Development of an ELISA for Evaluation of Swab Recovery Efficiencies of Bovine Serum Albumin

After a potential biological incident the sampling strategy and sample analysis are crucial for the outcome of the investigation and identification. In this study, we have developed a simple sandwich ELISA based on commercial components to quantify BSA (used as a surrogate for ricin) with a detection range of 1.32–80 ng/mL. We used the ELISA to evaluate different protein swabbing procedures (swabbing techniques and after-swabbing treatments) for two swab types: a cotton gauze swab and a flocked nylon swab. The optimal swabbing procedure for each swab type was used to obtain recovery efficiencies from different surface materials. The surface recoveries using the optimal swabbing procedure ranged from 0–60% and were significantly higher from nonporous surfaces compared to porous surfaces. In conclusion, this study presents a swabbing procedure evaluation and a simple BSA ELISA based on commercial components, which are easy to perform in a laboratory with basic facilities. The data indicate that different swabbing procedures were optimal for each of the tested swab types, and the particular swab preference depends on the surface material to be swabbed.     

An in situ method for determining bacterial survival on food preparation surfaces using a redox dye

A simple method is described for the direct enumeration of viable bacteria dried on test surfaces. Inoculated surfaces were overlayed with agar and after incubation nitroblue tetrazolium solution (pale yellow) was used to stain colonies (purple) at the agar-test surface interface. Stained colonies could be readily detected and counted even against the opaque background of ceramic tile or stainless steel or when present within opaque films of milk or serum. Recovery of bacteria by this method was approximately fivefold greater than using a conventional swabbing procedure. The method was used to demonstrate the marked effect of the composition of the suspension fluid, in which bacteria were dried, and the length of surface exposure upon bacterial survival.     

Disinfection of wooden structures contaminated with Paenibacillus larvae subsp. larvae spores

AIMS: The aim of the study is to examine the disinfection of wood contaminated with Paenibacillus larvae subsp. larvae spores, in order to find a practical decontamination method for hive materials. METHODS AND RESULTS: The number of viable spores recovered after the treatment, on the surface by swabbing, and in the deeper parts of the wood by scraping, was used to test the efficiency of the disinfection. Our results indicate that chemical disinfection is only complete when high concentrations (> 50%) of the disinfectant are used. Heat treatment in general was found to be very effective. The scorching of wood was not satisfactory as it only killed spores at the surface. CONCLUSION: Complete disinfection is only possible with some heat treatments or by using high concentrations of chemical disinfectants. SIGNIFICANCE AND IMPACT OF THE STUDY: This study puts forward some methods that can provide complete decontamination, which is necessary for an effective control of American foulbrood disease.     

First comprehensive view on a magnetic separation based protein purification processes: From process development to cleaning validation of a GMP‐ready magnetic separator

Magnetic separation processes are known as integrated bioanalytical protein purification method since decades and are well described. However, use of magnetic separation processes in a regulated industrial production environment has been prevented by the lack of suitable process equipment and prejudice against the productivity of the process and its qualification for cleaning‐in‐place operation. With the aim of overcoming this prejudice, a comprehensive process development approach is presented, based on a GMP‐compliant magnetic separator, including an optimization of the batch adsorption process, implementation into a technical‐scale, and the development and validation of cleaning routines for the device. By the implementation of a two‐step counter‐current binding process, it was possible to raise the yields of the magnetic separation process even for very low concentrated targets in a vast surplus of competing proteins, like the hormone equine chorionic gonadotropin in serum, from 74% to over 95%. For the validation of the cleaning process, a direct surface swabbing method combined with a total organic carbon analysis was established for the determination of two model contaminants. The cleanability of the process equipment was proven for both model contaminants by reliably meeting the 10 ppm criteria.     

