Delafield'S Hematoxylin and Safranin for Staining Plant Materials

An improved schedule is suggested for staining plant materials in Delafield's hematoxylin and safranin. Tissues are stained first in Delafield's hematoxylin. A short bath in acidulated water (1 or 2 drops concentrated HCl to 100 cc.) removes objectionable precipitates, and at the same time serves as a destaining agent. The acid bath must be followed quickly by a thoro wash in tap water, or dilute lithium carbonate solution, to restore the original dark blue color (made reddish in the acid bath) of the hematoxylin and to “set” the stain. Once the hematoxylin solution is satisfactory, none of the reagents ordinarily used will remove it—unless they contain acid. Tissues are counterstained in rapid safranin (5 drops analin in 100 cc. of 1% safranin 0 in 50% ethyl alcohol); this materially lessens the time necessary for staining. The safranin is de-stained in 50% ethyl alcohol (which does not affect the hematoxylin) until sharp differentiation is secured. If destaining is too slow, or differentiation poor, a quick rinse in acidulated 50% alcohol usually sharpens contrast of the stains. This must be followed quickly by a wash in 50% alcohol containing lithium carbonate to neutralize the acid. Dehydrate, and mount as usual. This schedule allows each stain to be individually, and independently, controlled at the will of the operator.     

A Rapid Safranin-Metal Phthalocyanine Double Staining Technique for Plants

Pure metal 4.4',4',4'-tetxa-substituted, sulfo-, carboxy- and nitrophthalocyanines were synthesized. Mounted, deparaffinized and partially dehydrated sections of plant tissues were stained with 0.5% safranin in 50% alcohol for 5-10 min. Excess safranin was removed with a series of 70%, 95% and absolute alcohol washes. The sections were then stained for 2-3 min using metal 4,4',4',4'-phthalocyanine tetracarboxylic acid (MPTC, 0.5% (V/V) containing a few drops of dilute sodium hydroxide), metal 4,4',4',4'-tetra-sulfophthalocyanine (MPTS, 0.5% (V/V)) or metal tetranitrophthalocyanine (MPTN, 0.5% (V/V) in dimethyl sulfoxide). The sections were washed with 95%, then absolute alcohol; however, the metal tetranitrophthalocyanine section was washed only with absolute alcohol. Stained sections were treated briefly with xylene, then mounted on a coverslip. Bright peacock blue (MPTC and MPTS using Cu, Co or Ni), turquoise blue (MPTN using Cu or Ni) or parrot green (zinc phthalocyanine tetracarboxylic acid-ZnPTC, zinc phthalocyanine tetranitro derivative-ZnPTN) colors were obtained. Lignin-containing cells were stained red by safranin and the remaining cell structures were stained by the metal phthalocyanine complex with color brightness superior to that of fast green. Uniform staining, no color fading after a year, reliability, brief staining times, high color contrast (log ε = 4.0-4.9) and ease of use make this double staining combination ideal for routine use and photomicrography.     

Studies in Gram Staining

Gram-negative bacteria stained with crystal violet are decolorized by 95% alcohol within 2 min, whereas Gram-positive bacteria require at least 3 min treatment. Aqueous solutions of safranin, neutral red, and fuchsin replace crystal violet from stained Gram-positive bacteria more quickly than alcohol alone, and alcoholic solutions of these counterstains are in most cases still more effective. Treatment of crystal viokt-stained organisms with alcoholic safranin (0.25%) for 15 scc will distinguish Gram-positive bacteria (viokt) from Gram-negative bacteria (pink).

Alcohol containing very low concentrations of iodine generally decolorizes crystal violet-stained Gram-positive bacteria more quickly than alcohol alone. Increasing concentrations of iodine in alcohol reduce the rate of decolorization of stained bacteria, but stained Gram-negative bacteria are still readily dccolorized. The addition of 0.1% iodine to alcohol increases the rate of extraction of crystal violet by alcohol from Gram-negative organisms, but delays extraction of dye from Gram-positive organisms, and this applies when counterstain is also present. A two-solution modification of Gram staining is described in which crystal violet-stained bacteria are treated with an alcoholic solution of safranin, fuchsin, and iodine.     

Studies in gram staining.

