Enumeration of micro-organisms in processed soy products with an automated most probable number method compared with standard plate method

Aim: The automated TEMPO system (bioMerieux) is based on the most probable number (MPN) method for the enumeration of micro‐organisms in foods. In this study, we evaluated the performance of the TEMPO system as a diagnostic tool in comparison with the standard method in processed soy products. Methods and Results: A verification study was conducted using artificially contaminated soy product samples such as soy protein isolate, water‐soluble soy polysaccharides, soy milk and processed soy food. Five types of micro‐organisms were analysed using the automated MPN method (total aerobic bacteria, total coliforms, Enterobacteriaceae, yeast and mould and Staphylococcus aureus) vs the standard plate method. The results from each of the methods were highly correlated (r > 0·95). Naturally contaminated processed soy products on the market were also studied. There were no discrepancies observed between the respective methods. Conclusions: TEMPO methods were equivalent to the corresponding standard plate methods with very good rates of agreement. Significance and Impact of the Study: The automated MPN method is more practical and reliable for in‐house microbiological testing in processed soy products.     

Antimicrobial and antiadhesive properties of a biosurfactant isolated from Lactobacillus paracasei ssp. paracasei A20

Aims: The aim of this study was to determine the antimicrobial and antiadhesive properties of a biosurfactant isolated from Lactobacillus paracasei ssp. paracasei A20 against several micro‐organisms, including Gram‐positive and Gram‐negative bacteria, yeasts and filamentous fungi. Methods and Results: Antimicrobial and antiadhesive activities were determined using the microdilution method in 96‐well culture plates. The biosurfactant showed antimicrobial activity against all the micro‐organisms assayed, and for twelve of the eighteen micro‐organisms (including the pathogenic Candida albicans, Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis and Streptococcus agalactiae), the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) were achieved for biosurfactant concentrations between 25 and 50 mg ml^?1. Furthermore, the biosurfactant showed antiadhesive activity against most of the micro‐organisms evaluated. Conclusions: As far as we know, this is the first compilation of data on antimicrobial and antiadhesive activities of biosurfactants obtained from lactobacilli against such a broad group of micro‐organisms. Although the antiadhesive activity of biosurfactants isolated from lactic acid bacteria has been widely reported, their antimicrobial activity is quite unusual and has been described only in a few strains. Significance and Impact of the Study: The results obtained in this study regarding the antimicrobial and antiadhesive properties of this biosurfactant opens future prospects for its use against micro‐organisms responsible for diseases and infections in the urinary, vaginal and gastrointestinal tracts, as well as in the skin, making it a suitable alternative to conventional antibiotics.     

Determination of microbial diversity in meju,fermented cooked soya beans,using nested PCR‐denaturing gradient gel electrophoresis

Aims: To identify the microbiota in meju, fermented cooked soya beans, that may directly affect the microbial communities of Korean fermented soya bean foods. Methods and Results: Using conventional bacterial 16S rDNA, bacilli‐specific 16S rDNA or fungi 18S rDNA‐specific primers, PCR products were amplified through a series of PCRs using the DNA extracted from ten meju samples. The amplicons were analysed using denaturing gradient gel electrophoresis (DGGE), which showed that Enterococcus durans was commonly detected in nine of ten meju samples. Bacillus subtilis was shown to be the major strain of bacilli in the samples tested. Based on the DGGE analysis of fungi in meju, we determined that Absidia corymbifera, Aspergillus sp. and Candida rugosa were the main fungi in the tested samples. Conclusions: A variety of bacterial and fungal micro‐organisms were identified in meju samples, in addition to the micro‐organisms already known to be present. Significance and Impact of the Study: This is the first report showing the differences and similarities in the populations of micro‐organisms in meju samples using nested PCR‐DGGE, a culture‐independent method. The results may be applicable to the development of improved meju, in which the indigenous micro‐organisms required for fermentation can be standardized.     

