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At least 19 records

Viral Enumeration Using Cost-Effective Wet-Mount Epifluorescence Microscopy for Aquatic Ecosystems and Modern Microbialites

Enumeration is a fundamental measure of community ecology in which viruses represent the most numerous biological identities. Epifluorescence microscopy (EFM) has been the gold standard method for environmental viral enumeration for over 25 years. Currently, standard EFM methods using the Anodisc filters are no longer cost-effective (>$15 per slide) and have yet to be applied to modern microbialites. Microbialites are microbially driven benthic organosedimentary deposits that have been present for most of Earth’s history. We present a cost-effective method for environ mental viral enumeration from aquatic samples, microbial mats, and exopolymeric substances (EPSs) within modern microbialites using EFM. Our integrated approach, which includes filtration, differential centrifugation, chloroform treatment, glutaralde hyde fixation, benzonase nuclease treatment, probe sonication (EPS and mat only), SYBR Gold staining, wet mounting, and imaging, provides a robust method for modern microbialites and aquatic samples. Viral abundances of modern microbialites and aquatic samples collected from Fayetteville Green Lake (FGL) and Great Salt Lake (GSL) did not differ across ecosystems by sample type. EPS and microbial mat samples had an order of magnitude higher viral-like particle abundance when compared to water regardless of the ecosystem (107 vs 106). Viral enumeration allows for estimates of total viral numbers and weights. The entire weight of all the viruses in FGL and GSL are ~598 g and ~2.2 kg, respectively. Further development of EFM methods and software is needed for viral enumeration. Our method provides a robust and cost-effective (~$0.75 per sample) viral enumeration within modern microbialites and aquatic ecosystems.

Madeline Bellanger

Ensemble Weight Enumerators for Protograph LDPC Codes

Recently LDPC codes with projected graph, or protograph structures have been proposed. In this paper, finite length ensemble weight enumerators for LDPC codes with protograph structures are obtained. Asymptotic results are derived as the block size goes to infinity. In particular we are interested in obtaining ensemble average weight enumerators for protograph LDPC codes which have minimum distance that grows linearly with block size. As with irregular ensembles, linear minimum distance property is sensitive to the proportion of degree-2 variable nodes. In this paper the derived results on ensemble weight enumerators show that linear minimum distance condition on degree distribution of unstructured irregular LDPC codes is a sufficient but not a necessary condition for protograph LDPC codes.

enumerators

A direct viable count method for the enumeration of attached bacteria and assessment of biofilm disinfection

This report describes the adaptation of an in situ direct viable count (in situ DVC) method in biofilm disinfection studies. The results obtained with this technique were compared to two other enumeration methods, the plate count (PC) and conventional direct viable count (c-DVC). An environmental isolate (Klebsiella pneumoniae Kp1) was used to form biofilms on stainless steel coupons in a stirred batch reactor. The in situ DVC method was applied to directly assess the viability of bacteria in biofilms without disturbing the integrity of the interfacial community. As additional advantages, the results were observed after 4 h instead of the 24 h incubation time required for colony formation and total cell numbers that remained on the substratum were enumerated. Chlorine and monochloramine were used to determine the susceptibilities of attached and planktonic bacteria to disinfection treatment using this novel analytical approach. The planktonic cells in the reactor showed no significant change in susceptibility to disinfectants during the period of biofilm formation. In addition, the attached cells did not reveal any more resistance to disinfection than planktonic cells. The disinfection studies of young biofilms indicated that 0.25 mg/l free chlorine (at pH 7.2) and 1 mg/l monochloramine (at pH 9.0) have comparable disinfection efficiencies at 25 degrees C. Although being a weaker disinfectant, monochloramine was more effective in removing attached bacteria from the substratum than free chlorine. The in situ DVC method always showed at least one log higher viable cell densities than the PC method, suggesting that the in situ DVC method is more efficient in the enumeration of biofilm bacteria. The results also indicated that the in situ DVC method can provide more accurate information regarding the cell numbers and viability of bacteria within biofilms following disinfection.

