The Use of Salicylic Acid as a Fixative

The following salicylic acid-containing fixatives are useful for cytological studies in plants. The first, here designated HFC, is recommended for studies on somatic mitosis and chromosome individuality. The second, denoted HFP, is recommended for studies on plastids.

HFC is made up in two solutions. Sol. A: 100 cc. sat. aq. sol. salicylic acid, slight excess copper hydroxide, 20 cc. formaldehyde, 30 cc. normal ortho-phosphoric acid, 200 cc. water, 1 g. saponin; pH 1.8 to 1.9. Fix in Sol. A 15 to 30 minutes in partial vacuum of 35 cms. Then add Sol. B: 1% aq. chromic acid in equal parts. Continue fixation for period of 18 to 24 hours.

HFP is also made up in two solutions which are used in equal parts. Sol. A: 100 cc. sat. aq. sol. salicylic acid, slight excess copper hydroxide, 10 cc. normal ortho-phosphoric acid, 1/2 g. saponin. Sol. B: 187.5 cc. 1% aq. chromic acid, 50 cc. 2% osmic acid. Fixation technic as HFC.

Dehydrate and infiltrate with paraffin after Zirkle. Stain with crystal-violet-iodine.     

Further Studies on a Modification of the Gram Stain

In perfecting the modification of the Gram-stain previously proposed, the following points are of interest:

1. Acetone is too strong a decolorizer for Gram-positive organisms and alcohol too weak for Gram-negative organisms. Consequently, it is now recommended that equal parts of acetone (100% c.p.) and ethyl alcohol (95%) be used as a decolorizing agent. The time of application should not ordinarily exceed 10 seconds.

2. Aqueous basic fuchsin (0.1%) serves as a strongly contrasting counterstain. Prolonged application renders Gram-positive organisms doubtful or Gram-negative, while short application renders Gram-negative organisms doubtful or Gram-positive. Twenty (20) seconds is therefore recommended as the time of application of the counterstain.

3. The method here described, with due regard for its limitations, is of value in Gram-staining pure or mixed cultures as well as for organic materials, such as Acidophilus milk, feces, etc., either for research purposes or classroom use. The method is as follows:

Air-dry film and fix with least amount of heat necessary.

Flood with dye for 5 minutes. Previously mix 30 drops of a 1% aqueous solution of crystal violet or methyl violet 6B with 8 drops of a 5% solution of sodium bicarbonate. Allow the mixture to remain for 5 minutes or more.

Flush with iodine solution for 2 minutes. Two grams iodine dissolved in 10 cc. normal sodium hydroxide solution and 90 cc. water added.

Drain without blotting but do not allow film to dry.

Add a mixture of equal parts of acetone and alcohol drop by drop until the drippings are colorless. (10 seconds or less.)

Air-dry slide.

Counterstain for 20 seconds with 0.1% aqueous solution of basic fuchsin.

Wash off excess stain by short exposure to tap water and air-dry. If slide is not clear immersion in xylol is recommended.     

A Controllable Carmine Technic for Plants with Small Chromosomes

Anthers of small chromosome plants (Antirrhinum, Brassica, Capsicum etc.) were fixed 12 hours or longer at 0-3° C. in: ferric acetate in glacial acetic acid (sat. soln.), 1 part; absolute alcohol, 3 parts. They were transferred to: ferric acetate (sat. soln.) in 45% acetic acid, 3 parts; 45% acetic acid, 5 parts; 1% formalin (aq.), 2 parts, and allowed to remain 5-15 minutes at room temperature for mordanting. The amount of iron introduced into the specimens was controllable by the time in the mordanting fluid. After rinsing the specimen in 45% acetic acid and macerating in a drop of Belling's acetocarmine on a slide, a cover slip was applied followed by warming and pressing with blotting paper to flatten the pollen mother cells and expel excess stain. Preparations stored temporarily by sealing the edges of the cover slip with rubber solution were best made permanent by removing the cover slip after 1-2 days, dehydrating and mounting in euparal.     