Development and evaluation of a new device to effectively detach micro-organisms from food samples

Aims: To develop a new instrument of great versatility for recovering micro‐organisms from all types of food samples and to compare the effects with existing sample preparation methods. Methods and Results: To detach micro‐organisms from large‐size unbroken food samples such as apples, carrots, potatoes and tomatoes without preprocessing, the Spindle apparatus was newly developed. The Spindle was used to effectively detach micro‐organisms from large‐size samples. In a comparative study involving 51 food samples, treatment with the Spindle and Stomacher showed that recovery of total aerobic micro‐organisms (naturally occurring mesophilic microflora) and foodborne pathogens (from samples inoculated with Escherichia coli O157:H7, Salmonella Typhimurium and Listeria monocytogenes) for both methods was highly correlated (R² = 0·98). Furthermore, diluents treated by the Spindle contained much less food debris than those treated by stomaching. Conclusions: These results indicate that Spindle is a novel, effective alternative method for detaching micro‐organisms from food samples including four kinds of large‐size samples without the need for preprocessing. Significance and Impact of Study: The Spindle might be used to widely detaching micro‐organisms from all types of food samples for microbiological assay.     

Phylogenetic diversity of gene sequences isolated from the rumen as analysed using a self‐organizing map (SOM)

Aims: To determine the origins of DNA sequences isolated from the rumen microbial ecosystem using a self‐organizing map (SOM). Methods and Results: DNA sequences other than 16S small subunit ribosomal RNA (SSU rRNA) gene sequences that were detected from the rumen were analysed by the SOM method reported by Abe et al. [2000, Self‐Organizing Map (SOM) unveils and visualizes hidden sequence characteristics of a wide range of eukaryote genomes. Gene 365, 27–34]. Because query sequences positioned by SOM were scattered on the master drawing of SOM, it was suggested that many DNA sequences isolated from the rumen were collected from a broad range of micro‐organisms. Although the results obtained by SOM were similar to those obtained by the neighbour‐joining (NJ) method, SOM was able to presume the phylotypes of the query sequences without information about the 16S SSU rRNA gene sequences and homology searches, and to reveal existence of novel micro‐organisms deduced to be cellulolytic bacteria, archaea and methanotrophic bacterium. Conclusions: As the SOM method defined phylotypes of unreported rumen micro‐organisms, it is presumed that these phylotypes would be involved in rumen fermentation in cooperation with known rumen micro‐organisms. Moreover, it is demonstrated that SOM is a useful tool for affiliating DNA sequences, which have no matches in databases. Significance and Impact of Study: Through SOM analysis, a better means of identifying rumen micro‐organisms and estimating their roles in rumen function was provided.     

Colonisation of soft lining materials by micro‐organisms

doi:10.1111/j.1741‐2358.2009.00300.x
Colonisation of soft lining materials by micro‐organisms Objective: This study evaluated the in vitro adherence of pathogenic micro‐organisms, Candida albicans, Staphylococcus aureus and Pseudomonas aeruginosa, to soft lining materials and their inhibitory effect on these micro‐organisms. Materials and Methods: To measure adherence, specimens of Molloplast B and Ufi Gel P were inoculated [10⁷ colony‐forming units per millimetre (cfu/ml)] with TSB media containing the micro‐organisms. To determine the number of micro‐organisms in the 10^?2–10^?5 dilutions, 25 μl of the suspension were transferred to plates of selective media. Colony counts of each specimen were quantified (cfu/ml). The surface roughness was measured with a perfilometer to assess the relationship between the adherence of micro‐organisms and surface roughness of each material. For the inhibition test, specimens of materials were placed in agar plates inoculated individually with the micro‐organisms. After 48 h, the inhibition zones around the specimens were measured. Results: None of the materials exhibited inhibition zones. The number of cfu/ml of S. aureus and P. aeruginosa were significantly greater than C. albicans for both materials. The Ufi Gel P exhibited greater adherence of C. albicans than Molloplast B. No correlation was observed between the adherence of micro‐organisms and surface roughness. Conclusion: The surface roughness of the materials is not the only factor governing micro‐organism adherence.     

