration of Alkylbenzenesulfonate-Degrading Microorganisms

A simple method for detection and enumeration of alkylbenzenesulfonate (ABS)-degrading microorganisms by using agar plates was developed and used in microbiological studies of coastal marine and polluted river waters. The method depends upon the color responses of neutral red in alkaline medium. Neutral red changes from pink, when it enters into ABS micelles, to yellow, when the ABS is degraded, and does not form micelles. When neutral red-tris(hydroxymethyl)-aminomethane buffer solution and then cationic surfactant solution were sprayed onto the agar surface of ABS-nutrient agar cultures, transparent haloes appeared around the colonies of ABS-degrading microorganisms against a pink background. Viable counts of ABS-degrading bacteria isolated from both seawater and freshwater environments were considerably higher in polluted waters than in less polluted areas. Viable counts of ABS-degrading bacteria averaged 1.5 x 105/ml in samples from the surface water of polluted Tokyo Bay and 3.0 x 104/ml in samples from the surface water of polluted Tamagawa River but were fewer in number in samples from less polluted waters.     

厌氧菌预还原琼脂平板培养方法

为简化厌氧菌分离培养方法,使其在普通实验条件下于固体培养基上形成单菌落,本研究增加庖肉培养基无氧溶液体积,用作无氧倍比稀释液,在琼脂柱下进行倍比稀释,将皿盖带有胶塞孔的厌氧琼脂平板进行预还原,注射接种倍比稀释菌液,通过厌氧指示剂监测无氧效果,初步试用于肠道厌氧菌分离培养。结果显示,该方法整个操作过程厌氧效果良好,无需专门厌氧设备即可以分离纯化培养肠道乳酸杆菌,甚至无芽胞专性厌氧菌,如双歧杆菌和韦荣球菌。     

Agar Dish Isopiestic Equilibration Method for Controlling the Water Potential of Solid Substrates

Maintenance of substrate water potential in petri dishes is achieved by using vapor-pressure controlling, solute-amended agar gel discs attached to the inside of the top halves of the dishes.     

Evaluation of the Accuracy and Precision of Enumerating Aerobic Heterotrophs in Water Samples by the Spread Plate Method

Factors associated with accuracy and precision in the enumeration of aquatic aerobic heterotrophs by the spread plate method were evaluated by using a nested analysis of variance experimental design. Variances associated with individual components of the spread plate procedure were isolated, and optimal replications of each step were allocated. A practical scheme for optimal allocation of resources is proposed, consisting of four subsamples and two plates per subsample and yielding a total variance decrease of 70% from a single-subsample, 10-plate series. Data transformation was, in general, unnecessary for intraexperiment or intrasample statistical analysis, whereas interexperiment or intersample comparisons may require transformation of data. Rapid changes in the numbers of organisms in stored water samples were observed that were not reproducible and did not follow detectable trends, with increases or decreases in counts occurring in samples regardless of whether they were stored at room temperature or refrigerated, or stored in plastic or glass containers. Rapid sample handling is strongly recommended to minimize variations in the microbial populations of samples for aquatic environments.     

Use of Ultrasounds to Reduce the Count of Campylobacter coli in Water

The present study aimed to evaluate the effectiveness of low-frequency ultrasounds applied to eliminate Campylobacter spp. from water. The strains used in this research were isolated from water contaminated with sewage. Campylobacter coli alone was detected in the samples and used for further research. The reference strain C. coli ATCC 33559 was simultaneously tested. The isolate was exposed to ultrasounds at frequencies of 37 kHz and 80 kHz in a continuous operation device with ultrapure deionized water. After 5 min of sonication, the count of C. coli decreased by 5.78% (37 kHz) and 6.27% (80 kHz), whereas the temperature increased by 3°C (37 kHz), and 6°C (80 kHz). After 30 min of sonication, the death rates of bacterial cells were 40.15% (37 kHz) and 55.10% (80 kHz), whereas the temperature reached the maximum values of 36°C (37 kHz), and 39°C (80 kHz). Sonication at the frequency of 80 kHz reduced the bacterial count from 6.86 log CFU/ml to 3.08 log CFU/ml, whereas the frequency of 37 kHz reduced the bacterial count from 6.75 log CFU/ml to 4.04 log CFU/ml. Despite significant differences (p < 0.05) in the number of C. coli cells, the cell death rate remained at the same level. Open in a separate window     

使用血细胞计数板测定微生物数量实验的改进

血细胞计数板是微生物数量测定实验所使用的特制载玻片。通常做细菌、酵母菌等数量测定实验时,先是让学生在显微镜下找到血细胞计数板上的方格网及其计数室,认识计数室的构造,之后再进行加样、计数等实验步骤。然而在实验中发现,由于构成方格网的线条加工的非常细而浅,且无色透明。     