Effectiveness of cleaning techniques used in the food industry in terms of the removal of bacterial biofilms

The effectiveness of cleaning was investigated through food factory trials and laboratory experiments using a naturally occurring biofilm from a food factory environment and generated biofilms. The efficacy of factory cleaning and disinfection programmes was assessed by swabbing and total viable count (TVC) analysis of surfaces before cleaning, after cleaning and after disinfection. Cleaning produced a 0.91 log reduction in the attached population. Investigation of the effectiveness of a variety of cleaning methods in the removal of a naturally occurring food factory biofilm showed that the high pressure spray and the mechanical floor scrubber, which use a high degree of mechanical action, were most effective. Cleaning trials with biofilms of Pseudomonas aeruginosa or Staphylococcus aureus showed that spraying with water at pressures of 34.5, 51.7 and 68.9 bar did not significantly increase the removal, as assessed by direct epifluorescent microscopy (DEM) and swabbing and TVC analysis, beyond the three log reduction observed at 17.2 bar. The effect of spray time at 17.2 bar showed that increasing spray time from 1 to 10 s did not significantly increase removal of Ps. aeruginosa biofilm. Investigation of the optimum distance of the spray lance from the surface at 17.2 bar was found to be between 125 and 250 mm. The use of an alkaline, acidic or neutral detergent prior to spraying with water at 17.2 bar did not significantly increase the removal of Ps. aeruginosa or Staph. aureus. However, the acidic and alkaline products significantly (P = 0.05) affected the viability of Staph. aureus and Ps. aeruginosa, respectively, thereby minimizing the potential for the spread of contamination.     

Problems associated with traditional hygiene swabbing: the need for in-house standardization

AIMS: To investigate factors influencing the recovery of micro-organisms from surfaces using traditional swabbing techniques. METHODS AND RESULTS: Stainless steel squares were inoculated with known levels (approx. 2.7x10(2)-2.7x10(4)) of either Escherichia coli or Staphylococcus aureus and sampled using different swab/solution combinations. Overlaying the coupons with agar allowed colonies remaining on the surface to be enumerated. Conventional cultivation was used to determine the ease with which the bacteria were released from the swabs and the viability of the organisms within the solutions over a 24-h period. Minimal bacterial growth occurred when the samples were stored at 4 degrees C. At room temperature, whilst the presence of nutrients significantly increased bacterial numbers over time, the addition of Tween 80 to nutrient depleted environments significantly reduced the viability of Staph. aureus. The percentage of bacteria released from directly inoculated swabs was significantly higher than that recovered from surface swabs, highlighting the importance of effectively removing bacterial contaminants from a surface. Increasing the level of mechanical energy generated during swabbing increased the number of bacteria removed from a wet surface. However, it is hypothesized that cellular damage, perhaps caused by the swabbing action itself, may have reduced recoverability from a dry surface. Nonetheless, an increased ability to effectively remove bacteria from a surface did not necessarily correlate with higher bacterial recovery, implying that an equally important factor in terms of swabbing efficiency is the ability of a swab to effectively release bacteria into a diluent. CONCLUSIONS: Both swab and wetting solution can influence the number of bacteria recovered. Under the experimental conditions described here, the use of swabs coated with a brush-textured nylon flock in combination with a non-growth-enhancing wetting solution appeared the best system to use when sampling a wet surface. However, this combination may not always be ideal and proper consideration must be given to how the sample is to be taken, transported and, if necessary, stored prior to analysis. SIGNIFICANCE AND IMPACT OF THE STUDY: Careful selection of swabbing materials can increase the sensitivity of traditional microbiological analysis. However, any improvements made are likely to be insignificant in relation to the overall poor performance of the swabbing technique.     

A non-lethal technique for detecting the chytrid fungus Batrachochytrium dendrobatidis on tadpoles

Batrachochytrium dendrobatidis (Bd) infection on post-metamorphic frogs and salamanders is commonly diagnosed using polymerase chain reaction (PCR) of skin scrapings taken with mildly abrasive swabs. The technique is sensitive, non-lethal, and repeatable for live animals. Tadpoles are generally not sampled by swabbing but are usually killed and their mouthparts excised to test for the pathogen. We evaluated a technique for non-lethal Bd diagnosis using quantitative PCR (qPCR) on swabs scraped over the mouthparts of live tadpoles. The sensitivity of non-lethal (swabbing) and lethal (removal of mouthparts) sampling was assessed using 150 Bd-infected Rana subaquavocalis tadpoles. Swabbing was consistently less sensitive than lethal sampling, but still detected Bd. Experimental Bd prevalence was 41.1% when estimated by destructively sampling mouthparts and 4.7 to 36.6% (mean = 21.4%) when estimated with swabs. Detection rates from swabbing varied with investigator and time since infection. The likelihood of detecting Bd-infected tadpoles was similar regardless of size and developmental stage. Swabbing mouthparts of live tadpoles is a feasible and effective survey technique for Bd, but, because it is less sensitive, more tadpoles must be sampled to estimate prevalence at a confidence level comparable to destructive sampling.     

