Sudan Black B And Acetocarmine as a Combination Stain

Epidermis stripped from either fresh or fixed plant organs, or sections of paraffin-embedded or fresh material are placed on a slide and covered with a drop or two of iron-acetocarmine. The stain is intensified by warming the slide over a flame. After a few minutes a drop or two of a saturated solution of Sudan black B in 45% acetic acid is added and a cover slip applied. The preparations cannot be made permanent, but last a few weeks if sealed with a compound such as gum mastic-paraffin, or if the combined stain is drained off and a drop of Karo syrup is added before the cover slip is applied. The acetocarmine produces its usual staining effects, i.e., nuclei dark red and some components of the cytoplasm of certain cells a less intense red. The Sudan black B colors lipid structures an intense blue.     

A simple method of preparing permanent squash preparations using cellophane]

A technique to prepare permanent squashed preparations of cell nuclei and chromosomes is proposed. Fix a piece of material on the slide with acetic alcohol (1:3), macerate with a 45% acetic acid, cover with hydrophilic cellophane previously soaked in a 45% acetic acid and then with a cover slip and filter paper to squash finally as it is routinely performed. After that soak off the cover slip with alcohol, post-fix the squashed preparation together with cellophane in alcohol for 5-10 min, unstick the cellophane, pass the preparation through alcohol once again and dry it. The subsequent treatment of the squashed preparation depends on the purpose of investigation. The slide may be tinctured overlaid with photoemulsion for autoradiography, or processed by different ways.     

Simple Methods for Mammalian Chromosomes

The ordinary Feulgen-squash technic, after acetic-alcohol fixation, provides a simple and reliable method of preparing many mammalian tissues for chromosome counts. Tissues best suited to the technic were the seminiferous tubules. Small pieces of tissue about 3-6 mm. long and 1-2 mm. wide were immersed in a freshly made fixative (composed of 3 parts absolute ethyl alcohol and 1 part glacial acetic acid) immediately after removal by biopsy or from a freshly killed animal. After fixation for 3-12 hours the tissue was stained by the standard Feulgen procedure after hydrolysis for 12 minutes in normal HCl at 60 °C A 1-2 mm. piece was then teased apart on a slide in 45% acetic acid with a pair of mounted needles, and the teased tissue was squashed between the slide and the cover slip. During squashing the pressure was applied by hand and was regulated so as to avoid any excessive scattering of the chromosomes. The preparations were made permanent by dehydrating and mounting in Euparal.     

An Acetocarmine Technic for Cucurbita

Staminate Cucurbita buds undergoing meiosis are fixed for 12-24 hours in a solution containing 3 parts of 95% alcohol, 1 part of acetocarmine to which iron acetate has been added, and a flake of rusted iron. After fixation the buds are washed in 95% alcohol and stored in 95% alcohol with the iron flake for 5-10 days. A stain containing 10 drops of 45% acetic acid, 10 drops of acetocarmine, and 10 drops of brown storage solution is prepared. A small piece of anther is placed in a drop of stain on a slide. At the moment the anther is macerated, the debris is removed, and when the cells turn grey to dark brown a cover slip is applied. The stain is differentiated by gentle heat and the cover slip is sealed with paraffin.     

Simple Aids to Microscope Illumination

The following rapid but reliable method of making permanent preparations from temporary mounts has proved to be very useful.

Pollen mother-cell smears: Smeared anthers are treated hi the usual way with Belting's acetocarmine, except that the cover slip is left off. When correct differentiation is attained the stain is thoroly washed off with 50% acetic acid and the slide flooded with dioxan. This is followed by 2 changes of dioxan for 2 minutes each. A drop of Canada balsam dissolved in dioxan is added and a cover slip applied. In cases where a cover slip has been used at the acetocarmine stage it can be floated off in a staining jar of 50% acetic acid and dehydration with dioxan carried out as above.

Insect salivary gland chromosome smears: The glands are crushed under a cover slip in acetocarmine on a slide coated with dried egg albumen. After 20 minutes the area around the cover slip is flooded with 50% acetic acid and the cover slip floats loose so that it can be removed. The above described dioxan dehydrating procedure is then employed.

Squash preparations: Root tips are fixed in some suitable fixative and the Feulgen technic applied. The stained root tips can either be dehydrated by passing thru 3 changes of dioxan and mounting in dioxan-balsam where they are divided into small longitudinal sections by sharp needles, or they can be put immediately into a mixture of 1 part of 50% acetic acid to 1 part of corn syrup where shredding with needles is carried out. A cover slip is put on and separation of the cells completed by tamping or by applying pressure to the cover. This squash method is useful with anthers which are difficult to smear when in the early prophase stages of meiosis.     