An approach to integrated data assessment in a proficiency test on the enumeration of Escherichia coli

Aims: To develop appropriate statistical approaches to plan and evaluate proficiency tests for the enumeration of Escherichia coli, addressing, in particular, a possible but frequently unavoidable lack of test sample homogeneity. Methods and Results: Each of 50 laboratories analysed two samples of a stabilized suspension of E. coli in duplicate, using various media, inoculation methods, and incubation times and conditions. In parallel, the E. coli suspension was tested by the organiser for homogeneity and stability. Escherichia coli counts followed a log‐normal distribution. After eliminating, by Youden analysis, two data sets that were considered outliers and eight data sets for underperformance of the laboratories (substantial lack of repeatability), the standard deviation of the mean was about 0·06 log₁₀ units. There was no evidence of bimodality of the data. Lack of homogeneity of distribution of bacteria had a strong effect on measurement uncertainty, in addition to laboratory bias and method repeatability. The homogeneity decreases during storage of the individual test vials; this effect could be modelled by the known kinetics of inactivation of micro‐organisms. The results were confirmed by Monte Carlo simulations. Conclusions: By a tailored analysis of proficiency testing data, it is possible to distinguish the effect of lack of homogeneity, laboratory bias and method repeatability, on the measurement uncertainty. Significance and Impact of the Study: A statistic tool is provided to solve problems related to lack of stability of microbiological test material and to separate the effects of sample inhomogeneity from the performance of the individual laboratory.     

Concurrent Proteomic Fingerprinting and Molecular Analysis of Cyathostomins

Rapid, cost‐effective, efficient, and reliable helminth species identification is of considerable importance to understand host–parasite interactions, clinical disease, and drug resistance. Cyathostomins (Nematoda: Strongylidae) are considered to be the most important equine parasites, yet research on this group is hampered by the large number of 50 morphologically differentiated species, their occurrence in mixed infections with often more than 10 species and the difficulties associated with conventional identification methods. Here, MALDI‐TOF MS, previously successfully applied to identify numerous organisms, is evaluated and compared with conventional and molecular genetic approaches. A simple and robust protocol for protein extraction and subsequent DNA isolation allowing molecular confirmation of proteomic findings is developed, showing that MALDI‐TOF MS can discriminate adult stages of the two closely related cyathostomin species Cylicostephanus longibursatus and Cylicostephanus minutus. Intraspecific variability of proteomic profiles within morphospecies demonstrated an identification of morphospecies with an accuracy of close to 100%. In contrast, three genospecies within C. minutus and sex‐specific profiles within both morphospecies could not be reliably discriminated using MALDI‐TOF MS. In conclusion, MALDI‐TOF MS complemented by the molecular protocol is a reliable and efficient approach for cyathostomin species identification.     

Phenotypic persistence and external shielding ultraviolet radiation inactivation kinetic model

Aim: To develop an inactivation kinetic model to describe ultraviolet (UV) dose‐response behaviour for micro‐organisms that exhibit tailing using two commonly referenced causes for tailing: physical shielding of micro‐organisms and phenotypic persistence. Methods and Results: Dose‐response data for Escherichia coli, Mycobacterium terrae and Bacillus subtilis spores exposed to UV radiation were fit to the phenotypic persistence and external shielding (PPES) model. The fraction of persistent micro‐organisms in the original population (N_persistent/N_total) that exhibited reduced sensitivity to UV radiation was estimated by the PPES model as approx. 10^?7, 10^?5 and 10^?4 for E. coli, B. subtilis spores and Myco. terrae, respectively. Particle shielding effects were evaluated for Myco. terrae and resulted in additional reduction in UV sensitivity. Conclusions: Tailing occurred in laboratory experiments even when clumping and shielding were eliminated as major factors in UV resistance, suggesting that phenotypic persistence in addition to shielding may be important to consider when evaluating dose‐response curves for disinfection applications. Significance and Impact of the Study: The PPES model provides a mechanistically plausible tool for estimating the dose–response behaviour for micro‐organisms that exhibit tailing in dispersed and aggregated settings. Accurate dose‐response behaviour (including the tailing region) is critical to the analysis and validation of all UV disinfection systems.     

