Comparison of assay techniques for beta-lactamase activity

Two chemical, one physical and two microbiological techniques for the assay of β-lactamase activity have been compared using three partially purified β-lactamases of different specificities. Seven cephalosporins and three penicillins with a wide range of susceptibilities have been used as substrate. The different methods gave results that were in general agreement with the exception of the hydroxylamine assay. The cephalosporins were most rapidly assayed by the uv method. Its suitability for use in the assessment of new cephalosporin analogues was confirmed, but it could not be used for penicillins. The iodometric assay was rapid and reliable but required large amounts of substrate. Where the amount of substrate available was limited, the microbiological techniques were preferred. The results obtained for enzyme activities against two of the cephalosporins and against the penicillins were higher with the hydroxylamine assay than with the other methods and it had no apparent advantage.     

Chemical and biological indicators of soil quality in organic and conventional farming systems in Central Italy

In the past decade, there has been increased scientific interest in the so-called organic farming, especially in comparison with conventional agriculture. Many recent studies compare these two fundamentally different systems for soil properties, in different regions of the world. In this study, two adjacent fields in Central Italy, one managed according to organic, and the other according to conventional farming methods, were studied to determine the effects of these two agricultural systems on soil quality indicators at the farm level. Chemical and microbiological properties were chosen as indicators of soil quality and were measured at soil depth intervals of 5–20 and 20–35 cm, after 7 years of organic certified and conventional management methods. The field under organic management showed significantly better soil nutritional and microbiological conditions; with increased level of total nitrogen, nitrate and available phosphorus, and an increased microbial biomass content, and enzymatic activities (acid phosphatase, protease and dehydrogenase). No consistent increase in total organic carbon was observed. Results of the study suggest that, over the period of 7 year, organic management method strongly affects soil quality indicators. Large differences between the two soils were found in terms of microbiological properties, which are sensitive soil indicators of changes occurred under the different farming systems.     

Interval estimation of the density of organisms using a serial-dilution experiment

The serial-dilution assay is a standard microbiological method for estimating the density of organisms in a solution. The commonly used interval-estimation procedures of Woodward and deMan are described and compared. A new method for the calculation of two-sided confidence intervals, which is superior to the standard procedures, is presented. The computations are illustrated for a decimal dilution assay with three samples at each of three dilutions.     

A quantitative microbiological determination of 6-aminopenicillanic acid

Several complicated chemical and microbiological methods have been described for the quantitative assay of 6-aminopenicillanic acid in the presence of benzyl- and phenoxymethylpenicillin. In all these methods the penicillins must be removed since they interfere with the assay. A specific microbiological plate assay withSerratia marcescens D 217 (ATCC 27117) is described for estimating even small amounts of 6-APA in benzyl- and phenoxymethylpenicillin samples. The contents of this paper were presented at the Xth International Congress for Microbiology at Mexico City 1970. The author wishes to express his gratitude to Mrs. I. Struyk-Hoette and Messrs. A. P. Struyk and R. G. W. Spickett for their valuable help and discussions.     

In support of null hypothesis significance testing

Many criticisms have been levelled at null hypothesis significance testing (NHST). It is argued here that although there is reason to doubt that data subjected only to NHST have been subjected to sufficient analysis, the search for clear answers to well-formulated questions derived from substantive hypotheses is well served by NHST. To reliably draw inferences from data, however, NHST may need to be complemented by additional methods of analysis, such as the use of confidence intervals and of estimates of the degree of association between independent and dependent variables. It is argued that these should be seen as complements of, rather than as substitutes for, NHST since they do not directly test the strength of evidence against a null hypothesis.     

A novel method for sampling bacteria on plant root and soil surfaces at the microhabitat scale

This study reports the first method for sampling bacteria at a spatial scale approximating a microhabitat. At the core of this method is the use of tungsten rods with laser-cut tips of known surface area (0.013 mm(2)). Exposed plant root or soil surfaces were viewed with a dissecting microscope and micro-sampling rods were guided to sample sites using a micro-manipulator. Bacteria that adhered to the sampling tips were then recovered for microbiological analyses. The efficiency of this method for removing bacteria from root surfaces was similar to that with which bacteria are recovered from dissected root segments using the conventional technique of washing. However, as the surface area of the micro-sampling tips was known, the new method has the advantage of eliminating inaccuracy in estimates of bacterial densities due to inaccurate estimation of the root or soil surface sampled. When used to investigate spatial distributions of rhizoplane bacteria, the new technique revealed trends that were consistent with those reported with existing methods, while providing access to additional information about community structure at a much smaller spatial scale. The spatial scale of this new method is ca. 1000-times smaller than other sampling methods involving swabbing. This novel technique represents an important methodological step facilitating microbial ecological investigations at a microhabitat scale.     