Permanent Preparations from Rapid Cytological Technics

The following rapid but reliable method of making permanent preparations from temporary mounts has proved to be very useful.

Pollen mother-cell smears: Smeared anthers are treated hi the usual way with Belting's acetocarmine, except that the cover slip is left off. When correct differentiation is attained the stain is thoroly washed off with 50% acetic acid and the slide flooded with dioxan. This is followed by 2 changes of dioxan for 2 minutes each. A drop of Canada balsam dissolved in dioxan is added and a cover slip applied. In cases where a cover slip has been used at the acetocarmine stage it can be floated off in a staining jar of 50% acetic acid and dehydration with dioxan carried out as above.

Insect salivary gland chromosome smears: The glands are crushed under a cover slip in acetocarmine on a slide coated with dried egg albumen. After 20 minutes the area around the cover slip is flooded with 50% acetic acid and the cover slip floats loose so that it can be removed. The above described dioxan dehydrating procedure is then employed.

Squash preparations: Root tips are fixed in some suitable fixative and the Feulgen technic applied. The stained root tips can either be dehydrated by passing thru 3 changes of dioxan and mounting in dioxan-balsam where they are divided into small longitudinal sections by sharp needles, or they can be put immediately into a mixture of 1 part of 50% acetic acid to 1 part of corn syrup where shredding with needles is carried out. A cover slip is put on and separation of the cells completed by tamping or by applying pressure to the cover. This squash method is useful with anthers which are difficult to smear when in the early prophase stages of meiosis.     

Autoradiography of Water-Diffusible Substances in Sections of Whole Baby Rats

Rats, 7 days postnatal which had been injected with a radioactive nuclide, were quick frozen and sectioned in the frozen state. An adhesive cellulose tape (Sellotape) was used to support the section during cutting, through freeze-drying, and attaching to slides. Dehydration of the frozen sections consisted of 1 hr in a chilled desiccator containing silica gel, then at reduced pressure of 2-3 mm Hg until quite dry. The exposed side of the section was sprayed with celloidin dissolved in amyl acetate and allowed to dry. This side of the section was attached to a slide, previously coated with 1% gelatin containing 0.1% chrome alum, by means of an adhesive consisting of 4% gelatin and 5% formalin in 60% glycerol. In applying this adhesive it is mandatory that a border of about 3 mm of bare glass be left outside the adhesive, to allow intimate contact between the sticky side of the tape and the glass. The adhesive was allowed to set for 20 min, the slide immersed in water lor 50 sec, and the cellulose layer of the tape peeled off. The rubber base from the tape was removed with chloroform, the slide dried, and the exposed surface of the section coated with celloidin in amyl acetate, by dipping. After this treatment, the slides could be coated by dipping in autoradiographic emulsion without affecting water-soluble radioactive substances in the tissue.     

Insects did it first: a micropatterned adhesive tape for robotic applications

Based on the structural and experimental studies of more than 300 insect species from different lineages, we have developed and characterized a bioinspired polymer material with the ability of multiple glue-free bonding and debonding. The material surface is covered with a pattern of microstructures, which resembles the geometry of tenent hairs previously described from the feet of flies, beetles, earwigs and other insects. The tape with such a microstructure pattern demonstrates at least two times higher pull-off force per unit apparent contact area compared to the flat polymer. Additionally, the tape is less sensitive to contamination by dust particles than a commercially available pressure-sensitive adhesive tape. Even if the 'insect tape' is contaminated, it can be washed with a soap solution in water, in order to completely recover its adhesive properties. We have successfully applied the tape to the 120 g wall-climbing robot Mini-Whegs. Furthermore, the tape can be used for multiple adhering of objects to glass surfaces or as a protective tape for sensitive glass surfaces of optical quality. Another area of potential applications is gripping and manipulation of objects with smooth surfaces.     

An Adhesive Transfer Method of Cutting and Mounting thin Sections for Autoradiography

A short strip of adhesive transfer cellophane tape is applied to the face of the block to provide backing for the section during cutting and handling. The tape is sealed to the nuclear track plate with the section against the dry emulsion. After exposure the cellophane backing is removed by immersion in water, and the adhesive is dissolved from the section in unleaded gasoline. The section is hydrated and photographic and histological processing are carried out in the usual manner.     

The problem of proprietary dyes,with special reference to alamar blue,a proprietary dye revealed

A short strip of adhesive transfer cellophane tape is applied to the face of the block to provide backing for the section during cutting and handling. The tape is sealed to the nuclear track plate with the section against the dry emulsion. After exposure the cellophane backing is removed by immersion in water, and the adhesive is dissolved from the section in unleaded gasoline. The section is hydrated and photographic and histological processing are carried out in the usual manner.     

