Comparison of culture-based methods to enumerate Escherichia coli in tropical and temperate freshwaters

Aims: The specificity of a method for the enumeration of Escherichia coli (chromocult agar, CC) was tested using freshwater samples from a tropical area (Cuba Island) by isolating colonies and identifying them with API (Appareillage et procédé d’identification) strips. Enumerations of E. coli by the most probable number (MPN) microplate method were compared with counts on chromogenic and fluorogenic agar media [CC, rapid E. coli (REC), fluorocult] in tropical and temperate freshwater samples. Methods and Results: A high percentage of specificity (95·7%) for the CC agar enumeration of E. coli was observed. High regression coefficients (log‐log linear regressions) were found between E. coli counts on agar media and by the MPN method. In the tropical environment, counts with REC medium were significantly different from those obtained with the other methods. MPN counts were found to be significantly higher than those obtained using the plate counts methods in the temperate environment. Conclusions: Escherichia coli enumeration methods based on glucuronidase activity appear to be suitable for the evaluation of microbiological quality in the tropical environment featured in this study. Significance and Impact of the Study: The methods for the enumeration of E. coli tested in this study should help improve the evaluation of microbiological contamination of Cuban freshwaters.     

Survival ofLegionella pneumophila in the aquatic environment

Survival ofLegionella pneumophila SG 1 in seawater and river water was assessed using plate counts on buffered charcoal yeast extract agar amended with α-ketoglutarate (BCYEα) and [³H]thymidine-labeling. The [³H]thymidine-labeling method for assessing survival ofL. pneumophila in aquatic environments was compared with viable counts, direct fluorescent microscopy (DFA), and acridine orange direct counts (AODC). Protozoa were isolated from the samples employed in the study and identified by characteristic trophozite and cyst morphology. Selective filtration employing 2.0 μm Nucleopore filters was used to determine the effect of grazing on survival ofL. pneumophila in seawater and river water.Legionella viability as measured by plate counts (CFU/ml), declined to a greater extent than cell lysis, assessed by thymidine, DFA, and AODC counts, suggesting thatL. pneumophila survives in aquatic habitats to a greater extent than revealed through culturable counts.     

Comparison of swabbing and destructive methods for microbiological pig carcass sampling

Aims: To compare the Belgian swabbing sampling method for pig carcasses with the reference destructive method with regard to Escherichia coli and aerobic plate counts, Salmonella and Campylobacter prevalence and their relationship. Methods and Results: Recovery was significantly lower for the swabbing method and corresponded to a recovery of 36% for E. coli counts and 81% for aerobic plate counts in comparison with the destructive method. There was no significant difference between the swabbing and destructive sampling methods for the prevalence of Salmonella or Campylobacter. A higher median for E. coli counts was detected for samples where Salmonella or Campylobacter were detected. The same association was also observed between the median for aerobic plate counts and the presence of Campylobacter. Conclusions: The method of swabbing used, covering 600 cm² on each half‐pig carcass, is efficient for the sampling of pig carcasses in comparison with the reference destructive method. Significance and Impact of the Study: This study describes an efficient method for microbiological pig carcass sampling. The Belgian swabbing method should continue to be used to allow the follow up of bacterial contamination in the Belgian meat production chain.     

Rapid drone semi-automated counts of wintering Greater Flamingos (Phoenicopterus roseus) as a tool for amateur researchers

Drone-conducted aerial counts of waterbirds are at least as accurate as traditional ground counts and drastically reduce fieldwork resource expenditures, but the image processing can be very time-consuming. The aim of the present study was to compare the accuracy, precision and time requirement of semi-automated counts, using drone-derived imagery, carried out with the open-access ImageJ software, in comparison with conventional ground methods for counting wintering Greater Flamingos Phoenicopterus roseus. Semi-automated counts were easy to execute, affordable and safe for birds. They were faster and yielded more reliable and consistent results than traditional ground count approaches, providing a simple, accessible method for use by amateur ornithologists.     

