Centrifugal cytology. III. The utilization of centrifugal cytology for the preparation of fixed stained dispersions of cells separated by bovine serum albumin bouyant density centrifugation.

This paper describes the modification of Centrifugal Cytology for the preparation of permanent, fixed, stained dispensions for both light and scanning electron microscopy of cells which have been isolated on bovine serum albumin (BSA) boyant density gradients. The principal problem with BSA gradient fractions is that the albumin which is present even after dilution is precipitated by the glutaraldehyde fixative. This problem has been solved by the layering of an intermediate D2O solution under the BSA and subsequent removal of the BSA solution and the underlaying with D2O containing glutaraldehyde. A special layering machine facilitates and expedites these operations. This technique has also been applied to BSA-seperated guinea pig and chicken bone marrow cells, as well as Ehrlich ascites tumor cells, hen and human blood cells. The number of celll present in each area of the slide is maintained at a constant value by utlizing a table of dilution factors. This table was generated by a computer program which calculates the concentration of cells present in the rractions and divides it by the number of celll desired.

Buoyant density separation of cells. I. The buoyant distribution of guinea pig bone marrow cells.

Guinea pig bone marrow cells were separated by buoyant density utilizing linear gradients of bovine serum albumin (BSA). It has finally become possible to characterize the cells present in the density fractions in terms of classical morphology. The development of the Cell Type computer program which calculates the percentages of the individual types of cells present in the fractions and their buoyant density distributions and plots the data has greatly facilitated and improved the accuracy of these studies. Approximately 40 cell types were observed in guinea pig bone marrow. Cells with definitive morphologies such as erythrocytes, the neutrophilic series, the binucleate blast megakaryocyte precursor and cells in mitosis band as virtually single peaks. Cells which are parts of continua or can easily be wrongly classified are found in multiple peaks. The small lymphocytes which are known to be polydisperse are found as five peaks. Because of the very strong benzidine staining by the glutaraldehyde-fixed hemoglobin, some of the erythroblasts were wrongly staged, resulting in a multimodal distribution. The presence of macrocytes further complicated these distributions. The rule that the younger cells are always less dense than the mature cells was adhered to in those cases where the cells could be definitively characterized, such as the neutrophilic series and the blasts. These results indicate that morphology is a good first approximation of reality.

Centrifugal Cytology, IV. The Prearation of fixed stained dispersions of gynecological cells.

The Centrifugal Cytology technique has been utilized to produce glutaraldehyde fixed stained dispersions of both conventional Ayre scrapes and Davis pipet (PAPette) samples. Light microscope studies of dispersions of both types of cells on conventional microscope slides indicated that both the tinctorial and morphological appearance of the cells after Papanicolaou staining was very similar to that observed with conventional smears and that the same criteria could be utilized with the Centrifugal Cytology dispersions to screen the cells for cancer as had previously been used with the smears. A preliminary study indicated that six out of six positives with no false negatives or false positives were found. The Centrifugal Cytology technique appears to have promise as a method for preparing suspension samples such as pipets of gynecologic cells. Scanning electron microscope studies reveal that the squamous epithelial cells are very thin and at least some of them are covered by a network structure.

Electrodeposited iridium oxide pH electrode for measurement of extracellular myocardial acidosis during acute ischemia.

In the present paper, fabrication, characterization, and physiological applications of a solid-state pH electrode are described. The pH sensing layer was based on an anodic electrodeposited iridium oxide film (AEIROF). Sputtered platinum electrodes (1 mm diameter) fabricated on flexible Kapton films or platinum wires were used as planar or cylindrical supports. Each electrode site was coated with Nafion to attenuate the interference of anionic redox species and to protect the electrode surface during in vivo measurements. Performance of the AEIROF was evaluated, for the first time, as a pH electrode and proved to have a slightly super-Nernstian response with slope of -63.5 +/- 2.2 mV/pH unit for both wire and planar sputtered platinum electrodes. Linear pH responses were obtained in the pH range 2-10. The electrodes have a working lifetime of at least 1 month with accuracy of about 0.02 pH unit and fast response time. The electrodes showed very low sensitivities for different species, such as Na+, K+, Li+, NH4+, Ca2+, Mg2+, dissolved oxygen, lactate, ascorbate, and urate, which are important for physiological applications. The electrodes were applied in extracellular pH measurements during brief regional ischemia in a swine heart and no-flow ischemia in an isolated rabbit papillary muscle. A first report on extracellular pH, K+, and lactate simultaneous measurements during no-flow ischemia using the AEIROF pH electrode and the previously described K+ and lactate electrodes is presented as well.