Distribution and sources of microbial contamination on beef carcasses

Three beef dressing lines of different capacity (160, 440 and 800 head d(-1)) were investigated with respect to contamination associated with carcass/hide and carcass/faeces contacts, the distribution of microbial contamination on carcasses and the antimicrobial efficacy of cold water carcass washes. Swab samples were taken from up to 17 sites for determination of Aerobic Plate Counts at 37 degrees C (APC 37 degrees C) and Escherichia coli enumeration using the Petrifilm procedure. The three beef dressing systems produced virtually identical patterns of microbial contamination. High contamination was found at those sites associated with opening cuts and/or subject to hide contact during hide removal. Where contamination is intermittent, the use of mean microbial data tended to obscure evidence of faecal or hide contact. Consequently, worst-case results, as represented by the 95th percentile value, were used to identify probable instances and sources of contact contamination. Sites not subject to faecal contamination or hide contact typically had swab sample APC (37 degrees C) values of less than log 2.00 cfu cm(-2) accompanied by the occasional detection of E. coli at levels below log 1.00 cfu cm(-2). Sites contacted by 'clean' hide typically had APC (37 degrees C) counts of log 3.00 cfu cm(-2) or greater accompanied by occasional E. coli counts not exceeding log 2.00 cfu cm(-2). Sites contaminated by direct faecal contact or contact with faecally contaminated hides typically had APC (37 degrees C) counts equal to, or greater than, log 4.00 cfu cm(-2) accompanied by E. coli counts exceeding log 2.00 cfu cm(-2). Cold water carcass washing was ineffective in removing microbial contamination and tended to bring about a posterior to anterior redistribution, resulting in increased counts at forequarter sites.     

石油污染地土壤微生物群落的碳源利用特性

应用Biolog微平板技术,研究了大庆油田开采36年的石油污染土壤不同土层（0～10 cm、10～20 cm、20～30 cm）土壤微生物群落对碳源的利用特性.结果表明: 石油污染明显提高了土壤微生物群落的代谢活性,3个土层的微生物代谢强度均高于无污染土壤（CK）,不同土层之间的微生物代谢强度存在显著差异,其中20～30 cm土层的碳源代谢能力最强,其次为10～20 cm土层,0～10 cm土层最弱.石油污染使10～20 cm和20～30 cm土层土壤微生物群落对底物碳源利用种类增多,代谢功能的多样性增强,而0～10 cm土层则无明显变化.10～20 cm土层土壤微生物对底物碳源的利用由对照土壤的羧酸类居多转为石油污染土壤的碳水化合物最多,而20～30 cm土层土壤微生物对底物碳源的利用以羧酸类居多.说明石油污染土壤的微生物群落具有独特的群落结构和特点.     

Sterility Test Method for Petrolatum-Based Ophthalmic Ointments

A sensitive sterility testing procedure for the detection of microbial contamination in petrolatum-based ointments is described. The method involves dissolving the ointment in filter-sterilized isopropyl myristate and filtering through a membrane filter. Improved sensitivity is obtained by blending the membrane in Trypticase Soy Broth before incubation. Filter-sterilized isopropyl myristate is shown to be less toxic to microorganisms than heat-sterilized isopropyl myristate. The isopropyl myristate method is more sensitive than the polyethylene glycol-ether method for the detection of microbial contamination.     

Taxonomic identification of microorganisms by capture and intrinsic fluorescence detection

Quick and accurate detection of microbial contamination is accomplished by a unique combination of leading edge technologies described in this and the accompanying article. Microbe capture chips, used with a prototype fluorescence detector, are capable of statistically sampling the environment for pathogens (including spores), identifying the specific pathogens/exotoxins, and determining cell viability where appropriate.     

