Dilute Malachite Green: A Background Stain for the Millipore Filter

A rapid method for increasing the visual contrast of colonies on the Millipore filter consists of flooding the filter with a 0.01% solution of malachite green for about 3 sec. The excess is then immediately poured off. The colonies remain unstained but the filter area not covered by the colonies becomes light green in color. The procedure requires only a few seconds and counts can be made immediately. Counting on crowded surfaces is facilitated since small transparent colonies appear white and are counted easily without the aid of magnification. The difficulty of differentiating between minute vegetable particles and small colonies is eliminated because the foreign particles become stained the same as or darker than the filter.     

Development and application of computer software for cell culture laboratory management

Prototype computer software for a Cell Culture Laboratory Management System (CCLMS) has been developed to relieve cell culture specialists of the burden of manual recordkeeping. Conventional data archives in cell culture laboratories are prone to error and expensive to maintain. The reliance upon cell culture to provide models for biochemical and molecular biological research serves to magnify errors at great expense. The CCLMS prototype encapsulates a modular software application that manages the many aspects of cell culture laboratory recordkeeping. A transaction-based database stores detailed information on subcultures, freezes and thaws, prints waterproof labels for culture vessels, and provides for immediate historical trace-back of any cultured cell line. Linked database files store information specific to an individual culture flask while removing redundancy between similar groups of flasks. A frozen cell log maintains locations of all vials within any type of cryogenic storage unit, locates spaces for newly frozen cell lines, and generates alphabetical or numerical reports. Finally, modules for maintaining cell counts, user records, and culture vessel specifications to support a comprehensive automation process are incorporated within this software. The developed CCLMS prototype has been demonstrated to be an adaptable, reliable tool for improving training, efficiency, and historical rigor for two independent cell culture facilities.     

Rapid Determination by Light Scattering of Growth Parameters of Mycoplasma laidlawii in Liquid Media

An impediment to progress in the study of the course of growth, the effects of medium components, antibiotics, etc., of Mycoplasma has been the cumbersome methods of growth measurement currently in use. Heretofore, it required the plating of numerous samples during growth, at least in triplicate, after appropriate dilution, followed by a delay of 2 to 3 days before the colonies developed so that counts could be made. We applied the technique of light scattering to measure the growth of Mycoplasma laidlawii in liquid culture continuously in a manner analogous to the use of absorbancy for bacteria. Scattered light measurements precisely paralleled data obtained by the tedious method of plate counts and were available immediately during the development of the culture. The lower limit of sensitivity with the system described is 10⁵ Mycoplasma per ml. The presence of serum in the medium lowers sensitivity somewhat. However, concentrations of serum up to 10% are easily tolerated. Higher serum content may require calibration curves. Thus the technique may be used with many pathogens, etc., that require serum to develop. One can easily and rapidly measure differences in growth rates as well as final cell yields during the course of growth, rather than 3 days later, after colonies have developed.     

人肺巨细胞癌蛋白质组的二维电泳和计算机图象分析

为优化用于蛋白质组研究的二维电泳技术和计算机图象分析技术 ,以及初步分析比较与肿瘤细胞转移相关的蛋白质 ,以人肺巨细胞癌 (PLA- 80 1 - D、C)高、低转移株作为研究对象 ,应用 IPG-phor进行第一向等电聚焦 ,随后 ,在 Protein IPG conversion Kit上进行垂直 SDS- PAGE的分离 .利用光密度仪对银染的凝胶扫描 ,通过 PDQuest软件进行蛋白斑点检测和配比 .结果表明 :(1 )应用 IPGphor,采用样品直接加入重泡胀溶液的形式 ,增大了溶解性 ,缩短聚焦时间、增大样品负荷量 (分析型 ) ,提高了分辨率 .(2 )比较宽 (p H=3～ 1 0 L)、窄 (p H=4～ 7L)范围 IPG胶条 ,窄 p H范围的 IPG胶条具有较高的分辨率 .(3)比较 PLA- 80 1 - C、D细胞蛋白图谱之间的差异 ,其相关系数为 0 .7339± 0 .0 2 91 ;仅在 PLA- 80 1 - C株出现的蛋白为 1 79个 .     

