Multicolour digital image analysis system for identification of bacteria and concurrent assessment of their respiratory activity

AIMS: To develop a rapid and simple multicolour digital image analysis system for simultaneous identification of bacteria and assessment of their metabolic activity. METHODS AND RESULTS: We developed an image analyser capable of distinguishing triple-stained bacterial cells. Bacteria were stained with a nucleic acid stain, a fluorescent antibody and a fluorescent metabolic indicator for enumeration, species identification and assessment of metabolic activity. This multicolour image analyser was used to simultaneously identify Escherichia coli O157:H7 in milk samples and assess their respiratory activity. The images of the triple-stained bacteria were captured using a combination of blue light and u.v. excitation and an epifluorescence microscope and were processed by our image analyser. We found a good correlation between the counts of actively respiring (r = 0.93) and total (r = 0.94) E. coli O157:H7 measured by digital image analysis and visual observation. CONCLUSION: The multicolour digital image analysis system described here was able to quantify active pathogenic micro-organisms within 2 h. SIGNIFICANCE AND IMPACT OF THE STUDY: This multicolour image analysis allows the rapid and simultaneous quantification of bacteria, identification of species and assessment of metabolic activity.     

Image analysis for monitoring the barley tempeh fermentation process

AIMS: To develop a fast, accurate, objective and nondestructive method for monitoring barley tempeh fermentation. METHODS AND RESULTS: Barley tempeh is a food made from pearled barley grains fermented with Rhizopus oligosporus. Rhizopus oligosporus growth is important for tempeh quality, but quantifying its growth is difficult and laborious. A system was developed for analysing digital images of fermentation stages using two image processing methods. The first employed statistical measures sensitive to image colour and surface structure, and these statistical measures were highly correlated (r=0.92, n=75, P<0.001) with ergosterol content of tempeh fermented with R. oligosporus and lactic acid bacteria (LAB). In the second method, an image-processing algorithm optimized to changes in images of final tempeh products was developed to measure number of visible barley grains. A threshold of 5 visible grains per Petri dish indicated complete tempeh fermentation. When images of tempeh cakes fermented with different inoculation levels of R. oligosporus were analysed the results from the two image processing methods were in good agreement. CONCLUSION: Image processing proved suitable for monitoring barley tempeh fermentation. The method avoids sampling, is nonintrusive, and only requires a digital camera with good resolution and image analysis software. SIGNIFICANCE AND IMPACT OF THE STUDY: The system provides a rapid visualization of tempeh product maturation and qualities during fermentation. Automated online monitoring of tempeh fermentation by coupling automated image acquisition with image processing software could be further developed for process control.     

Automated image analysis to improve bead ingestion toxicity test counts in the protozoan Tetrahymena pyriformis

AIMS: To improve bead ingestion counts in Tetrahymena pyriformis by automated image analysis as an alternative to direct-counts. METHODS AND RESULTS: Fluorescent latex beads were added to T. pyriformis cultures for ingestion tests. The number of beads ingested by 25 cells was counted directly by epifluorescence microscopy and compared with similar data from image analysis. anova indicated that counts were not significantly different (P < 0.05). The image analysis particularly provided advantages in terms of speed. CONCLUSIONS: The image analysis is superior to direct beads counting in T. pyriformis particularly in terms of speed of analysis. SIGNIFICANCE AND IMPACT OF THE STUDY: The image analysis method is very rapid and will allow many more toxicological analyses to be undertaken with less operator error.     

Carbon steady-state model of the planktonic food web of Lake Biwa, Japan

1. A steady‐state model of carbon flows was developed to describe the summer planktonic food web in the surface mixed‐layer of the North Basin in Lake Biwa, Japan. This model synthesised results from numerous studies on the plankton of Lake Biwa. 2. An inverse analysis procedure was used to estimate missing flow values in a manner consistent with known information. Network analysis was applied to characterise emergent properties of the resulting food web. 3. The system strongly relied on flows related to detrital particles. Whereas primary production was mainly by phytoplankton >20 μm, microzooplankton were active and mainly ingested detritus and bacteria. 4. The main emergent property of the system was strong recycling, through either direct ingestion of non‐living material by zooplankton, or ingestion of bacteria after degradation of detritus to release dissolved organic carbon.     

