The Effect of the Quantitative Protargol Stain and Lugol's and Bouin's Fixatives On Cell Size: A More Accurate Estimate of Ciliate Species Biomass

ABSTRACT. The quantitative protargol stain (QPS) is used to estimate ciliate biomass and species composition from mixed field samples. Length, width, breadth and volume of live Euplotes sp., Eutintinnus sp., Strobilidium spiralis, Strombidium acutum , and Gymnodinium sanguineum were compared with 0.6% acid Lugol's fixed, 5% Bouin's fixed, and QPS cells. Cells shrank due to treatments (ANOVA and Tukey's test, α= 0.05). Protistan post-fixation cell volume (as a percentage of live volume) was 55%-80% for acid Lugol's fixed, 40%-70% for Bouin's fixed, and 30%-65% for QPS. Each species shrank to a different extent; cytostructural elements apparently alter the effect of fixation. Egestion is likely not the main cause of shrinkage since the autotroph, G. sanguineum , shrank to the same extent as the heterotrophs when stained by QPS. If field studies do not consider fixation effects on cell size, biomass may be underestimated. We recommend, for studies on planktonic ciliates, either acid Lugol's and QPS be used concurrently or QPS be used alone and biovolume values divided by 0.4 to correct for shrinkage. We stress that this is a rough estimate as this value ranges from 0.3 to 0.45 for planktonic protists.     

The Use of Picric Acid with the Gram Stain in Plant Cytology

The technic described involves the use of a saturated solution of picric acid in absolute alcohol in the process of dehydration following the gentian-violet-iodine stain as applied to plant cytological material. The method is suitable for both paraffin sections and smears of pollen mother cells fixed in Navashin's or Flemming's solutions. Differentiation in clove oil is very easy since cytoplasm destains immediately, while chromatic material destains very slowly following picric acid. Chromosomes are stained more distinctly than with the usual Gram stain and do not fade.     

Removal of Acid Hematin-Type Pigments from Sections; Efficacy of Alcoholic Picric Acid Solution

Paraffin sections from tissues fixed in nonneutralized 10% formalin or Carnoy's fluid were treated with saturated solutions of picric acid in each of the following solvents: water, 80%, 90%, 95% and absolute ethanol, and with half-saturated solutions in 95% ethanol for 5 min. Tests with the supernatant fluid 30-60 min after mixing the solid and the solvent showed that adequate concentration had not yet occurred; hence more time is needed, with 15 hr recommended. Only saturated solutions of picric acid in absolute and 95% ethanol removed all acid hematin-type pigment. The effectiveness of saturated solutions decreased with decreasing alcohol concentration.     

Staining of Acid Mucopolysaccharides after Chromatography on Filter Paper

Acid mucopolysaccharides obtained both from commercial sources and by isolation from human urine have been chromatographed on Whatman No. 1 filter paper, using propanol or ethanol in pH 6.5 M/15 phosphate buffer as solvent systems. The chromatograms are then fixed by immersion in 95% alcohol and in diethyl ether. After drying, they are stained in 0.06% toluidine blue O in 0.5% aqueous acetic acid. A final rinsing in 2% aqueous acetic acid removes the excess dye from the paper and exposes the stained mucopolysaccharide to a pH favoring orthochromasia.     

Staining of Acid Mucopolysaccharides after Chromatography on Filter Paper

Acid mucopolysaccharides obtained both from commercial sources and by isolation from human urine have been chromatographed on Whatman No. 1 filter paper, using propanol or ethanol in pH 6.5 M/15 phosphate buffer as solvent systems. The chromatograms are then fixed by immersion in 95% alcohol and in diethyl ether. After drying, they are stained in 0.06% toluidine blue O in 0.5% aqueous acetic acid. A final rinsing in 2% aqueous acetic acid removes the excess dye from the paper and exposes the stained mucopolysaccharide to a pH favoring orthochromasia.     

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.     

