Some Synthetic Resins IN Combined Fixing, Staining and Mounting Media

Many of the recently devised plasticizers and resins can be utilized to advantage in cytological technics. Some of them have solubilities which enable us to incorporate them in such fixing and staining solutions as aceto-carmine and propiorric-carmine. They are non-volatile, do not alter the fixation images of the fluids with which they are mixed, and serve as mounting media as the volatile components evaporate. Thus it is possible to make a permanent slide in a single operation. These newer compounds are better adapted for this technic than are the natural balsams which have been used previously, as their greater tolerance for water provides a much greater margin of safety. Procedures are described for the utilization of (1) Rezyl 7020, a water-soluble resin (now, unfortunately, not available), which dries to form a water insoluble film, (2) Amberol 750 and (3) Bakelite BR-7160, two alcohol soluble resins, more miscible in solutions containing water than are the natural balsams. Formaldehyde can be included in the aceto-carmine and propionic-carmine fluids with the result that more nuclear detail is preserved. Lacto-gelatin has some valuable properties as a mounting medium and can be used when the specimen is stained with orcein. Carmine, which gives a permanent stain in Rezyl 7020, Amberol 750 and Bakelite BR-7160 fades in lacto-gelatin.     

Production of Pine Resjn Balsams for Microscopical and Optical Uses

The authors describe a method of obtaining balsam from the crude resin of Yugoslav pines. The quality and the refraction of those balsams are very favorable and they may be used instead of Canada balsam and cedar oil for microscopic work and in some cases for the optical industry. When placed in the above mentioned balsams, histological preparations dyed with very sensitive colors remained unaltered. The authors are of the opinion that the best of all is Balsamum Pini peuce. The balsams of Pinus halpensis, Pinus leucodermis, Pinus nigrae and Pinus silvestris are very similar in their properties to the balsam of Pinus peuce. Tables are given to show: some physical properties of these balsams; the yield obtained in preparing them; their composition in respect to resin and turpentine.     

Dissection, Staining, and Mounting of the Embryos of Conifers

A statement is given of the advantages of this special technic and its place in embryological investigations, including directions for selecting the proper stages in collecting conifer cones and ovules, their methods of dissection from living material and their preservation for later dissection. The choice of dissecting microscopes and dissecting instruments, as well as directions for staining embryos with phloxine which may be combined with slow dehydration in glycerin, or for staining with Delafield's or Heidenhain's hematoxylin which may be followed by the glycerin dehydration are described. Glycerin affords a convenient break for a temporary stopping place in this technic.

Directions are given for transfer from glycerin thru 95% and absolute ethyl alcohol into other solvents such as diaphane solvent, essence of euparel or an easily prepared sandarac solvent. Other mounting media which have been used for conifer embryos are discussed—glycerin jelly, Venetian turpentine and Canada balsam—emphasizing the special advantages found in the media employing sandarac.     

Simplified Permanent Aceto-Carmine Smears with A Water-Miscible Mountant

By employing a water-miscible mountant with a good refractive index, permanent aceto-carmine smears can be made from any of a variety of killing, fixing, and staining schedules. Clear-col, a commercial preparation, orginally developed for mounting fungi from various water-containing media, is employed for anther smears by placing the medium directly on the final stage of aceto-carmine staining schedule. Thus, no dehydration and clearing are necessary. By controlling amounts of acetic acid either mixed with Clearcol or on smears, clear, well-stained preparations can be produced.     

The Piccolyte Resins as Microscopic Mounting Media

A new series of synthetic resins, the “Piccolytes” (β-pinene polymers), is recommended for permanent mounting media in histological work. These resins, which are available with various melting points, are of correct refractive indices, very low acid numbers, are pale, non-yellowing, have good adhesion to glass, and are freely soluble in xylene. Of a considerable variety of newer synthetic resins which were tried (stimulated by the recent scarcity of Canada balsam), only these terpene resins were found entirely satisfactory. Being of controlled manufacture, they are uniform in characteristics as well as readily available and very cheap.

A partial review of the literature of synthetic resin mountants is included.     

Preparation of sand fly (Diptera: Psychodidae: Phlebotominae) specimens for histological studies

Phlebotominae sand fly specimens were prepared for histological and physiological studies. Different fixatives were tested on sectioned and whole bodied adult females in order to obtain good fixation and provide satisfactory penetration of the embedding media. All fixed specimens were infiltrated (up to seven days under 5 C) and embedded in hydroxyethyl metacrylate. Two-three m sections were stained, mounted in Canada balsam and observed by light microscopy. Best results were achieved when whole bodied insects were double fixed in Bouin's and Carnoy's fluids (4 h/2 h) and stained in Hematoxilin/Eosin or fixed in calcium formaldehyde and stained in mercury bromophenol blue.     

