A New Methylene Blue Technic for Permanent Preparations

1. Tissues stained intra vitam with methylene blue are fixed in a 10% ammonium molybdate solution in physiological saline (or sea water if the tissue is from a marine animal). Fixation time is kept to a minimum. Washing also is reduced to a minimum.

2. Excess fluids are removed from tissues by blotting with a paper or cloth towel before they are put into the succeeding solution. Tissues are taken from the wash water, blotted and placed in a mixture of equal parts of absolute ethyl alcohol and n-butyl alcohol for 30 minutes. They are then blotted and transferred to n-butyl alcohol for 30 minutes. After blotting they are placed in a mixture of one part methyl salicylate and four parts xylene until cleared. Tissues may be mounted whole or prepared for sectioning by embedding in paraffin in the usual way.

3. Tissues fixed, washed, dehydrated and cleared as described retain nearly all of the stain; the time required is greatly reduced; there is no need to chill the dehydrating solutions; cell distortion is much reduced.     

The Differentiation of Placoid, Ctenoid and Cycloid Scales by Means of Alizarin Red S

This technic has been used successfully by the author for staining, in situ, the placoid scales of selachians and the smaller forms of cycloid and ctenoid scales of teleosts. Sections of skin are dissected from the ventrum of the specimen, cleaned of fascia and muscle tissue and fixed in a 10% formalin solution. The section is macerated in several changes of a 3% KOH solution until translucent. Staining is accomplished in a fresh 2% KOH solution to which is added a saturated alcoholic solution of alizarin red S. The section remains in the stain for 24 hours. If necessary, the tissue may be quickly destained in acid alcohol (1% H₂SO₄ in 95% alcohol). The skin is dehydrated in cellosolve or the alcohol series and cleared in methyl salicylate. Placoid and teleost scales prepared in this manner are stained a brilliant red. The various parts of the denticle are well differentiated.     

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.     

Permanent Stained Preparations of Thick Blood Films

Fixing thick films in alcoholic solution of dye after the usual staining-and-laking procedure preserves the appearance of parasites and blood elements very similar to that of the usual thick films (not fixed) for the diagnosis of malaria and relapsing fever.

Procedure recommended: Films are stained and laked for 15 minutes in diluted Giemsa—1 to 3 drops of stock solution (0.4 g. in 60 ml. equal parts absolute methyl alcohol and glycerin) per ml. distilled water; rinsed in water and allowed to dry. They are then immersed in, or flooded with, May-Griinwald's stain (0.5% in absolute methyl alcohol) for 30 seconds, rinsed in water and allowed to dry. Solutions of MacNeal's tetrachrome stain in methyl alcohol and glycerin may be substituted for Giemsa and a solution in methyl alcohol may be substituted for May-Griinwald. With slight modification of the procedure, both thick and thin films on the same slide may be stained together.

Films stained and fixed as described, and mounted in Diaphane, have shown no evidence of fading in 3 years.     

The Use of Bismarck Brown Y in some new Stain Schedules

The staining quality of Bismarck brown Y may be improved and sterility maintained by adding 5% phenol to a 1% aqueous solution. Use the phenolic Bismarck brown in combination with iron alum hematoxylin except for stripped epidermis in the following procedures:

Stem and Root Schedule: Mordant sections from water in 4% iron alum for 10 minutes. Rinse in distilled water and stain in 0.5% aqueous hematoxylin for 1 minute or until darkly stained. Rinse in distilled water and destain in 2% iron alum until a gray color appears. Rinse thoroly in distilled water and intensify hematoxylin by transferring sections to 0.5% aqueous lithium carbonate until the desired black color appears. Rinse thoroly in distilled water and stain for 1-5 minutes in phenolic Bismarck brown. Rinse in distilled water, dehydrate successively in 30, 50, 70, 95 and 100% alcohol. Clear in methyl salicylate for 5 minutes, then to xylene for 3-5 minutes, and mount in balsam.

Middle Lamellae in Wood: Destain more thoroly in 2% iron alum than for the general stem and root schedule, and intensify in lithium carbonate for a longer period (about 1 hour).

White Potato Tuber Sections: Modify above schedule by reducing time of destaining in 2% iron alum to about 30-60 seconds and intensify hematoxylin until starch grains appear bluish in color. Stain in phenolic Bismarck brown for 1-2 minutes.

Wheat Grain Sections: Fix grain for sectioning when in “dough” stage. Use schedule the same as for potato tuber except for reducing time of staining in phenolic Bismarck brown to about 45 seconds.

Tradescantia zebrina Epidermis: Strip epidermis from leaf while submerged in water. Fix in 100% alcohol 10 minutes, pass thru 95, 70, 50, 30, and 10% alcohol to water. Stain in phenolic Bismarck brown for 10-20 minutes. Dehydrate, clear in methyl salicylate and mount in balsam.     