A Controllable Carmine Technic for Plants with Small Chromosomes

Anthers of small chromosome plants (Antirrhinum, Brassica, Capsicum etc.) were fixed 12 hours or longer at 0–3° C. in: ferric acetate in glacial acetic acid (sat. soln.), 1 part; absolute alcohol, 3 parts. They were transferred to: ferric acetate (sat. soln.) in 45% acetic acid, 3 parts; 45% acetic acid, 5 parts; 1% formalin (aq.), 2 parts, and allowed to remain 5–15 minutes at room temperature for mordanting. The amount of iron introduced into the specimens was controllable by the time in the mordanting fluid. After rinsing the specimen in 45% acetic acid and macerating in a drop of Belling's acetocarmine on a slide, a cover slip was applied followed by warming and pressing with blotting paper to flatten the pollen mother cells and expel excess stain. Preparations stored temporarily by sealing the edges of the cover slip with rubber solution were best made permanent by removing the cover slip after 1–2 days, dehydrating and mounting in euparal.     

Method for Staining Bacterial Flagella

The following method of staining bacterial flagella is ecommended for use on smears made from suspensions of 10 to 16-tour agar slant cultures, incubated 30 minutes at 37°C before spreadng on thoroly cleaned and named slides:

Cover with fixative (100 cc. of 1/4 sat. aqu. solution picric acid, with 5 g. tannic acid and 7.5 g. ferrous sulfate).

Wash with tap water, dry and cover with Fontana spirochaete stain; heat to steaming and allow to act for 1 to 2 minutes. Wash in ap water. The stain is prepared as follows: To 25 cc. 2% AgNO₃ add dilute ammonia till the precipitate which forms redissolves; then add more AgNO₃ till a faint turbidity results. A clear solution is useess.     

Injection of Lymphatics: With Colored Cedar Oil; With Plastic

(1) The oil mass consists of: cedar oil, 1; color in oil (a paint pigment, e.g., Prussian blue), 1; and toluene, 2, parts by volume. To use, add 1 ml of diethyl ether to each 10 ml of mass, mix thoroughly and inject into the fresh organ with a very fine glass or metallic needle. Heat the organ in water at 50-60° C before starting the injection, massage gently after injection, then fix. For macroscopic studies, fix 5 days in 5% formalin, and dissect. For microscopic studies, fix at least 5 days in: formalin, 10 ml; Al₂(SO₄)₃, 2 gm; ZnSO₄, 2 gm; acetic acid, 4 ml; and distilled water, 90 ml. Dehydrate with dioxane, embed in paraffin and section at 10-20 μ. Stain with hematoxylin-eosin or with one of the following modifications of Van Gieson's formula: 1. 1% acid fuchsin, 10; picric acid (sat. aq.), 50; and 5% ZnSO₄, 40 volumes. 2. 1% acid fuchsin, 20; picric acid (sat. aq.), 80; and 5% CoSO₄, 40 volumes.

(2) The plastic mass consists of a 5-10% solution of Rhodopas (a vinyl copolymer) in acetone. Injection is made as with the oil mass except that a plastic squeeze-bottle and glass needle is preferable to a syringe. Indirect injection is used for both procedures, i.e., into the organ substance; not into a cannulated lymphatic vessel. After the plastic has hardened (24 hr), the unfixed tissue is subjected to corrosion by 5-10% NaOH in water.     

Cytological Methods for Crepis Species

Root tips of Crepis species are fixed in La Cour's “2BE” and dehydrated thru a butyl alcohol series. They are stained in 1% crystal violet for 1 hour, with chromic acid and iodine as pre-and post-staining mordants, respectively, and passed thru dehydrating alcohols containing picric acid and ammonium hydroxide. Differentiation is done in clove oil. The method is rapid; the chromosomes are dark purple; the centromere is not stained; and the cytoplasm is clear. By further controlled destaining the hetero-chromatic segments within the chromosomes may be located.

Pollen mother cells are fixed in acetic alcohol (1:4) and squashed in aceto-carmine. A method is described for making semi-permanent preparations mounted in diaphane.

Pollen grains are mounted in lacto-phenol with acid fuchsin or anilin blue W. S. as the dye.     

Simple and durable staining of thick sections of the human brain for macroscopic study.

Observations on gross morphology of the brain is greatly facilitated by enhancing the contrast between gray and white matter. The proposed technique is much more simple than the generally recommended Mulligan method and it variations. Moreover, there is no loss of stain since the fugitive surface impregnation, obtained by the Mulligan method, is replaced by a thoroughgoing block-staining procedure with the nonfading copper phathalocyanine dye astra blue. Staining procedure: wash formalin-fixed brain slices overnight in running tap water. Place slices in performic acid for 1 hour. Wash in running tap water. Place slices individually in staining solution consisting of 0.1 g astra blue (Merck) in 1000 cc distilled water and 1 cc HCl (37%), for 12-24 hours. Wash in running tap water. Embed in gelatin and mount in plastic cuvettes.     