Effects of copper and zinc on growth,morphology, and metabolism of Asterionella japonica (Cleve) 1

When exposed to elevated levels of copper or zinc, the diatom Asterionella japonica (Cleve) showed a reduced cell division rate and a marked increase in cell size. Metal-treated cells had greater cell volumes, dry weights, carbon, nitrogen, chlorophyll, and DNA contents, all in approximately the same proportion as control cells. Two protoplasts often appeared to be contained within one frustule. Metal-treated cells photosynthesized at near-normal rates on a per chlorophyll basis and above normal rates on a per cell basis. Excretion of photosynthetically fixed carbon was depressed by metal treatment; 10–22% of fixed carbon was excreted in control cells and typically less than 1% in treated cells. Thus, metal-treated cells showed an uncoupling of photosynthesis from cell division and continued to enlarge when fixed carbon could not be excreted or utilized in cell division.Uptake of sulphate and silicic acid proceeded at slower rates than other processes (e.g., nitrogen uptake or photosynthesis) in copper-treated cells. Free amino acids in copper-treated cells totalled ≈ 10% of control cell levels, with greatest proportional declines in methionine, cysteine, aspartic acid, valine, and isoleucine. Copper-treated cells resuspended in fresh medium shrank to normal size when exposed to methionine (which they accumulated), although cell division rates did not return to normal. These cells excreted 2–3 times as much fixed carbon as comparable EDTA-treated or untreated cells, neither of which decreased in size. Copper-treated cells appeared indistinguishable from silicon-limited cells (i.e., cells not dividing for lack of silicon) in a copper-free medium. Cells treated with the sulfhydryl binder PCMB divided at reduced rates and also swelled in a manner comparable to copper-treated cells. The results suggest that toxic metals may bind to sulfhydryl groups on cell membranes, impairing normal membrane function and reducing silicic acid uptake and amino-acid synthesis, thereby resulting in depressed cell division rates.     

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.     

Concurrent Demonstration of Desoxyribose Nucleic Acid and 1,2-Glycols

Sections from rat tissue were fixed in 10% formalin in 90% alcohol and placed in a 1.0% suspension of sodium bismuthate (NaBiO₃) in 40% phosphoric acid for 40 minutes at room temperature. Bismuth phosphate crystals were removed with 2N HCl. The sections were next placed in the Schiff reagent for 20 minutes. By this method the DNA was hydrolyzed by the phosphoric acid and the 1,2-glycols were oxidized by the NaBiO₃. In both cases aldehyde groups were released and subsequently stained by the Schiff reagent. A photomicrograph is included demonstrating the nuclei, goblet cells, striated border and basement membrane stained by this combined method.     

Differential Staining Of Tissue In The Block With Picric Acid And The Feulgen Reaction

The authors have found a modification of the Feulgen reaction to be a satisfactory stain for tissue in the block.

Pieces of fresh mammalian tissue not thicker than 5 mm. are fixed for approximately 48 hours at 25° C. in a mixture of equal parts of 5% aqueous sulfosalicylic acid and saturated aqueous picric acid. They are washed for 30 minutes in three ten-minute changes of distilled water and placed in Feulgen's staining solution diluted to one-half strength with distilled water. The staining solution is allowed to act for 24 hours (2 to 3 mm. thick blocks) up to 48 hours for 5 mm. thickness. After staining, the specimens are transferred to a mixture of sodium bisulfite, 0.5 g. and N hydrochloric acid, 5 ml. in' 100 ml. of distilled water. Two changes of IS to 30 min. each in the acid sulfite are given and these are followed by dehydration through 50%, 70% and 95% alcohol. One to two hours are allowed for each change except the last 95%, in which the stained tissue is allowed to remain overnight. The dehydration is completed in two changes of absolute alcohol with subsequent clearing in xylene and embedding in paraffin. Sections may be cut 10 μ or other thickness desired, mounted on slides, paraffin removed, and covered in the usual manner. Nuclei stain reddish violet against a lemon yellow background when the stain is typical. Orange G, 200 mg. per 100 ml. may be added to the fixing fluid if a more polychromatic effect is desired.     

The Value of Alcohol for Fixation of Skin

Stained sections of skin fixed in 70% alcohol were compared with others from pieces fixed in 4% formaldehyde-saline. The sections of alcohol-fixed material were much more susceptible to the action of deoxyribonuclease and lipase than those from formalin-fixed, as demonstrated by a standardized hematoxylin staining method and by fluorescence microscopy. After formalin, cytoplasmic basophilia was increased, presumably because formalin fixation caused ribonucleic acid to diffuse from nuclei to cytoplasm. Both types of fixation damaged collagen, as seen in fluorescence induced by 5-anvmo-2-chloro-7-methoxyacridine, but alcohol caused less distortion than formalin. Probably fluorochroming of fresh tissue is the only satisfactory method for studying collagen in pathological conditions.     

