Count Procedure Research Articles

Differential enumeration of subpopulations in concentrated frozen and lyophilized cultures of Lactobacillus delbrueckii ssp. bulgaricus

Differential enumeration of subpopulations in concentrated frozen and lyophilized cultures of Lactobacillus delbrueckii ssp. bulgaricus ND02 derived from 2 propagation procedures was determined. The subpopulations consisted of 3 categories (physiological states): viable cells capable of forming colonies on agar plates (VC+), viable cells incapable of forming colonies on agar plates (VC-), widely referred to as viable but nonculturable (VBNC) cells, and nonviable or dead cells (NVC). Counts of VC+ were recorded using a conventional plate count procedure. A fluorescent vital staining procedure that discriminates between viable (VC+ and VC-) and NVC cells was used to determine the number of viable and nonviable cells. Both propagation procedures had 2 variables: in procedure (P)1, the propagation medium was rich in yeast extract (4.0%) and the pH was maintained at 5.7; in P2, the medium was devoid of yeast extract and the pH was maintained at 5.1. The results showed that post-propagation operations-concentration of cells by centrifugation and subsequent freezing or lyophilization of cell concentrate-induced different degrees of transience from VC+ to VC- states in cells derived from P1 and P2. Compared with cells derived from P2, cells from P1 were more labile to stress associated with centrifugation, freezing, and lyophilization, as revealed by differential counting.

Sediment size distribution at three rivers with different types of land use in Endau Catchment Area, Kluang, Johor, Malaysia

Land use along the river will determine the substrate size and distributions. Substrate particle size will influence aquatic life because substrate is one of the main components forming aquatic habitat. This aquatic habitat is very important in determining river water quality as well as river health. Therefore, identifying the sediment size distribution based on land use is very crucial in river maintenance. The objective of this study is to determine the particle size variation between upper reach and lower reach of the sampling station as well as variation from different land use cover at different sub-catchment areas. The pebble counts was conducted at upper reach station and lower reach station by applying the Pebble Count Protocols developed by the West Virginia Department of Environmental Protection and Wolmen Pebble Count Procedure. The average median (D50) for Dengar River ranged between 4.5 mm for upper portion and 2.4 mm for lower portion. Similar observations were found at the Mengkibol River, where particle size ranged between 3.0 mm for upper portion and 2.4 mm for lower portion. As for Madek River which represents land use area for logging activity, the results obtained were different in which the upper portion the particle size was smaller (D50 = 4.5 mm) than the lower portion (D50 = 41.5 mm). The finding of this study will be a basis for river catchment management study and can be used by river management authorities in the country for river management planning.Keyword: pebble count, sediment size, average median, fine gravel, very fine gravel, urban, oil palm, logging, land use

English

Bacterial biofilms forming on indwelling urinary catheters continue to represent a public health problem because they are associated with urinary tract infections (UTIs). This study was undertaken to evaluate the ability of immobilized Lactobacillus acidophilus cells to inhibit biofilm formation on catheter surfaces. Urine bacteria and Lactobacillus species were isolated from urine and vaginal swabs (HVS), respectively. Immobilization of L. acidophilus on catheter samples was achieved using sodium alginate and the inhibition of urine bacteria by the immobilized Lactobacillus cells was evaluated by microscopy and viable cell count procedures following co-culture of the immobilized cells and urine bacteria. Results showed that pre-coating of catheter surfaces with L. acidophilus before exposure to urine bacteria significantly (p<0.05) reduced attachment of some urine bacteria to the catheter surfaces. Staphylococcus aureus, Klebsiella and Escherichia coli were significantly inhibited, while Pseudomonas aeruginosa was not inhibited. Furthermore, crude bacteriocin preparations from the Lactobacillus cells had antimicrobial activity against the urine bacteria. This study shows that pre-coating of catheter surfaces with L. acidophilus could be an effective strategy for controlling biofilm formation on urinary catheters. Key words: Biofilm, catheter, Lactobacillus, immobilization, sodium alginate. &nbsp

