The Use of Acenaphthene in Pollen Tube Technic

A procedure is described for growing pollen tubes in such a manner that a large number of clearly analyzed figures can be obtained. The pollen grains are sown on an artificial medium of sugar, agar, gelatin, and water, the proportions of each varying with the species of pollen grain used. The medium is smeared on the slide while still hot to insure a thin covering, and the pollen grains are dusted on when the medium has sufficiently cooled and hardened. The slides are placed in a staining dish provided with slide slots and a cover, the inside of the cover and the bottom of the dish being lined with moist, but not wet, filter paper. Acenapthene crystals are lightly sprinkled on the bottom of the dish. The developing pollen tubes are thus exposed to the fumes given off by these crystals with consequent disturbance to the spindle mechanism. As a result, the chromosomes are not crowded on a metaphase plate but are widely separated in the tube facilitating any observations to be made.     

MORPHOLOGICAL AND KINETIC ASPECTS OF MITOTIC ARREST BY AND RECOVERY FROM COLCEMID

The effect of Colcemid on the in vivo system of regenerating rat liver and on the in vitro system of HeLa cell cultures was studied to determine some of the morphological and kinetic aspects of metaphase blockage and recovery. The results indicated that under certain conditions the blocking effects of the drug were reversed; a functional bipolar spindle reorganized, and normal division resulted. Individual HeLa cells were followed by time-lapse cinemicrography prior to, during, and after Colcemid treatment. There was no apparent effect on cells in interphase. Cells entered mitosis at a normal rate and passed through prophase. A spindle was formed in most cells, albeit deformed, stunted, or shrunken. Within a certain range of drug concentrations, the spindle lengths showed characteristic unimodal distributions. After a 2-hr exposure to the drug followed by 1 hr in fresh medium, spindle lengths were restored to normal. Cells arrested in metaphase for periods as long as 5 hr were capable of reconstituting a normal functional spindle. Cells blocked for periods longer than 5 to 6 hr failed to recover.     

A Centrifugation Technique for Preparing Medium-Free Resinous Mounts of Feulgen-Stained Pollen Tubes

Air-dried pollen of Tradescantia paludosa and a colchicine doubled Allium ascalonium-fistulosum interspecific hybrid was sown in culture tubes on an inorganic salt-lactose liquid medium containing 0.02% colchicine. After a 16-20 hr incubation at 22 C, pollen tubes were harvested by centrifugation for 3-5 min at 1100-1400 rev/min and fixed with acetic-alcohol (1:3). Feulgen staining was carried out in the culture tubes with fluid changes made after the centrifugation following each step. Single drops of the final pollen-45% acetic acid suspension were flattened under silicone-treated coverglasses which were removed by the quick freeze technique prior to counterstaining with fast green, dehydration, and mounting in Diaphane or Canada balsam. Medium-free, Feulgen-stained, resin-mounted preparations of well-dispersed pollen tubes with arrested metaphases were obtained.     

A method for staining pollen tubes in pistil

A quadruple staining procedure has been developed for staining pollen tubes in pistil. The staining mixture is made by adding the following in the order given: lactic acid, 80 ml; 1% aqueous malachite green, 4 ml; 1% aqueous acid fuchsin, 6 ml; 1% aqueous aniline blue, 4 ml; 1% orange G in 50% alcohol, 2 ml; and chloral hydrate, 5 g. Pistils are fixed for 6 hr in modified Carnoy's fluid (absolute alcohol:chloroform:glacial acetic acid 6:4:1), hydrated in descending alcohols, transferred to stain and held there for 24 hr at 45 +/- 2 C. They were then transferred to a clearing and softening fluid containing 78 ml lactic acid, 10 g phenol, 10 g chloral hydrate and 2 ml 1% orange G. The pistils were held there for 24 hr at 45 +/- 2 C, hydrolyzed in the clearing and softening fluid at 58 +/- 1 C for 30 min, then stored in lactic acid for later use or immediately mounted in a drop of medium containing equal parts of lactic acid and glycerol for examination. Pollen tubes are stained dark blue to bluish red and stylar tissue light green to light greenish blue. This stain permits pollen tubes to be traced even up to their entry into the micropyle.     

