A membrane filter direct count technique for enumeratingBdellovibrio

A membrane filter direct count method was devised for enumeratingBdellovibrio cells in “clean” suspensions. The procedure involves filtering a specified volume of a diluted, Trisbuffered suspension ofBdellovibrio cells through a known area of a 100-nm-pore-size Millipore brand membrane filter. A clarification solvent was used to render the filter transparent, so that the bdeloyvibrios on the filter could be photomicrographed and counted either visually or by means of a Quantimet 720 Image Analyzing Computer. The number ofBdellovibrio cells per milliliter in the undituted suspension could be calculated from the mean number of cells per unit area of the filter, the dilution factor, and the volume of diluted suspension filtered. TheBdellovibrio cells were distributed on the filters in a Poisson manner when there were not more than about 3.5 cells per 100 μm² of filter surface. The membrane filter direct counts correlated well with direct counts obtained by the Petroff-Hausser method. The correlation of direct counts with plaque (“viable”) counts showed that 80 to 95% of the direct-countedBdellovibrio cells in the clean suspensions were capable of forming plaques on lawns of suitable substrate bacteria. *** DIRECT SUPPORT *** A01R4002 00007     

Variation in Vertebral Number in Galaxiid Fishes (Teleostei: Galaxiidae): A Legacy of Life History, Latitude and Length

Mean vertebral counts among species of Galaxiidae vary curvilinearly with fish size – large species have more vertebrae (pleomerism). The relationship with size breaks down among adults of diadromous species. There is mostly no relationship between vertebral count and body shape, although an inverse relationship between count and body depth in a series of divergent small galaxiids may reflect a shift in swimming mode. Diadromous populations have more vertebrae than non-diadromous (landlocked) conspecifics (around the same number as related non-diadromous species). Diadromous species have more vertebrae than related non-diadromous species and exhibit rising vertebral number with increasing latitude (Jordan's rule applies). Regressions of vertebral number against latitude differ among conspecific populations from different regions, perhaps reflecting different relationships between latitude and local climate. Differences in counts between diadromous and non-diadromous species are not simply a direct influence of environment on ontogeny, as embryonic development of species/populations undertaking the two distinct life history strategies occurs in similar, freshwater habitats; differences seem likely to be evolved, and to reflect an advantage of higher vertebral counts in marine environments, where diadromous species live as juveniles. This conclusion is supported by correlation between vertebral counts and size of juveniles of diadromous species at return from the sea, suggesting selection for vertebral number during marine life. Overall, vertebral count is thus influenced by a complex amalgam of fish size, life history, and environment. The explanation for pleomerism may well be related to the scaling relationship between length and cross-sectional area (area rises by the square of length increase) leading to increasing stiffness with growth – compensated for in larger species by having more vertebrae. This could also be driven by scaling in the surface area of the articular facets of the vertebrae themselves.     

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.     

Correlation of Direct Viable Counts with Heterotrophic Activity for Marine Bacteria

Viable-bacteria counts, heterotrophic activity, and substrate responsiveness of viable bacteria have been used to measure microbial activity. However, the relationship between these parameters is not clear. Thus, the direct viable count (DVC) method was used to analyze seawater samples collected from several different geographical locations. Samples collected from offshore waters of the South China Sea and western Pacific Ocean yielded DVC that indicated the presence of surface and subsurface peaks of viable, substrate-responsive bacteria which could be correlated with turnover rates of amino acids obtained by using uniformly ¹⁴C-labeled amino acids. DVC were always less than total viable counts (acridine orange direct counts), and the DVC subsurface peak occurred close to and within the chlorophyll a zone, suggesting algal-bacterial interactions within the layer. For comparison with the open-ocean samples, selected substrates were used to determine the response of viable bacteria present in seawater samples collected near an ocean outfall of the Barceloneta Regional Waste Treatment Plant, Barceloneta, Puerto Rico. The number of specific substrate-responsive bacteria at the outfall stations varied depending on the substrate used and the sampling location. Changes in the population size or physiological condition of the bacteria were detected and found to be associated with the presence of pharmaceutical waste.     