Gram-negative bacteria stained with crystal violet are decolorized by 95% alcohol within 2 min, whereas Gram-positive bacteria require at least 3 min treatment. Aqueous solutions of safranin, neutral red, and fuschsin replace crystal violet from stained Gram-positive bacteria more quickly than alcohol alone, and alcoholic solutions of these counterstains are in most cases still more effective. Treatment of crystal violet-stained organisms with alcoholic safranin (0.25%) for 15 sec will distinguish Gram-positive bacteria (violet) from Gram-negative bacteria (pink). Alcohol containing very low concentrations of iodine generally decolorizes crystal violet-stained Gram-positive bacteria more quickly than alcohol alone. Increasing concentrations of iodine in alcohol reduce the rate of decolorization of stained bacteria, but stained Gram-negative bacteria are still readily decolorized. The addition of 0.1% iodine to alcohol increases the rate of extraction of crystal violet by alcohol from Gram-negative organisms, but delays extraction of dye from Gram-positive organisms, and this applies when counterstain is also present. A two-solution modification of Gram staining is described in which crystal violet-stained bacteria are treated with an alcoholic solution of safranin, fuchsin, and iodine.     

Delafield's Hematoxylin and Safranin for Staining Meristematic Tissues

The following technic is suggested for staining permanent preparations of meristematic tissues: Prepare and mount the sections by the usual paraffin method. From water, stain them 2-10 minutes in a solution made by adding 2-4 cc. of Delafield's hematoxylin to a Coplin jar full of tap water. As staining is progressive, the sections should be examined from time to time with a microscope. When the cell walls have become a deep purple, transfer the preparations, thru the usual series, to a mixture of xylol-absolute-alcohol in equal parts, and from this to a counterstain made by adding 4-6 cc. of a saturated solution of safranin in absolute alcohol to a Coplin jar full of xylol (75%) with absolute alcohol (25%). This stains the nuclei. Leave the sections in the counterstain at least 2 hours and then rinse them in xylol-absolute-alcohol (1:1) to remove excess safranin. Transfer them to pure xylol and then mount them in neutral balsam.     

Staining of plant Materials Cleared in NaOH

Stains are listed which have proved suitable for staining the epidermis, mesophyll, and sclerenchyma and tracheary elements, respectively, of cleared leaf material of Mouriri and Linociera. Too rapid leaching is avoided by overstaining high in the dehydration series, destaining briefly in the same solvent, and moving through to xylene. Twenty to thirty minutes staining time is generally sufficient. Concentrations and solvents can be varied widely. If destained too much, the material can usually be replaced in the dye with no ill effects. A double stain schedule (Bonnett) of five to ten minutes in 1% Bismarck brown Y in 95% alcohol followed by one to two minutes in 1% fast green FCF in 100% alcohol may be advantageous for thin-walled cells in thick material. It may be preferable to treat thinner material with tannic-acid-iron-chloride followed by safranin (Foster). The effects of bleaches and clearing compounds other than NaOH on staining have not been investigated; however, Dr. Bonnett finds that lactic acid used after NaOH improves clearing and also improves the staining of his combination (above). Mordants can doubtless be used to advantage.     

Limitations of safranin 'O' staining in proteoglycan-depleted cartilage demonstrated with monoclonal antibodies

The intensity of safranin 'O' staining is directly proportional to the proteoglycan content in normal cartilage. Safranin 'O' has thus been used to demonstrate any changes that occur in articular disease. In this study, staining patterns obtained using monoclonal antibodies against the major components of cartilage proteoglycan chondroitin sulphate (anti CS) and keratan sulphate (anti KS), have been compared with those obtained with safranin 'O' staining, in both normal and arthritic tissues. In cartilage where safranin 'O' staining was not detectable, the monoclonal antibodies revealed the presence of both keratan and chondroitin sulphate. Thus, safranin 'O' is not a sensitive indicator of proteoglycan content in diseases where glycosaminoglaycan loss from cartilage has been severe.     

A New Method for Safranin Differentiation

A new method of differentiating safranin in pollen-mother-cell smears and paraffin sections is described in detail. Slides stained in safranin are dehydrated in a series of alcoholic solutions containing 1.5% picric acid with constantly decreasing percentages of water. Differentiation is principally effected in 83% alcohol containing 1.5% picric acid and completed in the final dehydration and clearing. Counterstains may be applied in clove oil if desired.     