Microbial growth in saline breast implants and saline tissue expanders

The subject of microbial growth within the saline medium of prosthetic breast implants has been one of great controversy in recent years. Although several articles have described microbial growth within the tissue surrounding implanted breast prostheses, few have attempted to determine the possibility of such contamination of the luminal saline. The authors studied the intraluminal saline medium of a series of explanted breast prostheses with the objective of identifying any microbial contamination. Over a 6-month period, a consecutive series of saline-filled breast implants and tissue expanders were removed from 37 patients. Under the supervision of a microbiologist, saline extracted from each implant was subjected to bacterial and fungal cultures, Gram staining, and acid-fast staining. A total of 24 saline-filled breast implants were removed from 15 patients, and 32 saline-filled tissue expanders were removed from 22 patients. The average length of implantation was 28.1 months for the implants and 7.1 months for the expanders. None of the saline within the implants or expanders within our series displayed any evidence of microbial contamination. These results suggest that microbial contamination of the luminal saline of prosthetic breast implants is an extremely unlikely event.     

Development of an apparatus and protocol for the efficient isolation of bacteria and yeasts that attach to Botrytis cinerea germlings

An apparatus and protocol for the efficient and consistent isolation of bacteria and yeasts with the ability to attach to germlings of Botrytis cinerea is described. The study focused on minimising microbial contamination by bacteria or yeasts which do not attach to the pathogen B. cinerea but which interact with materials used in the equipment and would otherwise be isolated along with target microbes. After development, the assay reduced the contamination rate to 1–2 cells per 100 added and this was found to be a satisfactory level for the selection of microbial attachers to fungal hyphae. One or more phenotypes of microbes that adhered to B. cinerea germlings were found in 97% of the 70 samples collected from phylloplane washings and processed using this assay.     

Aquatic microbial diversity associated with faecal pollution of Norwegian waterbodies characterized by 16S rRNA gene amplicon deep sequencing

Faecal contamination is one of the major factors affecting biological water quality. In this study, we investigated microbial taxonomic diversity of faecally polluted lotic ecosystems in Norway. These ecosystems comprise tributaries of drinking water reservoirs with moderate and high faecal contamination levels, an urban creek exposed to extremely high faecal pollution and a rural creek that was the least faecally polluted. The faecal water contamination had both anthropogenic and zoogenic origins identified through quantitative microbial source tracking applying host-specific Bacteroidales 16S rRNA genetic markers. The microbial community composition revealed that Proteobacteria and Bacteroidetes (70–90% relative abundance) were the most dominant bacterial phyla, followed by Firmicutes, especially in waters exposed to anthropogenic faecal contamination. The core archaeal community consisted of Parvarchaeota (mainly in the tributaries of drinking water reservoirs) and Crenarchaeota (in the rural creek). The aquatic microbial diversity was substantially reduced in water with severe faecal contamination. In addition, the community compositions diverge between waters with dominant anthropogenic or zoogenic pollution origins. These findings present novel interpretations of the effect of anthropo-zoogenic faecal water contamination on microbial diversity in lotic ecosystems.     

Utilization of Fluorescent Microspheres and a Green Fluorescent Protein-Marked Strain for Assessment of Microbiological Contamination of Permafrost and Ground Ice Core Samples from the Canadian High Arctic

Fluorescent microspheres were applied in a novel fashion during subsurface drilling of permafrost and ground ice in the Canadian High Arctic to monitor the exogenous microbiological contamination of core samples obtained during the drilling process. Prior to each drill run, a concentrated fluorescent microsphere (0.5-μm diameter) solution was applied to the interior surfaces of the drill bit, core catcher, and core tube and allowed to dry. Macroscopic examination in the field demonstrated reliable transfer of the microspheres to core samples, while detailed microscopic examination revealed penetration levels of less than 1 cm from the core exterior. To monitor for microbial contamination during downstream processing of the permafrost and ground ice cores, a Pseudomonas strain expressing the green fluorescent protein (GFP) was painted on the core exterior prior to processing. Contamination of the processed core interiors with the GFP-expressing strain was not detected by culturing the samples or by PCR to detect the gfp marker gene. These methodologies were quick, were easy to apply, and should help to monitor the exogenous microbiological contamination of pristine permafrost and ground ice samples for downstream culture-dependent and culture-independent microbial analyses.     