Fluorescent in situ hybridization applied on samples taken with adhesive tape strips

Fluorescent in situ hybridization (FISH), applied directly on samples taken with adhesive tape, is proposed as method to detect and identify microorganisms from the surfaces of valuable objects without being destructive. Results of tests carried out in laboratory conditions as well on samples taken from deteriorated surfaces of Roman Catacombs showed the feasibility of FISH when applied on adhesive tape. The potential as well as the limits of the technique were also discussed.     

Autoradiography of Water-Soluble Materials in Plant Tissues

Samples of tissue with water-soluble substances are lyophilized and doubly-infiltrated with parlodion and paraffin. Sections are mounted on an adhesive-coated cover slip with chloroform. The reverse side of the cover slip is coated successively with a thin layer of Harleco resin and a thick layer of Kodak “Opaque”. A corner of the cover slip is broken off as a marker. The cover slip is assembled with a covering glass slide on a nuclear track plate (Kodak NTB-2) with protective coating, the section being in contact with the emulsion, and held in place during radioactive exposure. Before development, the cover slip and plate are exposed briefly to light and then disassembled. Following development of the plate and removal of the layer of resin and opaque from the cover slip, staining of the section is optional. Lining up of the section with its autoradiograph can be accurate within 1-5μ.     

A Method for Making Aceto-Carmine Squashes Permanent Without Removal of the Cover Slip

Two modifications of Bridges' alcohol vapor method for making smear and squash preparations permanent are described. The variation of more general significance permits euparal to be applied without removal of the cover slip. Thus, possibility of loss, distortion or overlapping of desirable material is eliminated from this phase of preparation. The second modification is a reduction in duration of the alcohol vapor treatment. Although the latter alteration in schedule has no particular value in connection with material of many genera, it allows the vapor method to be applied to tissues of Nicotiana and may be useful in dealing with material of some other genera.     

Autoradiography of Water-Soluble Materials in Plant Tissues

Samples of tissue with water-soluble substances are lyophilized and doubly-infiltrated with parlodion and paraffin. Sections are mounted on an adhesive-coated cover slip with chloroform. The reverse side of the cover slip is coated successively with a thin layer of Harleco resin and a thick layer of Kodak “Opaque”. A corner of the cover slip is broken off as a marker. The cover slip is assembled with a covering glass slide on a nuclear track plate (Kodak NTB-2) with protective coating, the section being in contact with the emulsion, and held in place during radioactive exposure. Before development, the cover slip and plate are exposed briefly to light and then disassembled. Following development of the plate and removal of the layer of resin and opaque from the cover slip, staining of the section is optional. Lining up of the section with its autoradiograph can be accurate within 1-5μ.     

A Smear Technic for the Cucurbitaceae

It is very difficult to make satisfactory smear preparations of species in the Cucurbitaceae by ordinary methods because, (a) the differentiation between chromosomes and cytoplasm is poor, (b) the pollen mother cells are held together in tissue-like masses, (c) the chromosomes are comparatively small and numerous. Special procedures have been devised to overcome these difficulties. Staminate buds selected at the proper stage of maturity are fixed for a period of 12 to 24 hours in a mixture composed of 3 parts of 100% ethyl alcohol and 1 part acetocarmine to which a small quantity of iron acetate has been added. After prefixation the material is rinsed in several changes of 100% alcohol. It can then be transferred directly to the slide for smearing, or hydrated to 70% alcohol for storage. For smearing the anthers are dissected out, the extraneous flower parts discarded, and the storage fluid removed. A drop of acetocarmine diluted to one-half strength with 45% glacial acetic acid is added, and the anthers are macerated into small pieces and smeared. The anther debris is removed, and the cover slip added. It is necessary to carry out the above operations with a low power binocular microscope. After heating the cover slip can be sealed with Pyseal for temporary storage.     

Scanning Electron Microscopy of Pine Seedling Wood Tissue Sections Inoculated with the Pinewood Nematode Bursaphelenchus xylophilus Previously Prepared for Light Microscopy

Scanning electron microscopy (SEM) was applied to paraffin-embedded wood sections to study the histopathology of pine seedlings inoculated with the pinewood nematode (PWN), Bursaphelenchus xylophilus. The sections, which had been previously prepared and observed by light microscopy (LM) on glass slides, were originally obtained from experiments in which pine seedlings had been inoculated with PWN. The cover glass was removed by soaking the glass slide in xylene for 3 to 5 days. The glass slides were cut into small pieces so that each piece contained one wood section. Each piece of the glass slide was attached with double adhesive tape to an aluminum stub. The specimens were sputter-coated with gold and examined with a scanning electron microscope (JEOL-JSM 5200). Compared to LM (as documented in previous reports) SEM provided greater depth of focus and resolution of the damaged wood tissues, nematodes and associated bacteria. SEM made it possible to observe the relationship between bacterial distribution and nematode distribution in wood tissues. SEM observations also suggested the possibility of documenting the death of ray cells and other parenchyma cells in relation to disease development. Finally, the current study of PWN in pine seedlings demonstrated that glass slides prepared for LM observations more than 25 years earlier could be successfully processed for examination by SEM.     

