An improved formulation of the zirconyl hematoxylin stain for acidic mucins

Zirconyl hematoxylin stains acidic mucins darkly and specifically using a solution of 100 mg hematoxylin, 5 ml ethanol, 5 ml 0.5% sodium iodate, 400 mg zirconyl chloride octahydrate, and 30 ml 25% aqueous glycerol. The stain is especially advantageous for studying goblet cells and Paget cells.     

An improved formulation of the zirconyl hematoxylin stain for acidic mucins

Zirconyl hematoxylin stains acidic mucins darkly and specifically using a solution of 100 mg hematoxylin, 5 ml ethanol, 5 ml 0.5% sodium iodate, 400 mg zirconyl chloride octahydrate, and 30 ml 25% aqueous glycerol. The stain is especially advantageous for studying goblet cells and Paget cells.     

Zirconyl Hematoxylin: a New Stain for Acidic Mucins

Most stains for acidic mucins are time-consuming to prepare and have poor stability. Zirconyl hematoxylin is easily prepared and works for a year or more. It is made by adding 5 ml freshly-made 0.1% aqueous sodium iodate, 400 mg zirconyl chloride oc-tahydrate, and 40 ml 25% aqueous glycerol, in that order, to 100 mg of hematoxylin in 5 ml of absolute ethanol and stirring for 5 min. Stain 10 min and do not “blue” the stain. Chlorazole black or kernechtrot and fast green are good counterstains. Zirconyl hematoxylin stains acidic mucins violet or red violet, regardless of how they are fixed. It stains the same mucins as alcian blue in mouse and sheep salivary glands. It shows goblet cells in mouse rectum as well as alcian blue. It stains the same stomach regions in a lizard as alcian blue. Like alcian blue and colloidal iron, zirconyl hematoxylin stains the mucin of cancerous prostate, but not normal prostate.     

Zirconyl Hematoxylin: a New Stain for Acidic Mucins

Most stains for acidic mucins are time-consuming to prepare and have poor stability. Zirconyl hematoxylin is easily prepared and works for a year or more. It is made by adding 5 ml freshly-made 0.1% aqueous sodium iodate, 400 mg zirconyl chloride oc-tahydrate, and 40 ml 25% aqueous glycerol, in that order, to 100 mg of hematoxylin in 5 ml of absolute ethanol and stirring for 5 min. Stain 10 min and do not “blue” the stain. Chlorazole black or kernechtrot and fast green are good counterstains. Zirconyl hematoxylin stains acidic mucins violet or red violet, regardless of how they are fixed. It stains the same mucins as alcian blue in mouse and sheep salivary glands. It shows goblet cells in mouse rectum as well as alcian blue. It stains the same stomach regions in a lizard as alcian blue. Like alcian blue and colloidal iron, zirconyl hematoxylin stains the mucin of cancerous prostate, but not normal prostate.     

Assaying Actual Hematein Content of Commercial Hematoxylins and Hemateins

Hematein-free hematoxylin (HFH) was prepared by a modification of the procedure of Palmer and Lillie (Histochemie, 5: 44-54, 1965). Fifty mg of HFH were dissolved in 5 mg of ethylene glycol and then 45 nil of an aqueous solution of 2.25 gm KAl(SO₄)₂. 12H₂O and 5.445 mg KIO₃ were added. Since this amount of KIO₃ would be sufficient to oxidize 25 mg of HFH to hematein we have termed this half-oxidized hematoxylin (HOH). The peak absorbance (560 nm) of this purple solution remained constant for at least a week. With omission of the KIO₃ the solution was colorless. A curve was constructed by plotting absorbance against concentration of hematein in HOH at various dilutions. For analyses of hematein content of commercial hematoxylins 50 mg of sample and 100 mg of hydroquinone were dissolved in 5 ml of ethylene glycol and then 45 ml of a 5% solution of KAl(SO₄)₂. 12H₂O were added. The addition of the hydroquinone stabilized the absorbance for about 5 min. The hematein content could then be calculated by comparing the observed absorbance with the standard curve. Eleven samples of hematoxylin certified by the Biological Stain Commission had hematein concentrations varying from 0.01 to 0.43%. For analyses of the available hematein content of commercial hemateins, 50 mg of sample were dissolved in 10 ml of ethylene glycol, then 45 ml of water and 45 ml of 5% KAI(SO₄)₂. 12H₂O added. The hematein content could then be calculated by comparing the observed absorbance with the standard curve. In 9 samples of hematein from 4 different sources the active hematein content varied from 19 to 97%.     