An Acetic Acid Dissociation, Air-Drying Technique for Insect Chromosomes, With Aceto-Lactic Orcein Staining

The method differs from mammalian techniques for somatic chromosomes in that it uses very small amounts of material. Drosophila melanogaster and an ant, Dorymyrmex sp., are used as examples. Pretreatment with 0.05% Colcemid in insect Ringer solution is applied to mature Drosophila larvae for 5 hr, by feeding, but Dorymyrmex prepupae require puncture and a 15 hr exposure of the puncture to the solution. Organs are removed under 1% sodium citrate, tansferred to fresh citrate for 10-20 min, than fixed in acetic-methanol, 1:3, for 30 min. Transfer to a drop of 60% acetic acid on a clean warmed slide dissociates the cells, which are spread by adding a small drop of fixative and tilting the slide in all directions. After immersion in acetic ethanol, 1:3, for 4 hr, rinsing in the stain solvent and draining the slides then have 2-3 drops of aceto-lactic orcein placed on each, coverslips added, and warmed (at about 50 C) for about 12 hr or until staining is sufficient. They can then either be treated as semipermanent or made permanent by allowing the coverslips to slide off in acetic-ethanol, dehydrating, and mounting in Euparal, or a synthetic resin.     

In Situ Multiple Sampling of Attached Bacteria for Scanning and Transmission Electron Microscopy

An in situ electron microscope sampling technique for characterizing cells attached to smooth surfaces is demonstrated with an ultraviolet-induced mutant of Streptococcus mutans. The sterilized sampling unit consists of a 9 cm plastic Petri dish containing a glass slide, a 12 mm round coverglass, and a coverglass with Formvar-carbon coated copper grids. After the bacterial culture in a liquid medium is incubated in the Petri dish, the slide with attached bacteria is washed in double-distilled water, air-dried, coated with platinum and carbon, and processed for replicas and shadowed specimens for transmission electron microscopy. The coverglass is similarly washed, fixed in 2% glutaraldehyde, air- or freeze-dried, coated with palladium/gold, and examined in the scanning electron microscope. The coverglass with grids is rinsed in double distilled water, the grids are transferred to a filter paper and stained with a loopful of 2% phosphotungstic acid at pH 5.5. The bacteria growing on the surface of the plastic Petri dish are fixed, dehydrated, and embedded in situ with Epon. Sectioned and stained specimens are then examined in the transmission electron microscope. This procedure also appears useful with such other attached systems as normal or infected tissue culture cells.     

Description of a tissue culture technique using tissue from mussel (Mytilus edulis) for the preparation of chromosomes]

Explants from mantle and foot tissues of adult mussel were grown in culture tubes containing a medium composed of Eagle's Basal Medium supplemented with salts, Hepes buffer, egg yolk and antibiotics. The cultures were maintained at 18 degrees C and pH 7.50, without medium renewal. After 6-7 days, the cultures were stopped and harvested for slide preparation. Numerous metaphase spreads that were good enough for karyotyping were consistently obtained. This method may prove to be a reliable source of actively dividing cells that is a prerequisite for extensive chromosome structure analyses in the bivalves.     

Staining Germinating Pollen and Pollen Tubes

Germinating pollen on stigmas and pollen tubes in styles of Antirrhinum, Brassica, Oenothera, Raphanus, Rosa, solatium and Tagetes spp. were prepared for examination as follows: The styles were fixed in ethyl alcohol-acetic acid 3:1 for 1 hr, and hydrolyzed at 60°C for 5 to 60 min (depending on the species) in 45% acetic acid. The stigma with its attached strand(s) of stigmatoid tissue was then dissected out under a stereoscopic microscope, placed in a few drops of a staining solution made by dissolving 150 mg of safranin O and 20 mg of aniline blue in 25 ml of hot 45% acetic acid. After 5-15 min in this stain, the tissue was placed in a fresh drop of stain on a microscope slide and gently squashed under a cover glass. Because of a gradual precipitation of the aniline blue component, the stain had to be filtered regularly before use. However, a staining solution could be kept at room temperature for several weeks.     

