Image analysis as a mean to model growth of Escherichia coli O157:H7 in gel cassettes

AIMS: The potential of image analysis for rapid and quantitative determination of the effect of environmental parameters such as temperature and pH on the growth of colonies of Escherichia coli O157:H7 derived from immobilized cells in gel cassettes was investigated. METHODS AND RESULTS: The organism was grown in brain heart infusion agar contained within a cassette formed between sheets of PVC film. The medium was adjusted to pH 5, 6 or 7 and incubated at 10, 20, 30 or 40 degrees C. The primary model of Baranyi was used to fit the growth data obtained by conventional plate counting and changes in colony area (2-dimensional spread of colonies) by light microscopy to derive estimates of maximum specific growth rates (micromax and Area micromax) in both cases. Growth rate values from both measurements were correlated and a secondary quadratic model was developed to predict micromax obtained via image analysis in response to environmental factors (temperature and pH). A progressive decrease of micromax and Area micromax was observed at lower temperatures and pH values. Immobilized cells failed to initiate growth at a pH of 5.0 and 10 degrees C. There was high correlation between micromax values estimated by conventional plate counting and Area micromax values from microscopic observations in gel cassettes, regardless of temperature and pH. The values of micromax derived indirectly from the correlation with Area micromax values fitted well to the secondary model and gave realistic predictions of maximum specific growth rate values estimated by standard plate counting. CONCLUSIONS: The micromax of E. coli O157:H7 determined by plate counting was linearly correlated with Area micromax estimated by light microscopy, enabling indirect determination of micromax via the Area micromax. The estimates of micromax via the image analysis technique may be further modelled in response to environmental factors such as temperature and pH to predict the response of the organism in intermediate conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: Image analysis in combination with gel cassettes could be a potential tool for rapid and convenient data collection and construction of accurate mathematical models as an alternative to conventional plate counting methods.     

Microchamber array based DNA quantification and specific sequence detection from a single copy via PCR in nanoliter volumes

A novel method for DNA quantification and specific sequence detection in a highly integrated silicon microchamber array is described. Polymerase chain reaction (PCR) mixture of only 40 nL volume could be introduced precisely into each chamber of the mineral oil layer coated microarray by using a nanoliter dispensing system. The elimination of carry-over and cross-contamination between microchambers, and multiple DNA amplification and detection by TaqMan chemistry were demonstrated, for the first time, by using our system. Five different gene targets, related to Escherichia coli were amplified and detected simultaneously on the same chip by using DNA from three different serotypes as the templates. The conventional method of DNA quantification, which depends on the real-time monitoring of variations in fluorescence intensity, was not applied to our system, instead a simple method was established. Counting the number of the microchambers with a high fluorescence signal as a consequence of TaqMan PCR provided the precise quantification of trace amounts of DNA. The initial DNA concentration for Rhesus D (RhD) gene in each microchamber was ranged from 0.4 to 12 copies, and quantification was achieved by observing the changes in the released fluorescence signals of the microchambers on the chip. DNA target could be detected as small as 0.4 copies. The amplified DNA was detected with a CCD camera built-in to a fluorescence microscope, and also evaluated by a DNA microarray scanner with associated software. This simple method of counting the high fluorescence signal released in microchambers as a consequence of TaqMan PCR was further integrated with a portable miniaturized thermal cycler unit. Such a small device is surely a strong candidate for low-cost DNA amplification, and detected as little as 0.4 copies of target DNA.     

Measurement of marine picoplankton cell size by using a cooled, charge-coupled device camera with image-analyzed fluorescence microscopy.

Accurate measurement of the biomass and size distribution of picoplankton cells (0.2 to 2.0 microns) is paramount in characterizing their contribution to the oceanic food web and global biogeochemical cycling. Image-analyzed fluorescence microscopy, usually based on video camera technology, allows detailed measurements of individual cells to be taken. The application of an imaging system employing a cooled, slow-scan charge-coupled device (CCD) camera to automated counting and sizing of individual picoplankton cells from natural marine samples is described. A slow-scan CCD-based camera was compared to a video camera and was superior for detecting and sizing very small, dim particles such as fluorochrome-stained bacteria. Several edge detection methods for accurately measuring picoplankton cells were evaluated. Standard fluorescent microspheres and a Sargasso Sea surface water picoplankton population were used in the evaluation. Global thresholding was inappropriate for these samples. Methods used previously in image analysis of nanoplankton cells (2 to 20 microns) also did not work well with the smaller picoplankton cells. A method combining an edge detector and an adaptive edge strength operator worked best for rapidly generating accurate cell sizes. A complete sample analysis of more than 1,000 cells averages about 50 min and yields size, shape, and fluorescence data for each cell. With this system, the entire size range of picoplankton can be counted and measured.     