NASA Discipline Number 04-10

Wind Supply Chain Security: Hardware Enumeration and Analysis

This project, undertaken by Idaho National Laboratory (INL) for the Department of Energy (DOE) Wind Energy Technologies Office (WETO), focused on the enumeration and analysis of six key devices important to wind technologies. The devices analyzed included Beckhoff Bus Terminal Controllers (BK1120 and BC9000), a Beckhoff Economy Built-in Panel PC (CP6231), an N-Tron Managed Industrial Ethernet Switch (711FX3), a Bachmann M1 Gateway, and a Bachmann Smart Power Plant Controller. Device selection was driven by availability and budget constraints, with several components sourced from existing wind farms and others procured through a co-agreement with another WETO-funded project. The project's primary objective was to create a hardware bill of materials (HBOM) for each device, identifying and documenting all components to assess potential security and supply chain risks. A detailed analysis revealed over 750 unique components across the six devices, with 80% successfully identified and accompanied by datasheets. Notably, Texas Instruments emerged as the leading supplier, providing over 16% of the components, followed by ON Semiconductor at 11.3%, Analog Devices at 5.3%, and Renesas Electronics Corp at 4.1%. Other notable vendors included Toshiba Corporation, iC-Haus Corporation, Atmel, Vishay, and STMicroelectronics. The enumeration process involved thorough documentation of each component, including its designation, quantity, identifiers, pin package, description, vendor, model, and country of origin. This process provided valuable insights into the complexity and diversity of the electronic systems within these wind devices. It also highlighted the distinct separation of components between vendors, suggesting a trend of vendor-specific component usage. Key findings from the project emphasized the importance of broadening the scope of vendor analysis in future research to gain a comprehensive understanding of component distribution and commonality. The identification of vendor-specific component usage patterns offers new avenues for research and underscores the significance of continued investigation in this field. Overall, this project provides critical insights into the component composition of wind devices, aiding in the development of improved supply chain management and component sourcing strategies. The results contribute valuable knowledge to the wind technology sector, laying the groundwork for enhanced security and resilience in wind energy systems.

17 - WIND ENERGY

Apparatus and process for microbial detection and enumeration

An apparatus and process for detecting and enumerating specific microorganisms from large volume samples containing small numbers of the microorganisms is presented. The large volume samples are filtered through a membrane filter to concentrate the microorganisms. The filter is positioned between two absorbent pads and previously moistened with a growth medium for the microorganisms. A pair of electrodes are disposed against the filter and the pad electrode filter assembly is retained within a petri dish by retainer ring. The cover is positioned on base of petri dish and sealed at the edges by a parafilm seal prior to being electrically connected via connectors to a strip chart recorder for detecting and enumerating the microorganisms collected on filter.

Wilkins, J. R.

WHAMII - An enumeration and insertion procedure with binomial bounds for the stochastic time-constrained traveling salesman problem

This paper presents an algorithm (WHAMII) designed to solve the Artificial Intelligence Design Challenge at the 1987 AIAA Guidance, Navigation and Control Conference. The problem under consideration is a stochastic generalization of the traveling salesman problem in which travel costs can incur a penalty with a given probability. The variability in travel costs leads to a probability constraint with respect to violating the budget allocation. Given the small size of the problem (eleven cities), an approach is considered that combines partial tour enumeration with a heuristic city insertion procedure. For computational efficiency during both the enumeration and insertion procedures, precalculated binomial probabilities are used to determine an upper bound on the actual probability of violating the budget constraint for each tour. The actual probability is calculated for the final best tour, and additional insertions are attempted until the actual probability exceeds the bound.

Dahl, Roy W.

Rapid direct methods for enumeration of specific, active bacteria in water and biofilms

