Serological and conductimetric assays for the detection of Pseudomonas syringae pathovar pisi in pea seeds

Test protocols for detecting Pseudomonas syringae pv. pisi , the causal agent of bacterial blight, in pea seeds are generally based on dilution-plating assays. These assays are usually very specific and reliable, but are time-consuming and laborious. Tests suited for large scale screening of seed lots are therefore needed. Conductimetric assays, immunofluorescence microscopy (IF) and an enzyme-linked immunosorbent assay (ELISA) for detecting Ps. syr. pv. pisi in pea seed extracts were compared with dilution-plating by two extraction methods, viz. 6 h soaking of seeds and 2 h soaking of flour of ground pea seeds in water. In general, the detection of Ps. syr. pv. pisi with conductimetric, IF and dilution-plating assays in the suspension water of the ground and 2 h-soaked pea samples was less sensitive than detection in suspension water of the 6 h-soaked pea seeds. The detection threshold of these assays varied per seed lot between 0 and 4.08 log cfu ml^-1 for the 6 h soaking procedure. The detection threshold of ELISA varied for both extraction methods generally between 4.08 and 6.08 log cfu ml^-1. Detection times recorded in conductimetric assays correlated well (— 0.89 < r < —0.98) with the log colony-forming units of Ps. syr. pv. pisi added to seed extracts at 27 as well as 17°. However, confirmation of results by isolation on semi-selective media after conductimetry was more successful at 17° than at 27°, because of the relatively lower activity of saprophytic Pseudomonas spp. at this temperature.     

Errors in pH measurements in dilute or sulfhydryl-containing buffers

Large errors in pH measurements were found with most types of reference electrodes when used in buffers containing sulfhydryl reagents (either 1–5 mm 2-mercaptoethanol or 5–10 mm dithiothreitol), or when electrodes were used in dilute buffers (≤25 mm) without sulfhydryl reagents. These errors which are confined to the reference electrodes depend on its history, although new electrodes exhibit errors in sulfhydryl solutions. The errors appear independent of pH and buffer ions. Although high ionic strengths reduce dilution errors, sulfhydryl-induced errors persist. Numerous tests have been unsuccessful in clarifying the molecular sources of these errors. Sulfhydryl effects are usually discernible by the downward pH drift, whereas errors in dilute buffer are frequently stable. Due to the prevalence and magnitude of these errors, increased precautions in pH measurements are needed, as suggested.     

Detection of mutagenic pollution of natural environment using microbiological assays

One of the most important and serious ecological problems is mutagenic pollution of the natural environment. Therefore, detection of mutagenic compounds in samples taken from natural habitats is of special interest. Microbiological mutagenicity tests seem to be very useful tools for such detection. In this review article, a general view on the tests employing genetically modified bacterial strains designed for detection of low concentrations of mutagenic compounds is presented. Moreover, a comparison of advantages and disadvantages of selected assays, developed early on and more recently, and features of these assays are discussed. It appears that none of the currently available mutagenicity tests is perfect or optimal for all purposes. Thus, a choice for the particular assay must depend on the nature of studies and specific tasks of the experiments to be performed.     

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.     

Dynamic approach to predict pH profiles of biologically relevant buffers

Recently, dynamic approach has been applied to determine the steady state concentrations of multiple ionic species present in complex buffers at equilibrium. Here, we have used the dynamic approach to explicitly model the pH profiles of biologically relevant phosphate buffer and universal buffer (a mixture of three tri-protic acids such as citric acid, boric acid and phosphoric acid). The results from dynamic approach are identical to that of the conventional algebraic approach, but with an added advantage that the dynamic approach, allow for the modelling of complex buffer systems relatively easy compared to that of algebraic method.     

The use of environmental assays for impact assessment

The assessment of impacts of chemically contaminated aquatic environments on animal systems has a number of shortcomings. These include problems with analyses for toxic chemicals and the relevance of bioassays for predicting risk to ecosystems. Research is urgently needed to find better ways to solve these problems, particularly with respect to chronic exposures.     

Selection of quantum dot wavelengths for biomedical assays and imaging

Fluorescent semiconductor nanocrystals (quantum dots [QDs]) are hypothesized to be excellent contrast agents for biomedical assays and imaging. A unique property of QDs is that their absorbance increases with increasing separation between excitation and emission wavelengths. Much of the enthusiasm for using QDs in vivo stems from this property, since photon yield should be proportional to the integral of the broadband absorption. In this study, we demonstrate that tissue scatter and absorbance can sometimes offset increasing QD absorption at bluer wavelengths, and counteract this potential advantage. By using a previously validated mathematical model, we explored the effects of tissue absorbance, tissue scatter, wavelength dependence of the scatter, water-to-hemoglobin ratio, and tissue thickness on QD performance. We conclude that when embedded in biological fluids and tissues, QD excitation wavelengths will often be quite constrained, and that excitation and emission wavelengths should be selected carefully based on the particular application. Based on our results, we produced near-infrared QDs optimized for imaging surface vasculature with white light excitation and a silicon CCD camera, and used them to image the coronary vasculature in vivo. Taken together, our data should prove useful in designing fluorescent QD contrast agents optimized for specific biomedical applications.     