Mordant Blue 3: a Readily Availablx Substitute for Hematoxylin in the Routine Hematoxylin and Eosin Stain

Mordant blue 3 may be used as a suhstitute for hematoxylin in hematoxylin and eosin stains. The staining solution consists of 0.25 g dye, 40 ml of 10% iron dam, 5 ml of cone H₂SO₄, and 955 ml of dirtilled H₂O. Staining the is 5 minutes, followed by differentiation in acid water or acid alcohol. After blueing, the seaions are counterstained with emin. Results closely resemble the hematoxylin and eosin stain.     

A New Method Using a Modified Mayer's Hemalum at pH 6 for Demonstrating Mucous Neck Cells

The mucous neck cells of gastric glands were stained with a modified Mayer's hemalum adjusted to pH 6 with saturated aqueous lithium carbonate. One gram of hematoxylin was dissolved in 1000 ml distilled water and 200 mg sodium iodate, 3 g potassium alum, 50 g chloral hydrate and 1 g citric acid were added to the solution. Prior to staining, the solution was adjusted to pH 6 with saturated aqueous lithium carbonate. Bromine oxidation and urea abolished the alum hematoxylin reactivity of the mucous neck cells.     

An Evaluation and Modification of Cole's Hematoxylin

Consistency in staining with an alum hematoxylin is possible by the routine use of fresh staining solutions. A modification of Cole's hematoxylin is so easily prepared that fresh staining solutions present no problem. The staining solution consists of 100 ml 1.2% aqueous KA1(SO₄)₂ .12 H₂O, 1 ml 10% alcoholic hematoxylin and 2 ml 1% iodine. Mix, place in paraffin oven overnight and stain sections 5 minutes. The three solutions can be kept as stock solutions for years.     

A new method using a modified Mayer's hemalum at pH 6 for demonstrating mucous neck cells

The mucous neck cells of gastric glands were stained with a modified Mayer's hemalum adjusted to pH 6 with saturated aqueous lithium carbonate. One gram of hematoxylin was dissolved in 1000 ml distilled water and 200 mg sodium iodate, 3 g potassium alum, 50 g chloral hydrate and 1 g citric acid were added to the solution. Prior to staining, the solution was adjusted to pH 6 with saturated aqueous lithium carbonate. Bromine oxidation and urea abolished the alum hematoxylin reactivity of the mucous neck cells.     

A Single solution iron-hematoxylin Stain for Intestinal Protozoa

A single solution iron-hematoxylin stain is described for staining fecal smears rapidly and simply. The stain is prepared from the following solutions: Solution A: 1% hematoxylin in 95% alcohol, prepared by diluting a stock solution of 10% hematoxylin in 95% alcohol. Solution B: Ferric ammonium sulfate (violet crystals), 4.0 g.; glacial acetic acid, 1.0 ml.; concentrated sulfuric acid (sp. gr. 1.8),0.12 ml.; distilled water, 100 ml. Mix equal parts of Solution A and Solution B; allow to stand overnight, filter and use. For maximum length of staining life, store in full, air-tight bottles. To stain fecal smears, fix in Schaudinn's, pass through iodine alcohol to 50% alcohol, stain for three minutes, wash in running tap water 5 to 15 minutes, dehydrate and mount.     

A Single solution iron-hematoxylin Stain for Intestinal Protozoa

A single solution iron-hematoxylin stain is described for staining fecal smears rapidly and simply. The stain is prepared from the following solutions: Solution A: 1% hematoxylin in 95% alcohol, prepared by diluting a stock solution of 10% hematoxylin in 95% alcohol. Solution B: Ferric ammonium sulfate (violet crystals), 4.0 g.; glacial acetic acid, 1.0 ml.; concentrated sulfuric acid (sp. gr. 1.8),0.12 ml.; distilled water, 100 ml. Mix equal parts of Solution A and Solution B; allow to stand overnight, filter and use. For maximum length of staining life, store in full, air-tight bottles. To stain fecal smears, fix in Schaudinn's, pass through iodine alcohol to 50% alcohol, stain for three minutes, wash in running tap water 5 to 15 minutes, dehydrate and mount.     

Initial and Persisting Staining power of Solutions of Iron-Hematoxylin Lake

A study was made of factors affecting the initial staining power and the stability of iron-hematoxylin lake solutions. The findings were applied to the preparation of a superior hematoxylin staining solution. This is made up as follows: in 50 ml. water dissolve, in order, 1.0 g. ferric ammonium sulfate [FeNE₄ (SO₄)₂⋅ 12H₂O], 0.8 ml. sulfuric acid, 50 ml. 95% ethyl alcohol, 0.5 g. hematoxylin. Filter the solution to remove the insoluble, white crust of the ferric ammonium sulfate. The solution stains well ten minutes after it has been made. Peak performance is attained within 5 hours, and is maintained for 4 to 8 weeks. Staining time is 3 to 30 minutes. Excess stain can be rinsed off the slide and section by immersion in water, after which destaining, if necessary, can be accomplished with a solution of 50 ml. water, 50 ml. 95% ethyl alcohol, 0.18 ml. sulfuric acid. The slides may or may not be placed next in a neutralizing solution of 50 ml. water, 50 ml. 95% ethyl alcohol, 0.5 g. sodium bicarbonate. They may then be passed through 50 ml. water, 50 ml. 95% ethyl alcohol on the way to alcoholic counterstaining solutions, or through water leading to aqueous counterstains.

