Penetration of fluorescent microspheres into the NEEM (North Eemian) Greenland ice core to assess the probability of microbial contamination

The validation of microbiological results from non-aseptically drilled deep ice cores is challenging because exogenous microbial cells can be transported into the core interior and compromise the existing microbial populations. The NEEM (North Eemian) ice core in Greenland provided a first-time opportunity to use fluorescent microspheres as tracers for assessing potential microbial contamination of glacial ice. We developed specific procedures to coat the surfaces of selected NEEM core samples representing bubbly (93–100 m), brittle (633–644 m) and clathrated (1,730 and 2,050 m) ice with melamine-based carboxylated fluorescent microspheres and tracked periodically their penetration into the core interior for 2.5 years using flow cytometry. Sufficient ice surface coating was achieved by immersing retrieved cores in plastic bags containing suspensions of pre-counted 1- and 10-μm microspheres or by down-hole microsphere deployment in plastic sleeves attached to the drill barrel and liberated during drilling. We examined the relationship between microspheres penetration and ice core depth, structure and time after coating. One consistent observation for all cores (except the brittle ice) was that removing a few millimeters of the outer layer drastically reduced microsphere counts, independent of timing, indicating that penetration was mostly limited to the surface layers. Any deeper penetration was found to be microsphere size dependent. The brittle ice showed significant microsphere penetration possibly due to microfractures. Overall, the use of fluorescent microspheres as tracers and microbial surrogates proved to be a sensitive approach for testing potential contamination during deep core projects.     

Techniques for Contamination Assessment During Drilling for Terrestrial Subsurface Sediments

Details about the procedures for drilling a ca. 150 m long drill core in a terrestrial setting under contamination controlled conditions are presented. Different to previous studies we only used commercially available drilling equipment to reduce the cost of operation significantly. The goals were (1) to minimize, (2) to monitor and, if possible, to quantify the contamination of the recovered sediments, and (3) to identify the different sources of contamination. Both the potential contamination of the sample material by surface microorganisms and non-indigenous material was assessed. To estimate the infiltration of drill mud into the core, fluorescent microspheres, having about half the size as microorganisms, were added to the mud. The drilling technique used was mud rotary drilling. With the exception of the very beginning of the drilling operations, the drill mud was devoid of any allochthonous hydrocarbons potentially derived from the drilling equipment or drill additives, and its biomarker composition reflected the varying organo-facies that were penetrated. Due to the lack of allochthonous hydrocarbons in the drill mud, its infiltration into the sediment cannot be traced by organic geochemical biomarker analysis. Microspheres proved to be a sensitive tool for the assessment of infiltration of drill mud into the core. The concentration of microspheres in the drill mud decreased continuously during the drilling, most probably caused by seepage of mud through leaks and attachment of spheres to the surface scum in the mud pit. Microscopic enumeration of the microspheres showed great variability in the depth of penetration of mud into the core, apparently unaffected of lithology. The sampling of the core material in the laboratory was carried out inside an anaerobic chamber. Several techniques for subsampling were used, according to sediment properties. The overall results indicate that, if strict contamination control protocols are employed, it is possible to recover uncontaminated samples at reasonable cost with commercially available drilling equipment.     

Molecular analysis of bacterial diversity in kerosene-based drilling fluid from the deep ice borehole at Vostok, East Antarctica

Decontamination of ice cores is a critical issue in phylogenetic studies of glacial ice and subglacial lakes. At the Vostok drill site, a total of 3650 m of ice core have now been obtained from the East Antarctic ice sheet. The ice core surface is coated with a hard-to-remove film of impure drilling fluid comprising a mixture of aliphatic and aromatic hydrocarbons and foranes. In the present study we used 16S rRNA gene sequencing to analyze the bacterial content of the Vostok drilling fluid sampled from four depths in the borehole. Six phylotypes were identified in three of four samples studied. The two dominant phylotypes recovered from the deepest (3400 and 3600 m) and comparatively warm (-10 degrees C and -6 degrees C, respectively) borehole horizons were from within the genus Sphingomonas, a well-known degrader of polyaromatic hydrocarbons. The remaining phylotypes encountered in all samples proved to be human- or soil-associated bacteria and were presumed to be drilling fluid contaminants of rare occurrence. The results obtained indicate the persistence of bacteria in extremely cold, hydrocarbon-rich environments. They show the potential for contamination of ice and subglacial water samples during lake exploration, and the need to develop a microbiological database of drilling fluid findings.     