Adhesion of Pseudomonas aeruginosa,Achromobacter xylosoxidans,Delftia acidovorans,Stenotrophomonas maltophilia to contact lenses under the influence of an artificial tear solution

Abstract

Corneal infection is a devastating sight-threatening complication that is associated with contact lens (CL) wear, commonly caused by Pseudomonas aeruginosa. Lately, Achromobacter xylosoxidans, Delftia acidovorans, and Stenotrophomonas maltophilia have been associated with corneal infection. This study investigated the adhesion of these emerging pathogens to CLs, under the influence of an artificial tear solution (ATS) containing a variety of components commonly found in human tears. Two different CL materials, etafilcon A and senofilcon A, either soaked in an ATS or phosphate buffered saline, were exposed to the bacteria. Bacterial adhesion was investigated using a radio-labeling technique (total counts) and plate count method (viable counts). The findings from this study revealed that in addition to P. aeruginosa, among the emerging pathogens evaluated, A. xylosoxidans showed an increased propensity for adherence to both CL materials and S. maltophilia showed lower viability. ATS influenced the viable counts more than the total counts on CLs.     

Towards a standardized index of European rabbit abundance in Iberian Mediterranean habitats

European rabbits Oryctolagus cuniculus are a keystone species in Iberian Mediterranean ecosystems. However, the reliability of methods for estimating rabbit abundance, particularly when at low numbers, is not well understood. Further, better standardization of these methodologies would allow abundance estimates to be more reliably compared between areas and periods. Consequently, we compared several frequently used methods of estimating rabbit abundance and assessed their advantages and disadvantages. During the summers of 2008 and 2009, in 11 localities of central-southern Spain we undertook (a) driving transect counts of rabbits, either at dusk or at night, (b) linear transects on foot recording rabbit signs, (c) cleared-plot pellet counts at permanent plots, and (d) standing crop counts, both with and without habitat stratification. Density estimated at night from driving transects using the Distance Sampling method (the reference method against which all other indices were compared) varied from 0 to 2.69 rabbits ha⁻¹. Most pellet-count indices were significantly related to the reference method. In particular, cleared-plot pellet counts in permanent plots corrected for pellet persistence showed the best correlation with the reference method. In contrast, latrine counts were not related to the reference method index, and we recommend against their use. A standard methodology based on cleared-plot pellets counts could be used to monitor rabbit abundance on a large scale.     

Comparison of two pollen counting methods of slides from a hirst type volumetric trap

Two of the most frequently used methods of pollen counting on slides from Hirst type traps are evaluated in this paper: the transverse traverse method and the longitudinal traverse method. The study was carried out during June–July 1996 and 1997 on slides from a trap at Worcester, UK. Three pollen types were selected for this purpose: Poaceae, Urticaceae and Quercus. The statistical results show that the daily concentrations followed similar trends (p < 0.01, R-values between 0.78–0.96) with both methods during the two years, although the counts were slightly higher using the longitudinal traverses method. Significant differences were observed, however, when the distribution of the concentrations during 24 hour sampling periods was considered. For more detailed analysis, the daily counts obtained with both methods were correlated with the total number of pollen grains for the taxon over the whole slide, in two different situations: high and low concentrations of pollen in the atmosphere. In the case of high concentrations, the counts for all three taxa with both methods are significantly correlated with the total pollen count. In the samples with low concentrations, the Poaceae and Urticaceae counts with both methods are significantly correlated with the total counts, but none of Quercus counts are. Consideration of the results indicates that both methods give a reasonable approximation to the count derived from the slide as a whole. More studies need be done to explore the comparability of counting methods in order to work towards a Universal Methodology in Aeropalynology. This revised version was published online in June 2006 with corrections to the Cover Date.     