Development of a colorimetric colony‐screening assay for detection of defluorination by micro‐organisms

Aims: To develop a colorimetric colony‐screening assay to facilitate the isolation of micro‐organisms capable of defluorination. Methods and Results: A metal‐dye chelate, zirconium‐xylenol orange was used to detect fluoride ions released from a fluorinated substrate through microbial metabolism. Depolymerised zirconium reagent gave the greatest visual contrast for the presence of fluoride compared to more polymerised forms of zirconium reagent. The sensitivity of the assay was greatest when the molar ratio of depolymerised zirconium to xylenol orange was 1 : 2. Using depolymerised zirconium and xylenol orange (150 and 300 nmol l^?1 respectively), the assay could detect a fluoride application spot (5 mmol l^?1) containing 50 nmoles of fluoride ions. Most media constituents were well tolerated by the assay, although phosphate ions needed to be restricted to 0·1 g l^?1 and some proteins digest to between 1 and 5 g l^?1. A microbial enrichment culture growing on solidified medium containing 20 mmol l^?1 fluoroacetate was screened using the assay, and defluorinating bacteria belonging to the genus Burkholderia isolated. Conclusions: A method was developed that is sensitive, rapid and reliable for detecting defluorination by micro‐organisms growing on solidified medium. Significance and Impact of the Study: This method can be used to facilitate the isolation of micro‐organisms capable of defluorination.     

Screen of micro‐organisms for inducing the production of dragon’s blood by leaf of Dracaena cochinchinensis

Aims: To screen micro‐organisms for inducing the production of dragon’s blood, which is normally produced by stem xylem and by leaf of Dracaena cochinchinensis, and to evaluate the product by comparing with the standard. Methods and Results: Thirty microbial strains were isolated from D. cochinchinensis leaves. Three of them were confirmed to elicit the leaf of D. cochinchinensis producing dragon’s blood after inoculation. Upon elicitation, all of the 6‐month‐old leaves of the inducible trees produced dragon’s blood; 60–70% of the 1‐year‐old leaves elicited produced the resin. All the three strains were identified as Colletotrichum gloeosporioide by morphological and molecular methods. The leaf resin had a similar TLC profile and antioxidant activities to the standard resin. In particular, it had a higher total flavonol content and antimicrobial activity than the standard. Conclusions: Upon the induction of the screened C. gloeosporioide mycelia, D. cochinchinensis leaf produced dragon’s blood with higher total flavone content and antimicrobial activity than the standard dragon’s blood. Significance and Impact of the Study: This work has provided a strategy for producing dragon’s blood in a sustainable way using leaves of C. gloeosporioides by fungal elicitation.     

Evaluation of sample preparation methods for MALDI-TOF MS identification of highly dangerous bacteria

Aims: To propose a universal workflow of sample preparation method for the identification of highly pathogenic bacteria by MALDI‐TOF MS. Methods and Results: Fifteen bacterial species, including highly virulent Gram‐positive (Bacillus anthracis and Clostridium botulinum) and Gram‐negative bacteria (Brucella melitensis, Burkholderia mallei, Francisella tularensis, Shigella dysenteriae, Vibrio cholerae, Yersinia pestis and Legionella pneumophila), were employed in the comparative study of four sample preparation methods compatible with MALDI‐TOF MS. The yield of bacterial proteins was determined by spectrophotometry, and the quality of the mass spectra, recorded in linear mode in the range of 2000–20 000 Da, was evaluated with respect to the information content (number of signals) and quality (S/N ratio). Conclusions: Based on the values of protein concentration and spectral quality, the method using combination of ethanol treatment followed by extraction with formic acid and acetonitrile was the most efficient sample preparation method for the identification of highly pathogenic bacteria using MALDI‐TOF MS. Significance and Impact of the Study: The method using ethanol/formic acid generally shows the highest extraction efficacy and the spectral quality with no detrimental effect caused by storage. Thus, this can be considered as a universal sample preparation method for the identification of highly virulent micro‐organisms by MALDI‐TOF mass spectrometry.     