Agar Diffusion Method for the Assay of Colicins

An agar diffusion method for the assay of colicins A, B, D, E(2), E(3), and K is described. The assays were performed in large, square pyrex dishes that contained an agar layer seeded with an indicator organism sensitive to the colicin. The samples were applied to the agar in steatite beads positioned in a randomized sequence. The plates were stored at 4 C for 24 hr to allow the colicins to diffuse into the agar. After incubation at 37 C, the activity of each colicin preparation was estimated by measuring the diameter of the zone of inhibition of the growth of the indicator strain around each bead. The results of each assay were subjected to a statistical analysis, which included an analysis of variance and calculation of the theoretical regression and the confidence interval of the assay. The size of the inhibition zones, the form of the regression, and the slope of the regression of the responses were affected by the type and concentration of the agar, the depth of the agar layer, the indicator organism, the indicator inoculum density, and the time allowed for prediffusion of the colicins. Optimal conditions for the assay of each colicin were determined. Using a four-point assay design, the relative colicin concentration of unknown preparations was estimated in terms of a standard preparation of the same colicin. The experimental error of these assays (95% confidence interval) was about +/- 10%.     

Sorption of Heterotrophic and Enteric Bacteria to Glass Surfaces in the Continuous Culture of River Water

A natural population of heterotrophic bacteria, including enterics, was observed to sorb to glass surfaces and multiply during the continuous culture of river water. An initial rate of attachment equivalent to a doubling time of about 2 h was observed with a corresponding increase in the suspended population. After 24 h both the sorbed and suspended populations stabilized with a mass doubling time approximating 100 h at a dilution rate of 0.012/h. On the basis of respiration and degradative enzymatic data, the sorbed microorganisms appeared to be somewhat more metabolically active than the organisms in suspension.     

Agar Diffusion Method for the Differentiation of Bacillus anthracis

A method was developed for identification of Bacillus anthracis based on elaboration of protective antigen by individual colonies and its detection by double-diffusion precipitation in agar plates.     

Comparison of Two Optical-Density-Based Methods and a Plate Count Method for Estimation of Growth Parameters of Bacillus cereus

Quantitative microbiological models predicting proliferation of microorganisms relevant for food safety and/or food stability are useful tools to limit the need for generation of biological data through challenge testing and shelf-life testing. The use of these models requires quick and reliable methods for the generation of growth data and estimation of growth parameters. Growth parameter estimation can be achieved using methods based on plate counting and methods based on measuring the optical density. This research compares the plate count method with two optical density methods, namely, the 2-fold dilution (2FD) method and the relative rate to detection (RRD) method. For model organism Bacillus cereus F4810/72, the plate count method and both optical density methods gave comparable estimates for key growth parameters. Values for the maximum specific growth rate (μ_max) derived by the 2FD method and by the RRD method were of the same order of magnitude, but some marked differences between the two approaches were apparent. Whereas the 2FD method allowed the derivation of values for lag time (λ) from the data, this was not possible with the RRD method. However, the RRD method gave many more data points per experiment and also gave more data points close to the growth boundary. This research shows that all three proposed methods can be used for parameter estimation but that the choice of method depends on the objectives of the research.Food products are required to be safe and sufficiently stable within their given shelf-lives. For this reason, generally some kind of preservation is applied. An often-used preservation method is mild heat treatment, such as pasteurization, which inactivates vegetative microbial pathogens and spoilage microorganisms. Bacillus cereus is a Gram-positive, facultative anaerobic, spore-forming rod (17) that can be found in, for example, soil, food, and the human gastrointestinal tract (23). Viable spores present in a food product may germinate, and the vegetative cells may grow if conditions are favorable. In order to delay or prevent this, one or more hurdles for germination and outgrowth need to be present in the food or food environment. Examples of such hurdles are the acidification of food and addition of salt (12). When a combination of hurdles is used, generally the intensity of the hurdles may be lower to show a preservative effect comparable to the level of those hurdles when they are used individually (24).In this investigation, the use of acidification as a hurdle to prevent growth of B. cereus was studied. The amount of acid to be added to the food product is of importance for both safety reasons and organoleptic properties. B. cereus will not be able to grow at low pH values (pH 5 to 6, depending on the acidulant) (1), but, on the other hand, the food product should not be too acid, given consumer preferences. Research showed that children do not like orange juice with concentrations of citric acid above 0.02 M; for adults this value is 0.04 M (25). The lowest pH value allowing growth of B. cereus can be investigated by experiments culturing the microorganism in a suitable growth medium or food product with different pH values and using the viable plate count method for enumeration (33). Using such an approach to generate biological data for safety or shelf-life evaluations is considered both slow and human resource-intensive since experiments have to be repeated for every new condition. Quantitative microbiological modeling can speed up experimentation and reduce the resources required. Should modeling make it possible to predict the behavior of B. cereus over a wide range and a variety of conditions, then it would help to limit the need for experimentation to the point where just validation of predictions is needed.For successful use of modeling, the maximum specific growth rate (μ_max) and, preferably, also the lag time (λ) have to be known. These parameters can be determined using different techniques. Plate counting can be used to generate the data points μ_max and λ by fitting of the growth curve (15, 33). Often, automated optical density (OD) measurements are used to determine parameters for growth as they allow quicker data generation with less need for human resources (22). It is not possible, however, to directly translate OD values to μ_max values due to the high detection limit of OD readers of ∼10⁷ viable cells. When cells reach their maximum cell density, they proliferate at a progressively slower rate. Consequently, values for μ_max would be consistently underestimated by using OD values. For this reason, OD measurements are often used in combination with time-to-detection (TTD) measurements (5). Values for μ_max can be derived from OD measurements and TTD measurements by the 2-fold dilution (2FD) method (5, 28, 36). The 2FD method uses TTD and inoculum size variations to obtain values for both μ_max and λ. The relative rate to detection (RRD) method also uses OD measurements and TTD measurements to obtain parameters for growth. This method uses the ratio between TTD at the tested and the optimal conditions and can compute μ_max but not λ (20, 21, 37). Notably, plate count data allow both μ_max and λ to be derived, and this method is required as an addition to the RRD method.Although both the 2FD method and the RRD method make use of OD measurements, it is not known whether the two techniques result in comparable estimates of growth parameters and whether these values match values obtained with plate counts. Growth parameters determined by plate count and the 2FD method were previously compared in several studies (5, 27). The present study set out to compare the three different methods for the assessment of parameters for growth on the basis of the following, mainly measureable, criteria: the number of data points obtained per experiment, the measuring intensity, the time consumption per experiment, the reproducibility of the method as determined by comparing the standard deviations of the average μ_max at pH 7, the number of parameters obtained, and the number of criteria necessary for data analysis. B. cereus F4810/72 was used as the model organism. It was cultured at different pH values, a common hurdle in food products, to obtain a variety of growth rates.     

