Comparison of Papanicolaou staining and Feulgen staining for automated prescreening

Feulgen staining is considered to be a quantitative DNA-specific cytochemical procedure. The applicability of this staining in high-resolution cytometry was tested in comparison with a regressive Papanicolaou staining. Papanicolaou-stained or Feulgen-stained intermediate and carcinoma cells selected by a cytologist were examined with a Zeiss scanning microscope photometer at 546 and 560 nm, respectively. After cell image segmentation and feature extraction, a statistical data evaluation was carried out by computer. Cell distributions with respect to four selected nuclear features demonstrated the influence of the staining procedure on cell feature measurements. The discriminatory power of the classification system as related to both staining procedures was studied using discriminant analysis. Using only nuclear features, a 7.3% improvement of the overall correct classification rate (from 85.0% to 92.3%) was achieved using Feulgen staining. The misclassification rate was simultaneously reduced by 50%. Using cytoplasmic as well as nuclear features, a 98% rate of correct classification was achieved.     

Feulgen type staining of mammalian tissues with Dahlia-SO2.

The use of the basic dye, Dahlia, which belongs to triphenylmethane group but without a primary amino group in its molecule has been described as useful in the staining of aldehyde groups of acid hydrolysed DNA in tissue sections following the conventional Feulgen procedure. Dahlia-SO2 prepared with sodium hydrosulphite is highly suitable when used at pH 4-0 to 5-0. The absorption characteristics of the stained nuclei indicate on the peak of maximum absorption at 560 nm, whereas, that of the aqueous dye solution is at 590 nm.     

Increase in Feulgen staining intensity by pre-treatment with different reagents.

In the experiments sections of rat tissues fixed in 10 per cent buffered neutral formalin for 3 hour and treated for 15 minutes with different chemical reagents such as pyridine, tributylamine, urea, tris sodium nitrite and sodium hydroxide were subjected to hydrolysis in 6 N HCl at 25 degrees C for 20 minutes and stained by the UV Feulgen technique. The results reveal a far more intense staining of the nuclei in tissues treated with any of the chemicals mentioned than in untreated controls. The possible role of these chemicals in enhancing the staining intensity is discussed.     

Gram staining with an automatic machine

This study was undertaken to develop a new Gram-staining machine controlled by a micro-controller and to investigate the quality of slides that were stained in the machine. The machine was designed and produced by the authors. It uses standard 220 V AC. Staining, washing, and drying periods are controlled by a timer built in the micro-controller. A software was made that contains a certain algorithm and time intervals for the staining mode. One-hundred and forty smears were prepared fromEscherichia coli, Staphylococcus aureus, Neisseria sp., blood culture, trypticase soy broth, direct pus and sputum smears for comparison studies. Half of the slides in each group were stained with the machine, the other half by hand and then examined by four different microbiologists. Machine-stained slides had a higher clarity and less debris than the hand-stained slides (p<0.05). In hand-stained slides, some Gram-positive organisms showed poor Gram-positive staining features (p<0.05). In conclusion, we suggest that Gram staining with the automatic machine increases the staining quality and helps to decrease the work load in a busy diagnostic laboratory.     

A method for combined gross skeletal staining and Feulgen staining of embryonic chick tissues

This paper describes a combined technique for gross skeletal staining and Feulgen staining of avian embryonic limbs. The gross skeletal stain uses Victoria blue B, and the Feulgen stain is done en bloc before the skeletal stain is applied. The method has been useful in determining the cellular origins of supernumerary structures arising from experiments in which quail wing mesoderm is grafted into chick wing buds.     

Feulgen staining of rat liver fixed in different fixatives.

In the present study rat liver pieces fixed in 1) 10 per cent buffered neutral formalin, 2) 4 per cent glutaraldehyde, 3) Heidenhain's-Susa fixative and 4) Flemming's fluid, and following hydrolysis in 1-0 N HC1 at 60degreesC for varying time periods have been stained with the UV Feulgen procedure. The results of this study reveal that following hydrolysis for different time periods the tissue material fixed in formalin show the same staining pattern as those fixed in glutaraldehyde. The material fixed in Heidenhain's-Susa displays an intense Feulgen staining after two different times of hydrolysis, and that fixed in Flemming's fluid shows particular staining intensity for a prolonged time period thus indicating better preservation of DNA than in the materials fixed in the other three fixtatives.     

