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Evaluation of an Automated Colony Counter

W. A. GOSS , R. N. MICHAUD, AND M. B. McGRATH

432KB

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.

The Use of Adult Rat Liver Cultures in the Detection of the Genotoxicity of Various Polycyclic Aromatic Hydrocarbons

C. TONG, M. F. LASPIA, S. TELANG, AND G. M. WILLIAMS

The hepatocyte primary culture (HPC)-DNA repair test and the adult rat liver epithelial cell (ARL)-hypoxanthine-guanine phosphoribosyl transferase (HGPRT) mutagenesis assay are two in vitro short-term tests that possess intrinsic capability for xenobiotic biotransformation. Both assays detected the genotoxicity of a variety of carcinogenic polycyclic aromatic hydrocarbons. Thus, these two tests, which embody intact cellular metabolism, are useful for the evaluation of this class of carcinogens and provide results that strengthen those obtained in tests dependent upon subcellular metabolism.

Health Effects Of Chemicals: V. Computer-Assisted Genetic Toxicology Testing

LEON S. OTIS, ROSIE Mc CORMICK, AND BETTY STROMNESS

288KB

Until recently manual grain counting took hours for each slide and made routine testing with this procedure impractical. With the new automated system, grains are counted using a "colony counter," which can detect the exposed silver grains using a contrast-discriminator. Grain counting, therefore, can be accomplished in the time it takes to focus the microscope on a cell, set an electronic aperture over the desired counting region (which is displayed on a TV monitor), and push a count button on the colony counter. The data are fed directly into a file in the computer, eliminating the need to record raw data manually. This system has reduced the time required to score 50 cells per slide from a few hours to an average of less than 10 minutes. In addition, all subsequent processing of data is accomplished by computer programs with no additional entry of data required.

A Rapid, Semi-Automated Counting Procedure for Enumeration of Antibody-Forming Cells in Gell and Nucleated Cells in Suspension

DAVID H. KATZ, MERYL FAULKNER, LEE R. KATZ, ERIK LINDH, CHARLES C. LEONHARDT, KIMBERLEY HERR AND AMAR S. TUNG

Since the description by Jerne et al (1) of the hemolysis-in-gel technique for enumerating single antibody-producing cells, this procedure has become one of standard usage in most laboratories engaged in immunological research. Different laboratories use a variety of modifications of the technique, whether in a gel support medium as initially described or the suspension technique described by Cunningham and Szenberg (2). Irrespective of the modification employed, the final analysis involves enumeration of plaques which have developed in the indicator erythrocyte suspension. Typically, this has been done visually by examining either slides or petri dishes with an appropriate light source either with or without the aid of a suitable magnifying lens or stereozoom microscope.

Purification and Properties of a Rat Liver Protein that Specifically Inhibits the Proliferation of Nonmalignant Epithelial Cells from Rat Liver

JAMES B. McMAHON, JAMES G. FARRELLY, AND P. THOMAS IYPE

In inhibitor of cell proliferation was purified from rat liver by alcohol precipitation, ultrafiltration, and DFAE-cellulose chromatography. The hepatic proliferation inhibitor was shown to be pure by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, analytical isoelectric focusing, and high-performance liquid chromatography. The hepatic proliferation inhibitor was found to have a'molecular weight of 26,000 and an isoelectric point of 4.65. This protein inhibited the proliferation of nonmalignant rat liver cells in culture, and removal of the protein reversed the inhibition produced by low doses. It exerted no effect on the proliferation of malignant rat liver cells.

Semi-Automated Grain and Cell Counting

PAUL A. BRUNN JR., SHARON S. FORD, STANLEY E. SHACKNEY

A commercially available bacterial colony counter has been adapted for the counting of radioautographic grains over individual cells in smears, and for counting cells in histologic sections. For the counting of radioautographic grains, the correlation coefficients between counts obtained visually by two observers, and between counts obtained visually and using the instrument were similar (r = .999 and r = .998 respectively). The instrument counts were obtained more rapidly than the visual counts and were associated with less observer fatigue. While performance of the instrument in counting cells in mouse bone marrow sections was less accurate than in counting radioautographic grains, a good estimation of marrow cell number was obtained (r = .968). Data on bone marrow cellularity was obtained far more rapidly than with semi-quantitative methods.

Semi-Automated Autoradiographic Measurement of DNA Repair In Normal and Xeroderma Pigmentosum Cultured Human Fibroblasts

KENNETH H. KRAEMER, JOSEPH K. BUCHANAN, AND SHERMAN F. STINSON

Assessment of DNA repair in cultured human fibroblasts by autoradiography may be facilitated by using semi-automated grain counting instruments. The instrument determined number of autoradiographic grains per nucleus in cultured human skin fibroblasts was found to be linear in comparison to visual counts up to only 30 grains per nucleus. However, with two different instruments a greater range of linearity (100 to 120 grains per nucleus) was attained by measuring the grain surface area per nucleus. Semi-automated analysis of the grain surface area per nucleus yielded measurements of relative rates of unscheduled DNA synthesis after ultraviolet irradiation in xeroderma pigmentosum and normal human fibroblasts, which were reproducible and rapid.

From Image to Analysis: Object Detection and Measurement

MICHAEL K. BENDER

Image Analyzers, both manual and automated, allow users to identify objects and to categorize I them using various geometric parameters. Densitometric identification and categorization is also possible with some image analysis equipment. Although image analyzers are not a recent development, many of their applications are new. Technological developments in the last decade have extended the capabilities of image analyzers by increasing speed, accuracy, and flexibility. The instruments have also become significantly more affordable.

Detection, Enumeration, and Sizing of Planktonic Bacteria by Image-Analyzed Epifluorescence Microscopy

MICHAEL E. SIERACKI, PAUL W. JOHNSON, AND JOHN McN. SIEBURTH

Epifluorescence microscopy is now being widely used to characterize planktonic procaryote populations. The tedium and subjectivity of visual enumeration and sizing have been largely alleviated by our use of an image analysis system consisting of a modified Artek 810 image analyzer and an Olympus BHT-F epifluorescence microscope. This system digitizes the video image of autofluorescing or fluorochrome-stained cells in a microscope field. The digitized image can then be stored, edited, and analyzed for total count or individual cell size and shape parameters. Results can be printed as raw data, statistical summaries, or histograms. By using a stain concentration of 5 m g of 4'6-diamidino -2-phenylindole per ml of sample and the optimal sensitivity level and mode, counts by image analysis of natural bacterial populations from a variety of habitats were found to be statistically equal to standard visual counts. Although the time required to prepare slides, focus, and change fields is the same for visual and image analysis methods, the time and effort required for counting is eliminated since image analysis is instantaneous. The system has been satisfactorily tested at sea. Histograms of cell silhouette areas indicate that rapid and accurate estimates of bacterial biovolume and biomass will be possible with this system.

Automated Radiographic Grain Counting: Correction for Grain Overlap

W.H. SCHUETTE, S.S. CHEN, S.J. OCCHIPINTI, H.S. MUJAGIC, AND S.E. SHACKNEY

An algorithm is described for the calculation of radioautographic cell grain count from measurements of total cell nuclear area and total grain area. This algorithm provide-s a statistical correction for grain overlap that is based on the solution to the occupancy problem in probability theory. This method permits the use of automated grain counting over a wide range of grain counts/cell, and extends the useful dynamic range of radioautographic grain counting to well over 200 grains/cell.