Derivation of dynamic reference values for the classification of contaminant concentrations in bream (Abramis brama) of German rivers and lakes using data of the Environmental Specimen Bank

A direct assessment of contaminant concentrations in biota with respect to their biological relevance or importance for the ecosystem is rarely possible. Statistically derived reference values can serve as a guide to classify measured tissue concentrations, but they do not provide any information on the biological significance of the contaminant concentrations. However, previously such reference values did not exist for environmental-biomonitoring. Based on analytical data from muscle tissue of bream (Abramis brama) of the German Environmental Specimen Bank (ESB), we present a method on how to derive dynamic reference values. These show the general state of inland surface water pollution in Germany at a certain time. The reference values allow for classification of measured contaminant concentrations in muscle tissue of bream. This classification enables a comparative assessment in means of how the actual state of pollution in a specific ecosystem is, compared to the general, macro state of pollution.     

Adsorption-desorption of recombinant hepatitis B surface antigen (r-HBsAg) from P. pastoris on a diatomaceous earth matrix: Optimization of parameters for purification

Recombinant hepatitis B surface antigen (r-HBsAg) produced in yeast is adsorbed on a diatomaceous earth matrix for purification purposes. A pH dependence in the adsorption-elution behavior was found. The capacity of celite (Hyflo Super Cei) for adsorbing r-HBsAg increased with decreasing pH. Nonspecific proteins were also adsorbed, but a low pH dependence was found. Elution from the matrix was performed using a basic pH buffer, in which r-HBsAg is more specifically adsorbed/desorbed than contaminant proteins, permitting the purification of the r-HBsAg. A pH of 4.0 was used for adsorption and pH 8.2 was used for desorption. The described protocol allows a purification factor between three- and fivefold with respect to contaminant proteins and sixfold with respect to contaminant DNA. Finally, the adsorption step was successfully scaled-up for production purposes. (c) 1993 John Wiley & Sons, Inc.     

Quantitative capillary electrophoresis determination of oversulfated chondroitin sulfate as a contaminant in heparin preparations

A simple, accurate, and robust quantitative capillary electrophoresis (CE) method for the determination of oversulfated chondroitin sulfate (OSCS) as a contaminant in heparin (Hep) preparations is described. After degradation of the polysaccharides by acidic hydrolysis, the hexosamines produced (i.e., GlcN from Hep and GalN from OSCS) were derivatized with anthranilic acid (AA) and separated by means of CE in approximately 10 min with high sensitivity detection at 214 nm (limit of detection [LOD] of ∼200 pg). Furthermore, AA-derivatized GlcN and GalN showed quite similar molar absorptivity, allowing direct and simple quantification of OSCS in Hep samples. Moreover, a preliminary step of specific enzymatic treatment by using chondroitin ABC lyase may be applied for the specific elimination of interference in the analysis due to the possible presence in Hep samples of natural chondroitin sulfate and dermatan sulfate impurities, making this analytical approach highly specific for OSCS contamination given that chondroitin ABC lyase is unable to act on this semisynthetic polymer. The CE method was validated for specificity, linearity, accuracy, precision, LOD, and limit of quantification (LOQ). Due to the very high sensitivity of CE, as little as 1% OSCS contaminant in Hep sample could be detected and quantified. Finally, a contaminated raw Hep sample was found to contain 38.9% OSCS, whereas a formulated contaminated Hep was calculated to have 39.7% OSCS.     

Observations on the Value of a Swab Technique for Determining the Bacteriological State of Milking Equipment Surfaces

The reproducibility and efficacy of a swab technique for milking equipment in normal use was studied. Reproducibility was satisfactory when swabbing was done by trained personnel. It was confirmed that swabs pick up only a proportion of the microorganisms present on a surface; this proportion varies with the consistency of the contaminating material, but not with the type of surface on which it occurs. It is concluded that swabs allow the estimation of the maximum ability of a surface to contaminate the milk.
Rigid smooth surfaces of the milking machine are recontaminated by microorganisms originating from joints and complex parts of the machine during rinsing after cleaning. A procedure to evaluate the efficacy of cleaning these surfaces is proposed.     

