A comparison between formol saline and Mirsky's fluid as fixatives for nematodes

In order to evaluate the fixing properties of Mirsky's fluid on nematodes, a comparison was made with formaldehyde. On two occasions, nematodes recovered from the abomasa of calves were fixed in each of three of the following fluids: 5% formol saline, 10% formol saline, 5% Mirsky's fluid and 10% Mirsky's fluid. Based on measurements of length and microscopical examination of over 800 nematodes on each occasion, the authors concluded that Mirsky's fluid conferred no advantages over 5% formol saline or 10% formol saline for fixing nematodes.     

Use of Gallocyanin as a Myelin Stain for Brain and Spinal Cord

Tissue fixed in 10% formalin, formol saline, CaCO₃ or phosphate buffer neutralized formalin, Baker's formol calcium, Cajal's formol ammonium bromide, formalin-95% ethanol 1:9, formalin-methanol 1:9, Lillie's methanol-chloroform or Salthouse's formol cetyltrimethylammonium bromide was dehydrated and embedded in paraffin. Sections were attached to slides with either albumen or gelatine adhesive and processed throughout at room temperature of 22-25 C. Mordanting 30-60 min in 1% iron alum was followed by a 10 min wash in 4 changes of distilled water. Myelin was stained in a gallocyanin self-differentiating solution for 1-2.5 hr; thick sections requiring the longer time. The staining solution (pH approximately 7.4) consisted of Na₂CO₃, 90 mg; distilled water, 100 ml; gallocyanin, 250 mg; and ethanol, 5 ml. The ethanol was added to this mixture last, and after the other ingredients had been boiled and then cooled to room temperature. After a staining and thorough washing, Nissl granules were stained for 5-10 min in a solution consisting of: 0.1 M acetic acid, 60 ml; 0.1 M sodium acetate, 40 ml; methyl green, 500 mg. Washing, dehydration, clearing and mounting completed the process. Myelin sheaths were stained dark violet; neuronal nuclei, light green with dark granules of chromatin; nucleoli of motor cells and erythrocytes, dark violet; cytoplasm, green with dark green Nissl granules. The simple and reliable method can be adapted easily for use with automatic tissue processors.     

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.     

Preservation of differential staining of spermatozoa by formol citrate.

Semen from boar, bull, ram, rabbit, reindeer and stallion was diluted in formol citrate or formol saline and stained with eosinnigrosin. The proportion of eosinophilic spermatozoa did not differ from that in fresh semen after storage for 48 hr in the formol diluent at temperatures ranging from 4 degrees C to 40 degrees C. Some samples were kept for periods up to 3 weeks with very little increase in the proportion of eosinophilic spermatozoa.     

Comparison of different techniques to determine the percentage of intact acrosomes in frozen-thawed bull semen

Twenty-seven different batch dates of frozen bull semen from 26 bulls were used in this study. The semen was in 0.5-ml straws, and 23 of the batch dates were in whole milk extender while 4 were in egg yolk-tris extender. Straws from each batch of semen were incubated for 2 hours in a water bath at 37 degrees C. Following this, the percentage of progressive motility, the rate of motility, and the percentage of intact acrosomes were determined for each unfixed sample. Each batch of semen was fixed in 2 different solutions of 0.2% glutaraldehyde in phosphate-buffered saline (glutaraldehyde 1 and glutaraldehyde 2) and in 10% neutral buffered formol saline (formol saline). The percentage of intact acrosomes for each sample in these fixatives was determined at Day 0 and Day 7. There were no significant differences in the percentages of intact acrosomes among the unfixed samples and the samples in the 3 fixatives at Day 0. At Day 7, the samples in formol saline had a significantly higher percentage of intact acrosomes than those in glutaraldehyde 2. When the percentage of intact acrosomes for the unfixed samples at Day 0 was compared with the percentages of intact acrosomes for glutaraldehyde 1, glutaraldehyde 2, and formol saline at Day 7, only the percentage of intact acrosomes for formol saline was significantly higher than for the unfixed samples. Only one of the batches of semen in egg yolk-tris extender could be evaluated in formol saline because of a heavy precipitate that formed. There was a significant interaction between extender and storage. For the whole milk extender, the percentages of intact acrosomes at Day 7 were higher than for Day 0 for all the fixatives used. For the egg yolk-tris extender, the percentage of intact acrosomes decreased from Day 0 to Day 7. The correlations between the percentage of intact acrosomes for the unfixed samples and the post-incubation percentage of progressivé motility and rate of motility were 0.65 and 0.46, respectively.     