The novel method for isolating chitinolytic bacteria and its application in screening for hyperchitinase producing mutant of Alcaligenes xylosoxydans

AIMS: To develop a novel, rapid and effective screening method for chitinase producing bacteria. METHODS AND RESULTS: A simple and rapid technique for screening of potential chitinolytic bacteria has been developed using the chitin binding dye calcofluor white M2R in chitin agar. Microorganisms possessing high chitinolytic potential gave a clear zone under ultraviolet light after 24-48 h of incubation. This method was successfully applied for isolating the hyperchitinase mutant of Alcaligenes xylosoxydans. The mutant Alc. xylosoxydans EMS 33 was found to produce 3.4 times more chitinase than the wild type. CONCLUSIONS: In this study, the screening method for chitinase producing bacteria has been developed and it was applied to screen chitinase-overproducing mutant of Alc. xylosoxydans. SIGNIFICANCE AND IMPACT OF THE STUDY: The novel screening method for chitinase producer is more sensitive, rapid, user-friendly and reliable, which can also be used for screening of recombinants having chitinase gene.     

Identification of bacterial diversity in the oyster Crassostrea gigas by fluorescent in situ hybridization and polymerase chain reaction

AIMS: To carry out a rapid and reliable identification of bacterial diversity in the oyster Crassostrea gigas from Todos Santos Bay, México, in the current study we applied the molecular techniques of fluorescent in situ hybridization (FISH) and polymerase chain reaction (PCR). In order to reach this goal, genus and group-specific oligonucleotides targeted to 16S rDNA/rRNA were used. METHODS AND RESULTS: Oysters were collected and different tissues were analysed by means of culture-independent methodologies. In the digestive glands and gonads gamma-Proteobacteria and Gram-positive bacteria with a low G+C content, were identified as metabolically active by FISH. In the oyster gills a higher active diversity was observed, including Gram-positive bacteria with a low and high G+C content, members of the Cytophaga/Flavobacterium cluster and gamma-Proteobacteria. Consistent with FISH analysis, the amplification of 16S rDNA genes fragments with genus and group-specific oligonucleotides confirmed the presence of the same groups, as well as members of the alpha- and beta-Proteobacterias, Pseudomonas spp. and Bacillus spp. CONCLUSIONS: The combination of accurate and very easy-to-apply molecular methods allowed us to carry out a rapid screening of high bacterial diversity in oysters. SIGNIFICANCE AND IMPACT OF THE STUDY: This work is the first report about bacterial diversity in oyster tissues analysed by FISH and PCR, without using culture-dependent methods and allowed us to determine the phylogenetic diversity of the bacterial communities present in oyster cultures, including bacteria with and without metabolic activity, as well as uncultivable cells, which are generally underestimated by traditional identification.     

Algorithm to estimate cell biovolume using image analyzed microscopy

This paper describes an algorithm for calculating the biovolume of cells with simple shapes, such as bacteria, flagellates, and simple ciliates, from a 2-dimensional digital image. The method can be adapted to any image analysis system which allows access to the binary cell image--(i.e., the pixels, or (x,y) points, composing the cell. The cell image is rotated to a standard orientation (horizontal), and a solid of revolution is calculated by digital integration. Verification and a critical assessment of the method are presented. The algorithm accounts for irregularities in cell shape that conventional methods based on length, width, and geometrical formulas do not.     

Assessment of the metabolic activity of Acremonium chrysogenum using Acridine Orange

A method is described for the assessment of the metabolic activity of the filamentous fungus Acremonium chrysogenum using the fluorescent dye Acridine Orange. Changes in metabolic activity are indicated by a reversible red-green shift in the colour of the dye, quantifiable by image analysis. A. chrysogenum mycelia exhibited an overwhelmingly green colour in circumstances leading to high substrate uptake, while under carbon starvation they appeared orange-red. It is believed these colour changes reflected changes in internal pH. The method provides a visual tool for the investigation of the metabolic behaviour of filamentous fungi.     