A Standardized Cotton Blue Stain for Pollen Germination and Growth in Andropogoneae Grasses

Styles along with ovaries were dissected from spikelets of sorghum and other grasses and fixed in 1:3 acetic alcohol at 0.5 hr intervals for 3 hr after pollination. The material was hydrated and then macerated in 50% HCl for 10 min at 56 C, washed in running water for 30 min, cleared in lactophenol for 35 min at 60 C, stained in 1% cotton blue for 50 min at 50 C, and destained for 1 hr in lactophenol at room temperature. Styles were dissected from the ovary in a drop of lactophenol. Slight pressure on the coverslip with the blunt end of a needle and a little warming spreads the stylar tissue and reveals the darkly stained pollen tubes.     

An analysis of methods for permanently mounting ovules cleared in four-and-a-half type clearing fluids

Ovules cleared in benzyl benzoate-4 1/2 clearing fluid can be permanently mounted in Piccolyte or Permount by replacing the cleaning fluid with absolute ethanol, upgrading the ovules in mixtures of ethanol and xylene (3:1, 2:2, 1:3, and xylene), and mounting them in either mountant under the supported coverglass of a Raj slide. Optical saggittal sections through the ovules resemble microtome sections in that the protoplasts are slightly shrunken away from the cell walls. The artifact is common in permanently mounted sections; fixation and paraffin infiltration are usually cited as the causes--its appearance in the whole-mounted ovules is caused by xylene. Although miscible with the clearing fluid, Euparal is the least satisfactory of the standard mountants for permanent preparations of cleared ovules and is best used with an equal quantity of clearing fluid for semipermanent preparations. A large quantity of Euparal in the mountant produces pronounced shrinkage. A method for permanently mounting cleared ovules with the clearing image unaltered employs a mountant which contains the ingredients of Spurr low viscosity embedding medium. Vinylcyclohexene dioxide (10 drops) is combined with diglycidyl ether of polypropylglycol (6 drops) and nonenyl succinic anhydride (26 drops). Ovules treated for 24 hr in benzyl benzoate-4 1/2 clearing fluid are passed through a graded series of clearing fluid-epoxy medium mixtures (3:1, 2:2, 1:3, and pure epoxy medium) at intervals of 14 minutes. One drop of dimethylaminoethanol, the cure accelerator, is then added to the epoxy medium and the ovules are mounted and covered immediately on a Raj slide. The preparation is cured in an oven at 60 C for 24 hr and observed with phase contrast or Nomarski interference optics.     

An Analysis of Methods for Permanently Mounting Ovules Cleared in Four-and-a-Half Type Clearing Fluids

Ovules cleared in benzyl benzoate-4 1/2 clearing fluid can be permanently mounted in Piccolyte or Permount by replacing the clearing fluid with absolute ethanol, upgrading the ovules in mixtures of ethanol and xylene (3:1, 2:2, 1:3, and xylene), and mounting them in either mountant under the supported coverglass of a Raj slide. Optical sagittal sections through the ovules resemble microtome sections in that the protoplasts are slightly shrunken away from the cell walls. The artifact is common in permanently mounted sections; fixation and paraffin infiltration are usually cited as the causes—its appearance in the whole-mounted ovules is caused by xylene. Although miscible with the clearing fluid, Euparal is the least satisfactory of the standard mountants for permanent preparations of cleared ovules and is best used with an equal quantity of clearing fluid for semipermanent preparations. A large quantity of Euparal in the mountant produces pronounced shrinkage. A method for permanently mounting cleared ovules with the clearing image unaltered employs a mountant which contains the ingredients of Spurr low viscosity embedding medium. Vinylcyclohexene dioxide (10 drops) is combined with diglycidyl ether of polypropylglycol (6 drops) and nonenyl succinic anhydride (26 drops). Ovules treated for 24 hr in benzyl benzoate-4 1/2 clearing fluid are passed through a graded series of clearing fluid-epoxy medium mixtures (3:1, 2:2, 1:3, and pure epoxy medium) at intervals of 15 minutes. One drop of dimethylaminoethanol, the cure accelerator, is then added to the epoxy medium and the ovules are mounted and covered immediately on a Raj slide. The preparation is cured in an oven at 60 C for 24 hr and observed with phase contrast or Nomarski interference optics.     