Accumulation of lead and cadmium in upper parts of mustard (Brassica juncea) seedlings in response to putrescine

Lead and cadmium accumulation examined in shoot and leaf tissues of seedlings of mustard (Brassica juncea cv RH-30), at 7th day, treated with either putrescine (1 mM), or ammonium nitrate (10 mM) or IAA (10 microM). These were included in the nutrient medium, containing Pb or Cd (0.1 mM and 2 mM). Metal accumulation was more in shoot than in leaf tissues, which was increased manifold under saline conditions. However, Cd accumulation in tissues was higher than Pb. Chemical (putrescine, ammonium nitrate or IAA) treatment of the seedlings, decreased metal accumulation in leaf (10-20%) and in shoot (40 to 60%) tissues, depending upon external metal levels. Putrescine significantly decreased the metal accumulation and translocation under saline conditions.     

An acetylcholinesterase method for in toto staining of peripheral nerves.

Stomach, small intestine, uterus, urinary bladder, vagina, mesentery, mesometrium and joint capsule of rats, gall bladder, cystic duct and bile duct of dogs and uteri of young children are stained in toto. Procedure: Tissue is perfused with saline containing hyaluronidase, then pinned on a flat layer of Paraplast and fixed for 24 hr in cold sucrose formol solution. Stomach, urinary bladder and gall bladder are also fixed in toto. Rinse for 2 days in cold 0.22 M sucrose in a sodium cacodylate buffer pH 7.2. Incubate in medium consisting of 60 mM acetate-buffer pH 5.0 or pH 5.6 (for human material only), 2 mM acetylthiocholine iodide, 15 mM Na citrate, 3 mM Cu sulphate, 0.5 mM K3Fe(CN)6, 5 times 10-4 M iso-OMPA, 1% Triton X 100 at 37C. Rinse in doubly distilled water. Dehydrate in glycerine/water mixtures of increasing glycerine content. Store in glycerine or delaminate under dissecting microscope. Delaminated specimens are mounted on gelatinized object glasses, cleared in xylene and coverslipped with Malinol. Specimens stored in glycerine can be studied microscopically. Stained specimens can also be embedded in Paraplast and sections can be studied after counterstaining.     

Trypsin-Orcein Banding in Plant Chromosomes

A convenient and quick method using trypsin-orcein for handing plant chromosomes (O-banding) is suggested. The technique is directly applicable to meristematic tissues (e.g. root tip) and involves the treatment of root tip with 1-2% solution of trypsin either in buffer or in 05 N HCI for 5-10 minutes at 37 C or for 30-60 minuta near 0 C followed by staining with 1.5% acetic orcein: 1 N HCI (19:1). Dark staining bands are reproducible and species specific. These bands possibly represent specific DNA-protein-dye interaction.     

The staining of Brunner's gland and other neutral mucins by carmine, hematoxylin and orcein in alkaline solutions.

Brunner's glands and other neutral mucins may be stained red, brownish red, and violet, respectively, by carmine, hematoxylin, and orcein from appropriate alkaline solutions. Carmine and hematoxylin in concentrations of 0.2-1% are dissolved in 60-70% alcohol containing 1% potassium carbonate; orcein is used in a 0.2% alcoholic solution of sodium hydroxide. Staining times are 15 to 30 minutes. The stained sections are rinsed in 95% or absolute alcohol prior to xylene and mounting. The staining of these mucins is blocked by mild bromine oxidation. By using alcian blue 0.1% in 3% acetic acid for 5 minutes prior to the above stains, mucins may be characterized in the same preparation as acid, neutral or mixed.     

Trypsin-orcein banding in plant chromosomes.

A convenient and quick method using trypsin-orcein for banding plant chromosomes (O-banding) is suggested. The technique is directly applicable to meristematic tissues (e.g. root tips) and involves the treatment of root tips with 1-2% solution of trypsin either in buffer or in 0.5 N HCl for 5-10 minutes at 37 C or for 30-60 minutes near 0 C followed by staining with 1.5% acetic orcein: 1 N HCl (19:1). Dark staining bands are reproducible and species specific. These bands possibly represent specific DNA-protein-dye interaction.     