Effects of buffers and pH on rabbit red blood cell hexokinase

The kinetic properties of rabbit red blood cell hexokinase in different buffer systems have been studied. At pH 8.0 the reaction velocity (v) is about 30% higher in glycylglycine compared to Tris, Tea, Hepes or ammonium acetate buffers. The enzyme stability, heat-dependence and spectral properties of the enzyme are also affected by the buffer utilized. None of the following kinetic properties of red blood cell hexokinase varies with pH in the range 6.8-8.5: Km of glucose; Km of ATP and Ki of glucose 6-phosphate.     

Macroreticulate buffers: a novel approach to pH control in living systems.

It is possible to control the pH of growing living systems in vitro by adding, to the growth media, macroreticulate buffers, i.e. amphoteric resins made with buffering and titrant groups simultaneously affixed to the matrix. Such beads possess a very precise isoelectric point (pI) and are able to maintain the solutions' pH close to their pI values for extended growth periods. These pearls are made of a neutral polyacrylamide backbone containing up to 200 mM grafted weak acrylamido acids and bases. It is possible to produce such buffers with any desired pH value in the pH 2.5-11 scale. An example is given of conditioning the pH of endive plants grown hydroponically.     

Validation of assays for use with combination vaccines.

A general methodology is presented for the validation of assays used for testing combination vaccines. The presentation is detailed and technical as our intention is to address challenges that we have encountered in the design and statistical analysis of assay validation studies. There are several noteworthy features which render the approach particularly useful in practice. It employs a statistical experimental design approach to the investigation of assay ruggedness with respect to manufacturing variability; it makes use of the assay variability results to determine the level of test-run replication necessary to achieve precision compatible with the product specifications; and, it provides a generic approach to assay validation.With combination vaccines, as with other pharmaceuticals, the analytical methods for release and stability must be validated early in the development programme Several things, though, distinguish this task with combination vaccines: (1) assays are typically pre-existing and often have been validated for use with an established sample matrix, e.g. a monovalent formulation; (2) sample matrices are complex and therefore more subject to manufacturing variability and more likely to cause assay interferences; and (3) the analytical workload is considerable due to the number of antigens.The methodology presented here was developed jointly by Merck Research Laboratories (West Point, PA) and Pasteur Mérieux Connaught, Inc. (Swiftwater, PA). Many of the issues discussed here have application outside of combination vaccines and are common features of all assay validations.     

An azophenolic colorimetric reagent for use in enzyme-linked immunosorbent assays

A colorimetric reagent, 4-(4'-nitro-2'-methylsulfonylphenylazo)phenyl phosphate (NMPP), has been shown to be an effective substrate of alkaline phosphatase. NMPP and p-nitrophenyl phosphate were applied in comparative studies using enzyme immunoassays for the detection of viral antigens and antiviral antibodies. The new substrate exhibited similar, or even higher, sensitivity than p-nitrophenyl phosphate depending on the substrate concentrations used. Positive and negative reactions were easier to define, even without cumbersome equipment. The enzyme reaction was terminated by the uncompetitive inhibitor, theophylline.     

Photograph registration of petri dishes in high quality and image editing of microbiological assays

International Microbiology - Science is based on evidence that can be measured or observed through methodical techniques which are expressed in several ways, either quantitatively or qualitatively....     

Comparison of 18O exchange and pH stop-flow assays for carbonic anhydrase

The hydration velocity of CO2 (0.002 M) catalyzed by bovine carbonic anhydrase (BCA) was measured at 25 degrees C and pH 7.4 by three different techniques: two initial-rate (steady-state) stop-flow methods, one using a glass pH electrode (in Hannover, method 1) and one using spectrophotometric measurements of a pH indicator (in Philadelphia, method 2), and an exchange method in which the disappearance of C18O16O from a bicarbonate solution was determined at equilibrium (in Philadelphia, method 3). The Michaelis-Menten constant (Km) and the inhibition constants for chloride (Ki,Cl) and ethoxzolamide (Ki,ez) were the same for methods 1, 2, and 3. The turnover numbers were 270,000, 400,000, and 555,000 s-1 by methods 1, 2, and 3, respectively. Values for CO2 hydration velocity measured by methods 2 and 3 on the same solution of BCA at the same time were the same. Km, maximal reaction velocity (Vmax), Ki,ez, and Ki,Cl obtained from normal human hemolysate at 37 degrees C and pH 7.2 by methods 2 and 3 were the same. Km and Vmax of the carbonic anhydrase isozyme CA III of homogenate from rabbit soleus were also identical by methods 1 and 3. According to Michaelis-Menten theory, the values of Km and Vmax obtained by method 3 should have been significantly smaller than those obtained by methods 1 and 2. We conclude that the catalytic step itself is apparently not rate limiting under physiological conditions and that method 3 can be used to obtain Michaelis-Menten characteristics of carbonic anhydrase.