Method for Staining Bacterial Flagella

The following method of staining bacterial flagella is ecommended for use on smears made from suspensions of 10 to 16-tour agar slant cultures, incubated 30 minutes at 37°C before spreadng on thoroly cleaned and named slides:

1. Cover with fixative (100 cc. of 1/4 sat. aqu. solution picric acid, with 5 g. tannic acid and 7.5 g. ferrous sulfate).
2. Wash with tap water, dry and cover with Fontana spirochaete stain; heat to steaming and allow to act for 1 to 2 minutes. Wash in ap water. The stain is prepared as follows: To 25 cc. 2% AgNO₃ add dilute ammonia till the precipitate which forms redissolves; then add more AgNO₃ till a faint turbidity results. A clear solution is useess.

     

Simple and Durable Staining of Thick Sections of the Human Brain for Macroscopic Study

Observations on gross morphology of the human brain is greatly facilitated by enhancing the contrast between gray and white matter. The proposed technique is much more simple than the generally recommended Mulligan method and its variations. Moreover, there is no loss of stain since the fugitive surface impregnation, obtained by the Mulligan method, is replaced by a thoroughgoing block-staining procedure with the nonfading copper phthalocyanine dye astra blue. Staining procedure: wash formalin-fixed brain slices overnight in running tap water. Place slices in performic acid for 1 hour. Wash in running tap water. Place slices individually in staining solution consisting of 0.1 g astra blue (Merck) in 1000 cc distilled water and 1 cc HCl (37%), for 12-24 hours. Wash in running tap water. Embed in gelatin and mount in plastic cuvettes.     

Cleavage Of Oligodeoxyribonucleotides From Polymer Supports And Their Rapid Deprotection Under Microwaves

Abstract

Novel conditions for the cleavage of oligodeoxynucleotides from polymer supports and their complete deprotection under microwaves have been developed. The oligonucleotides synthesized using phosphoramidite synthons carrying base labile (Pac, Dmf and t-Bpac) and conventional (Bz for A and C and Pac for G) protecing groups for nucleic bases were deprotected using 0.2M sodium hydroxide (MeOH : H₂O:: 1:1, v/v) = Reagent A and 1M sodium hydroxide (MeOH: H₂O :: 1:1, v/v = Reagent B, respectively under microwaves. The deprotected oligonucleotides were found to be comparable with the corresponding oligonucleotides deprotected under standard conditions (aq. ammonia at 55°C).     

HPLC study on Fenton-reaction initiated oxidation of salicylic acid. Biological relevance of the reaction in intestinal biotransformation of salicylic acid

Abstract

Fenton-reaction initiated in vitro oxidation and in vivo oxidative biotransformation of salicylic acid was investigated by HPLC-UV-Vis method. By means of the developed high performance liquid chromatography (HPLC) method salicylic acid, catechol, and all the possible monohydroxylated derivatives of salicylic acid can be separated. Fenton oxidations were performed in acidic medium (pH 3.0) with two reagent molar ratios: (1) salicylic acid: iron: hydrogen peroxide 1:3:1 and (2) 1:0.3:1. The incubation samples were analysed at different time points of the reactions. The biological effect of elevated reactive oxygen species concentration on the intestinal metabolism of salicylic acid was investigated by an experimental diabetic rat model. HPLC-MS analysis of the in vitro samples revealed presence of 2,3- and 2,5-dihydroxybenzoic acids. The results give evidence for nonenzyme catalysed intestinal hydroxylation of xenobiotics.     