Influence of two different fixatives on the identification of plasma cells in human rectal mucosa

Plasma cells in sections of bisected human rectal biopsy specimens, fixed in two alternative fixatives, were enumerated after staining by an indirect immunoperoxidase procedure intended to demonstrate immunoglobulin-containing cells. The counts of immunoperoxidase-positive plasma cells were significantly higher after fixation in formol sublimate than after fixation in formol saline. Formol sublimate appears to be a more reliable fixative than formol saline for specimens of rectal mucosa in which quantitation of plasma cells, stained for intracellular immunoglobulin by an immunoperoxidase technique, is intended.     

Quantification of deep and superficial sensibility in saline-induced muscle pain-a psychophysical study

The aim of the present study was to study the sensibility in the area of saline-induced muscle pain. In three experiments, ten subjects were exposed to computer-controlled infusion of 0.5 ml isotonic (0.9%) or hypertonic (9%) saline into the anterior tibial muscle. The pain intensity was assessed on a visual analogue scale (VAS). The pain threshold (PT) to pressure and electrical stimulation in muscle and subcutaneous tissues was determined. Three experiments were performed in which infusion of hypertonic saline produced significantly higher VAS scores than isotonic saline. In all three experiments, there was no significant difference in PT obtained after infusion of isotonic saline compared with infusion of hypertonic saline. In experiment 1, the PT was determined at the infusion site and 4 cm from the infusion site. At the infusion site, the pressure PT decreased (- 19 2%) 1, 3, 5, 7 and 9 min after infusion of isotonic and hypertonic saline, but remained unchanged 4 cm from the infusion site. The intramuscular electrical PT at the infusion site and 4 cm from the infusion site increased significantly (29 6%) 5, 7 and 9 min after saline infusion. In experiment 2, the pressure PT and the intramuscular electrical PT were recorded after two infusions of saline separated by 1 day. The day after the first infusion, the pressure PT was decreased compared with the PT before the first infusion, but the electrical PT was not affected. Moreover, the hypertonic saline infusion given on the second day produced significantly higher (130 50%) VAS scores than the infusion given on the first day. In experiment 3, the PT was determined in the subcutaneous tissue, but no significant effects of saline infusion were found. The present placebo-controlled experiments failed to show muscular or subcutaneous hyperalgesia after saline-induced muscle pain per se.     

Enhanced Differentiation of Zaprionus Chromosomes by Prestaining Acid Hydrolysis

Two variations of orcein staining have been adapted to salivary gland chromosomes of Zaprionus. Method I: after dissection, glands are transferred to 1 N HCl at 60° C for 5 min, stained with 2.5% orcein in 60% acetic add for 15-20 min, and squashed in 60% acetic acid. Method II: after dissection, glands are transferred to 1 N HCl at 60° C for 5 min, transferred to a saturated solution of carmine in 45% acetic acid for 1 min, then to a mixture of 50 ml of 1% orcein in concentrated lactic acid and 50 ml of 30% acetic add for 5 min. They are squashed in the same mixture. The unproved differentiation of chromosomes from cytoplasm is attributed to the removal of cytoplasmic ribonucleic add by add hydrolysis.     

Exogenous Melatonin Alleviates Skeletal Muscle Wasting by Regulating Hypothalamic Neuropeptides Expression in Endotoxemia Rats

To investigate whether exogenous melatonin (MLT) could alleviate skeletal muscle wasting by regulating hypothalamic neuropeptides expression. Adult male Sprague Dawley rats were intraperitoneally injected with lipopolysaccharide (LPS) (10 mg/kg), followed by MLT (30 mg/kg/day) or saline for 3 days. Hypothalamic tissues and skeletal muscle were obtained on day 3. Skeletal muscle wasting was measured by the mRNA expression of two E3 ubiquitin ligases, muscle atrophy F-box and muscle ring finger 1 as well as 3-methylhistidine (3-MH) and tyrosine release. Three hypothalamic neuropeptides (POMC, AgRP, CART) expression were detected in all groups. POMC expression knockdown was achieved by ARC injection of lentiviruses containing shRNA against POMC. Two weeks after ARC viruses injection, rats were i.p. injected with LPS (10 mg/kg) followed by MLT (30 mg/kg/day) or saline for 3 days. Brain tissues were harvested for immunostaining. In septic rats, 3-MH, tyrosine release and muscle atrophic gene expression were significantly decreased in MLT treated group. POMC and CART expression were lower while AgRP expression was higher in MLT treated group. Furthermore, in septic rats treated with MLT, muscle wasting in those with lower expression of neuropeptide POMC did not differ from those with normal POMC expression. Exogenous MLT could alleviate skeletal muscle wasting in septic rats by regulating hypothalamic neuropeptides.