Excision vs sponge swabbing - a comparison of methods for the microbiological sampling of beef, pork and lamb carcasses

AIMS: The aim of this research was to compare excision sampling with polyurethane and cellulose acetate sponge swabbing for the recovery of total viable counts and Enterobacteriaceae on meat carcasses. METHODS AND RESULTS: Two sample types were used to compare the methods: (i) individual samples, taken from four sites on each carcass and (ii) composite samples, created by pooling the samples from four sites from an additional set of carcasses. When the polyurethane sponge and excision method were compared for individual sites, there were no significant differences in bacterial recovery on beef and pork carcasses and on two of four sites on lamb carcasses. However, when samples from each site were pooled, the excision method was more efficient than either swabbing method across the three animal species. CONCLUSIONS: Sampling using the polyurethane sponge represents an equivalent alternative method to excision for the bacteriological sampling of carcass surfaces which is nondestructive and less labour intensive. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides the scientific basis for using sponge swabbing instead of excision in compliance with 2001/471/EC.     

Patterns in the immunoenzyme analysis of bacterial cells

Some regularities of the enzyme immunoassay (EIA) of whole bacterial cells have been studied on one of the bacillary species of contaminant microflora. Early detection of this microorganism is highly important for the microbiological production of alpha-amylase and alkaline protease (produced by Bacillus subtilis). The effective kinetic and equilibrant parameters of the interaction of peroxidase-labeled antibodies with the cells of the contaminant microflora in the solution and on the surface of the polystyrene plates have been defined. Two methods for the separation of cells after their interaction with peroxidase-labeled antibodies have been optimized: filtration involving the use of special filter plates and centrifugation in plates. The method for the immobilization of cells in the wells of standard assay plates by centrifugation has been proposed. Four EIA methods for measurement of contaminant microflora have been developed and optimized. These methods permit the determination of the microflora at concentration of 5 X 10(5)-5 X 10(4) cells/ml, depending on the scheme of the assay, within 1-3.5 minutes.     

Modular estuarine mesocosm validation: ecotoxicological assessment of direct effects with the model compound endosulfan

This study represents the first in a series of validation experiments for the modular estuarine mesocosm testing direct pesticide effects. Endosulfan, an agricultural insecticide, was selected as a model contaminant for studying direct toxic effects as well as uptake of this model contaminant by estuarine biota. The grass shrimp, Palaemonetes pugio, and the mummichog, Fundulus heteroclitus, showed significant mortality (96 h LC₅₀: 0.12 and 2.2 μg/l, respectively) to endosulfan as predicted by laboratory bioassays. There was no effect on fiddler crabs and eastern oysters. The mesocosm was also useful in demonstrating the bioconcentration of endosulfan by eastern oysters (bioconcentration factor [BCF]=375) similar to results reported in field studies. This study illustrates the modular estuarine mesocosms' ability to detect direct effects of pesticide exposure and the uptake of a pesticide by estuarine fauna.     

Physico-chemical and biological dynamics of a sewage-polluted limestone aquifer

The spatio-temporal pattern of groundwater contamination at a 500 m² fractured limestone site polluted with sewage effluent was investigated over two years. In the context of a previously defined conceptual groundwater flow model, a multidisciplinary approach involving the collection of physico-chemical, bacteriological and biological (macro- and meiofauna) data was developed. Results showed that much of the sewage-polluted water infiltrated rapidly through some large subvertical fractures at the site and circulated with little or no contaminant attenuation through the solution-enlarged parts of a bedding joint. In such preferential contaminant paths, groundwater had low physico-chemical and bacteriological “stability”, highest average contaminant concentrations, and a high epigean organism component. Observation of the spatial distribution of organisms a short period after flooding also suggested that contaminants circulating through these conductive fractures entered the adjacent small-sized fissure matrix of the saturated zone. In this small aperture region of the site that was recharged by slow infiltrations of sewage-polluted surface water, groundwater had a greater physico-chemical and bacteriological “stability”, lowest average contaminant concentrations, and a high hypogean organism component. This lower sensitivity to surface pollution was partly due to attenuation processes such as retention of fecal indicator bacteria or nitrification of effluent ammonium which occurred in slow-moving water parts of the unsaturated zone.     