Use of nitrocellulose Synpor filters for counting soil bacteria by epifluorescence microscopy

A modified acridine orange staining method for estimating soil bacterial numbers by epifluorescence microscopy using Synpor filters (VCHZ Synthesia, Czechoslovakia) was elaborated. Comparing with the method of direct count of soil bacteria estimated in a Bürker chamber higher counts of bacteria and a lower variation of results were obtained. To verify the sensitivity of the method, microflora from various soil horizons was tested.     

Some recommendations for an accurate estimation of Lanice conchilega density based on tube counts

The tube building polychaete Lanice conchilega is a common and ecologically important species in intertidal and shallow subtidal sands. It builds a characteristic tube with ragged fringes and can retract rapidly into its tube to depths of more than 20 cm. Therefore, it is very difficult to sample L. conchilega individuals, especially with a Van Veen grab. Consequently, many studies have used tube counts as estimates of real densities. This study reports on some aspects to be considered when using tube counts as a density estimate of L. conchilega, based on intertidal and subtidal samples. Due to its accuracy and independence of sampling depth, the tube method is considered the prime method to estimate the density of L. conchilega. However, caution is needed when analyzing samples with fragile young individuals and samples from areas where temporary physical disturbance is likely to occur.     

Triaxial accelerometry for assessment of physical activity in young children

Objective: The purpose of the present study was to derive linear and non‐linear regression equations that estimate energy expenditure (EE) from triaxial accelerometer counts that can be used to quantitate activity in young children. We are unaware of any data regarding the validity of triaxial accelerometry for assessment of physical activity intensity in this age group. Research Methods and Procedures: EE for 27 girls and boys (6.0 ± 0.3 years) was assessed for nine activities (lying down, watching a video while sitting and standing, line drawing for coloring‐in, playing blocks, walking, stair climbing, ball toss, and running) using indirect calorimetry and was then estimated using a triaxial accelerometer (ActivTracer, GMS). Results: Significant correlations were observed between synthetic (synthesized tri‐axes as the vector), vertical, and horizontal accelerometer counts and EE for all activities (0.878 to 0.932 for EE). However, linear and non‐linear regression equations underestimated EE by >30% for stair climbing (up and down) and performing a ball toss. Therefore, linear and non‐linear regression equations were calculated for all activities except these two activities, and then evaluated for all activities. Linear and non‐linear regression equations using combined vertical and horizontal acceleration counts, synthetic counts, and horizontal counts demonstrated a better relationship between accelerometer counts and EE than did regression equations using vertical acceleration counts. Adjustment of the predicted value by the regression equations using the vertical/horizontal counts ratio improved the overestimation of EE for performing a ball toss. Discussion: The results suggest that triaxial accelerometry is a good tool for assessing daily EE in young children.     

Methodology matters when estimating deer abundance: a global systematic review and recommendations for improvements

Deer (Cervidae) are key components of many ecosystems and estimating deer abundance or density is important to understanding these roles. Many field methods have been used to estimate deer abundance and density, but the factors determining where, when, and why a method was used, and its usefulness, have not been investigated. We systematically reviewed journal articles published during 2004–2018 to evaluate spatio-temporal trends in study objectives, methodologies, and deer abundance and density estimates, and determine how they varied with biophysical and anthropogenic attributes. We also reviewed the precision and bias of deer abundance estimation methods. We found 3,870 deer abundance and density estimates. Most estimates (58%) were for white-tailed deer (Odocoileus virginianus), red deer (Cervus elaphus), and roe deer (Capreolus capreolus). The 6 key methods used to estimate abundance and density were pedestrian sign (track or fecal) counts, pedestrian direct counts, vehicular direct counts, aerial direct counts, motion-sensitive cameras, and harvest data. There were regional differences in the use of these methods, but a general pattern was a temporal shift from using harvest data, pedestrian direct counts, and aerial direct counts to using pedestrian sign counts and motion-sensitive cameras. Only 32% of estimates were accompanied by a measure of precision. The most precise estimates were from vehicular spotlight counts and from capture–recapture analysis of images from motion-sensitive cameras. For aerial direct counts, capture–recapture methods provided the most precise estimates. Bias was robustly assessed in only 16 studies. Most abundance estimates were negatively biased, but capture–recapture methods were the least biased. The usefulness of deer abundance and density estimates would be substantially improved by 1) reporting key methodological details, 2) robustly assessing bias, 3) reporting the precision of estimates, 4) using methods that increase and estimate detection probability, and 5) staying up to date on new methods. The automation of image analysis using machine learning should increase the accuracy and precision of abundance estimates from direct aerial counts (visible and thermal infrared, including from unmanned aerial vehicles [drones]) and motion-sensitive cameras, and substantially reduce the time and cost burdens of manual image analysis.     