Microbial diversity and activity through a permafrost/ground ice core profile from the Canadian high Arctic

Culture‐dependent and culture‐independent methods were used in an investigation of the microbial diversity in a permafrost/massive ground ice core from the Canadian high Arctic. Denaturing gradient gel electrophoresis as well as Bacteria and Archaea 16S rRNA gene clone libraries showed differences in the composition of the microbial communities in the distinct core horizons. Microbial diversity was similar in the active layer (surface) soil, permafrost table and permafrost horizons while the ground ice microbial community showed low diversity. Bacteria and Archaea sequences related to the Actinobacteria (54%) and Crenarchaeota (100%) respectively were predominant in the active layer while the majority of sequences in the permafrost were related to the Proteobacteria (57%) and Euryarchaeota (76%). The most abundant phyla in the ground ice clone libraries were the Firmicutes (59%) and Crenarchaeota (82%). Isolates from the permafrost were both less abundant and diverse than in the active layer soil, while no culturable cells were recovered from the ground ice. Mineralization of [1‐¹⁴C] acetic acid and [2‐¹⁴C] glucose was used to detect microbial activity in the different horizons in the core. Mineralization was detected at near ambient permafrost temperatures (?15°C), indicating that permafrost may harbour an active microbial population, while the low microbial diversity, abundance and activity in ground ice suggests a less hospitable microbial habitat.     

Synthesis and Characterization of Dual-Functionalized Core-Shell Fluorescent Microspheres for Bioconjugation and Cellular Delivery

The efficient transport of micron-sized beads into cells, via a non-endocytosis mediated mechanism, has only recently been described. As such there is considerable scope for optimization and exploitation of this procedure to enable imaging and sensing applications to be realized. Herein, we report the design, synthesis and characterization of fluorescent microsphere-based cellular delivery agents that can also carry biological cargoes. These core-shell polymer microspheres possess two distinct chemical environments; the core is hydrophobic and can be labeled with fluorescent dye, to permit visual tracking of the microsphere during and after cellular delivery, whilst the outer shell renders the external surfaces of the microspheres hydrophilic, thus facilitating both bioconjugation and cellular compatibility. Cross-linked core particles were prepared in a dispersion polymerization reaction employing styrene, divinylbenzene and a thiol-functionalized co-monomer. These core particles were then shelled in a seeded emulsion polymerization reaction, employing styrene, divinylbenzene and methacrylic acid, to generate orthogonally functionalized core-shell microspheres which were internally labeled via the core thiol moieties through reaction with a thiol reactive dye (DY630-maleimide). Following internal labeling, bioconjugation of green fluorescent protein (GFP) to their carboxyl-functionalized surfaces was successfully accomplished using standard coupling protocols. The resultant dual-labeled microspheres were visualized by both of the fully resolvable fluorescence emissions of their cores (DY630) and shells (GFP). In vitro cellular uptake of these microspheres by HeLa cells was demonstrated conventionally by fluorescence-based flow cytometry, whilst MTT assays demonstrated that 92% of HeLa cells remained viable after uptake. Due to their size and surface functionalities, these far-red-labeled microspheres are ideal candidates for in vitro, cellular delivery of proteins.     