The Venetian Turpentine Mounting Medium

As a mounting medium to follow aceto-carmine the following modification of Zirkle's is suggested: Venetian turpentine, 25 ml.; phenol, 50 ml.; propionic acid, 35 ml.; acetic acid, 10 ml.; water, 20 ml. The technic can be employed with either root-tip or pollen-mother smears, and has been used with quite a variety of plants. It is especially valuable where it is desired to make temporary mounts permanent. The method is simple, and with reasonable care no displacement of marked cells occurs.     

Interim Report of the Committee on Histologic Mounting Media: Resinous Media

The Fisher “Permount” naphthalene polymer, the Hartman Leddon “H.S.R.” terpene polymer resin, a Monsanto polystyrene P-1, the Will Corporation “Diaphane” and “Green Diaphane”, and the du Pont “Lucite” methyl methacrylate polymer were examined, and the possibility of use of some other plastics was also explored. The first 5 mentioned were tested for color preservation of a variety of stains in comparison with Canada balsam and Clarite X. From this point of view polystyrene, the Hartman Leddon “H.S.R.” and the Fisher “Permount” resins were the most satisfactory, then the “Diaphanes”. Both “Permount” and “H.S.R.” show some yellowing. The H.S.R. with a melting point of 115°C, the Permount with 150°C. melting point, and the Polystyrene with a thermal denaturation point above 220°C. all excell Canada balsam in heat resistance. Trimethylbenzene, cymene and monoamylbenzene appear to be the best solvents for polystyrene. Mounts made in a solution of 20 g. polystyrene in 100 ml. trimethylbenzene can be packed flat slide to slide in 24 hours after mounting without sticking together.

This report is not intended to deprecate the use of other resinous mounting media which have not as yet been tested or compared with those mentioned herein.     

Iron-Alum Aceto-Carmine Staining for Chromosomes and Other Anatomical Features of Rhodophyceae

The chromosomes, certain intracellular structures and gross anatomical details of many red algae, which, as a class, have proved technically difficult material, can be demonstrated by staining with aceto-carmine after a mordant bath of iron alum. Acetic-alcohol mixtures are used as nuclear fixatives and formalin-acetic-alcohol and other similar fluids for preservation of anatomical features. The tougher more cartilaginous thalli of some species can be softened, if squashes are desired, by prolonging fixation (24-48 hr.) in acetic alcohol and subsequent washing. The fixatives are washed out of the material before the latter is transferred to 0.5-5.0% ferric ammonium sulphate, the concentration of which may be altered according to the material. Excess mordant is removed by washing and the material stained in Belling's aceto-carmine containing a trace of ferric acetate as a “ripener”. The degree of heating before covering is critical as it controls the quality of the staining. Squashing must be very thorough to spread the chromosomes which are usually very small but only slight controlled pressure is necessary when diffuse structures such as carposporophytes, nemathecia or medullary filaments are being demonstrated. Paraffin sections mounted on slides can also be stained by this method.     

Impact of plant cover on the cavity‐nesting ant Temnothorax crassispinus

1. Plant communities influence the availability of important resources for ants, such as nest sites and food, as well as environmental conditions. Thus, plants affect the abundance and distribution of ants. 2. In a field experiment, the influence of plant cover on the settlement of nest sites and per‐capita productivity of sexual individuals by the ant Temnothorax crassispinus was analysed. In July 2014, in five areas with patches of alien balsam Impatiens parviflora, and another five of native balsam I. noli‐tangere, transects composed of artificial nests were established; the nest sites were situated inside patches of balsams, and outside of them. Four hundred and forty artificial nests were used. One year later, the nests were collected. 3. Colonies of the ants three times more often inhabited nest sites outside the patches of both balsams. Besides, colonies with queens were more frequently found in nest sites located away from balsams. The per‐capita productivity of sexual individuals was higher in nests collected in patches of balsam, and the colonies from patches of alien balsam produced a more female‐biased sex ratio. 4. In terms of the impact on the ant, no clear differences were found between the alien balsam and the native one. The most important factor affecting the fitness of ants in areas dominated by balsams is the presence of herbaceous plant cover rather than whether the plant is alien or native.     

An Apparatus for Fixation and Supravital Staining of Tissues by the Perfusion Method

An apparatus is described for the perfusion of the circulation which permits accurate control of the temperature, pressure and rate of flow of the perfusing fluids. Using this apparatus, the tissues can be perfused initially with saline and subsequently with a fixing solution or supravital stain without changing the cannula. The apparatus can be pre-set to any given requirement, and will thus give reproducible results. It is not suitable for the application of warm volatile fixatives, but these can be perfused cold after preliminary perfusion with warm saline.