ESTIMATING POPULATION SIZE IN ASYNCHRONOUS AGGREGATIONS: A BAYESIAN APPROACH AND TEST WITH ELEPHANT SEAL CENSUSES

Many organisms reproduce in temporary aggregations where estimates of colony size can be made by direct counts. When individuals are not synchronous, however, early breeders depart before the last arrive, so counts underestimate the total breeding population. We present a model describing a colony's census as a function of arrival, breeding tenure, and the correlation between them, and we use it to illustrate how variance in arrival and tenure affect the census. Counts of breeding female northern elephant seals ( Mirounga angustirostris ) from 1975 to 2007 were used to test the model. Four of the model's parameters—population size, mean and variance of arrival date, and the correlation between arrival date and breeding tenure—could be estimated from census data using a Bayesian approach; prior estimates of two other parameters—mean tenure and its variance—had to be used to avoid overparameterization. The model's predictions fit observed censuses well and produced reliable estimates of population size and arrival behavior, showing that the maximum census was 8%–16% below the total number of breeding females. This method could be used for estimating abundance in any asynchronous aggregation, given independent information on one of the defining distributions: arrival, tenure, or departure.     

Green fluorescent protein-based direct viable count to verify a viable but non-culturable state of Salmonella typhi in environmental samples.

The gfp-tagging method and lux-tagging method were compared to select a better method for verifying a viable but nonculturable (VBNC) state of bacteria in the environment. An environmental isolate of Salmonella typhi was chromosomally marked with a gfp gene encoding green fluorescent protein (GFP). The hybrid transposon mini-Tn5 gfp was transconjugated from E. coli to S. typhi. Using the same method, S. typhi was chromosomally marked with luxAB genes encoding luciferase. The survival of gfp-tagged S. typhi introduced into groundwater microcosms was examined by GFP-based plate count, total cell count, and a direct viable count method. In microcosms containing lux-tagged S. typhi, luminescence-based plate count and the measurement of bioluminescence of each microcosm sample were performed. In microcosms containing lux-tagged S. typhi, viable but nonculturable cells could not be detected by using luminometry. As no distinguishable luminescence signals from the background signals were found in samples containing no culturable cells, a VBNC state of S. typhi could not be verified in lux-based systems. However, comparison between GFP-based direct viable counts and plate counts was a good method for verifying the VBNC state of S. typhi. Because GFP-based direct viable count method provided a direct and precise estimation of viable cells of introduced bacteria into natural environments, it can be used for verifying the VBNC state of bacteria in environmental samples.     

Enumeration of Viable Bacteria in the Marine Pelagic Environment

The low percentage of living bacteria commonly obtained when comparing viable counts with total direct counts in seawater could be due more to inappropriate techniques for appreciating the growth ability of living cells than to unadapted culture conditions. The most-probable-number counts in filtered seawater cultures and the microscopic counts of 4(prm1),6-diamidino-2-phenylindole (DAPI)-stained aggregate-forming units grown on black polycarbonate filters appeared significantly correlated to the direct counts. Both these techniques show that in the superficial and intermediate water masses, the living cells may constitute an important (frequently higher than 20%) but highly variable part of the total populations. These viable counts appear more realistic than the conventional CFU counts, which provide only 0.001 to 0.2% of the total counts.     

THE EFFECT OF THE TETRACYCLINE COMPOUNDS ON THE STORAGE LIFE AND MICROBIOLOGY OF CHILLED EVISCERATED POULTRY

SUMMARY: Chlortetracycline, oxytetracycline and tetracycline, incorporated at 10 p/m in the ice slush used for cooling, were equally efficient in extending the storage life of eviscerated poultry which was subsequently frozen, thawed and stored at 1°. There was complete correlation between the sensory assessment of 'off'odour and the total numbers of micro-organisms present when these were determined by taking bulk samples of skin from the area under the wing and of the surface tissue of the visceral cavity near the vent. Direct microscopical counts confirmed that the latter area was the most heavily contaminated part of the visceral cavity. Non-pigmented strains of Pseudomonas were the main spoilage organisms of untreated chickens but Achromobacter strains and yeasts were responsible for the spoilage of the chlortetracycline treated chickens. Fluorescence was only observed in spoiled chickens where pigmented strains of Pseudomonas occurred in significant numbers. Spoilage in both control and antibiotic treated poultry was accompanied by a rise in pH in the contaminated muscles, but for various reasons this change could not be used as a direct measure of the extent of the microbial contamination.     