Use of dawn and dusk sight observations to determine colony size and family composition in Eurasian beaverCastor fiber

The methods used to determine family composition and colony size in Eurasian beaverCastor fiber Linnaeus, 1758 are often poorly described in published reports. Here we show how repeated counts of colony size in a random sample of colonies (n = 19) varied between dusk and dawn, between the months of August and September, and following successive counts. Mean counts at dusk and the following dawn did not vary significantly, though mean colony size was significantly greater in August than September. However, because all colony members are rarely seen during a single dusk or dawn count, successive counts often provided new information about the maximum number in each age class. This allowed us to adjust colony size following each count using the largest values thus far obtained, with the result that the mean adjusted colony size increased during six successive counts over seven weeks from 2.4 to 3.8. Family composition based on information from all six counts was 54% adults, 26% yearlings and 19% kits. Evidence suggests that kits in particular are undercounted by this method. Figures for colony size and composition in beaver should be viewed with caution if not obtained by methods tested for both precision and accuracy.     

Prediction of birch airborne pollen counts by examining male catkin numbers in Hokkaido,northern Japan

Birch pollen is a very common cause of pollinosis in Hokkaido, northern Japan. Birch airborne pollen concentrations vary each year; hence, the development of a method for predicting annual airborne pollen concentration is very important in preventing widespread symptoms of pollinosis. In the current study, we investigated airborne pollen counts and male catkin numbers (male flower index) of birch in four cities of Hokkaido between 2002 and 2008. Airborne pollen surveys were conducted using Durham’s sampler, and male catkin numbers determined for three major birch species (Betula platyphylla var. japonica, B. emanii, and B. maximowicziana). We found an annual variation in male flower index for all the three birch species investigated. This variation worked in combination with the amount of precipitation during the pollen season to influence total birch pollen counts. In conclusion, the male catkin numbers of three major birch species reliably predict airborne pollen counts in Hokkaido, but only when the effect of precipitation during pollen season is considered.     

Plant cover estimation based on the beta distribution in grassland vegetation

Cover is the most frequently used measure of abundance in vegetation surveys of grasslands, and various qualitative and semi-quantitative methods have been developed for visual estimation of this metric. Field survey is usually made with a point-grid plate. The frequency distributions of cover derived from point-grid counts follow a beta distribution. Combining point-grid counts from a field survey and the beta distribution for a statistical analysis, we developed an effort-saving cover-measurement method. Cover is measured with a transparent plastic plate on which, for example, 10 × 10 = 100 points are arranged in a lattice with 1-cm grid spacing (thus, one point count represents 1 cm² of cover). N quadrats are set out at randomly dispersed sites in a grassland, and, in each, the plastic plate is used for making counts. The number of grid points located above a given species is counted in every quadrat until the number of counted points reaches a given value c, which is determined in advance. If the number of counted points reaches c in a quadrat, the count is stopped and the quadrat is classified in the category “>c”. In quadrats where c is not attained, full point counts above the species bodies are made. Let g be the number of observed quadrats whose cover is ≤c. Using these g cover measurements and the number of quadrats (N − g) with cover >c, we can quantitatively estimate cover for each species and the spatial pattern index value based on the maximum likelihood method. In trial counts using this method, the time savings varied between 5% and 41%, depending on the shape of the cover frequency distribution. The mean cover value estimates agreed well with conventional measures without a stopping point (i.e., based on full counts of all points in each quadrat).     