The Effects of Sample Storage Conditions on the Microbial Community Composition in Hydraulic Fracturing Produced Water

The petroleum industry has an increasing interest in understanding the microbial communities driving biofouling and biocorrosion in reservoirs, wells, and infrastructure. However, sampling of the relevant produced fluids from subsurface environments for microbiological analyses is often challenged by high liquid pressures, workplace regulations, operator liability concerns, and remote sampling locations. These challenges result in infrequent sampling opportunities and the need to store and preserve the collected samples for several days or weeks. Maintaining a representative microbial community structure from produced fluid samples throughout storage and handling is essential for accurate results of downstream microbial analyses. Currently, no sample handling or storage recommendations exist for microbiological analyses of produced fluid samples. We used 16S rRNA gene sequencing to monitor the changes in microbial communities in hypersaline produced water stored at room temperature or at 4?°C for up to 7 days. We also analyzed storage at ?80?°C across a 3-week period. The results suggest ideal handling methods would include placing the collected sample on ice as soon as possible, but at least within 24?h, followed by shipping the samples on ice over 2–3?days, and finally, long-term storage in the ?20?°C or ?80?°C freezer.     

Isolation of nucleic acids and cultures from fossil ice and permafrost

Owing to their constant low temperatures, glacial ice and permafrost might contain the oldest nucleic acids and microbial cells on Earth, which could prove key to reconstructing past ecosystems and for the planning of missions to other planets. However, recent claims concerning viable cells and microbial nucleic acids obtained from ice- and permafrost cores from hundreds of thousands to millions of years old are not properly authenticated and the findings could be the result of contamination. Here, we discuss the processes that restrict the long-term survival of DNA and/or RNA molecules in ice and permafrost, and highlight sources of contamination that could result in false claims. Additionally, we present a set of precautions, controls and criteria to help ensure that future cultures and sequences are authentic.     

Evaluation of deep subsurface sampling procedures using serendipitous microbial contaminants as tracer organisms

Surface microbiological investigations are critically dependent on the procedures used to collect samples for study. It can be difficult to distinguish between indigenous organisms and those encountered as contaminants during the drilling process. We found that coliform bacteria contaminated drilling mud slurries. These bacteria proved useful as tracer organisms in evaluating the degree of microbial contamination accidentally encountered while drilling for subterranean samples. While these organisms were found in high numbers in both the circulating muds and in the mud reservoir, few subsurface samples harbored conforms. Subsurface slurries did not inhibit the growth of a known coliform inoculum. These results indicate that the methods used to collect and field‐process cores from Atlantic coastal plain sediments were sufficient to prevent a large degree of bacterial contamination in most samples. The microflora in drilling fluids did not quantitatively or qualitatively account for the number and diversity of bacteria in subsurface samples. We conclude that a large and viable bacterial community is present in deep regions of the terrestrial subsurface.     

Trends in Basalt and Sediment Core Contamination During IODP Expedition 301

Perfluorocarbon tracers (PFTs) are used during cruises of the Ocean Drilling Program (ODP) and Integrated Ocean Program (IODP) to measure sample contamination with drilling fluid. Drilling fluid is supplied with a constant PFT concentration that can then be detected and quantified in sediment and basalt core samples. During IODP Expedition 301, we used washing (2×) and flaming to effectively remove PFT from the exterior of basalt rocks. Near-complete removal from the exterior allowed us to demonstrate that the interior of basalts was only minutely, if at all, contaminated with drilling fluid. We examined horizontal and vertical trends in sediment core contamination. Contamination decreased greatly between the core exterior to halfway along the core radius, and slightly from halfway to the center of cores, and was generally very low in halfway and center portions. Clay cores were, on average, more contaminated than cores with fine sand. Contamination was typically highest in the two uppermost sections (sections 1 and 2) and lower below (sections 3–5). There was no relationship between depth of core origin and contamination. To determine mechanisms of contamination in halfway and interior parts of cores, we estimated the diffusive flux of PFT from the core liner towards the core center. Based on conservative estimates, we concluded that diffusion did not account for any of the PFT measured in halfway and interior parts of cores in this study. Any measurable PFT concentrations in halfway and center parts of cores were caused by advection.     

