Microscopy cell counting

Cells in log and stationary phases treated 24 hr with DFP (20-500 pAf). iri-o-ioJylphosphate (8-150 pj /), or dicrotophos (90-50(1 pAf) in medium wdlhoirt serumL cell counts and rates of incorporation of [ Clglucose and P Clleucine measured neurite morphology examined by light microscopy. 

Perhaps the most critical aspect of cellular materials is the structure of the cells—the size, shape, and proportion of open and closed cells. The size and shape of cells can be studied by microscopy or by projecting a very thin section, but to get numerical results is inevitably tedious. A British standard [82] determines cell count of flexible materials as the number of cells per linear 25 mm. which is considered a more convenient measure than cell size, particularly considering the variation in size for even uniform cell structures. 

Microscope slide methods A specimen smear on a glass slide followed by simple or differential staining remains one of the fastest ways to determine numbers of bacteria or yeasts. A minimum number of cells of 10" cm are needed. Both viable and nonviable cells may be enumerated and results can be obtained in 5 min. A number of slide counting devices exist with the Breed slide method being one of the most widely used. The Breed smear was one of the first methods to use microscopy for counting bacteria in milk and involved the preparation of milk films, staining with Methylene Blue, and then microscopic examination. 

A 39-year-old woman developed bilateral corneal edema after taking amantadine for 2 months [279 ]. Corneal thicknesses were 940 pm in the right eye and 802 pm in the left. There was diffuse stromal edema, folds in Des-cemet s membrane, and microcystic subepithe-lial edema. Specular microscopy showed significant pleomorphism and polymegathism with an endothelial cell count of 1504 cells in the right eye and 1596 in the left eye. 

Willey and Waterbury (1989) used a substantially different method to study chemotaxis in the marine cyanobacterium Synechococcus sp. This experiment involved the use of blind-well chemotaxis chambers (Neuroprobe, Inc., Cabin John, MD) that consisted of an upper (800- il) and lower (200- li1) acrylic chamber separated by a polycarbonate filter (3.0 pm). A cell suspension (165 pi) of the cyanobacterium was placed in the lower chamber over which the polycarbonate filter was placed. An air space was left between the cell suspension and the filter to control the starting time of the experiment. The upper chamber was filled with sterile seawater containing the compound to be tested and then inverted, allowing the cell suspension to contact the polycarbonate filter and the seawater/compound solution. The experiments were run for 65 min, after which time the chambers were inverted to stop the experiment. The number of cells crossing the filter into the seawater chamber was determined by direct cell counts using epifluorescence microscopy. The motile strain of Synechococcus sp. tested in this assay elicited positive chemotaxis to compounds such as ammonia, nitrate, urea, glycine, and P-alanine. Control chambers with the same concentration of chemoattractant in both the upper and lower chambers failed to elicit a chemotactic response. While the compounds tested in this study were relatively simple metabolites, one could... 

Figure Bl.24.17. An example of scanning transmission ion microscopy (STIM) measurements of a human oral cancer cell. The different images indicate different windows in the energy of transmitted helium ions as indicated in the figure. White indicate areas of high counts. The teclmique offers a thickness scan through the sample, and, in this case, the cell walls of one specific cell can be seen in the areas dominated by thicker structures (data from C A Pineda, National Accelerator Centre, Fame, South Africa).

Figure 18.3. Desulfovibrio vulgaris Hildenborough attachment kinetics to mild steel coupons under various Fe. Metal coupons (1.5 x 6.0 cm) were suspended into 24-h growing cultures under various Fe concentrations and removed at the times indicated. Coupons were washed with distilled water, dried, fixed with 5% gluteralde-hyde, and analyzed by scanning electron microscopy. The bacterial count at each datum point represents an average of 10 random sites on the coupon, counted from scanning micrographs and equated to a number (10 cells) per unit area (mm ) metal.

Duncan, R.R., Bergmann, A., Cousin, M.A., Apps, D.K., and Shipston, M.J. et al. 2004. Multidimensional time correllated single photon counting (TCSPC) fluorescence lifetime imaging microscopy (Aim) to Detect Fret in Cells. J. Microsc. 215 1. 

Xanthamonas campestris strain engineered to bioluminesce (Shaw et al., 1992). The strain was applied to cabbage plants and surrounding soils in a limited field study conducted in 1990. The fate of the organisms was monitored by both CCD-microscopy and plate counts, Recombinant cells were found for up to six weeks, with the CCD and plate counts giving quantitatively similar results. Bioluminescence in this case was no more sensitive than plate counts, and required a precision instrument,... 

In 1934, Andrew Moldavan in Montreal took a first step from static microscopy toward a flowing system. He suggested the development of an apparatus to count red blood cells and neutral-red stained... 

Light microscopy of immunoperoxidase-stained sections was done on Axiovert S100 microscope (Carl Zeiss, Tokyo, Japan) and digitized by a 3-CCD (charge-coupled device) digital camera (Fujifilm, Tokyo, Japan) on a personal computer (Fujitsu, Tokyo, Japan). Only cells in a single focal plane were counted on a computer screen to avoid oversampling. 

Samples for cell viability tests were taken three times during the different batch and fed-batch fermentations. A sample of 1 mL was diluted 104-105 times and 0.1 mL of diluted sample was applied to an agar plate. Five to six plates were prepared for every sample, and the plates were incubated for 24 h at 30°C. The total cell concentration in the reactor sample was determined with microscopy using a Biirker counting chamber. The viability, expressed as the fraction of cells able to form colonies, was calculated by dividing the concentration of colony-forming cells by the total cell concentration. 

Once the sample was prepared, meristems were scored with light microscopy using the x40 objective, and the mitotic index was estimated by counting up a total number of 1000 cells in three slides of the same sample. 

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