The Bielschowsky Staining Technic a Study of the Factors Influencing Its Specificity for Nerve FIBERS

By comparing results obtained with adult mammalian tissue from introducing variables into each separate step in block-staining by the Bielschowsky silver method, the following conclusions were reached:

1. No specific means for inhibiting the staining of connective tissue and still permitting complete staining of nerve fibers was found, but the avoidance of overstaining was very helpful toward such differentiation.
2. Overstaining could be corrected by reducing the concentration of the silver nitrate bath or by adding an excess of ammonia to the ammoniated silver bath.
3. Staining of fine fibers was favored by adding acetic acid to the formaldehyde used for fixation or by adding pyridin to the silver nitrate bath.
4. Addition of protein-precipitating organic acids (trichloracetic or sulfosalicylic) to the fixative was disadvantageous.
5. Prolonged fixation favored an increase in intensity of the stain. Four days' time was sufficient.
6. Extraction of lipids with ammoniated alcohol gave results similar to those obtained after extraction with pyridin, but the stain was lighter.
7. Ammoniated silver carbonate without excess ammonia had an action similar to ammoniated silver hydroxide with excess ammonia.
8. An excess of ammonia in the ammoniated silver solution (Ag 0.1 N) was tolerated, without apparent impairment of nerve-fiber staining, up to 6 M NH₃, altho the use of more than 3 M excess (2 cc. concentrated ammonia water added to 100 cc. of balanced ammoniated silver hydroxide solution) seemed unnecessary.
9. Impregnation with 1.7% (0.1 N) silver nitrate solution was quite satisfactory and variations in the concentrations of this bath suggested that the practical limits of concentrations that would be generally satisfactory lay between 0.3 and 3.0%.
10. The writers' experiences agreed with Agduhr's relative to the advantage of washing in 2.5% acetic acid between the ammoniated silver bath and formaldehyde reduction.

     

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.     

Biochemical studies of the chicken vitreous in light-induced avian glaucoma.

Changes in several macromolecular species of the vitreous are traced during post-hatching development in normal and glaucomatous avian eyes. 1. Total gel vitreous, and its hydroxyproline content, change little during development. 2. Sol vitreous increases parallel eye growth, the concentrations of hexosamine and hexuronic acid remaining constant. 3. In light-induced avian glaucoma, the sol, but not the gel, is further increased, although the hyaluronic acid components are not diluted in the pathologically enlarged eye.     

Studies on Kojic Acid and its Related γ-Pyrone Compounds

An excellent synthetic method of maltol from kojic acid has been established. By Mannich reaction with formaldehyde and dimethylamine, comenic acid gave the mono-Mannich derivative which was reduced to 6-methyl comenic acid. The acid was converted to maltol in good yield by decarboxylation with copper powder or KC–400 (tetra-chloro-diphenyl).

Hydroxymethylations of kojic, comenic and pyromeconic acids with formaldehyde in alkaline medium yielded the corresponding methylol derivatives which were converted to 6-methyl kojic acid, 6-methyl comenic acid and maltol in good yields, respectively, by reduction with stannous chloride. Oxidation of 6-methyl kojic acid with chromic anhydride and acetic acid gave 6-methyl comenic acid.     

The mechanism of the reaction between human plasminogen-activator inhibitor 1 and tissue plasminogen activator.

The structural events taking place during the reaction between PAI-1 (plasminogen-activator inhibitor 1) and the plasminogen activators sc-tPA (single-chain tissue plasminogen activator) and tc-tPA (two-chain tissue plasminogen activator) were studied. Complexes were formed by mixing sc-tPA or tc-tPA with PAI-1 in slight excess (on an activity basis). The complexes were purified from excess PAI-1 by affinity chromatography on fibrin-Sepharose. Examination of the purified complexes by SDS/polyacrylamide-gel electrophoresis (SDS/PAGE) and N-terminal amino acid sequence analysis demonstrated that a stoichiometric 1:1 complex is formed between PAI-1 and both forms of tPA. Data obtained from both complexes revealed the amino acid sequences of the parent molecules and, in addition, a new sequence: Met-Ala-Pro-Glu-Glu-. This sequence is found in the C-terminal portion of the intact PAI-1 molecule and thus locates the reactive centre of PAI-1 to Arg346-Met347. The proteolytic activity of sc-tPA is demonstrated by its capacity to cleave the 'bait' peptide bond in PAI-1. The complexes were inactive and dissociated slowly at physiological pH and ionic strength, but rapidly in aq. NH3 (0.1 mol/l). Amidolytic tPA activity was generated on dissociation of the complexes, corresponding to 0.4 mol of tPA/mol of complex. SDS/PAGE of the dissociated complexes indicated a small decrease in the molecular mass of PAI-1, in agreement with proteolytic cleavage of the 'bait' peptide bond during complex-formation.     