Comparative studies on the microbiological vitamin B12 assay at two laboratories.

The turbidimetric methods in routine use at two laboratories for the microbiological assay of vitamin B12 have been compared. Attempts were made to standardize some major parts of the method, i.e., assay design, test strain (Lactobacillus leichmannii), test medium, and reference standard. The laboratories used different approaches to achieve efficient assay procedures. During a 6-year period four comparative experiments were carried out. In these experiments the vitamin B12 content of five different products was determined in a series of independent assays at each laboratory. A satisfactory degree of agreement (difference less than 5%) was found for four of these products.     

Comparative studies on the microbiological vitamin B12 assay at two laboratories.

The turbidimetric methods in routine use at two laboratories for the microbiological assay of vitamin B12 have been compared. Attempts were made to standardize some major parts of the method, i.e., assay design, test strain (Lactobacillus leichmannii), test medium, and reference standard. The laboratories used different approaches to achieve efficient assay procedures. During a 6-year period four comparative experiments were carried out. In these experiments the vitamin B12 content of five different products was determined in a series of independent assays at each laboratory. A satisfactory degree of agreement (difference less than 5%) was found for four of these products.     

Comparison of gas chromatographic/mass spectrometric and microbiological assays for 5-fluorouracil in plasma

The concentrations of 5-fluorouracil in 99 plasma samples were determined by both microbiological and gas chromatographic/mass spectrometric assays. The values determined by the two methods were similar (correlation coefficient = 0.90). A regression of the natural logarithms of the concentrations (0.01-94 micrograms ml-1) determined by the two methods gave a line whose slope and intercept were not statistically different (p greater than 0.05) from 1 and 0 respectively. Thus, the microbiological assay has specificity over a sufficient concentration range to serve as a practical routine laboratory method for the determination of 5-fluorouracil.     

Determination of the fertility of the soil by microbiological methods

Summary A review is given of the determination of soil fertility by microbiological methods. The quantitative and the qualitative methods for the determination of the microflora were discussed as well as those methods in which micro-organisms are used for ascertaining a particular condition or a limiting factor in the soil. More especially the use ofAzotobacter cultures for this purpose was mentioned.Presented at the meeting of the Netherlands Society of Microbiology. Wageningen, May 13th, 1939.     

The allele-specific probe and primer amplification assay, a new real-time PCR method for fine quantification of single-nucleotide polymorphisms in pooled DNA

The evolution of fungicide resistance within populations of plant pathogens must be monitored to develop management strategies. Such monitoring often is based on microbiological tests, such as microtiter plate assays. Molecular monitoring methods can be considered if the mutations responsible for resistance have been identified. Allele-specific real-time PCR approaches, such as amplification refractory mutation system (ARMS) PCR and mismatch amplification mutation assay (MAMA) PCR, are, despite their moderate efficacy, among the most precise methods for refining SNP quantification. We describe here a new real-time PCR method, the allele-specific probe and primer amplification assay (ASPPAA PCR). This method makes use of mixtures of allele-specific minor groove binder (MGB) TaqMan probes and allele-specific primers for the fine quantification of SNPs from a pool of DNA extracted from a mixture of conidia. It was developed for a single-nucleotide polymorphism (SNP) that is responsible for resistance to the sterol biosynthesis inhibitor fungicide fenhexamid, resulting in the replacement of the phenylalanine residue (encoded by the TTC codon) in position 412 of the enzymatic target (3-ketoreductase) by a serine (TCC), valine (GTC), or isoleucine (ATC) residue. The levels of nonspecific amplification with the ASPPAA PCR were reduced at least four times below the level of currently available allele-specific real-time PCR approaches due to strong allele specificity in amplification cycles, including two allele selectors. This new method can be used to quantify a complex quadriallelic SNP in a DNA pool with a false discovery rate of less than 1%.     

Superiority of a soil debris isolation method over a beet seed colonization method for assay of Rhizoctonia solani at high soil inoculum densities.