Automatic quantitation of cell colonies on petri dishes by computerized image analysis

Clonogenic assay is one of the most sensitive assays, widely used to evaluate the effects of antineoplastic agentsin vitro. A computer program was developed on an IBAS 2.0 Image Analysis System for automated quantiation of cell colonies and clone area on Petri dishes. The sensitivity of the clonogenic assay can be greatly increased by evaluating the mean area of the clones. The program gives an objective, accurate and fast evaluation of large samples. It is simple to use and offers a high degree of flexibility. Special algorithms and techniques have been implemented for good quantitation of both connected and well-separated colonies and to reduce the background noise and the general error rate. The principles and solutions presented are applicable to any other image analysis system.Abbreviations FBS fetal bovine serum - ATCC American Type Culture Collection - DDATHF 5,10-dideazatetrahydrofolic acid - PBS phosphate buffered saline     

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)     

QuantWorm: A Comprehensive Software Package for Caenorhabditis elegans Phenotypic Assays

Phenotypic assays are crucial in genetics; however, traditional methods that rely on human observation are unsuitable for quantitative, large-scale experiments. Furthermore, there is an increasing need for comprehensive analyses of multiple phenotypes to provide multidimensional information. Here we developed an automated, high-throughput computer imaging system for quantifying multiple Caenorhabditis elegans phenotypes. Our imaging system is composed of a microscope equipped with a digital camera and a motorized stage connected to a computer running the QuantWorm software package. Currently, the software package contains one data acquisition module and four image analysis programs: WormLifespan, WormLocomotion, WormLength, and WormEgg. The data acquisition module collects images and videos. The WormLifespan software counts the number of moving worms by using two time-lapse images; the WormLocomotion software computes the velocity of moving worms; the WormLength software measures worm body size; and the WormEgg software counts the number of eggs. To evaluate the performance of our software, we compared the results of our software with manual measurements. We then demonstrated the application of the QuantWorm software in a drug assay and a genetic assay. Overall, the QuantWorm software provided accurate measurements at a high speed. Software source code, executable programs, and sample images are available at www.quantworm.org. Our software package has several advantages over current imaging systems for C. elegans. It is an all-in-one package for quantifying multiple phenotypes. The QuantWorm software is written in Java and its source code is freely available, so it does not require use of commercial software or libraries. It can be run on multiple platforms and easily customized to cope with new methods and requirements.     

Video Bioinformatics Analysis of Human Embryonic Stem Cell Colony Growth

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer software. Video bioinformatics is a powerful quantitative approach for extracting spatio-temporal data from video images using computer software to perform dating mining and analysis. In this article, we introduce a video bioinformatics method for quantifying the growth of human embryonic stem cells (hESC) by analyzing time-lapse videos collected in a Nikon BioStation CT incubator equipped with a camera for video imaging. In our experiments, hESC colonies that were attached to Matrigel were filmed for 48 hours in the BioStation CT. To determine the rate of growth of these colonies, recipes were developed using CL-Quant software which enables users to extract various types of data from video images. To accurately evaluate colony growth, three recipes were created. The first segmented the image into the colony and background, the second enhanced the image to define colonies throughout the video sequence accurately, and the third measured the number of pixels in the colony over time. The three recipes were run in sequence on video data collected in a BioStation CT to analyze the rate of growth of individual hESC colonies over 48 hours. To verify the truthfulness of the CL-Quant recipes, the same data were analyzed manually using Adobe Photoshop software. When the data obtained using the CL-Quant recipes and Photoshop were compared, results were virtually identical, indicating the CL-Quant recipes were truthful. The method described here could be applied to any video data to measure growth rates of hESC or other cells that grow in colonies. In addition, other video bioinformatics recipes can be developed in the future for other cell processes such as migration, apoptosis, and cell adhesion. Download video file.^{(111M, mp4)}     

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.     

Detection of multiple-strain carriers: the value of re-sampling

Bacteria may colonize a carrier with more than one strain of a species at any one time. Attempts to determine multiple colonization are labour intensive because of the large number of colonies per carrier which need to be tested. A possible solution -- in which only 3 colonies per carrier are initially tested and only multiple-strain carriers are re-sampled -- was recently described. We evaluated the accuracy of the "re-sampling method" devised by Cespedes et al. with 500,000 stochastic simulations per scenario. Re-sampling is acceptable where > or = 5 colonies are initially tested or where one strain predominates over others in colony counts. The method introduces bias towards overestimation which decreases with the number of available carriers, increases with the proportion of truly multiple carriers, with decreasing number of colonies available for testing, and with decreasing number of colonies tested per carrier. Initial testing of 5-8 colonies tested with re-sampling are adequate for a large study (>100 carriers), or a small study where it is suspected that no strain predominates over the other in colony counts. Testing 9-20 colonies with re-sampling is necessary for small studies where one strain predominates over others. Re-sampling is unnecessary where >20 colonies are tested.     