Evaluation of six sampling methods for recovery of bacteria from beef carcass surfaces

W.J. DORSA, C.N. CUTTER AND G.R. SIRAGUSA. 1996. Six bacterial sampling methods that might be used for rapid sampling of beef carcasses were evaluated in two separate studies. In Study 1, bacterial recovery from uninoculated beef rounds was 2.6, 2.3, 2.1 and 1.3 log₁₀ cfu cm^-2, respectively for excision (EX), and swabbing with cheesecloth (CC), sponge (SP) and cotton-tipped wooden swabs (CS). For Study 2, beef tissue was inoculated with bovine faeces at different levels and the mean recovery was 3.7, 3.0, 3.1 and 3.1 log₁₀ cfu cm^-2, respectively for EX, and swabbing with SP, griddle screen (GS) and 3M mesh (M). For both studies EX was determined to be the most consistently effective method while the initial study determined swabbing with CS was the least effective of the methods used. In both studies the most abrasive materials approached the effectiveness of EX even at low inoculation levels. As the inoculation levels increased, the additional effect of abrasiveness was lessened. When the carcasses were contaminated with bovine faeces, the bacterial populations that were rapidly recoverable from beef tissue using SP, GS or M were not significantly lower than those recovered using EX. Consequently SP, GS or M are an adequate method of beef carcass sampling for rapid, in-plant process monitoring to detect faecal contamination.     

Development of a Distributed Watershed Contaminant Transport,Transformation, and Fate (CTT&F) Sub-model

CTT&F is a physically based, spatially distributed watershed contaminant transport, transformation, and fate sub-model designed for use within existing hydrological modeling systems. To describe the fate of contaminants through landscape media as well as spatial variations of contaminant distributions, physical transport and transformation processes in CTT&F are simulated for each cell in the model and routed to the watershed outlet. CTT&F simulates contaminant erosion from soil and transport across the land surface by overland flow. The model also simulates contaminant erosion from stream bed sediment and transport through channels in addition to transport of contaminants inputs by overland flow. CTT&F can simulate solid (granular) contaminant transport and transformation, including partitioning between freely dissolved, dissolved organic carbon (DOC) bound, and particle-sorbed phases. To demonstrate model capabilities, CTT&F was coupled with an existing distributed hydrologic model and was tested and validated to simulate RDX and TNT transport using two experimental plots. These experiments examined dissolution of solid contaminants into the dissolved phase and their subsequent transport to the plot outlet. Model results were in close agreement with measured data. Such a model provides information for decision makers to make rational decisions relevant to the fate of toxic compounds.     

Effectiveness and mechanisms of dye adsorption on a straw-based biochar

A biochar (BC) generated from straw as a cost-effective substitute for activated carbon (AC) was tested for its adsorptive ability toward reactive brilliant blue (KNR) and rhodamine B (RB). BC and AC had similar surface areas but differed in porosity, surface acidity and point of zero surface charge. The two carbons were highly effective adsorbents for both dyes at pH 3.0 and 6.5. BC was slightly more effective than AC to adsorb RB due to the RB–BC electrostatic interactions and RB protonation at low pH. The two carbons reversed in their effectiveness to adsorb KNR for similar reasons. The π–π interactions between dye molecules and graphene layers of BC, the direct dye-BC electrostatic attraction/repulsion and the intermolecular hydrogen bonding are proposed to be the combined mechanisms for dye adsorption. Rich phenolic hydroxyls on the surface of BC are expected to enhance the π–π interactions.     

Buccal swabbing as a source of DNA from squamate reptiles

Buccal swabbing was compared with other tissues as a source of DNA for microsatellite genotyping from two squamate reptiles. For both species, the lizard Lacerta agilis and the snake Coronella austriaca, buccal swabbing proved more reliable than tissues including tail tips, toe clips and ventral scale clips.     

A covert contaminant of cultured plant cells: elimination of a Hyphomicrobium sp. from cultures of Datura innoxia (Mill.)

We have discovered a bacterial contaminant in some cell cultures of Datura innoxia (Mill.). The bacterium was tentatively identified as a species of Hyphomicrobium on the basis of its morphology and life cycle, and was isolated and grown in pure culture on a defined medium. The contaminant was not macroscopically observable in plant cell cultures. It caused neither a reduction of plant cell growth nor a noticeable increase in culture turbidity. Furthermore, it was not readily detectable by many standard assays for culture contamination: it would not grow alone in plant culture medium or yeast extract potato dextrose medium, and grew only very slowly on nutrient agar or beef-peptone medium. Repeated treatments with a combination of streptomycin (100 g/ml) and carbenicillin (100 g/ml) eliminated the contaminant from D. innoxia cell cultures without harming the plant cells.