Measurement of marine picoplankton cell size by using a cooled, charge-coupled device camera with image-analyzed fluorescence microscopy.

Accurate measurement of the biomass and size distribution of picoplankton cells (0.2 to 2.0 microns) is paramount in characterizing their contribution to the oceanic food web and global biogeochemical cycling. Image-analyzed fluorescence microscopy, usually based on video camera technology, allows detailed measurements of individual cells to be taken. The application of an imaging system employing a cooled, slow-scan charge-coupled device (CCD) camera to automated counting and sizing of individual picoplankton cells from natural marine samples is described. A slow-scan CCD-based camera was compared to a video camera and was superior for detecting and sizing very small, dim particles such as fluorochrome-stained bacteria. Several edge detection methods for accurately measuring picoplankton cells were evaluated. Standard fluorescent microspheres and a Sargasso Sea surface water picoplankton population were used in the evaluation. Global thresholding was inappropriate for these samples. Methods used previously in image analysis of nanoplankton cells (2 to 20 microns) also did not work well with the smaller picoplankton cells. A method combining an edge detector and an adaptive edge strength operator worked best for rapidly generating accurate cell sizes. A complete sample analysis of more than 1,000 cells averages about 50 min and yields size, shape, and fluorescence data for each cell. With this system, the entire size range of picoplankton can be counted and measured.     

Simultaneously Capturing Real-time Images in Two Emission Channels Using a Dual Camera Emission Splitting System: Applications to Cell Adhesion

Multi-color immunofluorescence microscopy to detect specific molecules in the cell membrane can be coupled with parallel plate flow chamber assays to investigate mechanisms governing cell adhesion under dynamic flow conditions. For instance, cancer cells labeled with multiple fluorophores can be perfused over a potentially reactive substrate to model mechanisms of cancer metastasis. However, multi-channel single camera systems and color cameras exhibit shortcomings in image acquisition for real-time live cell analysis. To overcome these limitations, we used a dual camera emission splitting system to simultaneously capture real-time image sequences of fluorescently labeled cells in the flow chamber. Dual camera emission splitting systems filter defined wavelength ranges into two monochrome CCD cameras, thereby simultaneously capturing two spatially identical but fluorophore-specific images. Subsequently, psuedocolored one-channel images are combined into a single real-time merged sequence that can reveal multiple target molecules on cells moving rapidly across a region of interest.     

A rapid BOD sensing system using luminescent recombinants of Escherichia coli

A biochemical oxygen demand (BOD) sensing system based on bacterial luminescence from recombinant Escherichia coli containing lux A-E genes from Vibrio fischeri has been developed. It was possible to use frozen cells of luminescent recombinants of E. coli as the bacterial reagents for measurement. Steady bioluminescence was observed during the incubation time between 90 and 150 min in the presence of a sole carbon source such as glucose, acetate, L-glutamate and BOD standard solution (GGA solution). This disposable bacterial reagent was applied to measure and detect organic pollution due to biodegradable substances in various wastewaters. The obtained values of this study showed a similar correlation with that of the conventional method for BOD determination (BOD5). Bacterial luminescence that was visualized with an imaging system using a charge coupled device (CCD) camera and a photomulti-counter demonstrated that this method could also be used for multi-sample detection of organic pollution due to biodegradable substances by using a microtiter plate. These results suggested for successful achievement of high-though-put detection of BOD in practical.     

Development of an on-line monitoring system of human keratinocyte growth by image analysis and its application to bioreactor culture

Human keratinocytes were cultured in serum-free medium for the purpose of on-line cell growth monitoring by image analysis. The validity of a process using a newly developed video microscopy system with image analysis for growth-rate monitoring in real time was verified by the measurement of the degree of confluence of keratinocytes in T-flasks and Petriperm dishes. The growth rate of keratinocytes was calculated subsequently from the linear relationship between average degree of confluence and cell concentration. This technique was applied to the culture in the bioreactor "KERATOR" in which a special video microscopy system using a CCD camera was built. The cell concentration evaluated by image analysis agreed well with that evaluated by conventional direct cell counting after enzymatic digestion, and the on-line monitoring of the specific growth rate allowed identification of both lag- and exponential-growth phases of the culture.     