Guideline Implementation: Prevention of Retained Surgical Items

A surgical item unintentionally retained in a patient after an operative or other invasive procedure is a serious, preventable medical error with the potential to cause the patient great harm. Perioperative RNs play a key role in preventing retained surgical items (RSIs). The updated AORN “Guideline for prevention of retained surgical items” provides guidance for implementing a consistent, multidisciplinary approach to RSI prevention; accounting for surgical items; preventing retention of device fragments; reconciling count discrepancies; and using adjunct technologies to supplement manual count procedures. This article focuses on key points of the guideline to help perioperative personnel provide optimal care during a procedure. Key points addressed include taking responsibility for RSI prevention as a team; minimizing distractions, noise, and interruptions during counts; using consistent counting methods; reconciling discrepancies; and participating in performance-improvement activities. Perioperative RNs should review the complete guideline for additional information and for guidance in writing and updating policies and procedures.

The Words of Violence: Autobiographical Narratives of Abused Women

Personal narratives tell the stories of people’s lives as well as provide insight into the meaning of those experiences. These narratives both reflect and are influenced by the relationships within which an individual is embedded. In this study, autobiographical narratives for two groups of women were compared: women who had experienced habitual gender-based domestic violence in their couple relationships and women who had not. The language of narratives was analyzed by LIWC (Language Inquiry and Word Count procedure). Results showed that the language and structure of narratives by women with a history of domestic violence indicated greater stress and trauma, more incoherent space-time organization, and poorer relationship quality. Women who experienced violence wrote longer narratives that contained proportionately more negative emotion words and more references to cognitions and physical/body issues, and indicated more disorganized structure by means of incoherent use of verbal tense, more impoverished use of connectives, and greater use of negative sentence syntax and discrepancy words. They also included proportionately more pronoun references to ‘I’,‘You’,’ and ‘He’, indicating self vs. partner conflictual relationships. However, women who had experienced relationship violence for longer decreased their references to the emotions of fear and anxiety, suggesting adaptation to violence over time.

Comparison of the Antimicrobial Properties of Silver Impregnated Vascular Grafts with and without Triclosan

The aim was to compare the antimicrobial efficacy of the silver impregnated collagen coated polyester vascular graft (IGS) with an identical graft combining silver and triclosan (IGSy). This was an in vitro study. A non-antimicrobial collagen polyester vascular graft served as control (IG). The IG, IGS, and IGSy grafts were contaminated separately with inoculates of each of the following micro-organisms: Staphylococcus epidermidis (SE), methicillin resistant Staphylococcus aureus (MRSA), and Escherichia coli producing extended spectrum beta-lactamase (ESBL-EC) or Candida albicans (CA). MRSA, ESBL-EC, and CA were obtained from retrieved infected grafts. The in vitro antimicrobial efficacies of the contaminated grafts were evaluated by time to kill assays over a 24 hour period in accordance with CLSI Guideline M26-A. All assays were repeated six times. Bacterial survival numbers were obtained at 1, 4, 8, and 24 hours using a standard plate count procedure. Bactericidal activity was defined as a 3 log10 reduction factor (logRF). To calculate the overall difference in the mean log10 CFU/mL within 24 hours, a one way ANOVA with a Bonferroni correction was calculated separately for each graft. The IG graft showed an increase in the number of viable organisms for the four strains tested. IGSy offered better antimicrobial properties than IGS for both ESBL-EC and MRSA, since only the IGSy graft achieved > 3 logRF and fulfilled the standard criteria for bactericidal activity at 24 hours with 3.78 and 4.08 logRF, respectively. For samples inoculated with SE and CA, both antimicrobial grafts achieved 24 hour bactericidal activity with > 3 logRF. However, for CA the one-way ANOVA analysis demonstrated that the IGSy graft performed differently in terms of speed of antimicrobial action, appearing more active as early as 4 hours following inoculation (p = .007). In the in vitro conditions, the Synergy vascular graft combining silver with triclosan demonstrated better short-term antimicrobial activity than the silver graft for all micro-organisms tested.

