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.     

Evaluation of an Automated Colony Counter

An automated colony counter was found to readily detect surface and subsurface bacterial colonies of 0.3-mm size or greater with a high degree of precision. On a logarithmic scale, counting efficiency consistently ranged from 89 to 95% of corresponding manual count determinations for plates containing up to 1,000 colonies. In routine application, however, automated plate counts up to approximately 400 colonies were selected as a more practical range for operation. The automated counter was easily interfaced with an automated data acquisition system.     

Evaluation of counting error due to colony masking in bioaerosol sampling.

Colony counting error due to indistinguishable colony overlap (i.e., masking) was evaluated theoretically and experimentally. A theoretical model to predict colony masking was used to determine colony counting efficiency by Monte Carlo computer simulation of microorganism collection and development into CFU. The computer simulation was verified experimentally by collecting aerosolized Bacillus subtilis spores and examining micro- and macroscopic colonies. Colony counting efficiency decreased (i) with increasing density of collected culturable microorganisms, (ii) with increasing colony size, and (iii) with decreasing ability of an observation system to distinguish adjacent colonies as separate units. Counting efficiency for 2-mm colonies, at optimal resolution, decreased from 98 to 85% when colony density increased from 1 to 10 microorganisms cm-2, in contrast to an efficiency decrease from 90 to 45% for 5-mm colonies. No statistically significant difference (alpha = 0.05) between experimental and theoretical results was found when colony shape was used to estimate the number of individual colonies in a CFU. Experimental colony counts were 1.2 times simulation estimates when colony shape was not considered, because of nonuniformity of actual colony size and the better discrimination ability of the human eye relative to the model. Colony surface densities associated with high counting accuracy were compared with recommended upper plate count limits and found to depend on colony size and an observation system's ability to identify overlapped colonies. Correction factors were developed to estimate the actual number of collected microorganisms from observed colony counts.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

High-Throughput Method for Automated Colony and Cell Counting by Digital Image Analysis Based on Edge Detection

Counting cells and colonies is an integral part of high-throughput screens and quantitative cellular assays. Due to its subjective and time-intensive nature, manual counting has hindered the adoption of cellular assays such as tumor spheroid formation in high-throughput screens. The objective of this study was to develop an automated method for quick and reliable counting of cells and colonies from digital images. For this purpose, I developed an ImageJ macro Cell Colony Edge and a CellProfiler Pipeline Cell Colony Counting, and compared them to other open-source digital methods and manual counts. The ImageJ macro Cell Colony Edge is valuable in counting cells and colonies, and measuring their area, volume, morphology, and intensity. In this study, I demonstrate that Cell Colony Edge is superior to other open-source methods, in speed, accuracy and applicability to diverse cellular assays. It can fulfill the need to automate colony/cell counting in high-throughput screens, colony forming assays, and cellular assays.     

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.     

Persistence of cell cycle times over many generations as determined by heritability of colony sizes of ras oncogene-transformed and non-transformed cells

Abstract. The persistence of cell lifetimes during about 10 successive cell generations was investigated by comparing the number of cells in primary colonies and in secondary colonies derived from primary colonies. Primary colonies were grown from single cells for 3 or 4 days (a time equivalent to an average of five cell generations) and the number of cells in each primary colony determined. Cells in each primary colony were dispersed to initiate secondary colonies, grown for the same time, and the number of cells in secondary colonies determined. Several criteria were used to compare primary and related secondary colonies, the most informative was found to be regression and correlation coefficients between number of cells in primary colonies and mean numbers of cells in related secondary colonies. For two non-transformed mouse fibroblast cell lines, NIH 3T3 and BALB 3T3, the regression and correlation coefficients of cell number in primary and secondary colonies were positive. This suggests inheritance of cell lifetimes over many cell generations. After the addition of an activated ras oncogene (human cellular Harvey ras , or viral Kirsten ras ) some regression and correlation coefficients changed in magnitude but all remained positive. Comparison of experimental data and the results of computer simulations suggest that several models of inheritance of cell lifetimes are not adequate to explain the results, including a model of independence between lifetimes of mother and daughter cells and the common model that describes daughter cells as inheriting the lifetime of their mother with deviation. Simulations do suggest that cell lifetimes are inherited within clones as deviation from the lifetime of the initial cell, and that the ras oncogene does not destroy persistence within clones but does increase heterogeneity of cell lifetimes.     