Acid hydrolysis characteristics of spleen colony cells under Feulgen staining

A cytophotometrical determination of DNA content was performed in cells of murine spleen colonies originating from bone marrow and in lymphocytes of axillary lymph nodes under various temperatures (22, 25 and 37 degrees C) of hydrolysis (5N HCl). It is shown that acid hydrolysis at 20 and 25 degrees C is most-preferable for proliferated cells of spleen clones and for non-proliferated lymphocytes. It is concluded that hydrolysis curves for clonal cell nuclei in different phases of mitotic cycle practically coincided.     

The staining behavior of oocyte cytoplasm with the alcoholic Feulgen variant and with methyl green

Summary A variant of the Feulgen reaction which has been proposed as a method for demonstrating cytoplasmic DNA in oocytes has been tested on ovarian material from a variety of species. While Schiff positive staining was developed, this was not removable by pretreatment with DNase and could be reproduced by using oxidants used in the pseudoplasmal reaction. This method was not considered useful for demonstrating cytoplasmic DNA.When chloroform extracted solutions of methyl green were used to stain ovaries, cytoplasmic staining identical in pattern to that obtained with other basic dyes was observed. The cytoplasmic staining was prevented by pretreatment of sections with RNase, but was not affected by DNase pretreatment. In somatic cells with high concentrations of cytoplasmic RNA, only nuclear staining was observed. This nuclear staining was labile to DNase but not to RNase.This work was supported by U.S. Public Health Service grants GM-10003-03 and K-3-6176-03.Contribution number 376 of the Bermuda Biological Station.     

Intensity of Feulgen staining of rat liver nuclei fixed with two different concentrations of formalin

Summary The results presented in this paper indicate that following fixation of rat liver in either 40% (w/v) or 10% formalin solution, Feulgen staining is far greater in the tissue fixed in the former fixative as compared with the same fixed in the latter. A possible mode of action of formalin towards fixation and subsequent Feulgen staining is suggested.     

Feulgen Hydrolysis with Phosphoric Acid

Sections from rat tissues fixed in a 10% solution of formalin in 90% alcohol were treated with phosphoric acid in concentrations varying from 30 to 85% at room temperature (28°), and subsequently stained with the Schiff reagent. Intense staining of the nuclear material was obtained in 5 to 15 minutes when 40 to 75% phosphoric acid was used. The intense staining after the higher concentrations of phosphoric acid may be due to the low concentration of water present, thus minimizing diffusion. The nucleoli in the rat-liver cells were well stained, especially the peripheral portion. The nucleoli of nerve cells, however, were only faintly stained and the Nissl substance was completely negative. The accompanying plate of photomicrographs shows the nuclei stained by this method in the Graafian follicle, the liver, intestinal villi of the rat and a metastatic carcinoma in the human pituitary.     

Feulgen Hydrolysis with Perchloric Acid

In tissue fixed with Carnoy's acetic alcohol (1:3), the hydrochloric acid hydrolysis performed as part of the Feulgen reaction is optimal for only a very short period of time. When 10% perchloric acid is used as the hydrolytic agent, the same color maximum is obtained, and the optimal hydrolysis time at 25°C. extends from 12 hours to 24 hours. During this time the intensity of color does not change. The events which take place during the period of suboptimal hydrolysis are the same as diose which take place during the corresponding period of hydrochloric acid hydrolysis. Part of the decrease in ultraviolet extinction of nuclei during the first 12 hours is due to the splitting off of purine bases from the desoxyribose nucleic acid. This is consistent with the increase in the amount of Feulgen dye bound by nuclei during this period of time. Between 12 hours and 24 hours no ultraviolet absorbing material is lost from nuclei, which is consistent with the fact that during this time the Feulgen color produced remains at a maximum.