Immunohistochemical and ultrastructural study of human melanoma colonies grown in soft agar

An immunohistochemical and ultrastructural study of human melanoma colonies grown in soft agar for up to 50 days was performed. Three morphological variants of developing tumor colonies are reported: 1) large light colonies, 2) small dark colonies, and 3) smooth-edged colonies. The large light colony variant is the most frequently observed in the soft agar assay (approximately 70%), followed by the dark colony variant (approximately 27%), and the smooth-edged colony variant (approximately 3%). Major morphological characteristics are associated with each variant, as shown with light microscopy (LM) and transmission electron microscopy (TEM). Both LM and TEM analyses demonstrated that the large light colony variant was hypomelanotic and contained a microfibrillar extracellular matrix (ECM). The small dark colony variant was found to be hypermelanotic and contained a less demonstrable ECM. The smooth-edged variant has an encapsulated periphery, no demonstrable ECM, and tightly packed cells with desmosome-like junctions. In order to characterize further the ECM in the most commonly observed variant, the large light colony, specific antibodies to fibronectin (FN) and collagen types IV and V (COLs IV and V) were applied and observed with immunofluorescence microscopy and immunoperoxidase. In paraffin sections of melanoma colonies, FN was observed associated with both the cell surface and the ECM. However, no specific staining was seen for COLs IV and V. In addition, ruthenium red was used to preserve and selectively bind to glycosaminoglycans (GAGs) and proteoglycans (PGs). TEM studies reveal GAG-like granules stained with ruthenium red in the fibrillar ECM and a dotted, punctate staining of the cell surface. Understanding the biological and architectural composition of developing melanoma tumor colonies in soft agar could contribute to the development of more efficient chemotherapeutic strategies.     

In vitro quantification by image analysis of inotropic and chronotropic effects of drugs on cultures of cardiac myocytes

Quantitative image analysis is used to measure the inotropic and chronotropic effects of drugs on cultured heart cells maintained at 37°C on the stage of an inverted light microscope, and sequentially superfused with control and treatment media. The beating of the cardiac myocytes is evaluated by simultaneously selecting up to eight areas, including cell edges, from digitized video image. The sizes and positions of these areas are controlled by the operator. To analyze the motion of cell edges in each area, the computer measures the shift of the mass center of pixels' grey levels. Finally, a few parameters are calculated for the eight areas and displayed graphically. In order to assess treatment effects, appropriate statistical tests are performed on the data. Image analysis is an efficient screening test for evaluating the pharmacologic or toxic effects of a substance on isolated or cultured cardiac myocytes from various species.Abbreviations MEM Minimum Essential Medium     

Optogenetic manipulation of neural activity in freely moving Caenorhabditis elegans

We present an optogenetic illumination system capable of real-time light delivery with high spatial resolution to specified targets in freely moving Caenorhabditis elegans. A tracking microscope records the motion of an unrestrained worm expressing channelrhodopsin-2 or halorhodopsin in specific cell types. Image processing software analyzes the worm's position in each video frame, rapidly estimates the locations of targeted cells and instructs a digital micromirror device to illuminate targeted cells with laser light of the appropriate wavelengths to stimulate or inhibit activity. Because each cell in an unrestrained worm is a rapidly moving target, our system operates at high speed (～50 frames per second) to provide high spatial resolution (～30 μm). To test the accuracy, flexibility and utility of our system, we performed optogenetic analyses of the worm motor circuit, egg-laying circuit and mechanosensory circuits that have not been possible with previous methods.     

Comparison of single and dual platform methodologies for the estimation of CD34+ hematopoietic progenitor cells: correlation with colony assay

In this study three assays for the enumeration of CD34+ progenitors were compared: 1) a modified version of the Milan protocol, used in the standard dual-platform format; 2) a dual-platform version of the ISHAGE protocol; 3) the ProCOUNT software version 2.0/ProCOUNT kit. The assays were compared to validate the accuracy of CD34+ cell counts in mobilized peripheral blood (PB), apheresis products (AP), and cord blood (CB). The ProCOUNT protocol uses reference beads for absolute CD34+ cell counting, whereas CD34 counts by other techniques are derived from a separate leukocyte count performed by a hematology analyzer. A good correlation between the ISHAGE and ProCOUNT methods was obtained for estimation of CD34+ counts in PB (n=42 samples analyzed) and AP (n=35)--except for samples having a leukocyte count >25 x 10(9)/L or a CD34 count <0.0025 x 10(9)/L)--while a suboptimal correlation between the methods was observed for CB (n=30). The ProCOUNT system proved to be effective in reducing the variability in CD34+ cell counting and appeared to be useful for intralaboratory methodology standardization. The main disadvantage of the ProCOUNT assay was its inability to calculate CD34 counts in leukopenic samples and in CB samples showing a high erythroblast count. As far as the correlation with hematopoietic colonies is concerned, data collected from apheresis samples showed a good correlation between the three flow cytometry methods and colony-forming unit granulocyte-macrophage (CFU-GM) counts, confirming the value of the flow cytometric test as a real-time, truly predictive test to measure the hematopoietic potential of the graft. In summary, all methods are suitable for enumeration of most PB samples, while the single-platform methodology should be preferred for the analysis of AP and CB. We also found the dual-platform format of the ISHAGE method precise and accurate for the estimation of CD34+ cells from CB samples. Based on these data it can be concluded that the single-platform flow cytometry assay format should be the preferred approach for CD34+ stem cell enumeration in different types of samples.     