Optical chamber system designed for microscopic observation of living cells under extremely high hydrostatic pressure

A novel pressure chamber system has been developed for the study of living cells under conditions of extremely high hydrostatic pressure up to 100 MPa (1 atm = 0.101325 MPa). The temperature in the chamber is thermostatically controlled in the range from 2 degrees to 80 degrees C. Two high-pressure pumps are employed for continuous perfusion of the chamber with culture medium and a chemical solution under high hydrostatic pressure conditions. The chamber has a 2-mm-thick glass window 2 mm in diameter, with a minimum working distance of 3.8 mm. The chamber system is designed to be adaptable to a variety of microscopic and imaging techniques. Using this chamber system, we successfully carried out real-time observations of elongated Escherichia coli and rounded HeLa cells under pressure.     

The correlation method for rapid monitoring of Escherichia coli in foods

AIMS: A new rapid method was developed to rapidly monitor Escherichia coli counts in foods. MATERIALS AND RESULTS: One ml of modified selective broth with 4-methylumbelliferyl beta-D-glucuronide and 1 ml of food sample were mixed in a sterile test tube and incubated at 37 degrees C. The positive reaction (fluorescence under u.v. light) was monitored at regular 30 min intervals. The positive reaction times in test tubes were compared with actual E. coli numbers from tested samples. The growth of E. coli in test tubes (broth) was much faster than growth on agar. The first experiment was performed to evaluate the rapid correlation method using pure E. coli cultures. The correlation between E. coli counts by the conventional plating method and positive reaction (fluorescence production) times in test tubes was highly agreeable (r(2) = 0 x 95). In the case of low E. coli numbers, such as 2 x 0 log10 cfu ml(-1), the rapid correlation method detected their presence after 10 h incubation. When highly contaminated samples were assayed (8 log10 cfu ml(-1)), the rapid correlation method detected the presence of E. coli after 4 h incubation. In the ground beef experiment, the correlation between fluorescence production time and actual E. coli numbers was also strongly agreeable (r(2) = 0 x 92). CONCLUSIONS: From these results, it is obvious that the new rapid method can rapidly monitor E. coli counts in foods. SIGNIFICANCE AND IMPACT OF THE STUDY: The results indicated that the new method saved about 10-14 h incubation time compared to conventional plating methods. The rapid correlation method required much shorter incubation times compared to conventional plating methods for monitoring E. coli.     

Rapid detection and counting of single bacteria in a wide field using a photon-counting TV camera

Using Escherichia coli as a model bacterium, we tested a photon-counting method for enumeration of bacteria. This method is based on the principle that microscopic sized luminous particles in a wide field can be directly detected and counted using a photon-counting TV camera without the use of a microscope. E. coli cells were labeled with peroxidase and luminescence induced by adding a luminol-based reaction mixture. The number of luminous spots in the TV images was in good agreement with the number of bacterial colonies grown from labeled cells. The results show that our method provides a rapid and easy microbial counting system for such purposes as clinical diagnosis, microbial analysis in food, and environmental assessment.     

Fully automated and fast image analysis of autoradiographs with a TAS-Leitz. Determination of size, Feulgen fluorescence and grain counts of individual nuclei and their evaluation by a simplified cluster analysis

