Microbiological Testing Methods Research Articles

Production of Anchovy and Mussel Pastes as Appetizer

Anchovy (Engraulis encrasicholus) is a species which mostly caught from Black sea by seine catch. This species represents the majority of the marine species consumed in Turkey. Mussel (Mytilus galloprovincialis) is a bivalve that has long been used as a marine food by boiling and stuffing with rice. It is harvested from Mediterranean, Aegean and Marmara sea and is mainly consumed in coastal towns. In this study, anchovy and mussel were used for raw materials. Subsequent to filleting the anchovy and extracting the meat of mussel, these two species were boiled in salty water and grinded. Ultimately, they were introduced separately to the mix of tomato paste, red pepper paste, swatted garlic, cumin, fenugreek powder, ground pepper, salt and olive oil. On account of its consistence, this developed product can be conserved in a paste tube as well as it can be bottled in a glass jar to preserve. The paste is suitable specially for use as appetizer in every repasts and can also be used as consistent intensifier and flavouring supplement during the cooking process. Aside from the production, the shelf life of the product was appointed by routine microbiological test methods (total aerobic mesophilic, psychrotrophic, coliform, Enterobactericeae, Micrococcus, Staphylococcus bacteria, yeast and moulds counts) and also the species of microorganisms found in pastes were identified. This is the first report about anchovy and mussel pastes describing microbiological flora.

Validation of qualitative microbiological test methods.

This paper considers a statistical model for the detection mechanism of qualitative microbiological test methods with a parameter for the detection proportion (the probability to detect a single organism) and a parameter for the false positive rate. It is demonstrated that the detection proportion and the bacterial density cannot be estimated separately, not even in a multiple dilution experiment. Only the product can be estimated, changing the interpretation of the most probable number estimator. The asymptotic power of the likelihood ratio statistic for comparing an alternative method with the compendial method, is optimal for a single dilution experiment. The bacterial density should either be close to two CFUs per test unit or equal to zero, depending on differences in the model parameters between the two test methods. The proposed strategy for method validation is to use these two dilutions and test for differences in the two model parameters, addressing the validation parameters specificity and accuracy. Robustness of these two parameters might still be required, but all other validation parameters can be omitted. A confidence interval-based approach for the ratio of the detection proportions for the two methods is recommended, since it is most informative and close to the power of the likelihood ratio test.

An improved method for microbiological testing of paper-based laminates used in food packaging

Food packaging materials fundamentally contribute to food quality and safety, as they protect the packaged food against external influences. In this context, the determination of the hygiene status of the packaging material is of great importance. However, European legislation neither sets any microbiological criteria nor provides any approved standard for the microbiological testing of food packaging materials. Nevertheless, reliable routine control is essential for guaranteeing high hygienic quality of packagings.With the aim to achieve a maximum recovery rate at low contamination levels, an improved experimental design was developed for the enumeration of the total colony count, yeasts and molds and Enterobacteriaceae on the surface of roll stock packaging materials. For this purpose, two different types of paper laminates were selected and exemplarily used as objects of investigation. Moreover, the performance of different growth media was compared for each microbiological parameter. This approach was followed by method validation using a selection of quantitative reference materials of representative microorganisms, including resistant forms of microbes such as bacterial endospores and fungal spores.

Verification of pharmaceutical power of generic meropenem vs innovator trough assessment by microbial potency test

This study presents the development of a microbiological test method with different microorganisms and different concentrations of antibiotics through microbial potency, which established the dose-response concentrations and test conditions. The assay method was based on the plate cylinder with the result that there is equivalence between the pharmaceuticals antibiotic such as generic meropenem and innovator for the microorganisms such as Bacillus subtilli ATCC6633, Staphylococcus aureus ATCC 29737, Pseudomonas aeruginosa ATCC 25619 and Escherichia coli ATCC 10536, and pharmaceutical equivalence does not exist between the generic antibiotic meropenem and innovative meronem for Microococcus luteus ATCC 9341. Key words: Microbial power, cylindrical methodin plate, antibiotics, pharmaceutical equivalence, Bacillus subtillis ATCC 6633, Staphylococcus aureus ATCC 29737, Pseudomonas aeruginosa ATCC 25619, Escherichia coli ATCC 10536, Microococcus luteus ATCC 9341.

Gluten Detection and Speciation by Liquid Chromatography Mass Spectrometry (LC-MS/MS).

