Hemocytometer

Abstract: The entomopathogenic fungus Neozygites parvispora (Entomophthorales: Zygomycetes) grows in vitro as irregularly rod-shaped hyphal bodies in a complex medium. In order to simplify the medium composition and determine growth-promoting compounds for the cultivation of this fungus, we were looking for a rapid and quantitative method to estimate the number of living cells in small volumes of liquid culture. A colorimetric method for the determination of cell densities using MTT [3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] proved to be more accurate and timesaving than conventional hemocytometer counting.

Abstract: Quantification of the differential cell count and total number of cells recovered from the lower respiratory tract by bronchoalveolar lavage is a valuable technique for evaluating the alveolitis of patients with inflammatory disorders of the lower respiratory tract. The most commonly used technique for the evaluation of cells recovered by lavage has been to concentrate cells by centrifugation and then to determine total cell number using a hemocytometer and differential cell count from a Wright-Glemsa-stained cytocentrifuge preparation. However, we have noted that the percentage of small cells present in the original cell suspension recovered by lavage is greater than the percentage of lymphocytes identified on cytocentrifuge preparations. Therefore, we developed procedures for determining differential cell counts on lavage cells collected on Millipore filters and stained with hematoxylin-eosin (filter preparations) and compared the results of differential cell counts performed on filter preparations with those obtained using cytocentrifuge preparations. When cells recovered by lavage were collected on filter preparations, accurate differential cell counts were obtained, as confirmed by performing differential cell counts on cell mixtures of known composition, and by comparing differential cell counts obtained using filter preparations stained with hematoxylin-eosin with those obtained using filter preparations stained with a peroxidase cytochemical stain. The morphology of cells displayed on filter preparations was excellent, and interobserver variability in quantitating cell types recovered by lavage was less than 3%.(ABSTRACT TRUNCATED AT 250 WORDS)

Abstract: We have established basic methods, using quantitative measures of EF5 binding, to estimate the actual pO2 of cells and tissues. In situations where the tissue can be dissociated into single cells, or for cell cultures, we can measure the distribution of cellular binding rates using flow cytometry and these can be compared with cells treated under pO2S controlled by the spinner vial or thin-film methods in vitro. The flow cytometer is calibrated by staining V79 cells treated with EF5 under "standard" conditions. For intact tissues treated with EF5 in vivo, we need to correct for possible variations in drug exposure (AUC). Frozen sections are stained for EF5 binding and are analyzed by a sensitive (cooled) CCD camera with linear output vs fluorescence [figure: see text] input. The camera has very consistent sensitivity, but the entire optical system, including the camera, can be calibrated by an absolute fluorescence standard (dye in hemocytometer). This system can also be used to measure the fluorescence of the flow cytometer standards, providing a direct link between the two assays. We can measure the maximum binding rate using the tissue cube method, but need to assume an "average" oxygen dependence of binding for intact tissues. The best-fit approximation for existing data is an inverse relationship between binding and pO2, with binding decreasing 50-fold between 0.1 and 10% oxygen. Using these methods, we routinely estimate the minimum pO2 (maximum binding) in experimental rodent and human tumors. In normal tissue models, an excellent correlation is found between near-maximal binding (severe hypoxia) and apoptosis (heart infarct and ductus arteriosus). Some normal tissues (e.g., skeletal muscle) are refractory to both cellular disaggregation and cube calibration methods. To extend the tissue imaging measurements to a complete two- or three-dimensional analysis of the distribution of tissue pO2s requires a substantial additional investment of imaging methods, which are currently being implemented.