Monitoring the microbial contamination of beef carcass tissue with a rapid chromogenic Limulus amoebocyte lysate endpoint assay

A chromogenic Limulus amoebocyte lysate (LAL) endpoint assay was found to be an accurate and rapid means of gauging levels of beef carcass microbial contamination within 10 min. The assay demonstrated a high correlation with the total mesophilic bacterial and coliform surface populations from inoculated beef carcass surface tissues. This assay was tested on a set of actual beef carcass surface samples (n = 121) demonstrating the utility of the chromogenic LAL test as a means of monitoring carcass microbial contamination in a near real-time fashion. Classifying the chromogenic LAL results into four contamination groups was found to be a sound means of utilizing the resultant chromogenic LAL data for detecting carcasses with high levels of microbial contamination. For beef carcass testing, this assay can be used with no instrumentation other than the required 37 degrees C incubator and, as an option, a microplate reader.     

Evaluating microbial contaminations of alternative heating oils

Since 2008, European and German legislative initiatives for climate protection and reduced dependency on fossil resources led to the introduction of biofuels as CO₂-reduced alternatives in the heating oil sector. In the case of biodiesel, customers were confronted with accelerated microbial contaminations during storage. Since then, other fuel alternatives, like hydrogenated vegetable oils (HVOs), gas-to-liquid (GtL) products, or oxymethylene ether (OME) have been developed. In this study, we use online monitoring of microbial CO₂ production and the simulation of onset of microbial contamination to investigate the contamination potential of fuel alternatives during storage. As references, fossil heating oil of German refineries are used. Biodiesel blends with fossil heating oils confirmed the promotion of microbial activity. In stark contrast, OMEs have an antimicrobial effect. The paraffinic Fischer–Tropsch products and biogenic hydrogenation products demonstrate to be at least as resistant to microbial contamination as fossil heating oils despite allowing a diversity of representative microbes. Through mass spectrometry, elemental analysis, and microbial sequencing, we can discuss fuel properties that affect microbial contaminations. In summary, novel, non-fossil heating oils show clear differences in microbial resistance during long-term storage. Designing blends with an intrinsic resistance against microbial contamination and hence reduced activity might be an option.     

An exometabolomics approach to monitoring microbial contamination in microalgal fermentation processes by using metabolic footprint analysis

The early detection of microbial contamination is crucial to avoid process failure and costly delays in fermentation industries. However, traditional detection methods such as plate counting and microscopy are labor-intensive, insensitive, and time-consuming. Modern techniques that can detect microbial contamination rapidly and cost-effectively are therefore sought. In the present study, we propose gas chromatography-mass spectrometry (GC-MS)-based metabolic footprint analysis as a rapid and reliable method for the detection of microbial contamination in fermentation processes. Our metabolic footprint analysis detected statistically significant differences in metabolite profiles of axenic and contaminated batch cultures of microalgae as early as 3 h after contamination was introduced, while classical detection methods could detect contamination only after 24 h. The data were analyzed by discriminant function analysis and were validated by leave-one-out cross-validation. We obtained a 97% success rate in correctly classifying samples coming from contaminated or axenic cultures. Therefore, metabolic footprint analysis combined with discriminant function analysis presents a rapid and cost-effective approach to monitor microbial contamination in industrial fermentation processes.     

Detection of microbial contaminants in mammalian cell cultures using a new fluorescence-based staining method

Aims: Microbial contamination of cell culture production processes is a current concern for biopharmaceutical industries. Traditional testing methods require several days to detect contamination and may advantageously be replaced by a rapid detection method. We developed a new method combining membrane filtration to microcolonies fluorescence staining method (MFSM) and compared it to epifluorescence microscopy. Methods and Results: Both methods were used to detect bacteria in CHO cells cultures. The epifluorescence microscopy showed to be limited by filterability, media interference and nonrobustness issues, whereas MFSM enabled consistent detection of Bacillus cereus, Staphylococcus epidermidis and Propionibacterium acnes after, respectively, 8, 9 and 48 h of incubation. Thanks to the nondestructive feature of the MFSM, stained membranes could be reincubated on culture media to yield visible colonies used for identification. Conclusions: The new method described in this study showed its ability to detect microbial contaminants in cell culture samples with time‐to‐results from 2–5 times shorter than the traditional testing method. Significance and Impact of the Study: The MFSM can be used as monitoring tool for cell cultures to significantly shorten detection times of microbial contamination, while preserving the ability to identify the contaminants and their viability.     