Seasonal movement and distribution of the grape mealybug (Homoptera: Pseudococcidae): developing a sample program for San Joaquin Valley vineyards

The grape mealybug, Pseudococcus maritimus (Ehrhorn), is an important pest of table grapes in California's San Joaquin Valley. The mealybug causes direct damage by infesting grape bunches, resulting in very low economic injury levels. To develop a sampling program to help growers predict damage and make control decisions, we destructively sampled six entire grapevines each month to determine mealybug abundance and within-vine distribution. These absolute counts were then used to evaluate several relative sampling methods: sticky tape barriers on canes, excised spur samples, standard-sized pieces of bark, timed counts, and nondestructive counts on spurs. At midseason we sampled additional vines to correlate mealybug numbers with economic damage at harvest. Finally, mealybug life stages and natural enemies were recorded throughout the study. Timed 5-min counts show the strongest correlation with total mealybug numbers, and a simple count of mealybugs on three spurs per vine at midseason is the best predictor of economic damage. Mealybugs completed approximately equals 2.5 generations in 1998. Comparison to data on mealybug development suggests that exceptionally long growing seasons could exacerbate infestations by allowing the completion of a third generation. No mealybugs were found on bunches before early August, when second-generation crawlers moved out of the bark. Grape bunches that touched old wood had significantly higher damage and mealybug densities. The majority of mealybugs were always found in protected locations (under the bark of the trunk, spurs or canes), indicating the need for chemical or biological controls that can penetrate these refugia.     

生物超微弱发光在猪肉新鲜度检测中的应用

为了寻找最佳测试条件来实现猪肉样品新鲜度的检测,采用单光子测试系统测试了不同条件下猪肉的自发辐射强度和延迟发光的变化。4天连续测试的噪声平均值为76.3 counts/s,平均偏差为1.1 counts/s;自发辐射测试中肥肉样品信号较强,其强度随放置时间的增加有增加过程;延迟发光测试中大排及肥肉信号较强;按照延迟发光动力学曲线拟合,拟合系数达0.996。随着测量时间的增加,延迟发光的特征时间t降低,反应了猪肉新鲜度下降。实验结果表明该系统能实现对生鲜猪肉新鲜度的直接检测,具有选材少,检测速度快的特点。     

Microbiology of Anaerobic Sludge Fermentation: I. Enumeration of the Nonmethanogenic Anaerobic Bacteria1

An anaerobic medium containing sludge supernatant fluid and glucose was used for enumeration of bacteria from the sludge fermentation. Comparison of viable counts from several separate samples consistently showed 10 to 100 times more anaerobic than aerobic bacteria. However, viable counts of the various samples differed by as much as 10 times; this variation probably reflects a change in the natural environment or sampling errors, or a combination of the two. Direct microscopic counts yielded values of about 10(10)/ml. The discrepancy between viable (10(8) to 10(9)/ml) and direct counts may be due to large numbers of dead cells. Random isolates of representative colonies from high dilutions exhibited the ability to ferment sugars and are not likely to be methane bacteria.     

Application of digital image analysis and flow cytometry to enumerate marine viruses stained with SYBR gold

A novel nucleic acid stain, SYBR Gold, was used to stain marine viral particles in various types of samples. Viral particles stained with SYBR Gold yielded bright and stable fluorescent signals that could be detected by a cooled charge-coupled device camera or by flow cytometry. The fluorescent signal strength of SYBR Gold-stained viruses was about twice that of SYBR Green I-stained viruses. Digital images of SYBR Gold-stained viral particles were processed to enumerate the concentration of viral particles by using digital image analysis software. Estimates of viral concentration based on digitized images were 1.3 times higher than those based on direct counting by epifluorescence microscopy. Direct epifluorescence counts of SYBR Gold-stained viral particles were in turn about 1.34 times higher than those estimated by the transmission electron microscope method. Bacteriophage lysates stained with SYBR Gold formed a distinct population in flow cytometric signatures. Flow cytometric analysis revealed at least four viral subpopulations for a Lake Erie sample and two subpopulations for a Georgia coastal sample. Flow cytometry-based viral counts for various types of samples averaged 1.1 times higher than direct epifluorescence microscopic counts. The potential application of digital image analysis and flow cytometry for rapid and accurate measurement of viral abundance in aquatic environments is discussed.     

Optimization of direct cell counting in sediment

This study reports a method for optimizing direct counts of bacteria in sediment, designed to reduce the masking by sediment particles. The protocol was designed to determine appropriate dilution factors by incorporating counting statistics and was used to measure depth-associated changes in microbial abundance in metal-impacted freshwater sediments. We demonstrated a direct method to determine appropriate sample dilution for accurate counting by adding a known amount of cells to the sediment. For accurate counting in our sediment samples, we determined that the average number of bacteria per microscope ocular field must be between 8.5 and 10. This is well below the 30 bacteria/field previously suggested for accurate counting. These results indicate that an optimal dilution rate must be determined before accurate direct counts in sediment can be achieved.     