A Method for Preparing Whole-Body Sections Suitable for Autoradiographic, Histological and Histochemical Studies

A method for the preparation of whole-body sections suitable for autoradiographic and histochemical study is described. Radioactive calcium chloride or [¹⁴Clproline was injected into the abdominal cavity of a rat. Thirty-five minutes after injection of calcium chloride or 40 min after injection of proline the rat was frozen in a mixture of hexane and solid carbon dioxide and blocked in 5% sodium carboxymethyl Cellulose. The carboxymethyl cellulose block was trimmed and a piece of copy paper was attached to the surface of the block with cellulose tape. Cryotome sections cut from the block were transferred from the paper to a glass slide coated with synthetic rubber adhesive. For wholebody autoradiography, sections were freeze-dried for 2 days and then placed against X-ray film. For light microscopic autoradiography, the freeze-dried sections were covered with a dried film of photographic emulsion. For histochemical use, the sections were fixed by raising the temperature to 4 C after immersion in 100% ethanol below -10 C. For histological observation, sections were postfixed with 2.5% glutaraldehyde and stained. Wholebody and light microscopic antoradiographs showed that sections so prepared could be used for the demonstration of soluble substances in wholebody sections and for detailed autoradiography at the light microscopic level, and the stained sections could be used for histological and histochemical studies.     

A method for preparing whole-body sections suitable for autoradiographic, histological and histochemical studies

A method for the preparation of whole-body sections suitable for autoradiographic and histochemical study is described. Radioactive calcium chloride or [14C]proline was injected into the abdominal cavity of a rat. Thirty-five minutes after injection of calcium chloride or 40 min after injection of proline the rat was frozen in a mixture of hexane and solid carbon dioxide and blocked in 5% sodium carboxymethyl cellulose. The carboxymethyl cellulose block was trimmed and a piece of copy paper was attached to the surface of the block with cellulose tape. Cryotome sections cut from the block were transferred from the paper to a glass slide coated with synthetic rubber adhesive. For whole-body autoradiography, sections were freeze-dried for 2 days and then placed against X-ray film. For light microscopic autoradiography, the freeze-dried sections were covered with a dried film of photographic emulsion. For histochemical use, the sections were fixed by raising the temperature to 4 C after immersion in 100% ethanol below -10 C. For histological observation, sections were postfixed with 2.5% glutaraldehyde and stained. Whole-body and light microscopic autoradiographs showed that sections so prepared could be used for the demonstration of soluble substances in whole-body sections and for detailed autoradiography at the light microscopic level, and the stained sections could be used for histological and histochemical studies.     

Karyological Leaf Squashes in Grasses, Aided by Pectinase Digestion at 45 C

Longitudinal sections of rapidly growing leaf shoots were soaked for 2-4 hr in 0.002 M 8-hydroxyquinoline at 25 C, blotted, and fixed in 3:4:1 ethanol, chloroform, acetic acid. A 30 min maceration at 45 C in a pectinase solution (Pectinal 59-L; Rohm and Haas) softened the material for staining and squashing. Excess pectinase was removed and 1% aceto-carmine stain was applied. After locating and gently tapping the cover clip to disperse the cells, heavy pressure was applied with a No. 9 rubber stopper and the heel of the hand. By the use of this procedure, karyotypes could be constructed in several genera of forage grasses. The karyotype of Paspalum notatum Flugge is illustrated.     

Sterility in unsterilized surgical adhesive tape

Providing a barrier to infectious organisms, sterile surgical adhesive tape has been used to close wounds for almost 20 years. The possibility that prepackaged unsterilized tape could be used for similar purposes is suggested by this study. Samples (480) were taken from 120 rolls that had been left in plastic surgical suite cabinets for 2 weeks. Aerobes and anaerobes were evaluated using tryptic soy agar with 5% sheep blood, while yeast and fungi were checked with Sabouraud dextrose agar. Sterility of sticky and smooth inner surfaces is shown to be a significant finding (p less than 0.01). It is concluded that prepackaged unsterilized surgical adhesive tape can be used to approximate wound edges without being a source of contamination (the sterile tape is 5600 percent more expensive). Wound closure in this manner would benefit the patient in the field, in the emergency room, or in third world countries where the supply of sterile tape is limited.