Comparative levels and types of microbial contamination detected in industrial clean rooms

The primary objective of this study was to determine quantitatively and qualitatively the predominant types of microbial contamination occurring in conventional and laminar flow clean rooms. One horizontal laminar flow, three conventional industrial clean rooms, and three open factory areas were selected for microbiological tests. The results showed that as the environment and personnel of a clean room were controlled in a more positive manner with respect to the reduction of particulate contamination, the levels of airborne and surface microbial contaminants were reduced accordingly. The chief sources of microbial contamination were associated with the density and activity of clean room personnel. In addition, the majority of microorganisms isolated from the intramural air by air samplers were those indigenous to humans. Studies on the fallout and accumulation of airborne microorganisms on stainless-steel surfaces showed that, although there were no significant differences in the levels of microbial contamination among the conventional clean rooms, the type of microorganism detected on stainless-steel surfaces was consistently and significantly different. In addition, the "plateau phenomenon" occurred in all environments studied. It was concluded that the stainless-steel strip method for detecting microbial accumulation on surfaces is efficient and sensitive in ultra-clean environments and is the most reliable and practical method for monitoring microbial contamination in future class 100 clean rooms to be used for the assembly of spacecraft which will be sterilized.     

Quick Counting Method for Estimating the Number of Viable Microbes on Food and Food Processing Equipment

A rapid method for estimating the extent of microbial contamination on food and on food processing equipment is described. Microbial cells are rinsed from food or swab samples with sterile diluent and concentrated on the surface of membrane filters. The filters are incubated on a suitable bacteriological medium for 4 hr at 30 C, heated at 105 C for 5 min, and stained. The membranes are then dried at 60 C for 15 min, rendered transparent with immersion oil, and examined microscopically. Data obtained by the rapid method were compared with counts of the same samples determined by the standard plate count method. Over 60 comparisons resulted in a correlation coefficient of 0.906. Because the rapid technique can provide reliable microbiological count information in extremely short times, it can be a most useful tool in the routine evaluation of microbial contamination of food processing facilities and for some foods.     

Ecology of Microbial Saprophytes and Pathogens in Tissue Culture and Field-Grown Plants: Reasons for Contamination Problems In Vitro

This review compares published surveys of microbial populations in plant tissue and cell cultures with the microbial saprophytes and pathogens found on field grown plants and microbial populations in the laboratory environment. From this comparison and the measured reduction in contamination after improvements in working practices in the laboratory, conclusions can be drawn about the importance of the explant and the laboratory as sources of contamination.

Mechanisms of pathogenicity in vitro are described to explain why bacteria, fungi, and yeasts that are not pathogenic to plants in the field become pathogens in plant tissue cultures. Conversely, plant metabolism and its effect on the tissue culture environment are described to explain why prokaryotes, viruses, and viroids that cause disease in the field can stay latent in vitro.

Detection methods for latent contaminants in plant tissue cultures are summarized, and the strategies and methods for prevention or treatment of contamination are discussed.     

Prediction of growth of Bacillus megaterium spores as affected by gamma radiation dose and spore load