A fully automatic analysis system based on television image analysis was developed to measure simultaneously three parameters in individual nuclei of microscopic autoradiographs prepared from mouse jejunal crypt cell squashes and ascites tumor cell smears: size, Feulgen fluorescence and reflection from silver grains. A dark light camera with an image intensified silicon tube (RCA-ISIT), an automatic scanning stage and an autofocus device were fitted to a Leitz-TAS microscope. The camera permitted localization of Feulgen stained nuclei and measurement of area and light intensity by means of incident of light fluorescence in the red. After automatic changes of the Opak-illuminator silver grains were determined by means of polarized incident light reflected from the grains in the blue. A 25 X oil objective (aperture 0.75) yielded sufficient resolution for measurements. The nadir between the proportions of labeled and unlabeled nuclei was calculated from the data of one specimen on a PDP-computer using a new algorithm based on the minimal variance of the logarithm of reflected light per nucleus. Labeling indices determined by visual grain counting and by automatic analysis of the autoradiographs were well correlated (r = 0.87 to 0.92). Visual grain counts/nucleus and reflected light/nucleus correlated well when individual nuclei were compared (r = 0.92 to 0.97) or means of labeled nuclei of various specimens prepared during a 5 year period (r = 0.90 to 0.93). Quenching of nuclear Feulgen fluorescence was minimal. The optimal labeling range is 30-100 grain counts/nucleus. The time interval between measurements of two specimens was 25 min for a squash of approximately 350 crypt cells within a 3 mm X 3 mm field, and 20 min for a meandering scan with 1,000 ascites tumor cells.     

Automatic cell counting with ImageJ

Cell counting is an important routine procedure. However, to date there is no comprehensive, easy to use, and inexpensive solution for routine cell counting, and this procedure usually needs to be performed manually. Here, we report a complete solution for automatic cell counting in which a conventional light microscope is equipped with a web camera to obtain images of a suspension of mammalian cells in a hemocytometer assembly. Based on the ImageJ toolbox, we devised two algorithms to automatically count these cells. This approach is approximately 10 times faster and yields more reliable and consistent results compared with manual counting.     

Expression of a thermostable xylanase gene from Bacillus coagulans ST-6 in Lactococcus lactis

AIMS: The aim of the study is to evaluate whether xylanase can be used as a potential reporter gene for cloning and expression studies in Lactococcus. METHODS AND RESULTS: The 750 bp xylanase gene was amplified and subcloned into the unique NheI restriction enzyme site of pMG36e and subsequently transformed into competent Escherichia coli XLI-blue MRF cells and Lactococcus lactis cells. Bacterial culture containing pMG36e-Xy has an enzyme activity of 390 microg xylose ml(-1) culture 30 min(-1), respectively, when compared with 40 microg xylose ml(-1) culture 30 min(-1) for the negative control (plasmidless strain). CONCLUSIONS: The thermostable xylanase gene was successfully expressed in both E. coli and L. lactis. The activity of xylanase can be easily detected by the formation of visible clearing zones around the transformed colonies on Remazol Brilliant Blue-Xylan (RBB-Xylan) agar media. However, there were some significant differences in the optimum growth temperature and plasmid stability in the new clones. SIGNIFICANCE AND IMPACT OF THE STUDY: The constructed reporter vector has the potential to be used as a reporter system for Lactococcus as well as E. coli, and it is an addition to the pool of lactococcal vector systems.     

Imaging the propagation of viruses

The propagation of viruses in a growing plaque has been measured using a digital image acquisition and analysis system. Plaques of phage T7 incubated at 37 degrees C and illuminated against a dark field emerged as dark growing spots against a background of host bacteria. Images of the growth were acquired using a charge-coupled device (CCD) camera at 1-h intervals over 24 h. The first 10 h of plaque development coincided with rapid growth of the agar-immobilized Escherichia coli host, measured as a reduction in gray value. Following this period, the average radial velocity of plaque growth remained constant at 0.059 mm/h while the standard deviation about this velocity increased. These results suggest the suitability of the system for spatially resolving the dynamics of viral evolution during plaque growth. (c) 1996 John Wiley & Sons, Inc.     

A "do-it-yourself" array biosensor

We have developed an array biosensor for the simultaneous detection of multiple targets in multiple samples within 15-30 min. The biosensor is based on a planar waveguide, a modified microscope slide, with a pattern of small (mm2) sensing regions. The waveguide is illuminated by launching the emission of a 635 nm diode laser into the proximal end of the slide via a line generator. The evanescent field excites fluorophores bound in the sensing region and the emitted fluorescence is measured using a Peltier-cooled CCD camera. Assays can be performed on the waveguide in multichannel flow chambers and then interrogated using the detection system described here. This biosensor can detect many different targets, including proteins, toxins, cells, virus, and explosives with detection limits rivaling those of the ELISA detection system.     

A model that uses the induction phase of lux gene-dependent bioluminescence in Pseudomonas fluorescens HK44 to quantify cell density in translucent porous media.