Use of 5-cyano-2,3-ditolyl tetrazolium chloride for quantifying planktonic and sessile respiring bacteria in drinking water.

Direct microscopic quantification of respiring (i.e., viable) bacteria was performed for drinking water samples and biofilms grown on different opaque substrata. Water samples or biofilms developed in flowing drinking water were incubated with the vital redox dye 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) and R2A medium. One hour of incubation with 0.5 mM CTC was sufficient to obtain intracellular reduction of CTC to the insoluble fluorescent formazan (CTF) product, which was indicative of cellular respiratory (i.e., electron transport) activity. This result was obtained with both planktonic and biofilm-associated cells. Planktonic bacteria were captured on 0.2-microns-pore-size polycarbonate membrane filters and examined by epifluorescence microscopy. Respiring cells containing CTF deposits were readily detected and quantified as red-fluorescing objects on a dark background. The number of CTC-reducing bacteria was consistently greater than the number of aerobic CFU determined on R2A medium. Approximately 1 to 10% of the total planktonic population (determined by counterstaining with 4,6-diamidino-2-phenylindole) were respirometrically active. The proportion of respiring bacteria in biofilms composed of drinking water microflora was greater, ranging from about 5 to 35%, depending on the substratum. Respiring cells were distributed more or less evenly in biofilms, as demonstrated by counterstaining with 4,6-diamidino-2-phenylindole. The amount of CTF deposited in single cells of Pseudomonas putida that formed monospecies biofilms was quantified by digital image analysis and used to indicate cumulative respiratory activity. These data indicated significant cell-to-cell variation in respiratory activity and reduced electron transport following a brief period of nutrient starvation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Genetic diversity and technological properties of Streptococcus thermophilus strains isolated from dairy products

AIMS: To evaluate the genetic diversity and the technological properties of 44 strains of Streptococcus thermophilus isolated from dairy products. Methods METHODS AND RESULTS: Strains were analysed for some relevant technological properties, i.e. exopolysaccharide (EPS) production, growth kinetic in skim milk medium, urease activity and galactose fermentation. The EPS production, determined by evaluating the colour of the colonies grown in ruthenium red milk agar, was observed in 50% of the analysed strains. Urease activity, determined by colorimetric and conductimetric methods, showed that 91% of the isolates, all except four, could hydrolyse urea. A conductimetric approach was also used for the evaluation of the overall metabolic behaviour in milk of Strep. thermophilus strains and the differences observed allowed grouping of the strains in seven different clusters. A total of 11 strains were able to produce acid in presence of galactose. Genetic diversity of Streptococcus thermophilus strains, evaluated by Random Amplified Polymorphic DNA fingerprinting (RAPD) and amplified epsC-D restriction analysis, allowed the identification of 21 different genotypes. CONCLUSIONS: Comparison between the genotypic and phenotypic data highlights an interesting correlation between some important technological properties and well-defined genotypes. SIGNIFICANCE AND IMPACT OF THE STUDY: The genetic and technological characterization carried out on several Strep. thermophilus strains of dairy origin should expand the knowledge on this important lactic acid bacteria species and lead to a simple, rapid, and reliable identification of strains on the basis of well-defined biotechnological properties.     

Behavior of sulfate reducing bacteria under oligotrophic conditions and oxygen stress in particle-free systems related to drinking water