Flow cytometric enumeration of colonies for in vitro chemotherapeutic drug evaluation

The percentage of 50-100 micron colonies formed by LX-T cells in medium containing agarose was determined microscopically, and this value was compared with the percentage determined by a flow cytometric method based on the forward and 90 degree light scatter of the colonies. As assessed by both in vitro methods, LX-T cells exposed to chemotherapeutic agents formed fewer colonies as the drug concentration increased. However, flow cytometric analysis indicated that a change in the number of colonies formed was a consequence of changing chemotherapeutic drug concentration, whereas microscopic colony counting did not always detect the corresponding change in colony number. These experiments demonstrate that measurement of a drug's chemotherapeutic potential by flow cytometric counting of colonies is an alternative to the enumeration of colonies microscopically.     

An improved method for staining cell colonies in clonogenic assays

Clonogenic assay is a widely used experimental approach to test for the effects of drugs/genes on the growth and proliferative characteristics of cells in vitro. Accurate quantitation of treatment effects in clonogeneic assays depends on the ability to visualize and count cell colonies precisely. We report a novel method (referred as ETeB) for staining cell colonies grown on plastic and specially coated substrates like collagen. Using colon cancer cell lines grown on plastic and collagen, we compared the colony staining efficiencies of the widely used methylene blue, and Ethidium bromide (ETeB) stains. Results show that the ETeB protocol works well on plastic and is extremely effective for staining colonies on collagen when compared to methylene blue. The key features and advantages of ETeB technique are; (a) reduction in background for colonies grown on collagen and possibly other substrates, (b) the whole procedure takes less than a minute, (c) no post-stain washing step is required which eliminates colony losses for cell lines that are loosely adherent, (d) colony visualization and counting can be done immediately following the staining procedure using a standard UV illuminator and software, and (e) the method works across a wide variety of cell lines. The simplicity and robustness of this procedure should warrant its usage in both small and large-scale clonogenic experiments.

Evaluation of an Automatic Electronic Device for Counting Bacterial Colonies

An automatic colony counter was tested extensively with colonies of two bacterial species, Serratia marcescens and Bacillus subtilis var. niger, grown on agar media. A stable relationship was established between machine counts and counts done visually by technicians. The calibration curve and estimates of the efficiency of the machine are presented and discussed. It is estimated that a 40% reduction in colony counting time is feasible through use of the machine.     

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.     

Genetic analysis of the clonal origin of regenerating mouse spermatogenesis following transplantation

Spermatogonial transplantation provides a straightforward approach to quantify spermatogonial stem cells (SSCs). Because donor-derived spermatogenesis is regenerated in the form of distinct colonies, the number of functional SSCs can be obtained by simply counting the number of colonies established in recipient testes. However, this approach is legitimate only when one colony arises from one stem cell (one colony-one stem cell hypothesis). In this study, we evaluated the validity of this hypothesis. Two populations of donor cells were obtained from the testes of two transgenic mouse lines and mixed at a 1:1 ratio. Following transplantation of the cell mixture, donor-derived colonies were visualized and individually excised, and genomic DNA was extracted from each colony. Based on unique marker genes of the two transgenic lines, the genotype of the cells contained in a colony was examined by polymerase chain reaction. A colony was determined to be clonal when only one transgene was detected. The results showed that 100% and 90% of colonies were clonal when <5 and 19 colonies were formed per recipient testis, respectively. However, the clonality of colonies decreased as the colony number per recipient testis or the length of each colony increased. These results support the one colony-one stem cell hypothesis and demonstrate that spermatogonial transplantation provides a highly quantitative assay for SSCs; however, these conclusions are applicable under a defined transplantation condition.     

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.     

An Evaluation of Procedures for Enumerating Bacteria in Activated Sludge

S ummary : A procedure for counting viable heterotrophic bacteria in activated sludge was evolved from a study of the effects of modifications to procedures at the different stages of enumeration. Optimal counts were obtained with Casitone-glycerol-yeast extract agar (CGY) with incubation for 6 days at 22°. Homogenization of mixed liquor was conveniently performed, with minimal lethal effect on the bacteria, by treating samples, diluted 1/10 in sodium tripolyphosphate solution (5 mg/1), in a boiling tube immersed in the Kerry ultrasonic cleaning bath for 1 min. Counts were significantly affected by the pH value of diluent and CGY, but not by the homogenization method or by treating homogenized samples with enzymes or N -acetyl cysteine, or by adding colloidal peptizing agents to the diluent. Replicate colony counts showed variances greater than the mean, although precision increased with increasing number of colonies/dish; there was a direct relationship between colony counts and volume plated for up to c. 1000 colonies/dish. Counts on spread plates tended to be higher and more precise than on dilution frequency plates, although the 2 methods showed satisfactory correlation. Counts were not significantly affected by the method of sampling and preparing the initial dilution, and it was considered prudent to examine samples immediately after collection.     