Liquid chromatography tandem mass spectrometry (LC-MS/MS) has been used historically in proteomics research for over 20 years. However, until recently LC-MS/MS has only been routinely used in food testing for small molecule contaminant detection, for example pesticide and veterinary residue detection, and not as a replacement of microbiological food testing methods, specifically allergen analysis. Over the last couple of years, articles have started to be published which describe the detection of allergens by LC-MS/MS. In this article we will describe how LC-MS/MS can be applied in the area of gluten detection and how it can be used to specifically differentiate the species of gluten used in food, where specific markers for each variety of gluten can be simultaneously acquired and detected at the same time. The article will discuss the effect of variety on the peptide response observed from different wheat grain varieties and will describe the sample preparation protocol which is essential for generating the peptide markers used for speciation.

Flow cytometry: is this a novelty in microbiological diagnostics?

Direct detection of pathogens is time- and labor-intensive. There is an increasing demand for new rapid microbiological testing methods, which would be faster and more sensitive than the conventional ones. Initally, automated methods were applied for the testing of bacteremia, urinary tract infections, characterization of antimicrobial susceptibility and quantitation of pathogen specific antibodies. Recently the nucleic acid-based detection methods have also become a routine. The molecular biological methods accelerate diagnosis, enhance specificity and provide an opportunity to identify pathogens with potential difficulties in culturing. However, they do not give any information about the immune status of the host. Yet it should also be borne in mind that detection of pathogen-specific nucleic acids is not equivalent to the presence of living microbes. The greatest advantage of FACS against these techniques is the capability to identify individual microbial cells as well. High speed FACS becomes a priority in the characterization of slow-growing microbes and identification of pathogens in mixed infections. Last but not least, it allows the monitoring of immune status and follow up of antimicrobial therapy. Orv. Hetil., 2013, 154, 1207–1218.

Quantitative relationship between biofilms components and emitter clogging under reclaimed water drip irrigation

Emitter clogging is a bottleneck to restrain the application and popularization of reclaimed water drip irrigation technology, and it is tightly related to the formation of biofilms attached on drip irrigation emitters. However, there is still no quantitative description about the relationship between biofilms components and emitter clogging. Based on the reclaimed water drip irrigation experiment, the characteristics of biofilms attached on 7 kinds of emitters under reclaimed water drip irrigation have been systematically explored with phospholipid fatty acids (PLFAs) and other microbiological testing methods, and quantitative relationship between biofilms components and emitter clogging was analyzed. The results showed that emitter clogging degree and biofilms components revealed an increasing trend with the drip irrigation system running, which could be regarded as the “quick-slow-quick-slow” increasing trend. Biofilms exhibited a rapid growth after restarting in the 2nd year. The dry weights (DWs), PLFAs, and extracellular polymers (EPS) of biofilms attached on the emitters could reach up to 0.133 g, 100.83 μg, and 1,645.04 μg, respectively, after accumulatively ran for 540 h. DWs of biofilms in different positions such as head, middle, and end parts of the lateral also revealed a increasing trend. Meanwhile, biofilms in the pressure-compensating emitters grew less than that in the non-pressure-compensating emitters. During the initial 540 h of the system running, DWs, PLFAs, and EPS of attached biofilms had a obvious negative linear correlation (R2 > 0.85) with emitter discharge ratio variation (Dra) and coefficient of uniformity (CU), which could describe the drip irrigation emitter clogging mechanism well. Furthermore, PLFAs reflected the type and quantity of microorganisms in attached biofilms, and sticky EPS secreted by microorganisms could link the microorganisms with absorbed particulates together and formed the stable biofilm structure. As PLFAs reflected the comprehensive effect of microorganisms, particulates and EPS on emitter clogging, and presented the highest negative linear correlation with Dra (R2 = 0.93), it is most appropriate to reflect emitter clogging mechanism. These results can provide a theoretical basis for exploring emitter clogging mechanism and promoting the application of reclaimed water drip irrigation technology.