Faecal pollution and solar purification of community water sources within Lake Naivasha basin, Kenya

As in other parts of Africa, and in other developing nations, the rise in the human population and anthropogenic activities within the Lake Naivasha basin is causing an increase in human health risks due to faecal contamination of domestic water sources. This study investigated faecal pollution of community water sources within the Lake Naivasha basin by measuring the densities of total coliforms, Escherichia coli, intestinal enterococci, Clostridium perfringens and heterotrophic bacteria in Lake Naivasha, the Malewa and Gilgil Rivers, and boreholes using membrane filtration techniques and heterotrophic plate count procedures. Selected physico-chemical parameters were also measured in situ from all the water sources sampled. Lakes and rivers had significantly higher microbial abundances than boreholes. Unlike boreholes, surface sources (rivers and lake) showed significant variation with respect to sampling sites for all the microbiological parameters (P < 0.05). The use of solar radiation in water disinfection with temperatures of 75 °C after 30 minutes from pasteurization point (time zero) fully eradicated E. coli and total coliforms from all the water sources. In conclusion, there is faecal pollution in water sources used by communities within the Lake Naivasha basin. The use of solar radiation is therefore recommended for water purification to reduce likely incidences of waterborne diseases.

Surgical swab counting: a qualitative analysis from the perspective of the scrub nurse.

The aim of the study was to conduct a qualitative exploration of the sociotechnical processes underlying retained surgical swabs, and to explore the fundamental reasons why the swab count procedure and related protocols fail in practice. Data was collected through a set of 27 semistructured qualitative interviews with scrub nurses from a large, multi-site teaching hospital. Interview transcripts were analysed using established constant comparative methods, moving between inductive and deductive reasoning. Key findings were associated with interprofessional perspectives, team processes and climate and responsibility for the swab count. The analysis of risk factors revealed that perceived social and interprofessional issues played a significant role in the reliability of measures to prevent retained swabs. This work highlights the human, psychological and organisational factors that impact upon the reliability of the process and gives rise to recommendations to address contextual factors and improve perioperative practice and training.

Holographic shell model: Stack data structure inside black holes?

Rather than tiling the black hole horizon by Planck area patches, we suggest that bits of information inhabit, universally and holographically, the entire black core interior, a bit per a light sheet unit interval of order Planck area difference. The number of distinguishable (tagged by a binary code) configurations, counted within the context of a discrete holographic shell model, is given by the Catalan series. The area entropy formula is recovered, including Cardy's universal logarithmic correction, and the equipartition of mass per degree of freedom is proven. The black hole information storage resembles, in the count procedure, the so-called stack data structure.

An experimental study on the conversion between IFPUG and UCP functional size measurement units

The use of functional size measurement (FSM) methods in software development organizations is growing during the years. Also, object oriented (OO) techniques have become quite a standard to design the software and, in particular, Use Cases is one of the most used techniques to specify functional requirements. Main FSM methods do not include specific rules to measure the software functionality from its Use Cases analysis. To deal with this issue some other methods like Kramer’s functional measurement method have been developed. Therefore, one of the main issues for those organizations willing to use OO functional measurement method in order to facilitate the use cases count procedure is how to convert their portfolio functional size from the previously adopted FSM method towards the new method. The objective of this research is to find a statistical relationship for converting the software functional size units measured by the International Function Point Users Group (IFPUG) function point analysis (FPA) method into Kramer-Smith’s use cases points (UCP) method and vice versa. Methodologies for a correct data gathering are proposed and results obtained are analyzed to draw the linear and non-linear equations for this correlation. Finally, a conversion factor and corresponding conversion intervals are given to establish the statistical relationship.

