Automated counting of bacterial colony forming units on agar plates

Manual counting of bacterial colony forming units (CFUs) on agar plates is laborious and error-prone. We therefore implemented a colony counting system with a novel segmentation algorithm to discriminate bacterial colonies from blood and other agar plates.A colony counter hardware was designed and a novel segmentation algorithm was written in MATLAB. In brief, pre-processing with Top-Hat-filtering to obtain a uniform background was followed by the segmentation step, during which the colony images were extracted from the blood agar and individual colonies were separated. A Bayes classifier was then applied to count the final number of bacterial colonies as some of the colonies could still be concatenated to form larger groups. To assess accuracy and performance of the colony counter, we tested automated colony counting of different agar plates with known CFU numbers of S. pneumoniae, P. aeruginosa and M. catarrhalis and showed excellent performance.     

Automated counting of mammalian cell colonies by means of a flat bed scanner and image processing.

BACKGROUND: Clonogenic assays are used frequently to measure the cell killing and mutagenic effects of radiation and other agents. Clonogenic assays carried out manually are tedious and time-consuming and involve a significant element of subjectivity. However, several commercial automatic colony counters are available. Based on CCD video imaging and image analysis they are relatively expensive and can analyze only one petri dish at a time. METHOD: We have developed a cheaper and more efficient device, which employs a flat bed scanner to image 12 60-mm petri dishes at a time. Two major problems in automated colony counting are the clustering of colonies and edge effects. By using standard image analysis and implementing an inflection point algorithm, these problems were greatly diminished. The resulting system was compared with two manual colony counts, as well as with automated counts with the Oxford Optronix ColCount colony counter for cell lines V79 and HaCaT. RESULTS: Comparisons assuming the manual counts to be correct showed that our automatic counter was slightly more accurate than the commercial unit. CONCLUSIONS: As a whole, our automated colony counter performed significantly better than the commercial unit with regard to processing time, cost and accuracy.     

A new formazan amplified clonogenic assay for cytotoxicity testing

Summary Recently, a tetrazolium salt known as MTT was developed to assess mammalian cell proliferation in vitro. Once reduced by active mitochondrial dehydrogenases it produces insoluble formazan crystals. These are usually dissolved with DMSO to give a colorimetric test. We took advantage of the insoluble formazan crystals production to amplify small colonies which are scored by means of a Biotran III automated colony counter. Throughout this study we tested whether or not this method could shorten the technical time applied to score colonies which have grown either in a T-flask or in soft-agar. Results presented below show that MTT may be used for colony enhancement in soft-agar assays. This amplification method was found to be reproducible and sensitive.     

ENUMERATING 3MTM PETRIFILMTM AEROBIC COUNT PLATES USING THE PETRISCAN® AUTOMATED COLONY COUNTER

In the food and dairy industries, aerobic plate counts are determined by a time-consuming and laborious hand-counting method. The PetriScan ® automated colony counter was developed to improve efficiency in the microbiology laboratory. In this study, colony counts of food, dairy, and milk products plated on 3M^TM Petrifilm^TM Aerobic Count Plates were compared using both automated and manual count plate methods. For sample variation, 16 different food, dairy, and milk products were used. Samples were prepared and serially diluted using Butterfield's diluent according to approved AOAC methods and APHA's Standard Methods. Plates were inoculated, incubated, and counted according to AOAC methods. For data collection, plates with counts between 5 and 300 colonies were included. A total of 55 low (5–30), 29 medium (31–100), and 23 high (101–300) count plates were used. Duplicate results were recorded for both methods; hand counts were tallied by two scientists. The duplicates of the mean log values for manual counts varied by 0.0005 and 0.0007, and the duplicates for the automated counts varied by 0.0011. The mean log value difference between the automated and manual counts for pooled data was 0.035. The correlation coefficient for the regression line comparing the automated and manual count methods for pooled data was 0.98. The regression equation was y = 0.9257x + 0.0781. These results demonstrate that the PetriScan® automated colony counter is a comparable and practical alternative to the standard method of manually counting plates.     