A quantitative soil debris isolation method (all debris from known weight of soil plated) and a garden beet seed saprophytic colonization method were compared over a 1-year period for assaying Rhizoctonia solani population. Four fields of different soil textures were selected. Within each field four areas of healthy and four areas of diseases (rhizoctonia root and crown rot) sugarbeets were sampled bimonthly from August 1976 until June 1977. The maximum numbers of R. solani colonies obtained by the debris method were 2 per gram of soil in areas of healthy beets, and 11 per gram of soil in areas of diseased sugarbeets. At such high inoculum densities the beet seed colonization method underestimated R. solani populations, because the inoculum per unit of soil exceeded the numbers of beet seeds per unit of soil available for colonization. Modifications of the beet seed method did not significantly alter results of colonization assays. Ranked correlation comparisons of assay methods yielded r = 0.81 for all data.     

Microbiological analysis of 5-formyltetrahydrofolic acid and other folates using an automatic 96-well plate reader

The growth of auxotrophic bacteria remains the method of choice for the determination of biologically active folate metabolites in plasma. This report describes a microbiological assay for folates adapted to use disposable 96-well plates and an automatic plate reader. The modifications in the assay decreased reagent costs and made the analysis of hundreds of samples per day possible with a sensitivity limit of 10 fmol of (6S)-5-formyltetrahydrofolic acid. This limit compares favorably with that of previously reported, more laborious methods. The unnatural 6R diastereomer of 5-formyltetrahydrofolic acid did not interfere with the microbiological assay of the natural 6S diastereomer.     

Immunofluorescent Assay for the Marine Ammonium-Oxidizing Bacterium Nitrosococcus oceanus

Nitrification is one of the important microbiological transformations of nitrogen in the ocean. Traditional enrichment-culture methods for enumerating the autotrophic bacteria which oxidize ammonium to nitrite are very time consuming (months) and are believed to seriously underestimate natural abundances. A fluorescent-antibody assay for a marine ammonium-oxidizing bacterium was developed to provide a rapid and direct means of identifying these microorganisms. Antibodies to Nitrosococcus oceanus were prepared and tested against pure cultures of marine, freshwater, and soil ammonium oxidizers and against bacteria from natural seawater samples. Cell counts of culture samples determined by the fluorescent-antibody assay agreed with hemacytometer and acridine orange counts. Our results demonstrated that the immunofluorescent assay is a powerful tool for the detection of Nitrosococcus in the marine environment.     

土壤微生物生态学研究中的非培养方法

土壤微生物种类和数量是评价土壤健康质量的重要指标之一。然而环境中90%以上的微生物不能够通过传统的培养基培养方法获得。最近发展起来的分析方法如磷脂脂肪酸(PLFA)、BIOLOG微孔板和分子生物学的方法可从不同方面对土壤微生物群落进行更为详尽的分析。论文就土壤微生物生态学研究中使用的主要方法进行了综述。     

Molecular identification of Salmonella isolates from poultry and meat productsin Irbid City,Jordan

The sensitivity and accuracy of molecular diagnosis of Salmonella from meat and poultry products using polymerase chain reaction (PCR) was compared with conventional microbiological methods. A total of 212 samples representing the most frequently used fresh and frozen meat and poultry products (whole, cut, ground, and processed) were collected from different locations within the city of Irbid. DNA was extracted directly from each food sample and amplified using Salmonella-specific primers. Samples were also analysed using conventional microbiological methods for the presence of Salmonella spp. Results showed that Salmonella was detected in 185 samples out of 212 (87%) by PCR technique, while 172 (81%) samples were detected Salmonella positive by conventional microbiological methods. On the other hand, 27 (12.7%) samples were negative by PCR and 40 (18.8%) samples were negative by conventional microbiological methods. PCR assay proved to be an effective method for Salmonella detection in meat and poultry products with high specificity and sensitivity and more importantly a less time-consuming procedure. Using PCR, Salmonella spp. detection could be achieved within 24–36 h compared to 3–8 days for the conventional microbiological methods.     