Comparison of Direct Microbial Count Procedures for Planktonics and Sessiles Enumeration

In the present investigation, the sensitivity of different direct microbial count procedures applied on systems containing both planktonics and sessiles was tested. The direct count pour plate was compared with direct epifluorescent microscopic enumerations in order to evaluate the efficiency of the studied techniques in giving information about microbial activity or viability. Our results indicate that the standard plate count procedure is the most sensitive method to estimate viable and cultivable planktonic cells. On the other hand, direct enumeration by epifluorescent microscopy may become an interesting alternative to count sessile cells.

Clinical Issues—July 2013

This MonthDisposal of unused medicationsKey words: pharmaceutical waste, medication, disposal, drug take‐back programs.Debris in sterile packKey words: event‐related sterilization, debris, sterile technique, breaks in sterile technique.Time‐out checklistKey words: documentation, Universal Protocol, time out, surgical checklist.Count sheetsKey words: count sheets, documentation, standardized count procedures.

A mode count procedure for mid-frequency analysis of complex vibro-acoustic systems

Satellites and space equipment are exposed to high diffuse acoustic fields during the launch process that are able to excite the structure. The use of adequate techniques to model the structure response to the acoustic loads is a fundamental task during the design and verification phases. Currently, the efforts are focused in the mid-frequency range that in this paper is defined as the frequency range in which a combination of models is required. To select the most accurate modelling technique in each frequency band, this paper proposes a procedure based on the modal count of each subsystem. The procedure is applied in an ordinary satellite case and the frequency response continuity and agreement with experimental results are shown.

Determination of the relative abundance and distribution of bacteria and fungi in Bonny light crude oil-contaminated sandy loam soil

This study was undertaken to investigate the effects of Bonny light crude oil (specific gravity = 0.81; API gravity 43.20) on the numerical composition of soil bacteria and fungi and relative distribution of Gram positive and Gram negative soil bacteria. Eight different levels of the crude oil (0.5, 1.0, 2.0, 2.5, 5.0, 10.0, 15.0 or 20.0% v/w of soil) were used for the controlled pollution of soil samples obtained from the Botanical Garden, University of Nigeria Nsukka. Studies on the effects of crude oil on bacterial and fungal populations were carried out by plate count procedures using nutrient agar and Sabouraud dextrose agar respectively. Crude oil significantly (p<0.05) inhibited bacterial and fungal growth in a dose-dependent manner. Bacterial counts in the control soil sample ranged between 2.32 × 109 and 2.80 × 109 cfu/g while those of the contaminated samples ranged between 2.00 × 108 and 2.77 × 109 cfu/g. Fungal counts ranged from 1.02 × 107 to 1.39 × 107 cfu/g in the control and 1.60 × 105 to 1.18 × 107 cfu/g in the contaminated samples. At 15.0 and 20.0% levels of crude oil, the growth inhibitory effects of crude oil were maximum for bacteria and fungi respectively. Microbial respiration decreased concomitantly with increase in crude oil pollution during the first four weeks of the study. There was a prevalence of Gram positive bacteria over Gram negatives in the unpolluted soil but a preponderance of Gram negative rods over Gram positives and other morphological forms of Gram negatives in the polluted samples. Key words: Bonny light crude oil, bacteria, fungi, contamination, hydrocarbon, biodegradation.

Assessment of equivalent initial flaw size estimation methods in fatigue life prediction using compact tension specimen tests

To assess the efficiency of the Equivalent Initial Flaw Size (EIFS) concept in the life estimation of mechanical components, a novel approach is outlined in this paper. For this purpose, experimental tests are conducted on the compact tension specimens made of 4340 steel and the number of cycles required for the crack to grow from the end of the notch up to the fracture of the specimen is counted. In fact, the fatigue cycling for the pre-crack initiation is a part of cycle count procedure and it is assumed that an initial micro-crack exists at the end of the notch, which grows due to fatigue loading, the length of which is estimated using the EIFS method. Three methods of back extrapolation, Kitagawa–Takahashi diagram, and time to crack initiation are used in order to estimate EIFS and their results are compared. For estimating EIFS by Kitagawa–Takahashi diagram, the threshold value of the stress intensity factor (ΔKth) is required. In this paper, ΔKth is estimated by both K-increasing and K-decreasing methods. The experimental results show that the value of ΔKth estimated by the K-increasing method is lower than that estimated by K-decreasing method. It is observed that the estimated EIFS by back extrapolation method and TTCI method is dependent on the loading amplitude, while the estimated EIFS by Kitagawa–Takahashi diagram is identical for all loading amplitudes and it can be considered as the material property. The predicted life based on the Kitagawa–Takahashi method is in relatively good agreement with the experimental results. However, the TTCI method does not have sufficient accuracy especially in low amplitude fatigue.