Improved Aerobic Colony Count Technique for Hydrophobic Grid Membrane Filters

The AOAC International official action procedure for performing aerobic colony counts on hydrophobic grid membrane filters (HGMFs) uses Trypticase soy-fast green FCF agar (FGA) incubated for 48 h. Microbial growths are various shades of green on a pale green background, which can cause problems for automated as well as manual counting. HGMFs which had been incubated 24 or 48 h at 35°C on Trypticase soy agar were flooded underneath with 1 to 2 ml of 0.1% triphenyltetrazolium chloride (TTC) solution by simply lifting one corner of the filter while it was still on the agar and adding the reagent. Microbial growths on HGMFs were counted after color had been allowed to develop for 15 min at room temperature. With representative foods, virtually all colonies stained pink to red. Automated electronic counts made by using the MI-100 HGMF Interpreter were easier and more reliable than control HGMF counts made by the AOAC International official action procedure. Manual counting was easier as well because of increased visibility of the microbial growths. Except in the case of dairy products, 24-h TTC counts did not differ significantly from 48-h FGA counts, whereas the FGA counts at 24 h were always significantly lower, indicating that for many food products the HGMF TTC flooding method permits aerobic colony counts to be made after 24 h.     

Application of Amido black staining to enumerating bacteria grown on membrane filters

A method to detect and enumerate bacterial colonies grown on membrane filters (MF) was described. The colonies were stained with an ethanolic solution of 0.1% Amido black 10B. The procedure yielded the rapid detection of colonies as compared to a conventional plate counting method.     

Genetic Analysis of the Drifting of Drones in Apis mellifera Using Multilocus DNA Fingerprinting

The presence of non-native drones in colonies of Apis mellifera was studied using multilocus DNA fingerprinting. Drones revealing a fingerprinting DNA banding pattern that did not correspond to the queen's genotype were classified as non-native animals. As previously reported for the drifting of workers, the position of the hive in relation to neighbouring colonies and the orientation of the flight entrance towards the sun showed significant correlations to the number of drifted drones in a colony. The frequency of non-native drones was low in central colonies (13 %) but high in marginal colonies (24 %). Furthermore, there was a significant correlation (r = 0.56) between the band-sharing coefficient of the non-native drones and the queen, and the number of drifted drones in the colony, which might indicate that genetically based nestmate recognition is involved in the drifting and/or acceptance of foreign drones in the colony.     

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.     

Murine mast cell colony formation supported by IL-3, IL-4, and recombinant rat stem cell factor, ligand for c-kit.

Recently, a novel cytokine designated stem cell factor (SCF) was isolated from medium conditioned by buffalo rat liver cells and proved to be the ligand for c-kit. We have examined the effects of recombinant rat SCF alone and in various combinations with interleukin-3 and interleukin-4 on murine mast cell colony formation in methylcellulose culture. As a source of connective tissue-type mast cells (CTMC), we used peritoneal mast cells. No individual factor supported colony formation by purified peritoneal mast cells. When cells were grown in combinations of two factors, significant mast cell colony growth was seen. When cells were grown in the presence of three factors, not only the number of colonies was increased but also the colonies were larger. Mast cells in these colonies contained safranin- and berberine sulfate-positive cells, but the proportions of positive and negative cells varied depending on the factor combinations. We then examined the effects of these factors on proliferation of bone marrow-derived mast cells (BMMC) by replating pooled mast cell colonies. As a single factor, only interleukin-3 supported mast cell colony formation. Combinations of two of the three factors supported mast cell colony formation. However, the most impressive synergism was seen again with the combination of the three factors. Not only was the number of colonies increased, but there was a significant increase in size. These results indicate that SCF is an important factor for the proliferation of both CTMC and BMMC.