Sample storage for soil enzyme activity and bacterial community profiles

Storage of samples is often an unavoidable step in environmental data collection, since available analytical capacity seldom permits immediate processing of large sample sets needed for representative data. In microbiological soil studies, sample pretreatments may have a strong influence on measurement results, and thus careful consideration is required in the selection of storage conditions. The aim of this study was to investigate the suitability of prolonged (up to 16 weeks) frozen or air-dried storage for divergent soil materials. The samples selected to this study were mineral soil (clay loam) from an agricultural field, humus from a pine forest and compost from a municipal sewage sludge composting field. The measured microbiological parameters included functional profiling with ten different hydrolysing enzyme activities determined by artificial fluorogenic substrates, and structural profiling with bacterial 16S rDNA community fingerprints by amplicon length heterogeneity analysis (LH-PCR). Storage of samples affected the observed fluorescence intensity of the enzyme assay's fluorophor standards dissolved in soil suspension. The impact was highly dependent on the soil matrix and storage method, making it important to use separate standardisation for each combination of matrix type, storage method and time. Freezing proved to be a better storage method than air-drying for all the matrices and enzyme activities studied. The effect of freezing on the enzyme activities was small (< 20%) in clay loam and forest humus and moderate (generally 20-30%) in compost. The most dramatic decreases (> 50%) in activity were observed in compost after air-drying. The bacterial LH-PCR community fingerprints were unaffected by frozen storage in all matrices. The effect of storage treatments was tested using a new statistical method based on showing similarity rather than difference of results.     

Quantification of biotin in feed, food, tablets, and premixes using HPLC-MS/MS

Two sensitive and specific methods for quantification of biotin in feed, food, tablets, and premixes based on HPLC-MS/MS have been developed and validated. Depending on sample matrix and biotin content different extraction procedures and HPLC conditions were applied. Key steps in sample preparation were an alkaline extraction or a hydrolysis with sulphuric acid followed by enzymatic digest with papain. For many samples with low biotin content the latter combination of extraction steps was shown to be necessary for an optimal release of biotin from the matrix. The first time synthesis of deuterated biotin for use as internal standard allowed the compensation of losses during sample work-up and ion suppression during HPLC-MS/MS analysis. The new methods are faster than the commonly used microbiological assay using Lactobacillus plantarum. Additionally, they have a higher specificity as results for biotin are based on determination of a chemically defined compound, and not of a biological activity. Quantification is applicable to samples with a biotin content >100 microg/kg. Results obtained with the new methods have been compared with those of the microbiological assay, and were in good agreement.     

Comparison of methods for measuring soil microbial activity using cotton strips and a respirometer

In order to develop a method of measuring the level of microbial activity in soil that is suitable for use by farmers, land managers, and other non-scientists, a simple method for determining soil microbial activity was evaluated and compared with two standard techniques. Soils sampled from vegetable farms in south east Queensland were incubated in the laboratory under controlled moisture and temperature conditions. Three methods were used to measure soil microbial activity, a respirometry method and two methods using the cotton strip assay (CSA) technique (image analysis and tensometer). The standard CSA method measured loss of tensile strength over a 35 day incubation period of buried cotton strips using a tensometer. The new CSA technique measured the intensity of staining by microbes using a flatbed scanner to create an image of the cotton strip whose staining percentage was determined using Photoshop software. The respirometry method used the substrate induced respiration rate (SIR) to determine microbial biomass in the soil at day 12 of incubation. The strong correlation between the image analysis method and the tensometer method (r(2)=0.81), a technique used by scientific researchers, suggests that the image analysis method could be used to monitor aspects of soil biological health by general community land-care groups and farmers. The image analysis method uses equipment which is readily available and, while not strongly correlated with more precise measurements of soil biological activity such as microbial biomass (r(2)=0.26), it can detect gross trends in biological health in a soil monitoring program. The CSA method using image analysis was the cheapest technique to measure soil microbial activity. CSA using image analysis can be a valuable tool in conjunction with other simple indicators of soil physical and chemical health such as slaking and pH to monitor soil amelioration or rehabilitation programs.     

The ATP-bioluminescence method for a rapid evaluation of the microbial activity in the stone materials of monuments

The examination of the state of conservation of works of art in stone includes the assessment of the presence of microbiological agents on the surface of the decayed monuments. These microorganisms can accelerate, via their metabolic activity, the decay process of the stone surface. At present this assessment is made with the traditional techniques for the microbiological examination of the soil, provides results only after a delay of 30 days. A bioluminescent ATP assay should provide rapid quantitation of actively growing organisms on the surface of a stone monument, and the applicability of this technique was verified on some samples of sandstone (Pietraforte) collected from a historic building (the Strozzi Palace) in Florence. These samples were evaluated for the amount of the ATP and the total number of microorganisms. The results obtained suggest that the bioluminescent assay could be suitable for detecting and quantitating the presence of microorganisms in a sample of stone.