An Aceto-Osmium Method for Demonstrating Nematodes in Citrus Roots

Difficulties are encountered in observing nematodes in citrus feeder roots because of the presence of suberin and other unsaturated compounds. To obviate these difficulties, infected citrus roots, either fresh or preserved, are immersed in a covered jar for 2 hr at 52° C in a solution composed of distilled water, 16 parts; 10% acetic acid, 10 parts; and 2% aqueous osmium tetroxide, 2 parts. The stained, blackened roots are washed in running water for at least 1 hr and then bleached in 10-30% hydrogen peroxide at 32°C for a few seconds until the color of the roots lightens perceptibly. After several washings in water to stop the oxidation reaction, roots are dehydrated in 70, 95, and absolute ethanol held at 52 °C for 30 min at each concentration. After dehydration, roots are cleared in methyl salicylate at 52°C. Examination for nematodes in most cases, can be made after 30 min.     

A Rapid Method for Making Permanent Mounts of Nematodes

A rapid method which can be used to mount and clear nematodes and their eggs is presented. Permanent mounts of certain nematodes and parasite eggs have been prepared using a medium consisting of 56 parts of a stock solution of polyvinyl alcohol (“PVA”), 22 parts phenol and 22 parts of lactic acid. The stock solution of PVA is prepared by dissolving 15 grams of PVA in 100 ml. of distilled water. This medium can be used on material killed and fixed in 10% formalin, any concentration of alcohol, alcohol-glycerin or glycerin. Results have been very satisfactory in most instances. An accompanying plate of photographs shows some of the preparations obtained by using this method.     

A Note on the Staining of the Skeleton of Cleared Specimens with Alizarin Red S

A selective, progressive method for staining the skeleton in cleared specimens, developed with rat material.

Fix in 95% alcohol for at least 48 to 96 hrs. Even longer fixation is desirable. Then place in a 1% solution of KOH until the bones are clearly visible through the surrounding tissues. Transfer directly to a dilute solution of alizarin in KOH, one part alizarin to 10,000 parts of 1% KOH. Allow the stain to act until the desired intensity is attained. Fresh stain may be added if necessary.

Complete the clearing process, (1) in Mall's solution, water 79 parts, glycerine 20 parts and KOH 1 part; (2) in increased concentrations of glycerine. Store in pure glycerine.

The success of the method depends on obtaining the proper degree of clearing before staining. If the specimen is insufficiently cleared, a general staining of all tissues usually occurs.     

Influence of salicylaldehyde and methyl salicylate on post-landing behaviour of Frankliniella occidentalis Pergande

This study investigates behavioural responses of adult western flower thrips (Frankliniella occidentalis Pergande; Thysan., Thripidae) females to direct contact with repellent phenylpropanoid plant compounds (salicylaldehyde and methyl salicylate) applied on bean and cucumber leaves. The residence time of F. occidentalis females until take off was significantly shorter on bean or cucumber leaf discs treated with salicylaldehyde at 1% concentration compared with control leaf discs. A methyl salicylate (1%) treatment of cucumber resulted in shorter time periods until thrips took off the treated leaf discs compared with the control leaf discs. In a choice experiment thrips avoided to settle on a 1% salicylaldehyde treatment of bean and cucumber leaf discs for a maximum of 3 h, on a 1% methyl salicylate treatment for a 5‐h period. Within a 24‐h period neither the egg‐laying nor the feeding activity of F. occidentalis was affected after salicylaldehyde application (0.1%, 1%) on bean or cucumber. In contrast, methyl salicylate (1%) applied on bean and cucumber significantly prevented thrips females from oviposition and reduced the percentage of damaged area caused by their feeding activity for 24 h. As olfactory repellent plant volatiles applied on crop plants may elicit diverse post‐landing responses of F. occidentalis, short‐ and long‐term effects should be considered when evaluating the factual applicability of secondary plant compounds in a successful thrips management strategy.     

Luxol Fast Blue Mbs: A Stain for Phase Contrast Microscopy

A staining method to increase the contrast of sectioned material for phase contrast microscopy is described. Two stock solutions of the stain are required. The first is made by dissolving 2 gm of luxol fast blue MBS in 100 ml of 95% ethanol. The second solution is made up of 4 ml of a 29% aqueous solution of FeCl₃, 95 ml of 95% ethanol, and 1 ml of concentrated HCl. The staining solution is made by mixing equal parts of the two solutions. Sections are deparaffinized and taken to 70% alcohol, stained for 1.5 hr, dehydrated, cleared and covered as usual.     