Quantitative image analysis of connective tissue progenitors

OBJECTIVE: To develop an image analysis system to automatically identify colony-forming units (CFUs) in in vitro cell cultures of connective tissue progenitors. This system was designed to quantitatively assess colony morphology and number of colonies in 4-cm(2) culture wells. STUDY DESIGN: Large field-of-view high-resolution fluorescence images of 4',6-diamidino-2-phenylindole (DAPI)- and alkaline phosphatase (AP)-stained bone marrow cell cultures were obtained using an epi-fluorescence microscope and automated scanning stage. Cell nuclei were identified in the DAPI-stained images after removal of fluorescent debris from the image. An Euclidean distance map (EDM) of the segmented cell nuclei was used to cluster cell nuclei into colonies. The automated system was evaluated using 40 tissue culture wells of bone marrow aspirate samples. The results of the automated analysis were compared to the manual tracings of colonies by 3 reviewers. RESULTS: The automated method agreed with all 3 reviewers on average 87.5% of the time. Additionally, reviewers identified other colonies not outlined by the reviewers on average 2.7 times more than the automated method. CONCLUSION: The automated method is a less biased method for identifying CFUs than individual reviewers, it provides more quantitative information about colony morphology than can be obtained manually and it is less time consuming.     

Automated counting of human tumour colonies in the Courtenay-Mills assay system

A procedure for using the Omnicon automated image analysis system for counting colonies grown from a human tumour cell line (COLO 205) in the Courtenay-Mills assay is described. This involves the transfer of the agar medium from culture tubes into petri dishes. Comparisons of observer and instrument counts were done on a blinded basis. Run-to-run correlation coefficient was 0.996 for automated counting and the inter-observer correlation coefficient was 0.984. Both assessments showed a linear relationship between the number of cells plated and the number of colonies grown. Automated colony counting is fast, reliable and provides additional information on colony size distribution, not obtainable with manual counting. This automated procedure will greatly facilitate in vitro drug sensitivity evaluation.     

Use of the IUL Countermat Automatic Colony Counter for Spiral Plated Total Viable Counts

An IUL Countermat automatic colony counter was used to enumerate colonies on spiral total viable count plates made with a wide variety of foods. The counter results exhibited a correlation with manual counting results similar to the reproducibility obtained with manually counted spiral plates. Use of this machine results in a large time saving compared with the conventional counting method and is recommended as a generally suitable method for counting spiral total viable count plates.     

Comparison of methods for assessing integrity of equine sperm membranes

Sperm membrane integrity (SMI) is thought to be an important measure of stallion sperm quality. The objective was to compare three methods for evaluating SMI: flow cytometry using SYBR-14/propidium iodide (PI) stain; an automated cell counting device using PI stain; and eosin-nigrosin stain. Raw equine semen was subjected to various treatments containing 20 to 80% seminal plasma in extender, with differing sperm concentrations, to simulate spontaneous loss of SMI. The SMI was assessed immediately, and after 1 and 2 d of cooled storage. Agreement between methods was determined according to Bland-Altman methodology. Eosin-nigrosin staining yielded higher (2%) overall mean values for SMI than did flow cytometry. Flow cytometry yielded higher (6%) overall mean values for SMI than did the automated cell counter. As percentage of membrane-damaged sperm increased, agreement of SMI measurement between methods decreased. When semen contained 50-79% membrane-intact sperm, the 95% limits of agreement between SMI determined by flow cytometry and eosin-nigrosin staining were greater (range = −26.9 to 24.3%; i.e., a 51.2% span) than for SMI determined by flow cytometry and the automated cell counter (range = −3.1 to 17.0%; 20.1% span). When sperm populations contained <50% membrane-intact sperm, the 95% limits of agreement between SMI determined by flow cytometry and eosin-nigrosin staining were greater (range = −35.9 to 19.0%; 54.9% span) than for SMI determined by flow cytometry and the automated cell counter (range = −11.6 to 28.7%; 40.3% span). We concluded that eosin-nigrosin staining assessments of percent membrane-intact sperm agreed less with flow cytometry when <80% of sperm had intact membranes, whereas automated cell counter assessments of percent membrane-intact sperm agreed less with flow cytometry when <30% of sperm had intact membranes.     