Application of Digital Image Analysis and Flow Cytometry To Enumerate Marine Viruses Stained with SYBR Gold

A novel nucleic acid stain, SYBR Gold, was used to stain marine viral particles in various types of samples. Viral particles stained with SYBR Gold yielded bright and stable fluorescent signals that could be detected by a cooled charge-coupled device camera or by flow cytometry. The fluorescent signal strength of SYBR Gold-stained viruses was about twice that of SYBR Green I-stained viruses. Digital images of SYBR Gold-stained viral particles were processed to enumerate the concentration of viral particles by using digital image analysis software. Estimates of viral concentration based on digitized images were 1.3 times higher than those based on direct counting by epifluorescence microscopy. Direct epifluorescence counts of SYBR Gold-stained viral particles were in turn about 1.34 times higher than those estimated by the transmission electron microscope method. Bacteriophage lysates stained with SYBR Gold formed a distinct population in flow cytometric signatures. Flow cytometric analysis revealed at least four viral subpopulations for a Lake Erie sample and two subpopulations for a Georgia coastal sample. Flow cytometry-based viral counts for various types of samples averaged 1.1 times higher than direct epifluorescence microscopic counts. The potential application of digital image analysis and flow cytometry for rapid and accurate measurement of viral abundance in aquatic environments is discussed.     

Quantitative determination of free-DNA uptake in river bacteria at the single-cell level by in situ rolling-circle amplification

Detection of plasmid DNA uptake in river bacteria at the single-cell level was carried out by rolling-circle amplification (RCA). Uptake of a plasmid containing the green fluorescent protein gene (gfp) by indigenous bacteria from two rivers in Osaka, Japan, was monitored for 506 h using this in situ gene amplification technique with optimized cell permeabilization conditions. Plasmid uptake determined by in situ RCA was compared to direct counts of cells expressing gfp under fluorescence microscopy to examine differences in detection sensitivities between the two methods. Detection of DNA uptake as monitored by in situ RCA was 20 times higher at maximum than that by direct counting of gfp-expressing cells. In situ RCA could detect bacteria taking up the plasmid in several samples in which no gfp-expressing cells were apparent, indicating that in situ gene amplification techniques can be used to determine accurate rates of extracellular DNA uptake by indigenous bacteria in aquatic environments.     

A note on 'plotless' methods for estimating bacterial cell densities

R oser , D., N edwell , D.B. & G ordon , A. 1984. A note on "plotless" methods for estimating bacterial cell densities. Journal of Applied Bacteriology 56 , 343–347.
'Plotless' techniques for determining population densities have been developed for, and applied to, higher plant populations. They can often be carried out more rapidly than techniques involving total counts of individuals in plots, or quadrants, but such plotless techniques have not been generally applied to the estimation of densities of bacterial cells. Direct microscopical counting of cell numbers in a field of view, an example of a plot-related method, has been traditionally used for micro-bial cell counts. In this study 'plot' and 'plotless' methods on a variety of bacterial samples are compared. Estimates of bacterial cell density were obtained by measuring the distance of cells from a fixed point in a field of view. The values, which were more rapidly obtained, were directly correlated with total cell counts. Although there was some apparent deviation from a perfect 1:1 relationship with total counts, as indicated by a correlation coefficient less than 1.0, there were no significant differences between the replicated counts of bacteria on samples of tissue from the surface of Hypholoma basidiocarps ( P < 0.05). This indicated that the methods of enumeration were comparable. The distance-related estimates could readily be obtained from fields of view with cell densities varying over several orders of magnitude. It was more rapidly applied, particularly at high density, and the method was applicable not only to random cell distributions but also to the non-random distributions encountered when microbial cells aggregated into micro-colonies. The method appears to be particularly well-suited for automated, digitized, direct counting procedures, as well as to estimating bacterial numbers on membrane filters and natural substrates.     

Sampling communities of ground-foraging ants: Pitfall catches compared with quadrat counts in an Australian tropical savanna

The relative abundances of ant species captured in pitfall traps was compared with those obtained by direct counts in quadrats at a savanna site in Kakadu National Park, Northern Territory. Two measures of abundance in traps were used, one based on total numbers of ants, the other on species frequency of occurrence. All species commonly recorded in quadrats were collected in traps, and their relative abundances were highly correlated on all occasions. Of the 20 most common species in quadrats, five occurred with a significantly different (in all cases lower) frequency in pitfall traps, but these species represented only 1.8–3.1% of total quadrat counts. Results from quadrats and pitfall traps were particularly similar (r > 0.8) when species-were classified into functional groups. Frequency data from traps may sometimes overestimate the abundance of widespread, solitary foraging species (e.g. ‘Chelaner’ and Tetramorium spp.) and underestimate species with large colony sizes (e.g. Iridomyrmex spp.). Data based on total numbers of ants in traps may be more prone to distortion arising from species differences in locomotor behaviour. Species counts in traps could be scaled to reduce these distortions. The finding that pitfall traps gave results comparable with those from quadrat counts provides support for the use of pitfall traps in studies of Australian ant communities in open habitats.     