Sensitivity of Agar Overlay Method for the Recognition of Enteroviruses

COMPARISON OF THE AGAR OVERLAY TECHNIQUE WITH THE FLUID CULTURE TECHNIQUE FOR ISOLATION AND IDENTIFICATION OF ENTEROVIRUSES SHOWED THE FORMER TO BE USEFUL: (i) for isolation of enterovirus when the number of virus particles was too small to produce detectable cytopathic effect (CPE) in fluid cultures, (ii) for isolation of echovirus 22 which did not produce detectable CPE in fluid cultures, (iii) as an aid to rapid differentiation of enteroviruses, and (iv) for differentiation of viruses in mixed infections. Nonpolio enterovirus isolation experience in the New Haven area over a 4-year period is presented. It was concluded that the agar overlay technique is both useful and relatively simple for routine examination of clinical specimens in a diagnostic laboratory.     

The Cost-Effectiveness of Wound-Edge Protection Devices Compared to Standard Care in Reducing Surgical Site Infection after Laparotomy: An Economic Evaluation alongside the ROSSINI Trial

Background

Wound-edge protection devices (WEPDs) have been used in surgery for more than 40 years to reduce surgical site infection (SSI). No economic evaluation of WEPDs against any comparator has ever been conducted. The aim of the paper was to assess whether WEPDs are cost-effective in reducing SSI compared to standard care alone in the United Kingdom.

Methods and Findings

An economic evaluation was conducted alongside the ROSSINI trial. The study perspective was that of the UK National Health Service and the time horizon was 30 days post-operatively. The study was conducted in 21 UK hospitals. 760 patients undergoing laparotomy were randomised to either WEPD or standard care and 735 were included in the primary analysis. The main economic outcome was cost-effectiveness based on incremental cost (£) per quality adjusted life year (QALY) gained. Patients in the WEPD arm accessed health care worth £5,420 on average and gained 0.02131 QALYs, compared to £5,130 and 0.02133 QALYs gained in the standard care arm. The WEPD strategy was more costly and equally effective compared to standard care, but there was significant uncertainty around incremental costs and QALYs. The findings were robust to a range of sensitivity analyses.

Conclusions

There is no evidence to suggest that WEPDs can be considered a cost effective device to reduce SSI. Their continued use is a waste of limited health care resources.