Effect of ultra high-pressure processing on microorganisms and ginsenosides of Panax ginseng

To study the feasibility of the application of ultra high-pressure processing (UHPP) as an anticorrosion and anti-mould method by comparing the total numbers of bacteria and mould colonies and the content of ginsenosides before and after UHPP. The total numbers of bacteria and moulds colony were determined by microbiological test method. The contents of 12 ginsenosides were determined by HPLC. Under the three selected conditions, the total number of bacterial colony decreased significantly, while the mould was not detected in UHPP samples; and the contents of 12 ginsenosides were increased significantly in methanol extracts and water extracts. UHPP not only shows anticorrosion and anti-mould effects, but also enhances the leaching rate of ginsenosides. It is a highly effective, safe and environmental friendly anticorrosion and anti-mould technique for Ranax ginseng worth in-depth study.

Validation of the Microbiological Testing of Tissue Preparations Using the BACTECTM Blood Culture System

Background: Since blood culture bottles are validated by the manufacturer for blood only, an additional validation for the use with fluids of tissue preparations is necessary. Methods: Two 10-ml samples of cornea culture medium, histidine-tryptophan-ketoglutarate (HTK) solution, or Ringer solution at the end of femur head thermo-disinfection were given into blood culture bottles (BD BACTEC^TM Plus Aerobic/F, Anaerobic/F for cornea culture medium and BD BACTEC^TM Standard Aerobic/ Anaerobic for HTK and Ringer solution) and subsequently spiked with 10–100 colony forming units (CFU) of bacteria or fungi (aerobic bacteria: Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa; anaerobic bacteria: Clostridium sporogenes; fungi: Candida albicans, Aspergillus brasiliensis) according to the European Pharmacopoeia Chapter 2.6.1. Results: All tested bacteria and fungi could be detected in all solutions. All positive and negative controls were tested correctly. Compared to the positive controls, the microbial growth was delayed in the antibiotic-containing cornea culture medium, and negative in two cases of B. subtilis spiking. Conclusion: The use of BACTEC^TM blood culture bottles seems to be a suitable method for microbiological testing of HTK solution, Ringer solution, and, with limitations, also for testing of the antibiotic-containing cornea culture medium.

A comparative in vitro microbiological evaluation of generic meropenem compounds against the innovator compound

Background: Meropenem is a broad-spectrum carbapenem widely used in the treatment of critically ill patients. A generic meropenem product has recently become available in South Africa and we aimed to compare the generic product with the innovator product using established in vitro microbiological testing methods. Method: Comparative minimum inhibitory concentrations (MICs) were determined for 115 clinically relevant isolates using the broth microdilution reference method. Comparative analysis of MIC was done using categorical and essential agreement. A subset of isolates was evaluated using minimum bactericidal concentration (MBC) testing. Results: The overall essential agreement exceeded the international standard of > 90%. A single major error and six minor errors were detected in 230 comparative MICs. For the 55 Enterobacteriaceae isolates tested, the MIC 50 and MIC 90 were 0.03 µg/ml and 0.06 µg/ml respectively, with no difference between extended–spectrum s-lactamase producers (ESBL) and non-ESBL isolates. Bactericidal activity was demonstrated for both generic and innovator products in all isolates tested. For eight of the 11 isolates, the MBC was only twice the MIC. Conclusion: Reference method MIC and MBC testing of a large sample of clinically relevant microorganisms against meropenem has demonstrated comparable in vitro activity between the innovator and generic products. Low MICs and bactericidal activity at concentrations close to the MIC indicate that meropenem remains a useful agent in the treatment of infections caused by ESBLproducing Enterobacteriaceae.

Antimicrobial testing for surface‐immobilized agents with a surface‐separated live–dead staining method

Modification of a traditional live-dead staining technique based on fluorescence microscopy has yielded an improved method capable of differentiating surface-immobilized antimicrobial agents from those agents acting via solution diffusion processes. By utilizing an inoculation chamber comprised of 50 µm polystyrene spheres as spacers between test substrate and coverslip control surfaces, three distinct bacterial cell populations can be probed by fluorescence microscopy for antimicrobial activity: (1) cells adhered to the coverslip, (2) cells adhered to the substrate, and (3) mobile cells in solution. Truly immobilized antimicrobial agents were found efficacious only at the substrate surface, while elutable agents were effective against all three populations. Glass surfaces derivatized with either quaternized poly dimethylaminoethylmethacrylate (pDMAEMA) or 3-(trimethoxysilyl) propyldimethyloctadecyl ammonium chloride (Si-QAC) were compared with bare glass control surfaces after contact and 4 h incubation with Staphylococcus aureus. pDMAEMA surfaces were both antimicrobial and immobilized, whereas the Si-QAC surfaces were only observed to be antimicrobial via active diffusion. In contrast to conventional thinking, Si-QAC surfaces showed no kill after removing all Si-QAC elutables via rinsing procedures. The semi-quantitative surface-separated live-dead staining (SSLDS) technique provides mechanistic insight and represents a significant improvement relative to current microbiological test methods for evaluating immobilized, antimicrobial agents.