In situ survival of Vibrio cholerae and Escherichia coli in tropical coral reefs

Vibrio cholerae and Escherichia coli were inoculated into membrane diffusion chambers and placed around two small coral reef islands in Puerto Rico and monitored for 5 days. Several chambers were also buried in the sands of one of the reefs. Both E. coli and V. cholerae densities declined by 2 orders of magnitude, as measured by direct particle counts with a Coulter Counter (Coulter Electronics, Inc., Hialeah, Fla.). However, the density of neither bacteria changed dramatically when the same samples were analyzed by epifluorescent direct counts. Differences in the two direct count methods were accounted for by changes in cell morphology that occurred in both bacteria after exposure to seawater. Morphological changes occurred more rapidly in E. coli compared with those in V. cholerae. Bacteria in chambers exposed to sediment did not show significant changes in morphology and had only a slight decline in density. Physiological activity declined by more than 40% for both bacteria within 24 h. The decline in activity was less severe in the sediments. Tropical coral reef sands and turtle grass beds were shown to be less stressful environments for V. cholerae and E. coli than would have been predicted from temperature and microcosm studies. V. cholerae can survive the in situ conditions of a tropical coral reef and could become a source of bacterial contamination for fish and shellfish in this environment. The simultaneous monitoring of E. coli levels established that this bacteria can not be used as an indicator of V. cholerae or other fecal-borne pathogens in coral reef environments because of the greater stress these environments put on E. coli. Both bacteria could be of greater public health importance in tropical marine areas than previously imagined.     

In situ survival of Vibrio cholerae and Escherichia coli in tropical coral reefs.

Vibrio cholerae and Escherichia coli were inoculated into membrane diffusion chambers and placed around two small coral reef islands in Puerto Rico and monitored for 5 days. Several chambers were also buried in the sands of one of the reefs. Both E. coli and V. cholerae densities declined by 2 orders of magnitude, as measured by direct particle counts with a Coulter Counter (Coulter Electronics, Inc., Hialeah, Fla.). However, the density of neither bacteria changed dramatically when the same samples were analyzed by epifluorescent direct counts. Differences in the two direct count methods were accounted for by changes in cell morphology that occurred in both bacteria after exposure to seawater. Morphological changes occurred more rapidly in E. coli compared with those in V. cholerae. Bacteria in chambers exposed to sediment did not show significant changes in morphology and had only a slight decline in density. Physiological activity declined by more than 40% for both bacteria within 24 h. The decline in activity was less severe in the sediments. Tropical coral reef sands and turtle grass beds were shown to be less stressful environments for V. cholerae and E. coli than would have been predicted from temperature and microcosm studies. V. cholerae can survive the in situ conditions of a tropical coral reef and could become a source of bacterial contamination for fish and shellfish in this environment. The simultaneous monitoring of E. coli levels established that this bacteria can not be used as an indicator of V. cholerae or other fecal-borne pathogens in coral reef environments because of the greater stress these environments put on E. coli. Both bacteria could be of greater public health importance in tropical marine areas than previously imagined.     

Verification of cleaning efficiency and its possible role in programmed hygiene inspections of food businesses undertaken by local authority officers

AIM: The aim of this study was to determine whether or not the assessment of surface cleanliness could make a contribution to visual inspections of food premises. METHODS AND RESULTS: Forty-five premises were studied with both rapid (ATP) and traditional microbiological swabbing being used to test surfaces that either come into direct contact with prepared foods or were likely to be touched by hands during food preparation. A significant link was found between aerobic colony counts and ATP measurements. In most cases, the visual appearance of surfaces could not be used to accurately predict either microbial or ATP results. CONCLUSION: This study suggests that ATP testing is a useful indicator of surface cleanliness and could be helpful to local authority officers as part of risk assessment inspections. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides further evidence that visual inspection alone may not always be adequate to assess surface cleanliness. In high-risk premises, ATP could, if appropriately targeted, help identify potential problem areas. The results are available at the time of the inspection and can be used as an on-the-spot teaching aid.