The nuclear stain produced is black, intense and very sharp and has proved to be consistently excellent on a variety of animal and human tissues following a number of different fixatives.     

Induction of pigmentation in nonproliferating cells of Serratia marcescens by addition of single amino acids

Addition of casein hydrolysate to suspensions of washed, nonpigmented, nonproliferating Serratia marcescens incubating at 27 C induced biosynthesis of prodigiosin. Four amino acids of casein hydrolysate, dl-aspartic acid, l-glutamic acid, l-proline, and l-alanine caused formation of pigment when added individually. dl-Ornithine also was effective. Optimal concentrations for maximal pigmentation were 5 to 10 mg/ml; at these high concentrations, d-serine also induced biosynthesis of some prodigiosin. dl-Alanine and -ornithine were as effective as the l-iosomers, but l-glutamic acid and l-proline gave better responses than their racemic mixtures. Kinetics of prodigiosin biosynthesis after addition of dl-alanine (20 mg/ml) were similar to those of cells suspended in 0.2% casein hydrolysate. The other amino acids were less effective. Addition of 5 mg of dl-alanine or casein hydrolysate per ml to minimal medium increased by 30% the amount of prodigiosin formed by growing cells after incubation for 7 days at 27 C. Cultures grown for 7 days at 27 C in 0.2% casein hydrolsate formed more prodigiosin than did suspensions of nonproliferating cells containing individual amino acids or casein hydrolysate. However, more pigment was produced by cells suspended in l-alanine (5 mg/ml) or l-proline (10 mg/ml) than when suspended in 0.4% natural or synthetic casein hydrolysate. Filtrates from suspensions of nonproliferating cells forming pigment in l-proline induced more rapid formation of prodigiosin, but filtrates from suspensions in dl-alanine did not. The data supported the hypothesis that pyrrole groups of prodigiosin may be synthesized from 5-carbon amino acids such as proline, ornithine, aspartic, and glutamic acids, but the role of alanine is unknown.     

A consistent phosphotungstic acid hematoxylin stain for glial fibers.

After deceration, celloidinization and hydration, oxidize 10 micron paraffin sections for 15 min in a solution containing 0.3 g KMnO4 and 0.1 ml conc. H2SO4 per 100 ml distilled water. Wash in water and reduce in 5% oxalic acid until the sections are colorless. Wash thoroughly in water and place in 4% iron alum solution for two hours. Wash briefly in water and stain for two hours in phosphotungstic acid hematoxylin. Rinse briefly in 95% ethanol and dehydrate in n-butyl alcohol or absolute ethanol for 4 min with two changes, clear and mount. Glial fibers, myofibrils, red blood cells, etc. are stained blue while astrocyte cell bodies, collagen, etc. are stained red. This stain has proven highly consistent in a wide variety of astrocytic derangements. Despite the intensity of this PTAH modification, false positive staining was not observed.     

Effects of some drugs on purified human erythrocyte CuZnSOD and in vitro inhibitiory effect of 5-fluorouracil on leukocyte total SOD activity

The inhibition and activation effects of some drugs on the activities of superoxide dismutase enzymes (SOD) in human erythrocyte and leukocyte cells was investigated. Firstly, CuZnSOD enzyme was purified 837–fold and 12% efficiency from human erythrocytes by ethanol-chloroform treatment to remove hemoglobin and then ion exchange chromatography (DEAE-Sepharose) and copper chelate affinity chromatography techniques. Inhibition or activation effects of fourteen drugs on CuZnSOD was investigated. None of the studied drugs except for 5-fluorouracil showed any effects on the enzyme. 5-fluorouracil showed activation effects on CuZnSOD at 3.33 mg/ml and 4 mg/ml concentrations with 33% and 32% activation, respectively. Leukocytes were isolated from healthy human blood, lysed in liquid nitrogen and the effect of 5-fluorouracil on the lysate SOD activity investigated. 5-Fluorouracil showed inhibition effects on total SOD activity of human leukocytes at 2 mg/ml and 4 mg/ml concentrations with 42% and 62% inhibition, respectively.     