The response of sulfate reducing bacteria (SRB) to oxygen stress under oligotrophic conditions in particle-free systems was studied in (i) sterile Berlin drinking water; (ii) mineral medium; and (iii) in coculture experiments with aerobic bacteria. Using a polyphasic approach including anaerobic cultivation, fluorescent in situ hybridization (FISH) and digital image analysis, the behavior of the strains zt3l and zt10e, isolated from Berlin groundwater and affiliated to the family Desulfovibrionaceae, was compared to the type strains Desulfomicrobium baculatum and Desulfovibrio desulfuricans. Anaerobic deep agar dilution series were performed for the determination of cell culturability. FISH and subsequent digital image analysis of probe-conferred fluorescence intensities were used for the assessment of metabolic activity. For the in situ identification of both isolates in coculture tests, two strain-specific oligonucleotides were developed and evaluated. The total cell counts of stressed SRB in drinking water decreased during the course of the assay dependent on the strain. Both environmental isolates could be cultured for a longer period than cells of D. baculatum and D. desulfuricans, respectively. The FISH intensities showed a strain-specific behavior. When exposed to simultaneous oxygen stress and carbon limitation in mineral medium, total cell counts of all four strains remained constant throughout a period of 72 days. The rate of culturability differed between the investigated strains. The decrease of metabolic activity as assessed by FISH was a strain-specific property. Exposure of SRB to oxygen stress and carbon starvation in coculture experiments with Aquabacterium commune resulted in strain dependent prolonged culturability and a delayed decrease of the metabolic activity compared to pure culture tests for all strains tested. Total cell counts of SRB were constant throughout the whole experiment.     

The abundance,biomass and acetylene reduction activity of bacteria associated with decomposing rhizomes of two seagrasses,Zostera marina and Thalassia testudinum

Bacteria growing on and in close association with the rhizome detritus of two seagrasses, Zostera marina L. and Thalassia testudinum Banks ex König, were examined using epifluorescence and scanning electron microscopy. The microbial community consisted of a diverse assemblage of bacteria dominated in biomass by large rod-shaped and filamentous cells. The large size of cells and the occurrence of measurable acetylene reduction activity suggested that a healthy, growing population of bacteria was associated with the rhizome detritus. Bacteria carbon biomass ranged betwee 5.2×10⁻⁵ and 1.7×10⁻³ g C gdw⁻¹ of rhizome detritus. Depending on cell doubling times, bacterial metabolism could account for a substantial portion of the turnover of rhizome detritus. Estimates of potential microbial production, nitrogen fixation and the physico-chemical nature of rhizome detritus are discussed and we propose hypotheses for the disposition of this detrital organic matter.     

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.     

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.     

Usefulness of epifluorescence for quantitative analysis of lactobacilli in probiotic feed

AIMS: Enumeration of total, active or viable probiotic micro-organisms from liquid or solid commercial feedstuffs was studied during processing and storage. METHODS AND RESULTS: After sample preparation, an epifluorescence microscopy technique and a plating method were investigated comparatively. It was shown that (i) on the day of manufacture, active or viable bacteria were in equivalent amounts and that viable numbers then decreased, depending on the different processing and storage factors enhancing ABNC production, (ii) the amount of total and active lactobacilli remained close and quite stable for months at a high level (>10(8) active fluorescent units). CONCLUSIONS: Processing and storage promoted ABNC cells in the products tested. Consequently, both techniques should be used to evaluate the viable-dead-active status of bacteria for which functional properties are claimed. SIGNIFICANCE AND IMPACT OF THE STUDY: Enumeration of the whole probiotic bacterial population should be take into account for guidelines and labelling since non-viable bacteria could have a probiotic effect.     

A comparative study of carboxyfluorescein diacetate and carboxyfluorescein diacetate succinimidyl ester as indicators of bacterial activity

Staining bacteria with esterified fluorogenic substrates followed by flow cytometric analysis offers a means for rapid detection of metabolically active bacteria. Flow cytometry (FCM) was used to assess carboxyfluorescein diacetate (CFDA) and carboxyfluorescein diacetate succinimidyl ester (CFDA/SE) as indicators of bacterial activity for cultured bacteria, including Aeromonas hydrophila, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus epidermidis and bacteria from environmental waters. In theory, CFDA/SE should be a better indicator of metabolic bacterial activity compared to CFDA due to greater intracellular retention of the fluorescent product. Qualitative and quantitative analysis of exponential phase cultures, mixtures of active and inactive cells and bacteria from environmental waters revealed CFDA was successful in detecting active bacteria, whereas CFDA/SE was not. CFDA/SE labelled inactive cells with intensities equal to that of the active population and could not even discriminate between bacteria in exponential phase growth and a fixed cell preparation. We propose that the specific mode of action of the succinimidyl ester (SE) group in combination with the nonenzymatic aqueous hydrolysis of the CFDA moiety results in the nonspecific labelling of all cells, irrespective of their metabolic state. This study shows that CFDA/SE is a poor marker of bacterial activity.     