Stochastic model for mast cell proliferation in culture of murine peritoneal cells

We recently identified two types of mast cell colonies derived from murine peritoneal cells: type 1 and type 2. Type 1 mast cell colonies consisted of berberine sulfate(+)- safranin(+) connective tissue-type mast cells (CTMC) and were derived from mature CTMC in the heaviest fraction obtained by Percoll density gradient centrifugation. In contrast, type 2 mast cell colonies consisted of alcian blue(+)- berberine sulfate(-)- safranin(-) mucosal mast cells (MMC) and were derived from immature progenitors in low density fractions. We replated a total of 60 type 1 and 60 type 2 mast cell colonies and examined their capability for producing secondary colonies. Although all of the primary colonies yielded secondary colonies, the replating efficiencies of individual colonies varied over a wide range. Cumulative distributions of secondary colonies from both type 1 and type 2 primary colonies could be fitted well by gamma distributions obtained by computer simulation. These findings are in agreement with the stochastic model for CTMC- and MMC proliferation. Cytological analyses of secondary colonies from primary type 1 colonies revealed heterogeneous distributions of alcian blue(+)- safranin(-)- berberine sulfate(-) mast cells, suggesting that transdifferentiation from mature CTMC to safranin(-)- berberine sulfate(-) mast cells is also governed by stochastic mechanisms.     

A Cluster Operating System Supporting Parallel Computing

The single factor limiting the harnessing of the enormous computing power of clusters for parallel computing is the lack of appropriate software. Present cluster operating systems are not built to support parallel computing – they do not provide services to manage parallelism. The cluster operating environments that are used to assist the execution of parallel applications do not provide support for both Message Passing (MP) or Distributed Shared Memory (DSM) paradigms. They are only offered as separate components implemented at the user level as library and independent servers. Due to poor operating systems users must deal with computers of a cluster rather than to see this cluster as a single powerful computer. A Single System Image of the cluster is not offered to users. There is a need for an operating system for clusters. We claim and demonstrate that it is possible to develop a cluster operating system that is able to efficiently manage parallelism, support Message Passing and DSM and offer the Single System Image. In order to substantiate the claim the first version of a cluster operating system, called GENESIS, that manages parallelism and offers the Single System Image has been developed.     

FlyCounter: a simple software for counting large populations of small clumped objects in the laboratory

While several software programs exist to count bacterial colonies on a Petri plate, no suitable solution is available for quick and reliable enumeration of small, live insects. We have written a program called FlyCounter that can obtain counts from images, even if insects are highly clumped in space. We also describe a simple and inexpensive system for anesthetizing and capturing high-quality images of the small insects. Taken together, our process is fast, fully automatic, and has a low percentage of error (~1%-4% on average). Although we have tested our software on fruit flies, it should be simple to extend to other organisms of similar size.     

Haemopoietic spleen colony growth: a versatile, parsimonious, predictive model

Abstract. Substantial support has been obtained for the stochastic model for stem cell differentiation first proposed by Till, McCulloch & Siminovitch (1964), over 20 years ago. By adding a cell maturation pathway, it is possible to predict (by computer simulation) the total number of cells and consequently the time at which individual colonies appear and disappear.
Only a few uncontroversial assumptions are required to predict that cells, uniform with respect to self-renewal, are capable of producing the high proportions of late disappearing and late appearing colonies observed experimentally in the spleens of irradiated mice that have been injected with normal haemopoietic cells. It is shown that differences in stem cell self-renewal only slightly influence the time of appearance of colonies; whereas changes in the kinetics of the maturing cells, by changing the size of colonies, has a marked effect on the time of appearance and disappearance of colonies and on the average doubling-time of colony-forming cells per colony (but not the doubling-time of individual colonies).
These results (1) seriously question the prevailing view that spleen colonies scored at 8 days measure a separate population (without the capacity for self-renewal), from those scored at 12 days; (2) argue against the existence of multiple sub-populations of stem cells with differing self-renewal and toxicity to cytotoxic agents; (3) help to identify those experiments for which it is obligatory to postulate heterogeneity, and (4) are consistent with self-renewal being regulated by a feedback control of stem cell differentiation, to which only proliferating stem cells can respond and where the stimulus for differentiation decreases at a time when the bone marrow is known to be depleted.