The dye-exclusion test for cell viability: Persistence of differential staining following fixation

Summary A method is described whereby the differential staining of viable and nonviable unfixed cells, as observed by the dye-exclusion method, can be reproduced in glutaraldehyde-fixed preparations by staining with alcian blue. The results suggest that the differential staining is due, at least in part, to structural differences that are retained following aldehyde fixation. This work was supported by grants from the National Cancer Institute of Canada and the National Research Council of Canada. Recipient of a Research Studentship from the National Cancer Institute of Canada.     

Evaluation of cell proliferation kinetics by the differential staining of the sister chromatids at 2 points of cell fixation

Lymphocyte proliferation in blood cultures from 23 normal individuals was studied. A simple method of evaluation of cell proliferative kinetics with sister chromatid differential staining using two different points of cell harvesting was suggested. It was shown that different composition of tissue culture medium plays a significant role during the cell exit from G0 stage, the mean doubling generation time of cellular population remaining constant.     

The Haematoxylin-basic Fuchsin-picric acid staining reaction as a test of myocardial viability in resuscitated and preserved hearts

Synopsis In the field of the transplantation of organs, there is a great need for anin vitro test of viability which would confirm that the organ was capable of performing its normalin vivo functions. Such a test should ideally be simple, rapid and reproducible. Preliminary studies using the Haematoxylin-Basic Fuchsin-picric acid (HBFP) staining reaction to assess myocardial ischaemia in resuscitated and preserved hearts would suggest that this test meets many of the requirements of a viability assay.The test has been employed in hearts which have been in a state of anoxic arrest for 30 min and then resuscitated and preserved as an autoperfusing heart-lung preparation. The positive response after 30 min anoxic arrest reverts to a negative response after 2 h myocardial perfusion. In hearts which have been preserved as an autoperfusing heart-lung preparation with no interim period of anoxic arrest the HBFP stain response remains negative throughout, confirming satisfactory myocardial perfusion.     

Fine structure of Lily pollen tubes following various fixation and staining procedures

Summary The cytoplasm of the growth zone at the tip of theLilium pollen tube contains numerous membrane-bound vesicles and an abundance of smooth endoplasmic reticulum. Covering the tip of the tube is a cap of compartmentalized wall material which arises from coalesence of vesicles with each other and with the existing cap.This organization, seen following glutaraldehyde fixation, is altered to varying degrees by different fixation procedures. Neither the cap nor the e. r. are preserved by osmium fixation. Formalin or permanganate preserve the cap but not its compartments. Preservation of the cap by permanganate is concentration-dependent but poor at best. Combined fixation by acrolein and glutaraldehyde results in better preservation of membranous elements than glutaraldehyde alone.Supported by grant RG 8827 from the National Institutes of Health, U. S. A.     

Kurloff cell ultrastructure after combined formaldehyde-cetylpyridinium chloride fixation and high-iron diamine staining

Summary This study deals with the ultrastructure of the chondroitin sulphate proteoglycans of the Kurloff body, a large lysosomal organelle that stains metachromatically with Toluidine Blue and which is present in Kurloff cells (a blood cell unique to the guinea pig). Splenic tissues were fixed with 1% cetylpyridinium chloride (CPC) added to 4% paraformaldehyde and examined either after Spicer's high-iron diamine staining for sulphated anionic sites followed by post-fixation with ferrocyanide-osmium tetroxide or after a simple post-fixation with ferrocyanide-osmium tetroxide. CPC-precipitated sulphated sites were preferentially located at the periphery of the Kurloff body but, unexpectedly, were absent in the central matrix. Although their electron opacity was lower, these anionic sites were readily observable in the absence of HID-staining after sole post-fixation by ferrocyanide-reduced osmium. CPC-precipitated sulphated anionic sites were either associated with the myelin figures or constituted unexpected structures. They contained (i) tightly-stacked lamellae, with a very regular 4 nm periodicity, and (ii) groups of 2, 3, 4 short dense lines with a 3–5 nm periodicity. By taking into account the susceptibility of these HID-reactive structures towards chondroitinase ABC, these different sulphated components were assumed to be related to the proteochondroitin-4-sulphate previously characterized as the only major sulphated glycoconjugate of the Kurloff cell. Their colocalization with phospholipidic structures was suggested following, observation of sections treated by a chloroform-methanol mixture.     

Analysis of regenerating hepatic cell replication in vivo by differential chromatid staining

The cell cycle of mouse hepatic cells was examined in vivo following partial hepatectomy, by differential chromatid staining in the presence of non-inhibitory concentrations of bromodeoxyuridine (BrdU). Using this technique, distribution curves were obtained for the appearance of metaphase cells in successive generations, and mean cell cycle time (11 hr) was determined. Cell cycle times derived with this technique are several-fold faster than previous reports of regenerating liver which used radionucleotide labelling.     

Analysis of regenerating hepatic cell replication in vivo by differential chromatid staining

Abstract. The cell cycle of mouse hepatic cells was examined in vivo following partial hepatectomy, by differential chromatid staining in the presence of non-inhibitory concentrations of bromodeoxyuridine (BrdU). Using this technique, distribution curves were obtained for the appearance of metaphase cells in successive generations, and mean cell cycle time (11 hr) was determined. Cell cycle times derived with this technique are several-fold faster than previous reports of regenerating liver which used radionucleotide labelling.     

An improved method to determine cell viability by simultaneous staining with fluorescein diacetate-propidium iodide

A rapid, simultaneous double-staining procedure using fluorescein diacetate (FDA) and propidium iodide (PI) is described for use in the determination of cell viability in cell suspension. Air-dried slide preparations can be made from the cell suspensions so that an accurate estimate of the viability of the cells in the original suspension can be made up to 1 week later. Viable cells fluoresce bright green, while nonviable cells are bright red. Furthermore, when FDA-PI staining is compared to trypan blue dye exclusion as a method to determine cell viability, FDA-PI is found to be more consistent over prolonged periods of exposure to the dyes. Therefore, double staining with FDA-PI is a rapid, convenient, and reliable method to determine cell viability.     

Periodic acid-Schiff-alcian blue: a method for the differential staining of glycoproteins

The staining mechanism underlying the periodic acid-Schiff (PAS)-Alcian Blue (AB) sequence has been investigated using a variety of glycoprotein-containing tissues from different organs of the monkey, rat and mouse. The results obtained suggest that reactive carbohydrates contain at least three types of chemical end-groups found in neutral and acidic glycoproteins: (1) PA-engendered aldehyde groups coloured magenta by the Schiff reagent; (2) PA-engendered aldehyde groups coloured blue bisulphite-AB; and (3) naturally occurring acidic (carboxyl and/or sulphate) groups coloured blue by AB only. The PAS-AB sequence showed heterogeneity of glycoprotein structures in the conjunctiva and the duodenal goblet cells. Thus, the PAS-AB sequence is not the simple reverse sequence of AB-PAS but has its own definite and unique staining selectivity and can hence be used as a reliable method for the histochemistry of glycoproteins at the light microscope level.