Conventional methods for detecting indicator and pathogenic bacteria in water may underestimate the actual population due to sublethal environmental injury, inability of the target bacteria to take up nutrients and other physiological factors which reduce bacterial culturability. Rapid and direct methods are needed to more accurately detect and enumerate active bacteria. Such a methodological advance would provide greater sensitivity in assessing the microbiological safety of water and food. The principle goal of this presentation is to describe novel approaches we have formulated for the rapid and simultaneous detection of bacteria plus the determination of their physiological activity in water and other environmental samples. The present version of our method involves the concentration of organisms by membrane filtration or immunomagnetic separation and combines an intracellular fluorochrome (CTC) for assessment of respiratory activity plus fluorescent-labelled antibody detection of specific bacteria. This approach has also been successfully used to demonstrate spatial and temporal heterogeneities of physiological activities in biofilms when coupled with cryosectioning. Candidate physiological stains include those capable of determining respiratory activity, membrane potential, membrane integrity, growth rate and cellular enzymatic activities. Results obtained thus far indicate that immunomagnetic separation can provide a high degree of sensitivity in the recovery of seeded target bacteria (Escherichia coli O157:H7) in water and hamburger. The captured and stained target bacteria are then enumerated by either conventional fluorescence microscopy or ChemScan(R), a new instrument that is very sensitive and rapid. The ChemScan(R) laser scanning instrument (Chemunex, Paris, France) provides the detection of individual fluorescently labelled bacterial cells using three emission channels in less than 5 min. A high degree of correlation has been demonstrated between results obtained with the ChemScan and traditional plate counts of mixed natural bacterial populations in water. The continuing evolution of these methods will be valuable in the rapid and accurate analysis of environmental samples.

Review, Tutorial

Blueprint: Open Worldwide Application Security Project Top 10 Common Weakness Enumerations for Electric Vehicle Supply Equipment

This report categorizes the top 10 security weaknesses in electric vehicle supply equipment (EVSE) using a similar approach by the Open Worldwide Application Security Project (OWASP) Top 10, and it describes example exploitations, methods for prevention, and related common weakness enumerations (CWEs) for each category. The weaknesses listed in this report could lead to security issues or vulnerabilities in EVSE if they are exploited by an attacker to compromise the confidentiality, integrity, or availability of a system. This report also recommends EVSE industry best practices and remediations for the identified weaknesses to use as guidelines for their secure products and systems.

33 ADVANCED PROPULSION SYSTEMS

SEAL /Subnetwork Enumeration And Listing/

SEAL /Subnetwork Enumeration And Listing/ computer program uses combinatorial techniques to generate all of the nonredundant subnetwork configurations derivable from an asymmetrical network or device. This is accomplished by a systematic shorting and opening of accessible terminals to obtain the desired allowable configurations.

Happ, W. W.

Finding the complete path and weight enumerators of convolutional codes

A method for obtaining the complete path enumerator T(D, L, I) of a convolutional code is described. A system of algebraic equations is solved, using a new algorithm for computing determinants, to obtain T(D, L, I) for the (7,1/2) NASA standard code. Generating functions, derived from T(D, L, I) are used to upper bound Viterbi decoder error rates. This technique is currently feasible for constraint length K less than 10 codes. A practical, fast algorithm is presented for computing the leading nonzero coefficients of the generating functions used to bound the performance of constraint length K less than 20 codes. Code profiles with about 50 nonzero coefficients are obtained with this algorithm for the experimental K = 15, rate 1/4, code in the Galileo mission and for the proposed K = 15, rate 1/6, 2-dB code.

Onyszchuk, I.

Rapid enumeration of viable bacteria by image analysis

A direct viable counting method for enumerating viable bacteria was modified and made compatible with image analysis. A comparison was made between viable cell counts determined by the spread plate method and direct viable counts obtained using epifluorescence microscopy either manually or by automatic image analysis. Cultures of Escherichia coli, Salmonella typhimurium, Vibrio cholerae, Yersinia enterocolitica and Pseudomonas aeruginosa were incubated at 35 degrees C in a dilute nutrient medium containing nalidixic acid. Filtered samples were stained for epifluorescence microscopy and analysed manually as well as by image analysis. Cells enlarged after incubation were considered viable. The viable cell counts determined using image analysis were higher than those obtained by either the direct manual count of viable cells or spread plate methods. The volume of sample filtered or the number of cells in the original sample did not influence the efficiency of the method. However, the optimal concentration of nalidixic acid (2.5-20 micrograms ml-1) and length of incubation (4-8 h) varied with the culture tested. The results of this study showed that under optimal conditions, the modification of the direct viable count method in combination with image analysis microscopy provided an efficient and quantitative technique for counting viable bacteria in a short time.

Non-NASA Center

Enumerating Spore-Forming Bacteria Airborne with Particles

A laboratory method has been conceived to enable the enumeration of (1) Cultivable bacteria and bacterial spores that are, variously, airborne by themselves or carried by, parts of, or otherwise associated with, other airborne particles; and (2) Spore-forming bacteria among all of the aforementioned cultivable microbes.

Lin, Ying