Optical method for monitoring of photodynamic inactivation of bacteria

Photodynamic inactivation is a new promising approach to treat bacterial infections. Usually, the evaluation of the efficacy of this method is done through time-consuming and labor-intensive microbiological test methods. This paper describes the development and implementation of an optical method to evaluate the photodynamic inactivation of bacteria based on non-invasive diffuse reflectance measurements. Five Staphylococcus aureus cultures and 15 mice have been used in this study. A skin lesion was created on the back of all animals, and it was contaminated with S. aureus (5.16 ± 0.013 log CFU/ml). Toluidine Blue O (c = 8.67 × 10 (- 3) M) has been used as a photosensitiser agent. The bacterial cultures and animals were exposed to laser radiation (λ = 635nm, P = 15mW, DE = 8.654J/cm(2)) for 20min. The photodynamic inactivation of bacteria was monitored by acquiring the wounds' reflection spectra at different time points and by microbiological exams on the bioptical material. The good correlation between the diffuse reflectance and colony-forming units demonstrates the value of this optical method based on diffuse reflectance measurements as a rapid technique to monitor photodynamic bacterial inactivation.

Etiology of Community‐Acquired Pneumonia: Increased Microbiological Yield with New Diagnostic Methods

BackgroundThe microbial etiology of community-acquired pneumonia (CAP) is still not well characterized. During the past few years, polymerase chain reaction (PCR)-based methods have been developed for many pathogens causing respiratory tract infections. The aim of this study was to determine the etiology of CAP among adults—especially the occurrence of mixed infections among patients with CAP—by implementing a new diagnostic PCR platform combined with conventional methods.MethodsAdults admitted to Karolinska University Hospital were studied prospectively during a 12-month period. Microbiological testing methods included culture from blood, sputum, and nasopharyngeal secretion samples; sputum samples analyzed by real-time quantitative PCR for Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis; nasopharyngeal specimens analyzed by use of PCR; serological testing for Mycoplasma pneumoniae, Chlamydophila pneumoniae, and viruses common in the respiratory tract; and urine antigen assays for detection of pneumococcal and Legionella pneumophila antigens.ResultsA microbial etiology could be identified for 67% of the patients (n = 124). For patients with complete sampling, a microbiological agent was identified for 89% of the cases. The most frequently detected pathogens were S. pneumoniae (70 patients [38]) and respiratory virus (53 patients [29]). Two or more pathogens were present in 43 (35%) of 124 cases with a determined etiology.ConclusionsBy supplementing traditional diagnostic methods with new PCR-based methods, a high microbial yield was achieved. This was especially evident for patients with complete sampling. Mixed infections were frequent (most commonly S. pneumoniae together with a respiratory virus).

The Utility of Rapid Microbiological and Molecular Techniques in Optimizing Antimicrobial Therapy

Early treatment of bloodstream infections with appropriate, definitive antimicrobial therapy has proven to reduce mortality, length of hospital stay, and healthcare costs. Culture-based testing methods require up to five days for final pathogen identification and susceptibility reporting, forcing use of broad spectrum empiric therapy. Recently, multiple rapid microbiological and molecular testing methods have been developed that reduce the time to identify the pathogen and susceptibility, allowing optimal antimicrobial therapy to be prescribed earlier. Real-time polymerase chain reaction and gene microarray have been described in literature, yet only peptide nucleic acid fluorescent in-situ hybridization has published data justifying its use based on clinical outcomes and cost savings. Target enriched multiplex polymerase chain reaction was developed to identify both the pathogen and multiple genes associated with resistance from blood within 6 hours and this methodology was studied in our hospital to assess effectiveness at optimizing antimicrobials in staphylococcal bloodstream infections.