Isolation of a benzoate-utilizing Pseudomonas strain from soil and production of catechol from benzoate by transpositional mutants

Pseudomonas sp. Ba-0511 was isolated from soil by enrichment cultivation on a medium containing 6 mg/ml of sodium benzoate. The bacterium could grow on a medium containing 20 mg/ml of sodium benzoate by a successive enrichment culture. One hundred and twelve transpositional mutants of the bacterium produced catechol from benzoate and accumulated it outside of the cells. Among the mutants, strain BA+63 produced a maximal amount of catechol (2.3 mg/ml) from 6 mg/ml of sodium benzoate after growing for 10.5 h. The conversion rate of benzoate to catechol was 50% on a molar basis. The catechol production by the resting cells increased in the presence of glycerol, and the maximal amount of catechol produced from 6 mg/ml of sodium benzoate reached 3.3 mg/ml at the conversion rate of 72% after 5 h of incubation. The resting cells converted m-methylbenzoic acid to 3- and 4-methylcatechol and m-chlorobenzoic acid to 3- and 4-chlorocatechol.     

Effects of some drugs on purified human erythrocyte CuZnSOD and in vitro inhibitiory effect of 5-fluorouracil on leukocyte total SOD activity

The inhibition and activation effects of some drugs on the activities of superoxide dismutase enzymes (SOD) in human erythrocyte and leukocyte cells was investigated. Firstly, CuZnSOD enzyme was purified 837-fold and 12% efficiency from human erythrocytes by ethanol-chloroform treatment to remove hemoglobin and then ion exchange chromatography (DEAE-Sepharose) and copper chelate affinity chromatography techniques. Inhibition or activation effects of fourteen drugs on CuZnSOD was investigated. None of the studied drugs except for 5-fluorouracil showed any effects on the enzyme. 5-fluorouracil showed activation effects on CuZnSOD at 3.33mg/ml and 4mg/ml concentrations with 33% and 32% activation, respectively. Leukocytes were isolated from healthy human blood, lysed in liquid nitrogen and the effect of 5-fluorouracil on the lysate SOD activity investigated. 5-Fluorouracil showed inhibition effects on total SOD activity of human leukocytes at 2 mg/ml and 4 mg/ml concentrations with 42% and 62% inhibition, respectively.     

Improved nuclear staining with mordant blue 3 as a hematoxylin substitute.

For progressive staining 1 g mordant blue 3, 0.5 g iron a alum and 10 ml hydrochloric acid are combined to make 1 liter with distlled water. Paraffin sections are stained 5 minutes blued in 0.5% sodium acetate for 30 seconds and counterstained with eosin. For regressive staining, 1 g dye, 9 g iron alum and 50 ml acetic acid are combined to make 1 liter with distilled water. Staining time is 5 minutes followed by differentiation in 1% acid alcohol and blueing in 0.5% sodium acetate. Counterstain with eosin. In both cases results very closely results very resemble a good hematoxylin and eosin.     

Improved Nuclear Staining with Mordant Blue 3 as A Hematoxylin Substitute

For progressive staining 1 g mordant blue 3, 0.5 g iron alum and 10 ml hydrochloric acid are combined to make 1 liter with distilled water. Paraffin sections are stained 5 minutes, blued in 03% sodium acetate for 30 seconds and counterstained with eosin. For regressive staining, 1 g dye, 9 g iron alum and 50 ml acetic acid are combined to make 1 liter with distilled water. Staining time is 5 minutes followed by differentiation in 1% acid alcohol and blueing in 0.5% sodium acetate. Counterstain with eosin. In both cases results very closely resemble a good hematoxylin and eosin.     

Orcein-Hematoxylin in Iodized Ferric Chloride as a Stain for Elastic Fibers, With Metanil Yellow Counterstaining

Autopsy and biopsy specimens of human skin were fixed overnight in alcoholic Bouin's solution, embedded in paraffin, cut at 7 μ, deparaffinized, hydrated to 70% alcohol, and treated as follows—stained 2 hours in a mixture consisting of: 0.2% orcein in 70% alcohol and 1% HC1 (conc.), 125 ml; 5% hematoxylin in absolute alcohol, 40 ml; 6% FeCl₃ in water, 25 ml; and aqueous I₂-KI (1:2:100), 25 ml—rinsed in distilled water until the excess stain was removed—differentiated in 1.2% FeCl₃, 5-15 sec—washed in running water, 5 min—differentiation completed in 0.01% HC1 acid-alcohol, 1 min—a dip in 95% alcohol—distilled water, 2 min—0.25% aqueous metanil yellow, 5-10 sec—a 95% alcohol dip—dehydrated in absolute alcohol, xylene, and mounted in a resinous medium. The technic combines the orcein of Pinkus' stain and the hematoxylin mixture of Verhoeff into a single staining solution and gives sharp and reliable results for both coarse and extremely delicate elastic fibers. These stain purple; nuclei, violet; and background, yellow. The stain allows the use of formalin, Bouin's fluid and Zenker-formol fixation. The results have been consistent in other primates as well as in man.