A new dynamic model for highly efficient mass transfer in aerated bioreactors and consequences for kLa identification

Gas–liquid mass transfer is often rate‐limiting in laboratory and industrial cultures of aerobic or autotrophic organisms. The volumetric mass transfer coefficient k_La is a crucial characteristic for comparing, optimizing, and upscaling mass transfer efficiency of bioreactors. Reliable dynamic models and resulting methods for parameter identification are needed for quantitative modeling of microbial growth dynamics. We describe a laboratory‐scale stirred tank reactor (STR) with a highly efficient aeration system (k_La ≈ 570 h^?1). The reactor can sustain yeast culture with high cell density and high oxygen uptake rate, leading to a significant drop in gas concentration from inflow to outflow (by 21%). Standard models fail to predict the observed mass transfer dynamics and to identify k_La correctly. In order to capture the concentration gradient in the gas phase, we refine a standard ordinary differential equation (ODE) model and obtain a system of partial integro‐differential equations (PIDE), for which we derive an approximate analytical solution. Specific reactor configurations, in particular a relatively short bubble residence time, allow a quasi steady‐state approximation of the PIDE system by a simpler ODE model which still accounts for the concentration gradient. Moreover, we perform an appropriate scaling of all variables and parameters. In particular, we introduce the dimensionless overall efficiency κ, which is more informative than k_La since it combines the effects of gas inflow, exchange, and solution. Current standard models of mass transfer in laboratory‐scale aerated STRs neglect the gradient in the gas concentration, which arises from highly efficient bubbling systems and high cellular exchange rates. The resulting error in the identification of κ (and hence k_La) increases dramatically with increasing mass transfer efficiency. Notably, the error differs between cell‐free and culture‐based methods of parameter identification, potentially confounding the determination of the “biological enhancement” of mass transfer. Our new model provides an improved theoretical framework that can be readily applied to aerated bioreactors in research and biotechnology. Biotechnol. Bioeng. 2012; 109: 2997–3006. © 2012 Wiley Periodicals, Inc.     

Enhanced germicidal effects of pulsed UV‐LED irradiation on biofilms

Aims: The major objective of the study was to evaluate the enhanced germicidal effects of low‐frequency pulsed ultraviolet A (UVA)‐light‐emitting diode (LED) on biofilms. Methods and Results: The germicidal effects of UVA‐LED irradiation (365 nm, 0·28 mW cm^?2, in pulsed or continuous mode) on Candida albicans or Escherichia coli biofilms were evaluated by determining colony‐forming units. The morphological change of microbial cells in biofilms was observed using scanning electron microscopy. After 5‐min irradiation, over 90% of viable micro‐organisms in biofilms had been killed, and pulsed irradiation (1–1000 Hz) had significantly greater germicidal ability than continuous irradiation. Pulsed irradiation (100 Hz, 60 min) almost completely killed micro‐organisms in biofilm (>99·9%), and 20‐min irradiation greatly damaged both microbial species. Interestingly, few hyphae were found in irradiated Candida biofilms. Moreover, mannitol treatment, a scavenger of hydroxyl radicals (OH^?), significantly protected viable micro‐organisms in biofilms from UVA‐LED irradiation. Conclusions: The study demonstrated that pulsed UVA‐LED irradiation has a strong germicidal effect (maximum at 100 Hz, over 5‐min irradiation) and causes the disappearance of hyphal forms of Candida. Significance and Impact of the Study: This study can assist in developing a low‐frequency pulsed UVA‐LED system to be applied to pathogenic biofilms for disinfection.     