     

Some histochemical reactions of mast cells of gerbils,hogs, armadillos and cats

Summary Mast cells of the Mongolian gerbil Meriones unguiculatus, the hog Sus scrofa, the cat Felis catus and the armadillo Pasypus novemcinctus were studied histochemically in relation to various fixation procedures, using azure A at pH 1 and 3, alcian blue at pH 1 and 2.5, diazosafranin at pH 3 and 7.8–8, and the PAS reaction. Fixations were performed in buffered 10% formol and 5% glutaraldehyde, in Kose's fluid, buffered sublimate (B4), lead nitrate and lead acetate formol.With azure A and alcian blue many mast cells were found in the gerbil with the aldehyde fixatives, fewer with the heavy metals. The diazosafranin reaction was present only in the aldehyde material, the PAS reaction was negative.In the hog, mast cells were more numerous after heavy metal fixation, fewer with aldehydes. Azure A stained metachromatically at pH 1 and 3, alcian blue reacted only at pH 1, the PAS reaction was negative, the pH 3 and 8 diazosafranin reactions were positive with all 4 fixations.In the cat, mast cells were moderately numerous with lead acetate formol, rare with formol and absent with glutaraldehyde. They stained with azure A at pH 1 and 3, with alcian blue at pH 1 and 2.5, with diazosafranin at pH 3 and 8 and by the PAS reaction.Armadillo mast cells were more numerous after heavy metal fixations, stained with azure A and alcian blue at pH 1 and 2.5 to 3, and with acid and alkaline diazosafranin.The mast cells of the 4 species vary in their requirements for aldehyde and heavy metal fixation, in their PAS reactivity and in their pH 2.5 alcian blue staining. All are sufficiently sulfated to react to cationic dyes at pH 1, but vary in PAS reactivity, indicating partial or complete sulfation. The presence of 5-hydroxytryptamine is indicated in all four species.Assisted by grant from National Cancer Institute C-04816.     

Cell-mediated immunity in mice infected with Acanthamoeba culbertsoni

Observations were made on the differences of cell-mediated responses in mice of three infection groups differently scheduled in their severity with pathogenic Acanthamoeba culbertsoni. Infections were done by dropping 5 microliters saline suspension containing 3 x 10(3), 1 x 10(4), or 1 x 10(5) trophozoites, respectively. Amoebae were cultured axenically in CGV medium and inoculated into the right nasal cavity of C3H/HeJ mice aging around 6-8 weeks, under the anesthesia by intraperitoneal injection of secobarbital. Delayed type hypersensitivity (DTH) responses in footpad and blastogenic responses of mouse spleen cells using (3H)-thymidine and the serum antibody titer were measured up to day 14 after infection, and natural killer cell activities were measured up to day 5 after infection. The results obtained in this study were as follows: 1. The mice infected with 3 x 10(3) trophozoites showed mortality rate of 17%, and 34% in the mice infected with 1 x 10(4) trophozoites and 65% with 1 x 10(5) trophozoites. 2. In regard to DTH responses in all experimental groups, the level increased on day 7 and declined on day 14 after infection, but their differences could not be noted between infected and control groups. 3. The blastogenic responses of splenocytes treated with amoeba lysates and lipopolysaccharides (LPS) showed no difference from the control group. The blastogenic responses of splenocytes treated with concanavalin A were declined significantly in the experimental group as compared with the control group, but the blastogenic responses of splenocytes treated with polyinosinic acid were not different from the control group. There was also no difference among three infected groups. 4. The cytotoxic activity of the natural killer cells was activated on day 1 after infection and declined to the level of control group on day 2 in all experimental groups. On day 5 after infection, the natural killer cell cytotoxicity was significantly suppressed as compared with the control groups. 5. The serum antibody titers of the infected mice increased after day 7, but there was no statistical difference between the three infected groups. In summary of the results, there was no difference in cell-mediated immune responses of three experimental groups scheduled with different infection intensities. But there was a significant difference in cell-mediated immune responses between infected and control mice. It is considered that cell-mediated immune responses should be involved in murine model infected with A. culbertsoni.     

Effect of formol on the fluorescence of staphylococci

Many strains ofStaphylococcus aureus give a strong immunofluorescence reaction with heterologous preparations of fluorescent antibodies. It is not known exactly whether this reaction is specific or unspecific. For diagnostic purposes it is important to reduce this fluorescence to low values without affecting the reaction of the homologous antigen with antibody. Formolisation of staphylococci, otherwise showing a strong fluorescence with the conjugates of heterologous antisera, for a longer period than 3 hours and at a formol concentration of 5%, lowers the fluorescence of the staphylococci to minimum. Formolisation proved suitable also when the staphylococci were contained in tissues (spleen, lymph nodes). In this case the fluorescence of the staphylococci disappears and the fluorescence of the tissues is suppressed. By treatment with formol in the concentrations used, the immunofluorescence reaction betweenPasteurella tuarensis and the corresponding preparation of the tularemy antiserum was not significantly suppressed.     