A Formalin-Wright Staining Technique for Avian Blood Cells

A rather concentrated alcoholic staining solution, an aqueous formalin-containing diluent, and a mixture of ethyl ether and absolute methyl alcohol are required. Formulas: A. Wright's stain (Harleco, Cert. No. LWr-52 was used), 3.3 gm; methyl alcohol, 500 ml. B. Formaldehyde solution 40% USP (Fisher's used), 0.25 ml; distilled water, 500 ml with its pH adjusted to 6.8 by addition of either 0.25% Na₂CO₂ or 0.25% HCl, as needed. C. A I:I mixture of ethyl ether and absolute methyl alcohol. Procedure: Prepare thin smears of normal or pathological avian blood, air dry, place the slides on a drying rack, cover with solution A, and let stand for about 8 min. Dilute the stain by dropping on a volume of B estimated to be equal to the volume of the partially evaporated stain, and let stand for 2-5 min, or until the surface is well covered by a metallic sheen. Wash with distilled water adjusted to pH 6.8 with the 0.25% Na₂CO₂ solution or 0.25% HCl. Dry the preparations quickly by blotting with filter paper. Differentiate and adjust the color intensities by dipping 6-10 times into C. Check the results microscopically and differentiate further if the colors are not properly balanced. Dry, uncovered preparations may be examined under oil; or, a cover glass can be applied with balsam or a synthetic resin for permanent mount. Results are similar to those described in textbooks, but have been more consistent than those obtained with other techniques for blood cells of chicken, pheasants, American and Indian partridge, quail, pigeon, turkey, goose, canary, and the Himalayan snow partridge.     

Staining Angiosperm Shoot Apices with Heidenhain's Iron Hematoxylin and Aniline Blue

Good differential staining of nuclei, cytoplasm and cell walls of angiosperm shoot apices is obtained by a hematoxylin-aniline blue sequence. First, follow a typical Heidenhain's iron-hematoxylin scheme so that the nuclei and cytoplasm are well stained, then bring the slides up through an ethyl alcohol series to absolute alcohol. Transfer to a saturated solution of aniline blue in methyl cellosolve for 10 minutes. Remove the excess aniline blue with absolute alcohol, and follow this with a mixture of 42 parts absolute alcohol, 25 parts methyl salicylate, and 33 parts xylene; next, a similar mixture but in the proportions of 1:2:1; then a xylene-alcohol mixture, 9:1, followed by pure xylene, 2 changes, and covering in balsam. Panchromatic plates or film are suitable for photomicrographic reproduction (Ilford Special Rapid Panchromatic plates were used).     

Enzymatic synthesis of water-soluble derivatives of salicylic acid in organic media

A novel enzymatic method for preparing water-soluble derivatives of salicylic acid catalyzed by immobilized lipase was described. This study is the first to describe the enzymatic transesterification of methyl salicylate in organic solvents with different hydroxyl donors. The acyl-transfer between methyl salicylate and sorbitol was best supported by solvents of log P values –0.33 to 1.4. With Candida antarctica lipase in tert-amyl alcohol, a sorbitol conversion yield of 98% can be obtained by transesterification with sorbitol and methyl salicylate in one step.     

Aphid Acceptance of Barley Exposed to Volatile Phytochemicals Differs Between Plants Exposed in Daylight and Darkness

It is well known that volatile cues from damaged plants may induce resistance in neighboring plants. Much less is known about the effects of volatile interaction between undamaged plants. In this study, barley plants, Hordeum vulgare cv. Kara, were exposed to volatiles from undamaged plants of barley cv. Alva or thistle Cirsium vulgare, and to the volatile phytochemicals, methyl salicylate or methyl jasmonate. Exposures were made either during natural daylight or darkness. Acceptance of exposed plants by the aphid Rhopalosiphum padi was assessed, as well as the expression of putative marker genes for the different treatments. Aphid acceptance of plants exposed to either barley or C. vulgare was significantly reduced, and an effect of the volatiles from undamaged plants was confirmed by the induction of pathogenesis-related protein, PR1a in exposed plants. However the effect on aphid acceptance was seen only when plants were exposed during darkness, whereas PR1a was induced only after treatment during daylight. Aphid acceptance of plants exposed to either methyl salicylate or methyl jasmonate was significantly reduced, but only when plants were exposed to the chemicals during daylight. AOS2 (allene oxide synthase) was induced by methyl jasmonate and BCI-4 (barley chemical inducible gene-4) by methyl salicylate in both daylight and darkness. It is concluded that (a) the effects on aphids of exposing barley to volatile phytochemicals was influenced by the presence or absence of light and (b) the response of barley to methyl salicylate/methyl jasmonate and to volatiles from undamaged plants differed at the gene and herbivore level.Key Words: methyl jasmonate, methyl salicylate, allelobiosis, barley, PR1, allene oxide synthase, Rhopalosiphum padi, light     

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.     

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.     

Luxol Fast Blue Mbs: A Stain for Phase Contrast Microscopy

A staining method to increase the contrast of sectioned material for phase contrast microscopy is described. Two stock solutions of the stain are required. The first is made by dissolving 2 gm of luxol fast blue MBS in 100 ml of 95% ethanol. The second solution is made up of 4 ml of a 29% aqueous solution of FeCl₃, 95 ml of 95% ethanol, and 1 ml of concentrated HCl. The staining solution is made by mixing equal parts of the two solutions. Sections are deparaffinized and taken to 70% alcohol, stained for 1.5 hr, dehydrated, cleared and covered as usual.     

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.