Application of the Papanicolaou Stain to Paraffin Sections

Routine paraffin sections from tissues fixed either in aqueous formalin, 80% alcohol (with or without 1% trichloracetic acid added), Carnoy's alcohol-chloroform-acetic (6:3:1) and Bouin's fixative were stained as follows: Harris' hematoxylin, 6 min; running water, 2-3 min; ascending grades of alcohol to 95%; orange G, 0.5% and phosphotungstic acid, 0.015% in 95% alcohol, 5 min; 95% alcohol, 2 changes; Papanicolaou's EA36, 2.5 min; dehydration, clearing, and covering in Permount. The results show morphology better than hematoxylin and eosin and the technic is recommended particularly for keratin, which always stains bright orange.     

A Rapid Combined Fixing and Staining Method for Plant Chromosome Counts

A new method for rapidly preparing slides suitable for chromosome counts by the use of a combined fixing and staining solution involves the substitution of anthraquinone for picric acid in Bouin's formula and the addition of alizarin red S with a metallic salt as a mordant. The fixed smears, after being dehydrated to 95% alcohol, are differentiated in 0.5% sulfuric acid in 95% alcohol saturated with picric acid, washed, cleared and mounted in xylol-balsam. Cymene may be used to intensify the stain. Root tips fixed in the above solution may be dehydrated in dioxan, a paraffin solvent; infiltrated, embedded, sectioned and mounted in the usual way. The sections are subsequently differentiated in picro-sulfuric acid alcohol and cymene. An alternative method of differentiation for this stain is also described.     

Acid Orcein-Iron and Acid Orcein-Copper Stains for Elastin

Paraffin sections of formol-fixed tissues stained 4-18 hr in 70% alcohol containing 1% orcein and 1% of concentrated (12 N) HCl by volume yield the familiar purple brown elastin and red nuclei on a pink background. When sections so stained are transferred directly from the stain to 70% alcohol containing 0.02% ferric chloride (FeCl₃·6 H₂O) or 0.02% copper sulfate (CuSO₄·5 H₂O) for a 15 sec to 3 min period, elastin coloration is changed to black or reddish black and chromatin staining to reddish black. The procedure can be counterstained with picro-methyl blue to yield blue collagen and reticulum or with our flavianic acid, ferric chloride, acid fuchsin mixture to give deep yellow background and deep red collagen.     

Regulation of Peroxidase-Dependent Oxidation of Phenolics by Ascorbic Acid: Different Effects of Ascorbic Acid on the Oxidation of Coniferyl Alcohol by the Apoplastic Soluble and Cell Wall-Bound Peroxidases from Epicotyls of Vigna angularis

Intercellular washing fluid (IWF) and washed cell walls obtainedfrom epicotyls of Vigna angularis catalyzed the oxidation ofconiferyl alcohol in the presence of hydrogen peroxide, indicatingthe presence of both soluble and bound peroxidases in the cellwalls. The products of oxidation of coniferyl alcohol were identicalin both cases. Ascorbic acid inhibited the oxidation of coniferylalcohol. The inhibition was due to the rapid reduction of anoxidized intermediate of coniferyl alcohol by ascorbic acid,with resultant regeneration of coniferyl alcohol. However, theinhibitory effects of ascorbic acid were different in the caseof IWF and cell walls. Ascorbic acid completely inhibited theoxidation of coniferyl alcohol by IWF peroxidase as long asascorbic acid was available, whereas the oxidation of coniferylalcohol by cell wall-bound peroxidase was competitively inhibitedby ascorbic acid. Ascorbic acid was present in cell walls andlignin was formed in cell walls during aging of stem. Basedon these results, a possible function for ascorbic acid in theregulation of oxidation of phenolics in cell walls is discussed. (Received March 19, 1993; Accepted May 24, 1993)     

Removal of Ribonucleic Acid from Bacterial Cells by Dilute Salt Solutions

Bacterial cells were impressed upon a clean glass slide, fixed in ethyl alcohol and immersed at 37°C in either of the following two salt solutions: (A) NaCl, 7.8 gm; KCl, 0.7 gm; distilled water, 1000 ml; adjusted to pH 7.0; or (B) 0.1M NaH₂PO₄, 400 ml; 0.1M Na₂HPO₄, 600 ml; KCl, 0.7 gm. After 1-5 hr soaking to remove ribonucleic acid, the slide was stained by Giemsa's method as usual. The staining revealed slender chromatinic bodies with reasonable clarity extending the whole diameter of the moderately swollen cell. The results of this method seemed to be much like those obtained after ribonuclease digestion.     