Safranin O Staining Using a Microwave Oven

We investigated the effects of microwave irradiation on a safranin O staining method for paraffin sections of formalin fixed rabbit larynx. The control sections were stained according to the conventional method, and the experimental sections were stained in microwave oven for 10 sec at 360 W in Weigert's iron hematoxylin, and for 30 sec at 360 W in fast green and 0.1% safranin O staining solutions. Light microscopic examination of the sections revealed that the microwave heating did not adversely affect the staining properties of cartilage tissue compared to the conventional staining method. Small differences such as darker staining of the matrix and shrinkage of the cytoplasm was observed in some microwave treated sections. The present study revealed that microwave application can be used safely for the safranin O method with the advantage of reduced staining time.     

Safranin O Staining Using a Microwave Oven

We investigated the effects of microwave irradiation on a safranin O staining method for paraffin sections of formalin fixed rabbit larynx. The control sections were stained according to the conventional method, and the experimental sections were stained in microwave oven for 10 sec at 360 W in Weigert's iron hematoxylin, and for 30 sec at 360 W in fast green and 0.1% safranin O staining solutions. Light microscopic examination of the sections revealed that the microwave heating did not adversely affect the staining properties of cartilage tissue compared to the conventional staining method. Small differences such as darker staining of the matrix and shrinkage of the cytoplasm was observed in some microwave treated sections. The present study revealed that microwave application can be used safely for the safranin O method with the advantage of reduced staining time.     

Thionin Staining of Paraffin and Plastic Embedded Sections of Cartilage

The usefulness of thionin for staining cartilage sections embedded in glycol meth-acrylate (GMA) and the effect of decalcification on cartilage sections embedded in paraffin and GMA were assessed. Short decalcification periods using 5% formic acid or 10% EDTA did not influence the staining properties or the morphology of cartilage matrix and chondrocytes. The standard stain safranin O-fast green for differential staining of cartilage was used as control in these experiments. Prolonged exposure of safranin P stained sections to fast green resulted in disappearance of the safranin O stained matrix, thereby hampering the quantitative measurement of negatively charged glycosaminoglycans (GAG). Thionin stained evenly throughout all cartilage layers, independent of the staining times. In contrast to safranin 0, thionin did not show meta-chromasia in nondehydrated cartilage sections, which made it more suitable for assessing cartilage quality in GMA embedded cartilage. To evaluate the selectivity of thionin staining in cartilage, chondroitinase ABC and trypsin digestions were carried out. Thionin staining was prevented by these enzymes in the territorial matrix, representing the interlacunar network and the chondrocyte capsule. Staining with thionin of the interterritorial matrix was only slightly reduced, possibly representing keratan sulfate and hyaluronic acid in cartilage of elderly patients. Comparison of thionin stained GMA embedded cartilage with safranin O stained paraffin embedded sections showed significant similarity in optical densitometry, indicative of the specificity of thionin bound to negatively charged GAG in cartilage. In GMA embedded cartilage morphology was relatively intact compared to paraffin embedded sections due to less shrinkage of chondrocytes and the interlacunar network.     

Thionin Staining of Paraffin and Plastic Embedded Sections of Cartilage

The usefulness of thionin for staining cartilage sections embedded in glycol meth-acrylate (GMA) and the effect of decalcification on cartilage sections embedded in paraffin and GMA were assessed. Short decalcification periods using 5% formic acid or 10% EDTA did not influence the staining properties or the morphology of cartilage matrix and chondrocytes. The standard stain safranin O-fast green for differential staining of cartilage was used as control in these experiments. Prolonged exposure of safranin P stained sections to fast green resulted in disappearance of the safranin O stained matrix, thereby hampering the quantitative measurement of negatively charged glycosaminoglycans (GAG). Thionin stained evenly throughout all cartilage layers, independent of the staining times. In contrast to safranin 0, thionin did not show meta-chromasia in nondehydrated cartilage sections, which made it more suitable for assessing cartilage quality in GMA embedded cartilage. To evaluate the selectivity of thionin staining in cartilage, chondroitinase ABC and trypsin digestions were carried out. Thionin staining was prevented by these enzymes in the territorial matrix, representing the interlacunar network and the chondrocyte capsule. Staining with thionin of the interterritorial matrix was only slightly reduced, possibly representing keratan sulfate and hyaluronic acid in cartilage of elderly patients. Comparison of thionin stained GMA embedded cartilage with safranin O stained paraffin embedded sections showed significant similarity in optical densitometry, indicative of the specificity of thionin bound to negatively charged GAG in cartilage. In GMA embedded cartilage morphology was relatively intact compared to paraffin embedded sections due to less shrinkage of chondrocytes and the interlacunar network.     