Simultaneous fluorescent gram staining and activity assessment of activated sludge bacteria

Wastewater treatment is one of the most important commercial biotechnological processes, and yet the component bacterial populations and their associated metabolic activities are poorly understood. The novel fluorescent dye hexidium iodide allows assessment of Gram status by differential absorption through bacterial cell walls. Differentiation between gram-positive and gram-negative wastewater bacteria was achieved after flow cytometric analysis. This study shows that the relative proportions of gram-positive and gram-negative bacterial cells identified by traditional microscopy and hexidium iodide staining were not significantly different. Dual staining of cells for Gram status and activity proved effective in analyzing mixtures of cultured bacteria and wastewater populations. Levels of highly active organisms at two wastewater treatment plants, both gram positive and gram negative, ranged from 1.5% in activated sludge flocs to 16% in the activated sludge fluid. Gram-positive organisms comprised <5% of the total bacterial numbers but accounted for 19 and 55% of the highly active organisms within flocs at the two plants. Assessment of Gram status and activity within activated sludge samples over a 4-day period showed significant differences over time. This method provides a rapid, quantitative measure of Gram status linked with in situ activity within wastewater systems.     

Physiological assessment of bacteria using fluorochromes

This minireview focuses on the application of fluorogenic compounds in the detection of bacteria with particular emphasis on the assessment of physiological activity using epifluorescence microscopy. Microbiological applications of several related methods will also be reviewed.     

A sensitive and rapid method to determine the viability of freeze-dried bacterial cells

AIMS: To determine the potential use of flow cytometry for viability asssessment of freeze-dried bacterial cells. METHODS AND RESULTS: Escherichia coli CIP 54.8T and Vibrio metschnikovii CIP 104262 were analysed. The viability of freeze-dried cells resuspended in a nutrient broth was evaluated by culture whereas activity was determined by flow cytometry analysis of both esterase activity and cell death. Activity assessment by flow cytometry was found to be a rapid and good indicator of cell viability and was very efficient for quality control. For V. metschnikovii the fraction of active cells varies greatly depending on the freeze-drying procedure and within a given procedure. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: Bacterial activity assessment by flow cytometry is very efficient for the control of freeze-dried cells.     

A simple method for measuring colour in wild animals: validation and use on chest patch colour in geladas (Theropithecus gelada)

Adaptive hypotheses about colour variation are widespread in behavioural ecology, and several methods of objective colour assessment have been proposed and validated for use in a wide variety of taxa. However, to date, the most objective and reliable methods of assessing colour are not readily applied to wild animals. In the present study, we present a simple method for assessing colour in unrestrained, wild subjects using digital photography. The method we describe uses a digital camera, a colour standard, and colour analysis software, and can be used to measure any part of the visible colour spectrum. We demonstrate that the method: (1) is accurate and precise across different light conditions; (2) satisfies previous criteria regarding linearity and red, green, and blue equality; and (3) can be independently validated visually. In contrast with previous digital methods, this method can be used under natural light conditions and can be readily applied to subjects in their natural habitat. To illustrate this, we use the method to measure chest colour in wild geladas ( Theropithecus gelada ). Unique among primates, geladas have a red patch of skin on their chest and neck, which, for males, is thought to be a sexually selected signal. Offering some support to this hypothesis, we found differences in chest 'redness' for males across different age groups, with males in their reproductive prime exhibiting the reddest chests.  © 2008 The Linnean Society of London, Biological Journal of the Linnean Society , 2008, 94 , 231–240.