Microbial Load of Drinking Water Reservoir Tributaries during Extreme Rainfall and Runoff

Hygienic and microbiological examinations of watercourses are usually not carried out during heavy rainfall and runoff events. After rainfall or snowmelt, there are often massive increases in turbidity in flooding creeks in mountain ranges, which are frequently interpreted as an indication of microbial contamination. The aim of this study was to quantify the microbial loads of watercourses during such runoff events and to compare these loads with loads occurring during regular conditions. In a 14-month monitoring period we investigated the microbial loads of three tributaries of different drinking water reservoirs. A total of 99 water samples were taken under different runoff conditions and analyzed to determine physical, chemical, bacterial, and parasitic parameters. Thirty-two water samples were considered event samples during nine measuring series. The criteria for events, based on duration and intensity of precipitation, water depth gauge measurements, and dynamics, had been fixed before the investigation for each creek individually. Of the physical and chemical parameters examined, only the turbidity, pH, and nitrate values differed clearly from the values obtained for regular samples. Most of the bacteriological parameters investigated (colony, Escherichia coli, coliform, fecal streptococcal, and Clostridium perfringens counts) increased considerably during extreme runoff events. If relevant sources of parasitic contamination occurred in catchment areas, the concentrations of Giardia and Cryptosporidium rose significantly during events. The results show that substantial shares of the total microbial loads in watercourses and in drinking water reservoirs result from rainfall and extreme runoff events. Consequently, regular samples are considered inadequate for representing the microbial contamination of watercourse systems. The procedures for raw water surveillance in the context of multiple-barrier protection and risk assessment ought to include sampling during extreme runoff situations.     

Investigating Acid Production by Streptococcus mutans with a Surface-Displayed pH-Sensitive Green Fluorescent Protein

Acidogenicity and aciduricity are the main virulence factors of the cavity-causing bacterium Streptococcus mutans. Monitoring at the individual cell level the temporal and spatial distribution of acid produced by this important oral pathogen is central for our understanding of these key virulence factors especially when S. mutans resides in multi-species microbial communities. In this study, we explored the application of pH-sensitive green fluorescent proteins (pHluorins) to investigate these important features. Ecliptic pHluorin was functionally displayed on the cell surface of S. mutans as a fusion protein with SpaP. The resulting strain (O87) was used to monitor temporal and spatial pH changes in the microenvironment of S. mutans cells under both planktonic and biofilm conditions. Using strain O87, we revealed a rapid pH drop in the microenviroment of S. mutans microcolonies prior to the decrease in the macro-environment pH following sucrose fermentation. Meanwhile, a non-uniform pH distribution was observed within S. mutans biofilms, reflecting differences in microbial metabolic activity. Furthermore, strain O87 was successfully used to monitor the S. mutans acid production profiles within dual- and multispecies oral biofilms. Based on these findings, the ecliptic pHluorin allows us to investigate in vivo and in situ acid production and distribution by the cariogenic species S. mutans.     

Investigation of the potential for microbial contamination of deep granitic aquifers during drilling using 16S rRNA gene sequencing and culturing methods

Total number of bacteria, viable counts of aerobic and anaerobic heterotrophic bacteria and 16S rRNA gene diversity were investigated during drilling of three boreholes in the walls of the Äspö hard rock laboratory tunnel, at depths ranging from 380 to 446 m below sea level. Water samples were taken from the drill water source, the drilling equipment and from the drilled boreholes. The drill water was kept under nitrogen atmosphere and all equipment was steam cleaned before the start of a new drilling. Total and viable counts of bacteria in the drilled boreholes were several orders of magnitude lower than in the samples from the drilling equipment, except for sulphate reducing bacteria. A total of 158 16S rRNA genes that were cloned from the drill water source, the drilling equipment and the drilled boreholes were partially sequenced. The drilled boreholes generally had a 16S rRNA diversity that differed from what was found in samples from the drilling equipment. Several of the sequences obtained could be identified on genus level as one of the genera Acinetobacter, Methylophilus, Pseudomonas and Shewanella. In conclusion, the tubing used for drill water supply constituted a source of bacterial contamination to the rest of the drilling equipment and the boreholes. The results show, using molecular and culturing methods, that although large numbers of contaminating bacteria were introduced to the boreholes during drilling, they did not establish in the borehole groundwater at detectable levels.     