AIMS: To provide data on the interaction of radiation dose (x1) and microbial contamination load (x2), as predictor variables, on the percentage of vials exhibiting growth of Bacillus megaterium spores (y). METHODS AND RESULTS: The influence of a wide range of spore loads (1-50 000 spores of B. megaterium vial-1) and gamma radiation doses (0.2-10 kGy) on the contaminated samples was determined. Each contamination load was studied by adding the specified number of spores to 100 vials containing nutrient broth and exposing them to various doses of gamma radiation. Curves representing the number of contaminated vials against the dose of radiation were sigmoidal in shape and the data showed an indirect relationship. Data were analysed by regression analysis which revealed a significant correlation (R2=0.85). The relationship among the tested variables is exponential and can be described by the following equation: y = 1 - (1 - e(0.0173x(1)))(x(2)) It was also estimated that, for each increase of 1 in the number of spores per vial, there is an increase of 1 in the number of contaminated vials. CONCLUSION: The two variables (x1 and x2) have great influence on the radiation sterilization efficiency and the proposed mathematical model is valid for the prediction of this efficiency. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of the present investigation can be of useful industrial application and can help to set acceptance and rejection limits for the production of materials vulnerable to microbial contamination.     

Seasonal dynamics of shallow-hyporheic-zone microbial community structure along a heavy-metal contamination gradient

Heavy metals contaminate numerous freshwater streams and rivers worldwide. Previous work by this group demonstrated a relationship between the structure of hyporheic microbial communities and the fluvial deposition of heavy metals along a contamination gradient during the fall season. Seasonal variation has been documented in microbial communities in numerous terrestrial and aquatic environments, including the hyporheic zone. The current study was designed to assess whether relationships between hyporheic microbial community structure and heavy-metal contamination vary seasonally by monitoring community structure along a heavy-metal contamination gradient for more than a year. No relationship between total bacterial abundance and heavy metals was observed (R(2) = 0.02, P = 0.83). However, denaturing gradient gel electrophoresis pattern analysis indicated a strong and consistent linear relationship between the difference in microbial community composition (populations present) and the difference in the heavy metal content of hyporheic sediments throughout the year (R(2) = 0.58, P < 0.001). Correlations between heavy-metal contamination and the abundance of four specific phylogenetic groups (most closely related to the alpha, beta, and gamma-proteobacteria and cyanobacteria) were apparent only during the fall and early winter, when the majority of organic matter is deposited into regional streams. These seasonal data suggest that the abundance of susceptible populations responds to heavy metals primarily during seasons when the potential for growth is highest.     

Sensitivity of biochemical test in comparison with other methods for the detection of mycoplasma contamination in human and animal cell lines stored in the National Cell Bank of Iran

Mycoplasma contamination in cell culture is considered as serious problem in the manufacturing of biological products. Our goal in this research is to find the best standard and rapid method with high sensitivity, specificity, accuracy and predictive values of positive and negative results for detection of mycoplasma contamination in cell cultures of the National Cell Bank of Iran. In this study, 40 cell lines suspected to mycoplasma contamination were evaluated by three different methods: microbial culture, enzymatic mycoalert^® and molecular. Enzymatic evaluation was performed using the mycoalert^® kit while in the molecular technique, a universal primer pair was designed based on the common and fixed 16SrRNA ribosomal sequences used. Mycoplasma contaminations in cell cultures with molecular, enzymatic and microbial culture methods were determined as 57.5, 52.5 and 40 %, respectively. These results confirmed the higher rate of sensitivity, specificity and accuracy for the molecular method in comparison with enzymatic and microbial methods. Polymerase chain reaction (PCR) assay based on fixed and common sequences in the 16SrRNA, is a useful valuable and reliable technique with high sensitivity, specificity and accuracy for detection of mycoplasma contamination in cell cultures and other biological products. The enzymatic mycoalert^® method can be considered as a substitution for conventional microbial culture and DNA staining fluorochrome methods due to its higher sensitivity, specificity and speed of detection (<20 min).     

啤酒腐败微生物与啤酒微生物稳定性研究进展

尽管生产环境和卫生条件已经得到大幅改善,但啤酒生产过程中仍然会发生微生物污染,因此,真正意义上的啤酒纯种酿制是很难实现的。为了有效控制生产过程中的微生物污染,本文系统介绍了啤酒微生物的多样性及其在生产工序中的分布,探讨了啤酒环境对抑制啤酒微生物污染的影响,讨论了啤酒微生物对啤酒质量与风味的积极贡献,提出合理控制外源微生物侵染是形成不同啤酒典型特征的关键。