Cytological Methods for Crepis Species

Root tips of Crepis species are fixed in La Cour's “2BE” and dehydrated thru a butyl alcohol series. They are stained in 1% crystal violet for 1 hour, with chromic acid and iodine as pre-and post-staining mordants, respectively, and passed thru dehydrating alcohols containing picric acid and ammonium hydroxide. Differentiation is done in clove oil. The method is rapid; the chromosomes are dark purple; the centromere is not stained; and the cytoplasm is clear. By further controlled destaining the hetero-chromatic segments within the chromosomes may be located.

Pollen mother cells are fixed in acetic alcohol (1:4) and squashed in aceto-carmine. A method is described for making semi-permanent preparations mounted in diaphane.

Pollen grains are mounted in lacto-phenol with acid fuchsin or anilin blue W. S. as the dye.     

Staining Nerve Fibers After Sublimate-Acetic and After Bouin'S Fluid

A silver staining method for paraffin sections of material fixed in HgCl₂, sat. aq., with 5% acetic acid is as follows. Process the sections through the usual sequence of reagents, and including I-KI in 70% alcohol, thiosulfate (5% aq.), washing and back to 70% alcohol containing 5% of NH₄OH (conc. aq.). After 3 minutes in the ammoniated alcohol, wash through tap water and 2 changes of distilled water and silver 5-10 minutes at 25°C. in 15% AgNO₃ aq. to which 0.02 ml. of pyridine per 100 ml. has been added. Blot the slide, but not the section and do not rinse. Reduce at 45°C. in 0.1% pyrogallol in 55% alcohol, then rinse in 55% alcohol and wash in water. The remainder of the process consists of gold toning, intensifying in oxalic acid, fixing in 5% Na₂S₂O₃, washing, dehydrating, clearing and covering. When the specimen contains much smooth muscle, the I-KI solution is acidified before use by adding 2 ml. of 1N nitric acid per 100 ml., and the sections treated for 3 minutes instead of the usual 2 minutes. Formalin should not be added to sublimate-acetic, but specimens that do not contain strongly argyrophilic nonneural tissue may be fixed in formalin or, preferably, Bouin's fluid. Sections of tissue after the latter type of fixation will not require the I-KI and thiosulfate but can go from 95% alcohol to the ammoniated alcohol. The advantages of fixing in HgCl₂-acetic acid are suppression of the staining of connective tissue and intensifying the staining of nerve fibers.     

Hot chromation oxyphilia

Summary On exposure of formol or methanol chloroform fixed sections to solutions of 2.5–5% potassium dichromate or 4.2% chromic acetate for 18 hours at 60° C a strong relative oxyphilia develops. As measured with buffered solutions of azure A eosin B the point at which various tissue elements pass from red to blue staining is elevated by 3 to 4 pH units. A similar change occurs in K₂Cr₂O₇ in about 21 days at 24° C and at 4° C only a slight elevation of protein pK appears in 6 weeks. Ferric chloride and ferrous sulfate solutions of comparable metal content produce a less marked shift (about 2 pH units) and relatively slight similar action is produced by aluminium and copper salts (about 1 pH unit). Heating in water, NaCl, KH₂PO₄ and K₂CrO₄ solutions is without effect. The effect is similar to that of methylation on phosphoric and carboxylic acid sites, and combination of the two procedures is not additive. Mast cell and cartilage staining are not significantly affected. Deoxyribonucleic acid is not extracted; the Feulgen reaction persists, though 1 hour hydrolysis is required vs 12–15 minutes on control formol material. Metal binding as revealed by staining in acetic hematoxylin remains demonstrable after 10 days heating in 2.5% K₂Cr₂O₇, but disappears in 6–16 hours in chromic, ferric and ferrous salt solutions. Chemical analysis reveals the continued presence of chromium at 4–5 % dry weight levels in liver and brain, alike after chromic acetate and potassium dichromate (24 hours, 60° C), though no histochemical Cr demonstration is obtained after Cr acetate. It is concluded that carboxyl and phosphoryl residues in tissue bind chromic (and iron) ions, which are then converted by heating to an unreactive insoluble form. Removal of demonstrable chromium with acid alcohol, sodium dithionite or ß-mercaptoethanol from dichromate treated tissues does not reverse the oxyphilia.Supported by National Cancer Institute Grant No. C4816, National Institutes of Health.