Development of a Fluorescent Based Immunosensor for the Serodiagnosis of Canine Leishmaniasis Combining Immunomagnetic Separation and Flow Cytometry

Background

An accurate diagnosis is essential for the control of infectious diseases. In the search for effective and efficient tests, biosensors have increasingly been exploited for the development of new and highly sensitive diagnostic methods. Here, we describe a new fluorescent based immunosensor comprising magnetic polymer microspheres coated with recombinant antigens to improve the detection of specific antibodies generated during an infectious disease. As a challenging model, we used canine leishmaniasis due to the unsatisfactory sensitivity associated with the detection of infection in asymptomatic animals where the levels of pathogen-specific antibodies are scarce.

Methodology

Ni-NTA magnetic microspheres with 1,7 µm and 8,07 µm were coated with the Leishmania recombinant proteins LicTXNPx and rK39, respectively. A mixture of equal proportions of both recombinant protein-coated microspheres was used to recognize and specifically bind anti-rK39 and anti-LicTNXPx antibodies present in serum samples of infected dogs. The microspheres were recovered by magnetic separation and the percentage of fluorescent positive microspheres was quantified by flow cytometry.

Principal Findings

A clinical evaluation carried out with 129 dog serum samples using the antigen combination demonstrated a sensitivity of 98,8% with a specificity of 94,4%. rK39 antigen alone demonstrated a higher sensitivity for symptomatic dogs (96,9%), while LicTXNPx antigen showed a higher sensitivity for asymptomatic (94,4%).

Conclusions

Overall, our results demonstrated the potential of a magnetic microsphere associated flow cytometry methodology as a viable tool for highly sensitive laboratorial serodiagnosis of both clinical and subclinical forms of canine leishmaniasis.     

Tracking Microbial Contamination in Retail Environments Using Fluorescent Powder - A Retail Delicatessen Environment Example

Cross contamination of foodborne pathogens in the retail environment is a significant public health issue contributing to an increased risk for foodborne illness. Ready-to-eat (RTE) processed foods such as deli meats, cheese, and in some cases fresh produce, have been involved in foodborne disease outbreaks due to contamination with pathogens such as Listeria monocytogenes. With respect to L. monocytogenes, deli slicers are often the main source of cross contamination. The goal of this study was to use a fluorescent compound to simulate bacterial contamination and track this contamination in a retail setting. A mock deli kitchen was designed to simulate the retail environment. Deli meat was inoculated with the fluorescent compound and volunteers were recruited to complete a set of tasks similar to those expected of a food retail employee. The volunteers were instructed to slice, package, and store the meat in a deli refrigerator. The potential cross contamination was tracked in the mock retail environment by swabbing specific areas and measuring the optical density of the swabbed area with a spectrophotometer. The results indicated that the refrigerator (i.e. deli case) grip and various areas on the slicer had the highest risk for cross contamination. The results of this study may be used to develop more focused training material for retail employees. In addition, similar methodologies could also be used to track microbial contamination in food production environments (e.g. small farms), hospitals, nursing homes, cruise ships, and hotels.     