Handling Germinated Pollen on Millipore Membrane During the Feulgen and Autoradiographic Procedures

To prevent loss of pollen during the Feulgen's procedure, the pollen was grown on an autoclaved membrane filter (Millipore AA WP 025 00) in contact with a sterilized medium containing agar 0.5-1%, sucrose according to the genus (Malus 0.3-0.5 M; Persica and Tulipa 0.4 M), and H₃BO₃, 0.01%. To fix the germinated pollen of most species, the membrane was placed for 2 hr to overnight at 2-4 C on filter paper wet with the following mixture: OsO₄, 1 gm; CrO₃, 1.66 gm; and distilled water, 233 ml. To fix Persica pollen, 10% of glacial acetic acid had to be added to the fixative. Washing with distilled water and bleaching with a mixture of 3% H₂O₂ and sat. aq. ammonium oxalate, 1:1, were performed also on filter paper. Similarly, the preparation was processed for Feulgen staining by use of pieces of filter paper wet with the required fluids. Hydrolysis preceding the Schiff's reagent was performed at room temperature with 5 N HCl for 18 min. The differentiation after the Schiff's action was with 2% K₂S₂O₅ buffered to pH 2.3 with 9 ml of phosphate buffer (KH₂PO₄, 1.4 gm; conc. HCl, 0.35 ml and distilled water to make 100 ml). The stained pollen was floated off the membrane with a drop of glacial acetic acid to a gelatinized or an albumenized slide, and squashed. When the coverslip is removed the preparation may be either dehydrated and mounted or coated with autoradiographic film.     

A Method for Staining Pollen Tubes in Pistil

A quadruple staining procedure has been developed for staining pollen tubes in pistil. The staining mixture is made by adding the following in the order given: lactic acid, 80 ml; 1% aqueous malachite green, 4 ml; 1% aqueous acid fuchsia, 6 ml; 1% aqueous aniline blue, 4 ml; 1 % orange G in 50% alcohol, 2 ml; and chloral hydrate, 5 g. Pistils are fixed for 6 hr in modified Carnoy's fluid (absolute alcohol:chloroform:glacial acetic acid 6:4:1), hydrated in descending alcohols, transferred to stain and held there for 24 hr at 45±2 C They were then transferred to a clearing and softening fluid containing 78 ml lactic acid, 10 g phenol, 10 g chloral hydrate and 2 ml 1% orange G. The pistils were held there for 24 hr at 45±2 C, hydrolyzed in the clearing and softening fluid at 58±1 C for SO min, then stored in lactic acid for later use or immediately mounted in a drop of medium containing equal parts of lactic acid and glycerol for examination. Pollen tubes are stained dark blue to bluish red and stylar tissue light green to light greenish blue. This stain permits pollen tubes to be traced even up to their entry into the micropyle.     