Evaluation of colony-forming ability experiments using normal and DNA repair-deficient human fibroblast strains and an automatic colony counter

An automated procedure for evaluation of colony-forming ability experiments has been developed using normal and xeroderma pigmentosum fibroblast strains. Technically this consists of an image analyzer equipped with a contrast-intensifying video camera, monitor, and a desk-top computer. Complex structures composed of overlapping colonies could be reduced to the constituent single colonies by special correction programs. Eighteen experiments were evaluated, and the D0 values for colony-forming ability were determined. A systematic but correctable deviation between visual and automated counting was found. The source of this deviation is described and a solution for correction is provided. The automatic procedure reduced the evaluating time by a factor of five to ten.     

Approaches,challenges and recent advances in automated bee counting devices: A review

For nearly 100 years, electronic bee counters have been developed using various technologies to track the foraging activity of mostly honey bee colonies. These counters should enable remote monitoring of the hives without disturbing natural flight behaviour while generating precise scientific data. However, there are few counters on the market that are able to fulfil this task. One main challenge is the lack of standardised methods to validate a counter's precision, as validation is crucial to categorise and judge the data produced by the counter, especially for scientific purposes. Another challenge is the interpretation of flight data to measure the effects of environmental or anthropogenic sources. Nevertheless, recent developments in the field are promising. This review describes the historic development of automated bee flight measurements and critically compares validation methods to encourage their improvement. To increase the comparability of future analyses of bee counters, current advances in data interpretation are also presented.     

Single-cell microliter-droplet screening system (MISS Cell): An integrated platform for automated high-throughput microbial monoclonal cultivation and picking

Solid plates have been used for microbial monoclonal isolation, cultivation, and colony picking since 1881. However, the process is labor- and resource-intensive for high-throughput requirements. Currently, several instruments have been integrated for automated and high-throughput picking, but complicated and expensive. To address these issues, we report a novel integrated platform, the single-cell microliter-droplet screening system (MISS Cell), for automated, high-throughput microbial monoclonal colony cultivation and picking. We verified the monoclonality of droplet cultures in the MISS Cell and characterized culture performance. Compared with solid plates, the MISS Cell generated a larger number of monoclonal colonies with higher initial growth rates using fewer resources. Finally, we established a workflow for automated high-throughput screening of Corynebacterium glutamicum using the MISS Cell and identified high glutamate-producing strains. The MISS Cell can serve as a universal platform to efficiently produce monoclonal colonies in high-throughput applications, overcoming the limitations of solid plates to promote rapid development in biotechnology.     

Direct identification of pure Penicillium species using image analysis

This paper presents a method for direct identification of fungal species solely by means of digital image analysis of colonies as seen after growth on a standard medium. The method described is completely automated and hence objective once digital images of the reference fungi have been established. Using a digital image it is possible to extract precise information from the surface of the fungal colony. This includes color distribution, colony dimensions and texture measurements. For fungal identification, this is normally done by visual observation that often results in a very subjective data recording. Isolates of nine different species of the genus Penicillium have been selected for the purpose. After incubation for 7 days, the fungal colonies are digitized using a very accurate digital camera. Prior to the image analysis each image is corrected for self-illumination, thereby gaining a set of directly corresponding images with respect to illumination. A Windows application has been developed to locate the position and size of up to three colonies in the digitized image. Using the estimated positions and sizes of the colonies, a number of relevant features can be extracted for further analysis. The method used to determine the position of the colonies will be covered as well as the feature selection. The texture measurements of colonies of the nine species were analyzed and a clustering of the data into the correct species was confirmed. This indicates that it is indeed possible to identify a given colony merely by macromorphological features. A classifier (in the normal distribution) based on measurements of 151 colonies incubated on yeast extract sucrose agar (YES) was used to discriminate between the species. This resulted in a correct classification rate of 100% when used on the training set and 96% using cross-validation. The same methods applied to 194 colonies incubated on Czapek yeast extract agar (CYA) resulted in a correct classification rate of 98% on the training set and 71% using cross-validation.     