Presence of pathogenic bacteria in ice cubes and evaluation of their survival in different systems

In this study, 60 samples of ice cubes produced at different levels (domestic, restaurant and industrial facilities), within a restricted geographical area, were investigated for their general microbiological characteristics through the analysis of populations other than enteric bacteria. Total mesophilic bacteria were in the range 1.01?×?10²–9.55?×?10³, 3.12?×?10²–6.31?×?10³ and 1.30?×?10²–3.99?×?10³?CFU/100 mL of thawed ice from domestic freezer (DF), stock boxes (SB) for self-production performed with ice machines in bars and pubs, and from sales packages (SP) of industrial productions, respectively. Some DF and SP samples were negative for the presence of total psychrotrophic bacteria, showing that there are no specific microbial groups associated with ice. Pseudomonads were found in the majority of ice samples analyzed. The levels of contamination of the ice samples were significantly different among the three ice cube production levels. The samples produced at domestic level and those collected from bars and pubs were characterised by the highest cell densities. The colonies representative for the different bacterial morphologies were randomly picked up from plates, purified to homogeneity and subjected to the phenotypic and genotypic characterisation. Fifty-two strains representing 31 species of eight bacterial genera were identified, with the most numerous groups included in Pseudomonas, Staphylococcus, Bacillus and Acinetobacter. A consistent percentage of the microorganisms identified from ice are known agents of human infections, and their presence indicate an environmental contamination. In order to evaluate the effectiveness of the ice cubes to transfer pathogenic agents to consumers, a bar consumption was simulated with different drink systems added with ice cubes artificially contaminated with the strains found at dominant levels (Acinetobacter lwoffii ICE100, Bacillus cereus ICE170, Pseudomonas putida ICE224 and Staphylococcus haemolyticus ICE182), and the results showed a consistent reduction of bacterial risk due to alcohol, CO₂, pH and antibacterial ingredients of vodka, whisky, Martini, peach tea, tonic water and coke.     

Can Volatile Organic Metabolites Be Used to Simultaneously Assess Microbial and Mite Contamination Level in Cereal Grains and Coffee Beans?

A novel approach based on headspace solid-phase microextraction (HS-SPME) combined with comprehensive two-dimensional gas chromatography–time-of-flight mass spectrometry (GC×GC–ToFMS) was developed for the simultaneous screening of microbial and mite contamination level in cereals and coffee beans. The proposed approach emerges as a powerful tool for the rapid assessment of the microbial contamination level (ca. 70 min versus ca. 72 to 120 h for bacteria and fungi, respectively, using conventional plate counts), and mite contamination (ca. 70 min versus ca. 24 h). A full-factorial design was performed for optimization of the SPME experimental parameters. The methodology was applied to three types of rice (rough, brown, and white rice), oat, wheat, and green and roasted coffee beans. Simultaneously, microbiological analysis of the samples (total aerobic microorganisms, moulds, and yeasts) was performed by conventional plate counts. A set of 54 volatile markers was selected among all the compounds detected by GC×GC–ToFMS. Principal Component Analysis (PCA) was applied in order to establish a relationship between potential volatile markers and the level of microbial contamination. Methylbenzene, 3-octanone, 2-nonanone, 2-methyl-3-pentanol, 1-octen-3-ol, and 2-hexanone were associated to samples with higher microbial contamination level, especially in rough rice. Moreover, oat exhibited a high GC peak area of 2-hydroxy-6-methylbenzaldehyde, a sexual and alarm pheromone for adult mites, which in the other matrices appeared as a trace component. The number of mites detected in oat grains was correlated to the GC peak area of the pheromone. The HS-SPME/GC×GC–ToFMS methodology can be regarded as the basis for the development of a rapid and versatile method that can be applied in industry to the simultaneous assessment the level of microbiological contamination and for detection of mites in cereals grains and coffee beans.     