Dynamic changes in white blood cell counts in uncomplicated Plasmodium falciparum and P. vivax malaria

Total and differential white blood cell (WBC) counts are basic and essential indicators in any type of illness resulting from infection. In malaria, WBC counts are generally characterized as low to normal during treatment. WBC-counts data, before and during treatment with artemisinin derivatives, was gathered for patients with either Plasmodium falciparum or Plasmodium vivax infection (at 28-day follow-up), to investigate dynamic changes in WBC count. We analyzed and compared the WBC counts of 1310 inpatients presenting with uncomplicated P. falciparum and P. vivax malaria at the Hospital for Tropical Diseases, in Bangkok, Thailand. Before-treatment, a statistically significant negative correlation was found between initial WBC count and highest temperature on admission. Before and during treatment, WBC counts were significantly lower in P. falciparum than P. vivax infection on days 0 and 7, but the numerical difference was small. We also found clinically significantly low WBC counts during the acute stages of both types of malaria, which subsequently normalized by day 28 follow-up. This finding has important clinical implications for the conventional method of estimating parasitemia using an assumed WBC count of 8000 cells/μL. The most significant finding in our analysis is that WBC counts in acute P. falciparum and P. vivax malaria are significantly lower than previously assumed for estimating malaria-parasite density. However, these abnormalities returned to normal within several weeks after artemisinin-derivative-based treatment.     

Characterization of some anaerobic bacteria from the liquid phase of a mesophilic anaerobic digester fed with a prefermented cheese whey substrate

Bacterial counts on the contents of an anaerobic fixed-bed digester receiving a whey substrate were conducted using the modified roll tube technique. Average anaerobic counts after 48 hours incubation on lactate containing media were 3.12 × 10⁹ and 3.7 × 10⁹ ml^–1, respectively. These counts were between 140 and 190 times higher than aerobic counts on the same media. Seventy-four strains from both media were isolated and characterized, and the relationship between the organisms was calculated according to the similarity coefficient of Sokal and Michener [20]. The organisms were clustered using the unweighted pair group method and the results were presented in the form of a simplified dendrogram. The isolates clustered in three major groups (A, B, and C) at a similarity level of 76%. A small diffuse group of five organisms was also found. The organisms in two (A and B) of the major clusters were obligate anaerobes. Cluster A represented 34% of the isolates, cluster B represented 50%, and the facultative streptococci in cluster C, 11%. The isolates in clusters A and B could only tentatively be identified as members of the generaBacteroides andPeptostreptococcus, respectively. The isolates could not be positively identified.     

Genomic identification of microbial species adhering to maxillofacial prostheses and susceptibility to different hygiene protocols

This study investigated the microbial colonization of maxillofacial prostheses and support tissues using the Checkerboard DNA–DNA hybridization method, and the efficacy of 0.12% chlorhexidine gluconate, 10% Ricinus communis solutions, or brushing, on colony forming unit (CFU) reduction in monospecies biofilms (Candida glabrata, Staphylococcus aureus, Streptococcus mutans, Escherichia coli, Enterococcus faecalis, and Pseudomonas aeruginosa) formed on two silicones (MDX 4-4210 and Bio-Skin). Biofilm was harvested from 43 maxillofacial prosthesis wearers for detection of 38 species of microorganisms. The CFU counts of the six above mentioned species were recorded after using the hygiene protocols. All 38 investigated species were identified in prostheses and tissues, with a higher prevalence in the prostheses. 0.12% chlorhexidine gluconate immersion showed the greatest antimicrobial effectiveness, followed by mechanical brushing protocols. MDX 4-4210 silicone produced lower CFU counts than Bio-Skin.     