Staining Methods For Studying Ingredient Distribution In Baked Cakes

A method is described for preparing cake crumb for sectioning and staining. Previous to embedding, the fat was stained and fixed by exposing small blocks of cake to the fumes from a 5%, freshly-prepared, aqueous solution of osmic acid (OsO₄). This was followed by dehydration in ethyl alcohol and tertiary butyl alcohol, removal of air under vacuum and infiltration with paraffin.

Sections were cut 20 and 9Op thick and mounted with water.

Wax was removed by immersion in xylene. The sections were rehydrated in a series of ethyl alcohol dilutions, from concentrated to dilute, then transferred to distilled water.

Protein was then stained pink by immersion of the slides in an acidified 0.04% water solution of eosin Y, or starch was stained blue with a dilute aqueous solution of iodine. Ten grams iodine and 10 g. KI were dissolved in 25 ml. distilled water. This stock solution was diluted for use one to two hundred times.

The relationship between protein and starch was demonstrated by staining the sections with eosin, differentiating in 50% alcohol and staining with iodine.

When slides of cake crumb were prepared in this way, the fat was stained black, the protein bright pink and the starch granules a dark blue.     

Safranin and Anilin Blue with Delafield's Hematoxylin for Staining Cell Walls in Shoot Apexes

The following technic is suggested for staining cell walls in shoot apexes: After the usual preliminary steps through 50% ethyl alcohol, stain in 1 % safranin 0 for 24 hours. Rinse in tap water and place in 2% aqueous tannic acid for 2 minutes. After rinsing in tap water, stain for 2 minutes in 1 part Delafield's hematoxylin to 2 parts distilled water and rinse in tap water. Remove excess hematoxylin with acidified water (1 drop cone. HC1 in 200 ml. water), then place slides in 0.5% lithium carbonate for 5 minutes. Dehydrate through an ethyl alcohol series, then transfer from absolute alcohol to a saturated solution of anilin blue in “methyl cellosolve” for 5-10 minutes. Wash in absolute alcohol, rinse in a solution of 25% methyl salicylate, 33% xylene, 42% absolute ethyl alcohol and clear for 10 minutes in a solution of 2 parts methyl salicylate, 1 part xylene, 1 part absolute ethyl alcohol. Transfer through two changes of xylene and mount in “clarite” or suitable alternate. The resulting preparations will have clearly defined, dark-staining cell walls and will photograph well when “Super Panchro-Press, Type B” film (Eastman Kodak Co.) is used in conjunction with suitable Wratten filters.     

Safranin and Anilin Blue with Delafield's Hematoxylin for Staining Cell Walls in Shoot Apexes

The following technic is suggested for staining cell walls in shoot apexes: After the usual preliminary steps through 50% ethyl alcohol, stain in 1 % safranin 0 for 24 hours. Rinse in tap water and place in 2% aqueous tannic acid for 2 minutes. After rinsing in tap water, stain for 2 minutes in 1 part Delafield's hematoxylin to 2 parts distilled water and rinse in tap water. Remove excess hematoxylin with acidified water (1 drop cone. HC1 in 200 ml. water), then place slides in 0.5% lithium carbonate for 5 minutes. Dehydrate through an ethyl alcohol series, then transfer from absolute alcohol to a saturated solution of anilin blue in “methyl cellosolve” for 5-10 minutes. Wash in absolute alcohol, rinse in a solution of 25% methyl salicylate, 33% xylene, 42% absolute ethyl alcohol and clear for 10 minutes in a solution of 2 parts methyl salicylate, 1 part xylene, 1 part absolute ethyl alcohol. Transfer through two changes of xylene and mount in “clarite” or suitable alternate. The resulting preparations will have clearly defined, dark-staining cell walls and will photograph well when “Super Panchro-Press, Type B” film (Eastman Kodak Co.) is used in conjunction with suitable Wratten filters.     