The role of visible faecal material as a vehicle for generic Escherichia coli,coliform, and other enterobacteria contaminating poultry carcasses during slaughtering

AIMS: A comparison of Enterobacteriaceae, coliform and Escherichia coli counts in chicken carcasses with and without visible faecal contamination was conducted to evaluate the role of contamination as a vehicle for generic E. coli, coliform and other enterobacteria contaminating broiler chicken carcasses when processed under routine commercial operations. METHODS AND RESULTS: Samples were removed from the processing line immediately after evisceration, inside-outside shower and chilling for microbiological analysis. After evisceration, mean counts were significantly different only for E. coli (P < or = 0.05) in chicken carcasses with and without visible faecal contamination. While the spray wash practice was not efficient enough for complete removal of the visible contamination from carcasses, leading to microbiological reduction percentages lower than expected, 25 ppm chlorinated water chilling did reduce the contamination level considerably in all samples. CONCLUSIONS: Carcasses with and without visible faecal contamination harboured E. coli and other potentially hazardous enterobacteria. E. coli was the predominant strain isolated in all samples, Enterobacter cloacae being next most frequent. SIGNIFICANCE AND IMPACT OF THE STUDY: The zero tolerance of visible faecal contamination requirement alone is not sufficient to assure safety and to improve the microbial quality of carcasses.     

Past and present vegetation of the Isle of Skye: H. J. B. Birks. Cambridge University Press,London, 1973, 415 pp., £13.50

Ice-wedge casts in southern Quebec are exclusive to deposits pertaining to two ice front positions during the retreat of the Laurentides ice sheet: the Highland front morainic system located at the southern margin of the St. Lawrence lowlands, and the St. Narcisse front morainic system located at the southern margin of the Laurentides highlands. Wedge casts filled with sand and gravel are relic of true ice wedges and give evidence of former permafrost conditions in the ground during the retreat of the ice sheet in that area, between 13 000 and 11 000 years B.P. Mean annual air temperature was lower than −6°C, a difference of 8–12° with the present-day temperature. Humidity was high enough to allow the growth of ice wedges in the permafrost. These rigorous climatic conditions prevailed during a few centuries only. There is no positive evidence of tundra conditions in southern Quebec young than 11 000 years ago.     

The origin, ultrastructure, and microbiology of the sediment accumulating in liquid nitrogen storage vessels

During long-term cryopreservation, ice sediment accumulates in storage Dewars and poses a risk of microbial contamination to stored samples. Ice accumulates in liquid nitrogen via two general processes: (1) ice forming in the atmosphere above an open Dewar falls into the vessel; and (2) ice forming on cold surfaces of the Dewar or inventory system enters the liquid nitrogen. These ice crystals aggregate and entrap other materials, such as bacteria, fungal spores, and general laboratory debris present within the liquid nitrogen. Measured changes in the ultrastructure of ice aggregates following long-term storage are consistent with transient warming events to temperatures of -100 degrees C. Bacteria were identified in all samples and filamentous fungi in 9 out of 10 samples. These micro-organisms are commonly found in the environment and would not be expected to have been derived from IVF samples. Some of the bacteria identified are associated with nosocomial infections in humans. The implications that the association of microbial contamination with ice crystals has on cryopreservation procedures are discussed.     

Microbial diversity in the samples from archeological complexes of the Pazyryk culture (IV–III centuries BC) in northwestern Mongolia

Prokaryotes were analyzed in the samples of ice and soils collected by the joint Russian-German-Mongolian expedition (2006) at the site of archeological excavations of two Pazyryk culture mounds in the upper Olon-Kurin-Gol River on the southern slope of the Saylyugem Mountains, Mongolian Altai. Phylogenetic analysis of the 16S rRNA gene fragments of the cultured bacteria revealed three major groups wide-spread in permafrost soils: Firmicutes, Actinobacteria, and Gammaproteobacteria. Analysis of the total DNA from the samples revealed nonculturable bacteria of the phyla Alphaproteobacteria, Gammaproteobacteria, Deltaproteobacteria, Actinobacteria, Chloroflexi, Gemmatoidetes, Firmicutes, and CFB (Cytophaga-Flavobacteria-Bacteroidetes) isolated from the samples of permafrost soils (Arctic, tundra, and from Tibet highlands), groundwater, and Arctic and Antarctic ice cores.