Metalworking fluids biodiversity characterization

Aims: Hypersensitivity pneumonitis of machinists associated with metalworking fluids (MWF) was recently linked to Mycobacterium immunogenum. In addition to Mycobacterium, impacts of continuous and massive contact to other micro‐organisms, such as Pseudomonas, were little studied. This report intended to quantify and characterize the microbial load of 44 in‐use MWF. Methods and Results: The main biodiversity of MWF was assessed using cultural methods, quantitative PCR (qPCR) and denaturing gradient gel electrophoresis (DGGE). Total bacteria concentrations ranged from undetectable to 10⁹ 16S rRNA gene copies per millilitre. Concentrations obtained by qPCR were up to five orders of magnitude higher than by culture, suggesting that MWF contamination is generally underestimated. Two samples showed high concentrations of Myco. immunogenum (1·55 × 10⁷ and 3·49 × 10⁵ 16S rRNA gene copies per millilitre). The overall biodiversity was low, as observed by culture and DGGE, and was comparable to data found in the literature. Pseudomonas pseudoalcaligenes was by far the main bacteria found in MWF samples (33 out of 44), followed by Ochrobactrum anthropi (32 out of 44). There was no significant relationship between the biodiversity profiles and the kind of MWF or equipment used, making it difficult to predict which micro‐organisms will colonize each particular MWF. Conclusions: Very high concentrations of bacteria were found in most MWF studied and limited biodiversities were observed. Many species of micro‐organisms were retrieved from MWF samples, but they were mostly colonized by Pseudomonas pseudoalcaligenes and Ochrobactrum anthropi. Significance and Impact of the Study: The major micro‐organisms observed or recovered in this study from in‐use MWF were present in very high concentrations, and thus further studies are needed to confirm their role in workers’ respiratory disorders or health‐related problems.     

The microbiology of Bandji,palm wine of Borassus akeassii from Burkina Faso: identification and genotypic diversity of yeasts,lactic acid and acetic acid bacteria

Aim

To investigate physicochemical characteristics and especially genotypic diversity of the main culturable micro‐organisms involved in fermentation of sap from Borassus akeassii, a newly identified palm tree from West Africa.

Methods and Results

Physicochemical characterization was performed using conventional methods. Identification of micro‐organisms included phenotyping and sequencing of: 26S rRNA gene for yeasts, 16S rRNA and gyrB genes for lactic acid bacteria (LAB) and acetic acid bacteria (AAB). Interspecies and intraspecies genotypic diversities of the micro‐organisms were screened respectively by amplification of the ITS1‐5.8S rDNA‐ITS2/16S‐23S rDNA ITS regions and repetitive sequence‐based PCR (rep‐PCR). The physicochemical characteristics of samples were: pH: 3·48–4·12, titratable acidity: 1·67–3·50 mg KOH g^?1, acetic acid: 0·16–0·37%, alcohol content: 0·30–2·73%, sugars (degrees Brix): 2·70–8·50. Yeast included mainly Saccharomyces cerevisiae and species of the genera Arthroascus, Issatchenkia, Candida, Trichosporon, Hanseniaspora, Kodamaea, Schizosaccharomyces, Trigonopsis and Galactomyces. Lactobacillus plantarum was the predominant LAB species. Three other species of Lactobacillus were also identified as well as isolates of Leuconostoc mesenteroides, Fructobacillus durionis and Streptococcus mitis. Acetic acid bacteria included nine species of the genus Acetobacter with Acetobacter indonesiensis as predominant species. In addition, isolates of Gluconobacter oxydans and Gluconacetobacter saccharivorans were also identified. Intraspecies diversity was observed for some species of micro‐organisms including four genotypes for Acet. indonesiensis, three for Candida tropicalis and Lactobacillus fermentum and two each for S. cerevisiae, Trichosporon asahii, Candida pararugosa and Acetobacter tropicalis.

Conclusion

fermentation of palm sap from B. akeassii involved multi‐yeast‐LAB‐AAB cultures at genus, species and intraspecies level.

Significance and Impact of the Study

First study describing microbiological and physicochemical characteristics of palm wine from B. akeassii. Genotypic diversity of palm wine LAB and AAB not reported before is demonstrated and this constitutes valuable information for better understanding of the fermentation which can be used to improve the product quality and develop added value by‐products.     