An Improved Hematoxylin-Eosin Stain for Sections of Marrow Units

The procedure recommended is: Fix “marrow units” (small functional structures of bone marrow) in 10% formol-saline solution for 1-2 hours and dehydrate in 80% alcohol, 95% alcohol and acetone 30 minutes each. Place in fresh 50° and 53°C. paraffin for 30 minutes each. Embed in fresh 53°C. paraffin. Serially section at 5μ thickness and mount with Schleicher's floating solution. Allow to dry for 1 hour in an oven and deparaffinize by passing through xylene I and II, absolute alcohol I and II, and 95% alcohol. Rinse in fresh distilled water and place in dilute Harris' hematoxylin (stock solution 50 ml., distilled water 200 ml.) for 2 to 3 minutes. Rinse well in distilled water and check staining under the microscope. Dip in acid-alcohol 5 times (1 dip to equal about 1 second). Rinse well in weak (0.02%) ammonia water and distilled water. Dip in 2% aqueous phosphotungstic acid about 3 to 5 times (equal to 3-5 seconds). Rinse in fresh distilled water and place in weak ammonia water for 1 minute. Rinse in fresh distilled water I and II. Place in 80% alcohol for 5 minutes and check under the microscope for “blueness” and nuclear differentiation. Place in dilute alcoholic eosin (0.5% alcohol-eosin stock solution 10 parts and 95% alcohol 90 parts) for 1 to 2 minutes. Rinse in 80% alcohol and place for 1 minute in 95% alcohol. Check under the microscope for staining quality. Place in absolute alcohol for 1 minute, alcohol-xylene (equal parts), 10 dips, and xylene I and II. Mount. This hematoxylin-eosin staining schedule brings out minute structural detail of bone marrow tissue heretofore not demonstrable.     

Orthochromatic, Species-Limited Staining of Mast Cells With Night Blue

Night blue will stain the mast cells of rat, mouse and hamster selectively if alcohol differentiation is controlled. The technical steps are: Dewax paraffin sections with xylene, 2 changes; air dry; 2% Na₂SO₄, 3-5 sec; 0.5% night blue in 10% ethanol, 1 hr at 60°C; rinse in water; 9% HNO₃, 15 sec; water 1-5 min; 70% ethanol, 2 changes, 30 sec each; wash; 0.01% safranin, 3-5 sec; rinse, blot, air dry, mount in synthetic resin. A clear orthochromatic stain of the mast-cell granules occurs. Acid fixation prevents the staining reaction.     

不同染色条件对淡黄绒泡菌Physarum melleum菌核形成过程的差异显示

为了了解非细胞黏菌菌核形成过程中原质团组分及形态的变化,探索在番红-固绿二重染色、番红-焦油紫-橙黄G三重染色条件下,淡黄绒泡菌Physarum melleum菌核形成过程中原质团形态及显微结构变化的显示差异。结果表明：三重染色法可以显示原质团中纤维结构及细胞核,并且菌核随着休眠胞成熟而对焦油紫具有更强的亲和力。     

The Use of Papain in Clearing Plant Tissues for Whole Mounts

Materials killed and fixed in FAA (formalin-acetic acid-alcohol) and similar fixatives frequently are difficult to clear for whole mounts because the denatured proteins will not become soluble in NaOH and other clearing agents. If tissues are washed for 3 days in running water, then incubated at 40 C for 5-7 days in 2% papain buffered to pH 7.2 and activated with 15 ml of .02 M Na₂S, cell contents are partly digested. Normal clearing with 5-10% NaOH followed by chloral hydrate (sat. aq.) can then effect complete solublility of cell contents and their removal. Permanent slides can be made after staining (1% safranin O in 50% alcohol for 12 hr is successful), by dehydration through alcohols, clearing in xylene, and mounting in resin.     

Thick Sections for Embryology

Night blue will stain the mast cells of rat, mouse and hamster selectively if alcohol differentiation is controlled. The technical steps are: Dewax paraffin sections with xylene, 2 changes; air dry; 2% Na₂SO₄, 3-5 sec; 0.5% night blue in 10% ethanol, 1 hr at 60°C; rinse in water; 9% HNO₃, 15 sec; water 1-5 min; 70% ethanol, 2 changes, 30 sec each; wash; 0.01% safranin, 3-5 sec; rinse, blot, air dry, mount in synthetic resin. A clear orthochromatic stain of the mast-cell granules occurs. Acid fixation prevents the staining reaction.