Application of an automated colony counter for evaluation of the viability of a yeast culture

Application of an automated colony counter for evaluation of the viability of microbial cultures was investigated with yeast cultures as a model. Statistical comparison of the results of automated and visual (“manual”) colony counting is presented, as well as the results of the application of the bundled software to digital images obtained by light microscopy for determination of the cell concentration in suspensions. Automated counting is concluded to significantly accelerate the evaluation of culture viability by colony-forming capacity, provided that a certain requirements of sample preparation and analysis are observed.     

Encephalitozoon cuniculi: quantitation of parasites and evaluation of viability

Two methods (manual and automated) for quantitation of viable versus dead Encephalitozoon cuniculi are reported. The manual method uses ethidium bromide and acridine orange to stain dead and viable organisms, respectively. The stained organisms are visually differentiated with the aid of a fluorescence microscope. The automated method uses propidium iodide to stain dead parasites, which are differentiated from viable unstained parasites with the aid of a flow cytometer. An automated cell counter (Coulter Counter) was used to count rapidly large numbers of samples and to improve the sensitivity of counting low concentrations of parasites. These methods will enhance investigators' abilities to conduct quantitative experiments on host defense mechanisms against E. cuniculi.     

COVASIAM: an Image Analysis Method That Allows Detection of Confluent Microbial Colonies and Colonies of Various Sizes for Automated Counting

In this work we introduce the confluent and various sizes image analysis method (COVASIAM), an automated colony count technique that uses digital imaging technology for detection and separation of confluent microbial colonies and colonies of various sizes growing on petri dishes. The proposed method takes advantage of the optical properties of the surfaces of most microbial colonies. Colonies in the petri dish are epi-illuminated in order to direct the reflection of concentrated light coming from a halogen lamp towards an image-sensing device. In conjunction, a multilevel threshold algorithm is proposed for colony separation and counting. These procedures improved the quantification of colonies showing confluence or differences in size. We tested COVASIAM with a sample set of microorganisms that form colonies with contrasting physical properties: Saccharomyces cerevisiae, Aspergillus nidulans, Escherichia coli, Azotobacter vinelandii, Pseudomonas aeruginosa, and Rhizobium etli. These physical properties range from smooth to hairy, from bright to opaque, and from high to low convexities. COVASIAM estimated an average of 95.47% (ς = 8.55%) of the manually counted colonies, while an automated method based on a single-threshold segmentation procedure estimated an average of 76% (ς = 16.27) of the manually counted colonies. This method can be easily transposed to almost every image-processing analyzer since the procedures to compile it are generically standard.     

Evaluation of leukocyte number by using an automated blood cell counter and a traditional hematological method in animals irradiated with gamma rays

Female mice were irradiated with a single whole body dose of 7 Gy of gamma-rays. Leucocyte numbers were monitored in the peripheral blood using automated blood cell counter Coulter counter and a traditional hematological method with a light microscope in the Bürker chamber. Reticulocyte numbers, RNA blood concentration, spleen weight and morphological changes in spleen and bone marrow were also studied. In the period between 15th-19th days after irradiation the numbers of leucocytes obtained by CC counting were manifold higher than those obtained by microscope counting. Since this period is characterised by a steep increase in the reticulocyte number and RNA concentration in blood as well as by increased weight of spleen as the result of marked regeneration of extramedullar erythropoiesis, leukocytes as well as reticulocytes are assumed to be additionally registered by the automated counter CC in this period, probably due to a higher resistance of reticulocytes to the lysing agent Zapoglobine.     