Predicting Physical Activity Energy Expenditure Using Accelerometry in Adults From Sub‐Sahara Africa

Lack of physical activity may be an important etiological factor in the current epidemiological transition characterized by increasing prevalence of obesity and chronic diseases in sub‐Sahara Africa. However, there is a dearth of data on objectively measured physical activity energy expenditure (PAEE) in this region. We sought to develop regression equations using body composition and accelerometer counts to predict PAEE. We conducted a cross‐sectional study of 33 adult volunteers from an urban (n = 16) and a rural (n = 17) residential site in Cameroon. Energy expenditure was measured by doubly labeled water (DLW) over a period of seven consecutive days. Simultaneously, a hip‐mounted Actigraph accelerometer recorded body movement. PAEE prediction equations were derived using accelerometer counts, age, sex, and body composition variables, and cross‐validated by the jack‐knife method. The Bland and Altman limits of agreement (LOAs) approach was used to assess agreement. Our results show that PAEE (kJ/kg/day) was significantly and positively correlated with activity counts from the accelerometer (r = 0.37, P = 0.03). The derived equations explained 14–40% of the variance in PAEE. Age, sex, and accelerometer counts together explained 34% of the variance in PAEE, with accelerometer counts alone explaining 14%. The LOAs between DLW and the derived equations were wide, with predicted PAEE being up to 60 kJ/kg/day below or above the measured value. In summary, the derived equations performed better than existing published equations in predicting PAEE from accelerometer counts in this population. Accelerometry could be used to predict PAEE in this population and, therefore, has important applications for monitoring population levels of total physical activity patterns.     

Estimating red deer Cervus elaphus populations: an analysis of variation and cost-effectiveness of counting methods

• 1 Different counting methods are currently used to estimate red deer populations in the open range in Scotland, but there are few data available to compare variation in estimates, or relative cost‐effectiveness.
• 2 While it is impossible to determine the accuracy of counts (as real numbers are unknown), variation within and between different methods can be measured by repeat counts of the same area within as short a period as possible.
• 3 This study aimed to quantify the variation observed from repeat counts using each of four methods (ground, helicopter, infrared helicopter and dung‐counting methods) at one of three study sites in late winters 2003, 2004 and 2005. Additional data from digital camera images of groups from counts in other areas of Scotland were also used to assess the accuracy of visual counts.
• 4 Coefficients of variation (CVs) within any method of between 5% and 16% were recorded, consistent with previous comparisons for red deer open range counts in Scotland. CVs were lowest for ground and helicopter counts. The infrequency of optimal conditions was likely to limit the applicability of infrared counts in Scotland.
• 5 In terms of cost‐effectiveness, helicopter counting was the least labour‐intensive, with costs of other techniques depending on the availability of existing manpower as an overhead cost.
• 6 It is concluded that helicopter counts are most likely to minimize errors while maximizing cost‐efficiency. Accuracy can be improved by the use of digital photography for counting larger deer groups. Estimates are likely to be improved further by increasing the frequency of counts and using the same methods, counters and routes for repeat counts.

     

Counting birds on farmland habitats in winter

Capsule Perimeter counts underestimate the number of birds using agricultural fields.

Aims To determine the degree to which farmland birds may be undercounted on field surveys and the factors influencing this.

Methods In a study of 96 fields, birds in the field were counted during a walk of the perimeter. Afterwards transects were walked across the field to determine the number of birds missed. Additionally in a national survey we looked at the birds seen in field transects expressed as a proportion of the total seen in the transect and on perimeter counts.

Results To obtain an accurate count of birds on a field requires the use of the whole-area search methodology especially for species such as Grey Partridge Perdix perdix, Snipe Gallinago gallinago, Skylark Alauda arvensis and Meadow Pipit Anthus pratensis. However, for a large number of species, including thrushes, flocking finches and buntings, over 90% of individuals can be recorded using the much less time-consuming perimeter count method.

Conclusions Perimeter counts are likely to be reliable for most species but special efforts may be required for a few cryptic species.