Rapid bioanalysis with chemical sensors: novel strategies for devices and artificial recognition membranes

The increasing importance of biological analytes in chemistry has triggered the development of a vast number of techniques for rapidly assessing them. Aside from microbiological test methods, a wide range of analytical sensor and detection methods are being developed. Within this article, we review the literature on this topic from the last five years, stressing two main aspects of method development. The first aspect is the design of novel analytical strategies and transducers to generate signals more sensitively, more rapidly and more efficiently. Most of the progress in this field has focused on electrochemical detection, although novel approaches to optical and mass-sensitive measurements have been reported. Second, we provide an overview of two main approaches to creating artificial interaction layers for sensors based on tailored interaction sites in polymeric or biomimetic systems. The most prominent of these approaches is (molecular) imprinting, where selectivity is achieved by directly templating a polymer material with the target analyte or a model compound, thus achieving biomimetic interaction sites within both thin films and particles.

Metalworking Fluid Microbes—What We Need to Know to Successfully Understand Cause-and-Effect Relationships

During the past decade we have witnessed a tumultuous debate over the disease risks posed by microbes that inhabit metalworking fluid (MWF) systems. Not infrequently, that debate has occurred in the absence of satisfactory data. This paper addresses the author's perspective on what types of data are needed in order to assess the actual disease risks posed by MWF microbes. The approach must be multidisciplinary and coordinated, including stakeholders with expertise in epidemiology, fluid management, immunology, industrial hygiene, microbiology, and public health medicine. Traditional microbiological sampling and test methods must be augmented by new, consensus methods that are adopted by industry stakeholders. Entities performing these tests should be participating in interlaboratory cross-check programs. The author will use Mycobacterium immunogenum to illustrate the general model for this strategy.

Obstacles to Developing a Multinational Report Card on Antimicrobial Resistance for Canada: An Evidence-Based Review

Many countries want to compare the results of their antimicrobial resistance programs to those of others nations to help gauge the effectiveness of their prevention and control practices. In our attempt to compare Canada with other nations, we encountered several challenges that must be addressed before meaningful multinational comparisons can be made. The fundamental barriers to comparison were the lack of shared targets for performance and predictive measures of success. Unique problems and policies within countries resulted in variations in goals, methods, pathogens, drugs, and priorities within and between jurisdictions. Other obstacles included: (1) lack of information on potential biases associated with different microbiological testing and sampling methods; (2) lack of information with which to conclude whether or not different programs examined comparable spectra of patients or outcomes; (3) inadequate description of the epidemiological rationale for sampling strategies; (4) use of aggregated national data that can hide regional or local variations; (5) rarity of studies designed explicitly for multinational comparison; and (6) lack of international agreement on methods, continuing education, and quality control needed to ensure program comparability. Comparison based on a country's ability to meet its internal goals for antimicrobial resistance control may be a more informative basis for a report card than specific resistance or drug use rates.

Incidence and characteristics of viral community-acquired pneumonia in adults

Background:In adults, viral causes of community-acquired pneumonia (CAP) are poorly characterised. The aims of this study were to characterise the viral aetiology of CAP in adults by using an extensive...

A Microbiological Test Method to Determine the Cleanability of Filter Media in Solid‐Liquid‐Separation Applications

AbstractOne of the major properties in the design and development of filter media for the food and drug industry is their cleanability. They should be designed according to hygienic criteria thereby decreasing residuals and reducing cleaning time and costs. A reproducible test method is necessary to determine or validate the degree of cleanability of filter media and to indicate areas of poor hygienic design. This work focused on the development of such a filter test and provides information about impurity, whether it occurs or not, and its local resolution. The growth of a microorganism, which is introduced as impurity, is used for the detection of residues which grow to the surface of the sample and offer a 2‐dimensional analysis. With this approach, different kinds of filter media can be compared with each other regarding their cleanability.

Redox potential measurement as a rapid method for microbiological testing and its validation for coliform determination

The redox potential is one of the most complex indicators of the physiological state of microbial cultures and its measurement could be a useful tool for the qualitative and quantitative determination of the microbial contamination. During the bacterial growth, the redox potential of the medium decreases. The shape of the redox potential curve is characteristic on the type of microorganism, and the rate of the change (d E/d t) is proportional to the living cell concentration. Defining the time required to reach a significant change in redox potential as Time to Detection (TTD), similarly to the impedimetric measurements, a strict linear correlation could be established between the TTD and the logarithm of the initial concentration of microorganisms. On the base of this calibration curve, the determination of living cell concentration could be simplified. For the experiments, a computer-controlled multi-channel measuring system and software was developed by the authors. The redox potential measurement method was tested and validated for the determination of coliform bacteria. The results have proved the high efficiency and reliability of the new method.