A new method of cloning mammalian tissue culture cells in suspension

Chinese hamster cells (line CHO) were cloned in suspension with a carboxymethylcellulose-containing medium, and the colonies were counted with an electronic particle counter after enzymatically degrading the carboxymethylcellulose with cellulase. Survival of x-irradiated cells was determined by both the standard plating technique and the suspension technique, and comparable results were obtained.     

Automation of cell line development

An automated platform for development of high producing cell lines for biopharmaceutical production has been established in order to increase throughput and reduce development costs. The concept is based on the Cello robotic system (The Automation Partnership) and covers screening for colonies and expansion of static cultures. In this study, the glutamine synthetase expression system (Lonza Biologics) for production of therapeutic monoclonal antibodies in Chinese hamster ovary cells was used for evaluation of the automation approach. It is shown that the automated procedure is capable of producing cell lines of equal quality to the traditionally generated cell lines in terms of colony detection following transfection and distribution of IgG titer in the screening steps. In a generic fed-batch evaluation in stirred tank bioreactors, IgG titers of 4.7 and 5.0 g/L were obtained for best expressing cell lines. We have estimated that the number of completed cell line development projects can be increased up to three times using the automated process without increasing manual workload, compared to the manual process. Correlation between IgG titers obtained in early screens and titers achieved in fed-batch cultures in shake flasks was found to be poor. This further implies the benefits of utilizing a high throughput system capable of screening and expanding a high number of transfectants. Two concentrations, 56 and 75 μM, of selection agent, methionine sulphoximine (MSX), were applied to evaluate the impact on the number of colonies obtained post transfection. When applying selection medium containing 75 μM MSX, fewer low producing transfectants were obtained, compared to cell lines selected with 56 μM MSX, but an equal number of high producing cell lines were found. By using the higher MSX concentration, the number of cell line development projects run in parallel could be increased and thereby increasing the overall capacity of the automated platform process. A. Salmén and K. Lindgren contributed equally to the work.     

Encephalitozoon cuniculi: Quantitation of Parasites and Evaluation of Viability1

Two methods (manual and automated) for quantitation of viable versus dead Encephalitozoon cuniculi are reported. The manual method uses ethidium bromide and acridine orange to stain dead and viable organisms, respectively. The stained organisms are visually differentiated with the aid of a fluorescence microscope. The automated method uses propidium iodide to stain dead parasites, which are differentiated from viable unstained parasites with the aid of a flow cytometer. An automated cell counter (Coulter Counter) was used to count rapidly large numbers of samples and to improve the sensitivity of counting low concentrations of parasites. These methods will enhance investigators' abilities to conduct quantitative experiments on host defense mechanisms against E. cuniculi.     

Environmentally adaptive fish or no-fish classification for river video fish counters using high-performance desktop and embedded hardware

Automated fish counters featuring robust, real-time computer vision capabilities can provide a cost-effective means to count migrating freshwater fish. In this work, we propose a four-stage process for automatically sorting videos with and without fish. Underwater fish counter videos provide a challenging range of environmental conditions including clear water, biofilm growth, bubbles, turbidity, low light and overexposure. To address this, our method also includes the automated classification of these six environmental conditions. The proposed methods are computationally efficient and can be implemented on servers, high-performance desktop computers and low-cost, energy-efficient embedded hardware. The models were trained, tested, and validated using a collection of 3000 videos taken from underwater fish counter installations in several alpine and lowland European rivers provided by commercial and governmental collaborators. This work demonstrates a fast, accurate, and robust computer vision workflow for large-scale automated freshwater fish counting systems.