Sticky Wax Infiltration in the Preparation of Sawed Undecalcified Bone Sections

The undecalcified bone specimen was surfaced by an ordinary motor-driven circular saw. After thorough drying in air, the specimen was infiltrated with melted Caulk sticky wax (L. D. Caulk Co., Milford, Del., 19963) without casting in a block. The specimen was affixed to the Gillings-Hamco thin-sectioning machine with cut surface parallel to the circular diamond blade. Prior to sawing each section, the specimen surface was blown dry and coated with a thin supporting layer of stick wax. The section was then attached to an albumen-coated glass slide with the newly cut surface facing the slide. After drying in room temperature, the slide was soaked in xylene to partially dissolve the sticky wax, and the loosened residue was removed subsequently by gentle brushing. The section was mounted and covered with a coverglass. Sections 50-100 μ thick were prepared satisfactorily by this method. The advantages of using sticky wax as an infiltration medium depend on its physical properties: it is gluey when melted, and holds the bony trabeculae together; it becomes hard and nonsticky at room tempperature, and can be sawed together with bone tissue. Since a new layer of wax blends readily with the old wax surface, it allows the important supportive coating of wax to be added to the sawing surface for each section cutting     

RNA content and chromatin structure of CHO cells arrested in metaphase by colcemid

To evaluate the stability of cells arrested in metaphase, cell viability, RNA content, and chromatin structure (the latter probed by the DNA in situ sensitivity to acid-induced denaturation) were studied in uniform-age mitotic CHO cell populations maintained either at 37 degrees C (in the presence of Colcemid) or at 0-4 degrees C for up to 6 h. No significant changes in cell viability and RNA content were seen throughout the experiment for both groups of cells. The sensitivity of DNA in situ to denaturation was significantly increased during the initial 40 min of cell arrest in mitosis. However, no further chromatin changes for up to 6 h were evident regardless of whether cells were kept at 37 degrees C with Colcemid or at 0-4 degrees C in its absence. The data indicate that neither significant deterioration of metaphase cells nor progressive chromatin changes are expected during stathmokinesis experiments in vitro or during the metaphase cell arrest in cytogenetic studies lasting up to 6 h. Also, no RNA turnover can be detected in mitotic cells during this time interval.     

Differential Staining of Aborted and Nonaborted Pollen

A single staining solution was made by compounding it in the following order (dyes were from British Drug Houses): ethanol, 10 ml; 1% malachite green in 95% ethanol, 1 ml; distilled water, 50 ml; glycerol 25 ml; phenol, 5 gm; chloral hydrate, 5 gm; acid fuchsin 1% in water, 5 ml; orange G, 1% in water 0.5 ml; and glacial acetic acid, 1-4 ml. For best results in differentiation to give green pollen walls and red protoplasm, the staining solution should be acidified with glacial acetic acid. The amount of acid to be added depends upon thickness of the pollen walls: for very thin-walled pollen, 1 ml; for moderately thin walls, 2 ml; and for thick-walled or spiny-walled pollen, 3 ml of acid. For pollen inside non-dehiscent anthers, 4 ml of acid should be used. Staining is hastened by flaming the slide (for loose thin-walled pollen) or by immersing thick-walled pollen or anthers for 24-48 hr at 50 C. In the typical stain, aborted pollen grains are green; nonaborted, red. The method is useful for pollen inside nondehiscent anthers if these are small and not too deeply coloured naturally. The stain is very durable, especially if the coverslips are sealed with param wax. The staining solution will keep well for about a month. It is useful both for angiosperms and gymnosperm microgametes.     

Novel system for improved control of filamentous microorganisms in continuous culture

The efficiencies of two 24-hr elevated-temperature tests to recover Escherichia coli from estaurine water were compared simultaneously with the 72-hr standard methods procedure of the American Public Health Association (APHA). From 1,710 tubes, E. coli was recovered 222 times in lauryl tryptose medium incubated at 44 +/- 0.2 C for 24 hr, 261 times in an experimental medium incubated at 44.5 +/- 0.2 C for 24 hr, and 257 times by the 72-hr APHA method. The number of false positives enumerated was similar in all three tests. The data indicated that E. coli in raw seawater could be determined in 24 hr without a significant loss of accuracy.     