Culturing marine bacteria – an essential prerequisite for biodiscovery

The potential for using marine microbes for biodiscovery is severely limited by the lack of laboratory cultures. It is a long-standing observation that standard microbiological techniques only isolate a very small proportion of the wide diversity of microbes that are known in natural environments from DNA sequences. A number of explanations are reviewed. The process of establishing laboratory cultures may destroy any cell-to-cell communication that occurs between organisms in the natural environment and that are vital for growth. Bacteria probably grow as consortia in the sea and reliance on other bacteria for essential nutrients and substrates is not possible with standard microbiological approaches. Such interactions should be considered when designing programmes for the isolation of marine microbes. The benefits of novel technologies for manipulating cells are reviewed, including single cell encapsulation in gel micro-droplets. Although novel technologies offer benefits for bringing previously uncultured microbes into laboratory culture, many useful bacteria can still be isolated using variations of plating techniques. Results are summarized for a study to culture bacteria from a long-term observatory station in the English Channel. Bacterial biodiversity in this assemblage has recently been characterized using high-throughput sequencing techniques. Although Alphaproteobacteria dominated the natural bacterial assemblage throughout the year, Gammaproteobacteria were the most frequent group isolated by plating techniques. The use of different gelling agents and the addition of ammonium to seawater-based agar did lead to the isolation of a higher proportion of Alphaproteobacteria. Variation in medium composition was also able to increase the recovery of other groups of particular interest for biodiscovery, such as Actinobacteria.     

Contaminations of laboratory surfaces with Staphylococcus aureus are affected by the carrier status of laboratory staff

Aim: As a biosafety laboratory, we take samples from surfaces in microbiological laboratories to survey the handling of micro‐organisms. Whereas contaminations with other micro‐organisms were rare, Staphylococcus aureus was found in the working environment of many laboratories. As 20–60% of the healthy population are carriers of S. aureus we wanted to asses the effect of carriers on our sampling results. Methods and Results: Nasal swabs of staff members in nonmicrobiological laboratories and offices as well as surface samples from their personal work environment were taken and analysed for S. aureus DNA. In addition S. aureus strains were isolated using S. aureus‐specific agar plates and analysed by randomly amplified polymorphic DNA (RAPD)–PCR and multilocus sequence typing (MLST). Our data show that contaminations with S. aureus in nonmicrobiological environments are common with 29% of the surface samples containing S. aureus DNA. In the working environment of carriers, the number of contaminations was significantly increased compared to the environment of noncarriers. Conclusion: The carrier status of staff members significantly affects the number of contaminations on laboratory surfaces. Therefore, even in the absence of intentional handling of S. aureus, contaminations can be detected on a substantial amount of surfaces. Significance and Impact of the Study: Sampling procedures need to be adapted based on these results with respect to the locations where samples are taken and the threshold for significant contaminations. Because of its wide distribution, S. aureus can serve as a marker for hygienic standards in laboratories.     

Technical note: Efficiency of total demineralization and ion‐exchange column for DNA extraction from bone

We investigated whether a combination of recently introduced methods, total demineralization and ion‐exchange columns, would increase DNA recovery from old bone. Ten bone samples taken after a burial period of ∼60 years were used in this study. Bone powder was digested using total or incomplete demineralization. DNA was extracted by the standard organic method. The DNA extract was purified with ion‐exchange columns or QIAquick® spin columns. The efficiency of different DNA extraction methods was compared in terms of DNA concentration, inhibitors generated by real‐time PCR, and conventional STR typing results. The mean DNA concentration using the total demineralization method is ∼3 times higher than that using the incomplete demineralization method. For DNA purification, the method using QIAquick® spin columns appeared to yield approximately double the DNA than the method using ion‐exchange columns. Furthermore, 2 out of 10 samples showed higher levels of inhibition with C_T values of IPC ≥30 cycles when using only ion‐exchange columns. In STR results, total demineralization yielded more locus profiles by 4.2 loci than incomplete demineralization, and QIAquick® spin columns also yielded more locus profiles by 3.5 loci than ion‐exchange columns. Total demineralization of bone powder significantly increased DNA yield and improved STR typing results. However, the use of ion‐exchange columns was not efficient when compared with the method using QIAquick® spin columns. It is suggested that the combination of total demineralization and QIAquick® spin columns lead to greatly improved STR typing results. Am J Phys Anthropol 2010. © 2009 Wiley‐Liss, Inc.