A cooled charge-coupled device (CCD) camera was used to follow the kinetics of induction of lux gene-dependent bioluminescence in Pseudomonas fluorescens HK44 held either in aqueous suspensions minus sand, saturated or unsaturated translucent sand (0.348 and 0.07 cm(3) H(2)O/cm(3) of sand, respectively), and at cell densities ranging between 1 x 10(6) and 8.5 x 10(8) cells/ml. Before O(2) availability became a limiting factor, the rate of light emission (L) increased with the square of time (t) and linearly with increasing cell density (c). A nonlinear model was developed that contains a "rate of increase in light emission" constant, B', which is determined directly from the slope of a plot of radical L/c against t. The model predicted the behavior of lux induction in HK44 under a variety of conditions. Similar B' values were determined [49.0-57.6 x 10(-10) light units/(cell min(2))] for cell suspensions held in aqueous medium minus sand, in saturated or unsaturated 40/50 grade sand (0.36 mm grain diameter) and in two other textural classes of translucent sand. Although both the growth phase, and the presence of glucose during lux induction affected the first detectable time (FDT) of bioluminescence by HK44 in sand, the kinetics of induction of light emission were similar among treatments (stationary phase cells plus glucose, B'=61.6+/-3.2, log phase cells plus glucose, B'=63.2+/-7.2). The potential exists to use a combination of a CCD camera system, an inducible lux gene containing bioluminescent bacterium, and a light transmission chamber to nonintrusively visualize and quantify in real time the interactions between bacterial growth and unsaturated flow of water and solutes in porous media.     

A simple chemiluminescence-based method for rapid enumeration of Listeria spp. microcolonies

AIMS: Listeria monocytogenes is capable, under certain conditions, of producing chemiluminescence which is amplified by luminol. This property was used to detect and count microcolonies of Listeria spp. in a few hours, without the use of a microscope. METHODS AND RESULTS: After trapping Listeria cells on polyvinylidene fluoride membranes, a chemiluminescence mixture was sprayed onto the membrane. The chemiluminescent spots emitted were analysed by a charge-coupled device camera connected to a data-processing system, which restored the intensity of the signals into three dimensional images. The intensity of the luminescence of microcolonies was improved by addition of cellobiose, and by brief exposure to u.v. light. CONCLUSION: Microcolonies of Listeria spp. can be imaged and counted by luminol-enhanced chemiluminescence with a photon-counting system. SIGNIFICANCE AND IMPACT OF THE STUDY: This method can be applied to the rapid detection and counting of Listeria spp. in raw milk.     

Quantification of chemotactic response of quiescent and proliferating fibroblasts in Boyden chambers by computer-assisted image analysis

The chemotactic response of human gingival fibroblasts to platelet-derived growth factor (PDGF) was investigated in 48-well modified Boyden chambers. Results were quantified using computer-assisted image analysis of propidium iodidestained cells and were compared with results obtained using the conventional method of quantification by direct counting. Quantification by image analysis was found to be rapid and accurate, and correlated closely with results obtained by direct counting. Bromodeoxyuridine (BrdU)-labeled proliferating cells were double stained with propidium iodide and fluorescein isothiocyanate (FITC)-conjugated anti-BrdU antiserum. The cycling cells showed a markedly reduced chemotactic response to PDGF, whereas cells in S-phase of the cell cycle did not show any response. This method of quantification of results of Boyden chamber assays is simple and reliable, and allows the use of double labels to investigate the chemotactic response of subpopulations of cells within a heterogeneous population of cells.     

Initial evaluation of a direct detection device detector for single particle cryo-electron microscopy

We report on initial results of using a new direct detection device (DDD) for single particle reconstruction of vitreous ice embedded specimens. Images were acquired on a Tecnai F20 at 200 keV and a nominal magnification of 29,000×. This camera has a significantly improved signal to noise ratio and modulation transfer function (MTF) at 200 keV compared to a standard CCD camera installed on the same microscope. Control of the DDD has been integrated into Leginon, an automated data collection system. Using GroEL as a test specimen, we obtained images of ∼30 K particles with the CCD and the DDD from the same specimen sample using essentially identical imaging conditions. Comparison of the maps reconstructed from the CCD images and the DDD images demonstrates the improved performance of the DDD. We also obtained a 3D reconstruction from ∼70 K GroEL particles acquired using the DDD; the quality of the density map demonstrates the potential of this new recording device for cryoEM data acquisition.