A Versatile Stain for Pollen Fungi, Yeast and Bacteria

A versatile stain has been developed for demonstrating pollen, fungal hyphae and spores, bacteria and yeasts. The mixture is made by compounding in the following order: ethanol, 20 ml; 1% malachite green in 95% ethanol, 2 ml; distilled water, 50 ml; glycerol, 40 ml; acid fuchsin 1% in distilled water, 10 ml; phenol, 5 g and lactic acid, 1-6 ml. A solution has also been formulated to destain overstained pollen mounts. Ideally, aborted pollen grains are stained green and nonaborted ones crimson red. Fungal hyphae and spores take a bluish purple color and host tissues green. Fungi, bacteria and yeasts are stained purple to red. The concentration of lactic acid in the stain mixture plays an important role in the differential staining of pollen. For staining fungi, bacteria and yeasts, the stain has to be acidic, but its concentration is not critical except for bacteria. In the case of pollen, staining can be done in a drop of stain on a slide or in a few drops of stain in a vial. Pollen stained in the vial can be used immediately or stored for later use. Staining is hastened by lightly flaming the slides or by storing at 55±2 C for 24 hr. Bacteria and yeasts are fixed on the slide in the usual manner and then stained. The stock solution is durable, the staining mixture is very stable and the color of the mounted specimens does not fade on prolonged storage. Slides are semipermanent and it is not necessary to ring the coverslip provided 1-2 drops of stain are added if air bubbles appear below the coverslip. The use of differentially stained pollen mounts in image analyzers for automatic counting and recording of aborted and nonaborted pollen is also discussed.     

SEPARATION OF HELA CELLS BY COLLOIDAL SILICA DENSITY GRADIENT CENTRIFUGATION : I. Separation and Partial Synchrony of Mitotic Cells

Using a colloidal silica density gradient, HeLa cells in mitosis were found to have a density of 1.040–1.046 g/cc, lighter than the remaining interphase cells. The mitotic cells could be harvested and cultured after centrifugation, showing growth synchrony by measurement of a peak in mitotic index 21 hr after establishing the culture. By using Colcemid or vinblastine sulfate, HeLa cells were arrested in metaphase and centrifuged on the colloidal silica density gradient. The blocked metaphase cells were lighter in density than the interphase cells but somewhat more dense than untreated cells selected by the density gradient centrifugation. Near-equilibrium conditions were established during the centrifugation of cells so that cell density measurements could be made, and the gradient medium employed was not measurably toxic to those cells tested.     

PROTANDRY,INCOMPATIBILITY, AND SECONDARY POLLEN PRESENTATION IN CEPHALANTHUS OCCIDENTALS (RUBIACEAE)

Cephalanthus occidentalis L. is protandrous and presents pollen secondarily on the stigma surface. Because self-pollen is present on the stigma, the degree of selling vs. outcrossing in this species will depend on 1) the phenology of pollen presentation and stigma receptivity; 2) whether the species is self-incompatible; and 3) the rates of self vs. crossed pollen tube growth. This study describes floral morphology and phenology, self-incompatibility, and pollen tube growth rates in self- and crosspollinations of C. occidentalis. Light and scanning electron microscopy were used to study stigma morphology after flower opening, while controlled pollinations tested for incompatibility. Stigmas were unreceptive initially but became receptive by the second day after flower opening. Ninety-two percent of cross-pollinated flowers set fruit, compared to 12% fruit set in self-pollinations. Pollen tubes from selfed and out-crossed pollen initially had similar growth rates. Out-crossed pollen tubes began to grow rapidly ca. 5 hr after pollination of a receptive stigma, whereas selfed pollen tubes ceased growth or grew slowly after this time. Pollen tubes from out-crossed pollen grew the length of the style within 24 hr after pollination, while selfed pollen tubes were inhibited at the stigma-style junction. Our results indicate that C. occidentalis has selfincompatibility, in addition to protandry and secondary pollen presentation. Protandry allows removal of self-pollen from the unreceptive stigma, while self-incompatibility prevents fertilization by unremoved self-pollen.