Single‐cell analysis of cell viability after a biocide treatment unveils an absence of positive correlation between two commonly used viability markers

Discrimination among viable/active or dead/inactive cells in a microbial community is a vital question to address issues on ecological microbiology or microbiological quality control. It is commonly assumed that metabolically active cells (ChemchromeV6 [CV6] procedure) correspond to viable cells (direct viable count procedure [DVC]), although this assumption has never been demonstrated and is therefore a matter of debate. Indeed, simultaneous determination of cell viability and metabolic activity has never been performed on the same cells. Here, we developed a microfluidic device to investigate the viability and the metabolic activity of Escherichia coli cells at single-cell level. Cells were immobilized in a flow chamber in which different solutions were sequentially injected according to different scenarios. By using time-lapse microscopy combined with automated tracking procedures, we first successfully assessed the ability of cells to divide and their metabolic activity at single-cell level. Applying these two procedures on the same cells after a hypochlorous acid (HOCl) treatment, we showed that the ability of cells to divide and their metabolic activity were anticorrelated. These results indicate that the relation between CV6 uptake and cell viability may be partially incorrect. Care must be taken in using the terms “CV6-positive” and “viable” synonymously.

In vitro evaluation of the antimicrobial efficacy of a new silver-triclosan vs a silver collagen-coated polyester vascular graft against methicillin-resistant Staphylococcus aureus

Vascular graft infection is a rare but serious complication of vascular reconstructive surgery. This in vitro study investigated the antimicrobial efficacy of a new, silver-triclosan collagen-coated polyester vascular graft compared with a silver collagen-coated polyester vascular graft alone during the first 24 hours. The antimicrobial efficacy of the investigated vascular grafts was assessed by performing a time-kill kinetic assay following Clinical and Laboratory Institute Standards-approved guidelines M26-A. For the purpose of the experimental study, the ATCC 33591 strain of methicillin-resistant Staphylococcus aureus (American Type Culture Collection, Manassas, Va) was used. All assays were repeated sixfold. Bacterial survival numbers were obtained at 1, 4, 8, 12, and 24 hours using a standard plate count procedure. Bactericidal activity was defined as a 3 log(10) reduction factor (logRF), according to the approved guideline M26-A. Both antimicrobial vascular grafts achieved >3 logRF and fulfilled the efficacy criterion for bactericidal activity but performed differently in their speed of antimicrobial action. The silver-triclosan vascular graft achieved 3.37 logRF after 8 hours, and the silver vascular graft showed a 4.19 logRF after 24 hours. The silver-triclosan graft yielded significantly lower colony-forming units/mL counts after 4 hours compared with the silver graft (4.29 × 10(4) vs 1.03 × 10(6); P = .031). Both antimicrobial collagen-coated polymer vascular grafts showed bactericidal activity against methicillin-resistant Staphylococcus aureus in vitro. Although the silver-triclosan vascular graft showed a faster antimicrobial efficacy, the silver graft exhibited its antimicrobial properties after 24 hours. Which concept will protect an implanted vascular prosthetic graft better from bacterial contamination and subsequent infection needs to be investigated further in in vivo animal and clinical studies.