Immunofluorescence Microscopy of Cyanurated Tissues

Test tissues consisted of: (1) popliteal lymph nodes of rabbits, removed 6 hr after injection of hind footpads with 0.2 ml of 125 mg/ml solution of 5× crystallized chicken ovalbumin, and (2) lungs from guinea pigs, passively sensitized with rabbit antiovalbumin serum, then anaphylactically shocked by intracardial injection of a 1% chicken ovalbumin solution. Similar control tissues from normal rabbits, and lungs of passively sensitized guinea pigs, but shocked with histamine instead of ovalbumin, were included. Pieces of fresh tissue not exceeding 2 mm³ were fixed as follows: (1) Cyanuration—lymph nodes, 1 hr; lung, 0.5 hr; both at 23-27 C—in anhydrous methanol containing 0.5% w/v cyanuric chloride and 1% v/v N, N-diethylaminoethanol. (2) Control fixatives—all specimens 18-24 hr at 4—6 C—absolute methanol; 95% ethanol; neutral buffered 10% formalin; and an FAA mixture (formalin, conc., 6; glacial acetic acid, 2; 30% ethanol, 92). Freeze-dried material was either left unfixed (a control) or fixed in xylene or toluene containing 0.5% w/'v cyanuric chloride and 1% v/v N, N-diisopropylaminoethanol; time and temperature as for fresh tissues. All tissues were routinely dehydrated, cleared, and vacuum embedded in an ester wax, diethylene glycol distearate, or in paraffin at 52 C. Sections 2-4 μ thick were attached to gelatin-coated slides, the wax removed in petroleum ether, and stained 20 min at 23-27 C in a 0.10% solution of fluorescein isothiocyanate-conjugated rabbit antiovalbumin globulin, washed in phosphate buffered saline 10 min, dehydrated, cleared and covered in a nonfluorescent medium. With ultraviolet illumination, brightly immunofluorescent, anti-genically specific staining was obtained in cyanurated fresh and freeze-dried lymph node and lung tissues. In contrast, specific staining was diminished or absent in comparable tissues reacted in the control fixatives.     

Chromosome Preparatons of Bone Marrow Cells without Prior In Vitro Culture or In Vivo Colchicine Administration

Bone marrow (about 0.5 ml) from au erythropoietic region is freed of blood clots by washing 1-3 min in 1 μg/ml colchicine solution (2-3 ml) and then soaking 1-2 hr at 20-30° C in a second change. For mammalian or avian marrows, the colchicine is made up in phosphate-buffered (pH 7) physiological NaCl solution; for amphibian, Ringer's A solution. Next the specimens are soaked about 20 min in a hypotonic solution as follows: for mammalian, 1% Na-citrate; for avian, a 1:4 dilution of the buffered NaCl solution by distilled water; and for amphibian, Ringer's A-distilled water, 1:1. Then they are heated in a mixture of 2% orcein in 45% acetic acid and 1 N HCl, 9:1. Immediately after heating, squash preparations are made with 2% acetic-orcein in the usual manner. An alternative method is to dissociate the marrow cells by agitating after colchicine treatment. Then, recovering the cells between changes by low-speed centrifugation, to carry out the hypotonic treatment and subsequent fixation in Carnoy's solution I (alcohol acetic, 3:1) before drying the cells onto slides from the fixative. After thorough drying the slides may be stained 10-20 min in acetic orcein, or by other suitable technics.     

Demonstration of the Ground Substance of Cartilage, Bone, and Teeth

A slab of bone about 5 mm. thick is decalcified in 5% HCl, washed, and placed for several days to 2-3 weeks in 3% KOH in 20% glycerin (if the bone is medium sized); for small bones the KOH should be decreased to 1%, and for large bones it may be increased to 5%. The solution is changed frequently. When the bone begins to dissociate, it should be removed and washed in water till all traces of alkali are removed. The specimen is passed through 3 changes of dioxane into paraffin, and then through a second paraffin bath into the final paraffin. Sections are cut at 10-12 μ and stained with VanGieson's picro-fuchsin or with orcein.