A Comparative Study of some Dehydration and Clearing Agents

An experiment to determine the advantages of diozan, iso-butyl alcohol, tertiary butyl alcohol, and ethyl alcohol as dehydrants and chloroform, toluol, xylene, benzol, methyl benzoate, methyl salicylate, and acetone as clearers is described. Materials fixed in Bouin's fluid, Zenker formol, and 10% neutral formalin were dehydrated, embedded, sectioned, and stained. Bouin's fluid produces less hardening, shrinkage and distortion than the other fixatives employed. Slow dioxan is the best method of dehydration. All the picric acid need not be removed from tissues to be embedded in paraffin. Tissue blocks not more than 4 mm. thick may be dehydrated and impregnated with paraffin by slow dioxan in 13 hours, fast dioxan in 10 hours, iso-butyl alcohol and tertiary butyl alcohol in 14 hours, and ethyl alcohol-chloroform in 17 hours without incurring any distortion due to rapidity of dehydration and infiltration.     

Possible uses of Dioxan in Botanical Microtechnic

Dioxan has been well established as an advantageous dehydrating agent for plant tissues. It dehydrates equally well after fixatives containing formalin, acetic acid, chromic acid, chromates, mercuric chloride, osmic acid, and alcohol. Better infiltration of paraffin after dehydration may be obtained by passing the material thru (1) a cold bath composed of 30 cc. of dioxan, 5 cc. of xylol and 20 cc. of melted soft paraffin and, (2) a warm bath of 50 cc. of dioxan, 50 cc. of paraffin, and 10 cc. of xylol. Transfer from (2) to soft paraffin. A dioxan fixative consisting of dioxan 50 cc., formalin 6 cc., acetic acid 5 cc., water 50 cc. was devised for delicate subjects. The fixed material is transferred directly into dioxan and mounted in dioxan-diaphane or dioxan-balsam. Very delicate objects require dioxan dilution of the balsam and slow concentration of the mounting medium by evaporation.

Entire plant parts or epidermal peelings are fixed in any desired fixative, washed if necessary, transferred to dioxan and mounted in diluted dioxan-balsam or diaphane. Dioxan may be used to mount hyalin objects whose refractive indexes approach those of balsam in media of higher index than balsam. It may be used in place of alcohol in finishing parafin sections, and since it exhibits different stain solubilities than alcohol it offers an important new tool in obtaining and maintaining stain balances.     

Concise Synthesis of Valerena-4,7(11)-diene,a Highly Active Sedative,from Valerenic Acid

A concise synthesis of valerena-4,7(11)-diene with potent sedative activity was achieved in three steps involving, reduction of carboxylic acid, bromination of the resulting alcohol, and reduction of the bromide from valerenic acid in a 63% total yield. This synthetic method makes it possible to provide further materials for biological testing to realize comprehensive SAR studies.     

Effects of Ethanol and Other Alkanols on Transport of Acetic Acid in Saccharomyces cerevisiae

In glucose-grown cells of Saccharomyces cerevisiae IGC 4072, acetic acid enters only by simple diffusion of the undissociated acid. In these cells, ethanol and other alkanols enhanced the passive influx of labelled acetic acid. The influx of the acid followed first-order kinetics with a rate constant that increased exponentially with the alcohol concentration, and an exponential enhancement constant for each alkanol was estimated. The intracellular concentration of labelled acetic acid was also enhanced by alkanols, and the effect increased exponentially with alcohol concentration. Acetic acid is transported across the plasma membrane of acetic acid-, lactic acid-, and ethanol-grown cells by acetate-proton symports. We found that in these cells ethanol and butanol inhibited the transport of labelled acetic acid in a noncompetitive way; the maximum transport velocity decreased with alcohol concentration, while the affinity of the system for acetate was not significantly affected by the alcohol. Semilog plots of V_max versus alcohol concentration yielded straight lines with negative slopes from which estimates of the inhibition constant for each alkanol could be obtained. The intracellular concentration of labelled acid was significantly reduced in the presence of ethanol or butanol, and the effect increased with the alcohol concentration. We postulate that the absence of an operational carrier for acetate in glucose-grown cells of S. cerevisiae, combined with the relatively high permeability of the plasma membrane for the undissociated acid and the inability of the organism to metabolize acetic acid, could be one of the reasons why this species exhibits low tolerance to acidic environments containing ethanol.