A comparison of fluorescent stains for the assessment of viability and metabolic activity of lactic acid bacteria

Lactic acid bacteria (LAB) are used as starter or probiotic cultures in the food and pharmaceutical industries and, therefore, rapid and accurate methods for the detection of their viability are of practical relevance. In this study 10 LAB strains, belonging to the genera Enterococcus, Lactococcus, Leuconostoc, Lactobacillus, Streptococcus and Weissella, were subjected to heat and oxidative stresses and cell injury or death was assessed comparing different fluorescent probes (Syto 9; Propidium Iodide, PI; 4,6-diamidino-2-phenylindole, DAPI; 5,(6)-carboxyfluorescein diacetate, cFDA) to identify the stain combination which most reliably allowed the detection of live/metabolically active and dead cells. Protocols for specimen preparation and staining were optimized and a simple procedure for automated cell counts was developed using NIH ImageJ macros. Cysteine and semi-solid agar solution were efficiently used as anti-fading agent and mounting medium, respectively. The double staining cFDA-PI apparently offered the best and most versatile indication of both cell metabolic activity and membrane integrity. An excellent correlation between manual and automated cell counts for the majority of strain/stain combinations was found. This work provides a simple protocol for specimen preparation and staining based on the use of safe, easy to prepare and inexpensive reagents as compared to other methods. Additionally, the automated cell count procedure developed can be applied to several bacterial species and allows an increase in the number of experimental trials and the reproducibility and sensitivity of the analysis.

Confirmation of a natural hybrid species inPetrocosmea(Gesneriaceae) based on molecular and morphological evidence

Abstract A natural hybrid species inPetrocosmeanamedLongiantherain Yanshan County, Yunnan Province is confirmed for the first time based on molecular and morphological evidence. The character count procedure of the variable characters show thatLongiantherapopulations are characteristic of the intermediate morphological traits between its putative parentsYanshanandPetrocosmea martinii. The nuclear ribosomal internal transcribed spacer region and three chloroplast regions ofmatK,trnL‐F, andtrnT‐Lare sequenced in the putative hybrid and the related species. Both alignment of DNA sequences and the phylogenetic trees could exclude all the other species inPetrocosmeaas the parental species except forYanshanandP. martinii. Eight haplotypes in the 31 internal transcribed spacer sequences and six haplotypes in 42 cpDNA sequences were found from 14 individuals ofLongiantherapopulations. The analyses of DNA sequences, haplotypes, and phylogenetic trees indicate thatLongiantherais likely a hybrid species between its putative parentsYanshanandP. martinii, in whichYanshanmight be the most possible maternal parent. Several factors may contribute to the natural hybridization between these two parental species inPetrocosmea, such as the overlapped geographic distribution, habitats, flowering periods, and shared pollinators. Finally, the new species ofYanshanand the natural hybrid species ofLongiantheraare described.

Long-term Regulation of Genetically Modified Primary Hematopoietic Cells in Dogs

We report long-term results from a large animal model of in vivo selection. Nine years ago, we transplanted two dogs (E900 and E958) with autologous marrow CD34(+) cells that had been transduced with a gammaretrovirus vector encoding a conditionally activatable derivative of the thrombopoietin receptor. Receptor activation through administration of a chemical inducer of dimerization (CID) (AP20187 or AP1903) confers a growth advantage. We previously reported responses to two 30-day intravenous (i.v.) courses of AP20187 administered within the first 8 months post-transplantation. We now report responses to 5-day subcutaneous (s.c.) courses of AP20187 or AP1903 at months 14, 90, and 93 (E900), or month 18 (E958), after transplantation. Long-term monitoring showed no rise in transduced cells in the absence of drug. Retroviral insertion site analysis showed that 4 of 6 (E958) and 5 of 12 (E900) transduced hematopoietic cell clones persisted lifelong. Both dogs were euthanized for reasons unrelated to the gene therapy treatment at 8 years 11 months (E958) and 11 years 1 month (E900) of age. Three clones from E900 remained detectable in each of two secondary recipients, one of which was treated with, and responded to, AP1903. Our results demonstrate the feasibility of safely regulating genetically engineered hematopoietic cells over many years.