Perfusion is improved by the subcutaneous injection of heparin before death, and by the administration of amyl nitrite either in the perfusing fluid or as a vapor during anaesthesia.     

Handling Human Chromosomes by A Coumarin Technic

Small pieces of nonneoplastic and neoplastic human tissues, mainly from cervix uteri and soon after removal by biopsy, are transferred to a saturated aqueous solution of coumarin for 5-10 min, after which they are put into the regular aceto-carmine mixture for about 1 hr. A squash preparation is made with moderate pressure and the cover glass is sealed by a mixture made of equal volumes of Canada balsam and hard paraffin. These temporary preparations are suitable for the study of chromosomes for about 15 days, after which they can be made permanent by following one of the usual procedures that employ dehydration and clearing.     

Dissection and Preparation of Whole Mounts of Endosperm from the Seeds of Grevillea (Proteaceae)

It is sometimes very desirable to supplement the examination of serially cut microtome sections by dissection and study of whole mounts of objects. In the case of the seeds of Grerillea the endosperm forms a curious worm-like structure, the vermiform appendage, in elucidating the nature of which microtome sections alone are inadequate. The dissection of the endosperm for demonstrating this structure is very necessary; otherwise the structure is altogether overlooked. In dissecting out the endosperm, which is easily done, either fresh seeds or previously preserved seeds may be used; the former are easier for handling and yield more satisfactory results. In the case of fresh material, an immediate killing is necessary and any one of the ordinary killing fluids may be used; Bouin's fluid was tried by the writer and was entirely satisfactory. After killing, the material is stained and dehydrated, the several stages of which are best done on the slide alone on which the material is placed. Mounting is done in any of the mounting media, and Canada balsam answers the purpose quite well.     

Root-Tip Smear Method for Difficult Material

A technic is outlined for the preparation of difficult material for the study of chromosome number and morphology in root-tip smears. The chief objective is to obtain polar views of the metaphase plates rather than equatorial views. To achieve this end it is recommended that fresh root-tips be cut free-hand into thin cross sections. The important features are: thin freehand cross sectioning of the fresh root-tips; fixing in Belling's iron aceto-carmine solution; maceration for 2-5 minutes in 50% HCl in 95% alcohol; and mounting in “Diaphane.”     

Possible uses of Dioxan in Botanical Microtechnic

Dioxan has been well established as an advantageous dehydrating agent for plant tissues. It dehydrates equally well after fixatives containing formalin, acetic acid, chromic acid, chromates, mercuric chloride, osmic acid, and alcohol. Better infiltration of paraffin after dehydration may be obtained by passing the material thru (1) a cold bath composed of 30 cc. of dioxan, 5 cc. of xylol and 20 cc. of melted soft paraffin and, (2) a warm bath of 50 cc. of dioxan, 50 cc. of paraffin, and 10 cc. of xylol. Transfer from (2) to soft paraffin. A dioxan fixative consisting of dioxan 50 cc., formalin 6 cc., acetic acid 5 cc., water 50 cc. was devised for delicate subjects. The fixed material is transferred directly into dioxan and mounted in dioxan-diaphane or dioxan-balsam. Very delicate objects require dioxan dilution of the balsam and slow concentration of the mounting medium by evaporation.

Entire plant parts or epidermal peelings are fixed in any desired fixative, washed if necessary, transferred to dioxan and mounted in diluted dioxan-balsam or diaphane. Dioxan may be used to mount hyalin objects whose refractive indexes approach those of balsam in media of higher index than balsam. It may be used in place of alcohol in finishing parafin sections, and since it exhibits different stain solubilities than alcohol it offers an important new tool in obtaining and maintaining stain balances.     

Brilliant Cresyl Blue as a Stain for Plant Chromosomes

Methods are proposed for staining plant chromosomes with the dye brilliant cresyl blue, and for making these stained preparations permanent by using polyvinyl alcohol mounting medium.

The stain, which is composed of 2% brilliant cresyl blue in 45% aqueous acetic or propionic acid, is used with fixed material in making smear preparations. The technics for staining are similar to those employed in the aceto-carmine method.

The mounting medium is made by mixing 56% polyvinyl alcohol, which is diluted in water to the consistency of thick molasses, with 22% lactic acid and 22% phenol by volume. The permanent slides are made by floating off the cover slip of the temporary slide in 70% alcohol, then applying the mounting medium and replacing the cover slip.

The chief advantages of the methods described are:

1)The preparation of the stain is rapid and simple. The batch of stain will be good with the first try.

2)The staining procedure in some instances is shorter than when using aceto-carmine.

3)The stain shows a high degree of specificity for nuclear structures and gives better results than aceto-carmine when used on certain plant tissues.

4)A minimum number of cells is lost in making the slides permanent when using polyvinyl alcohol mounting medium as the slide and cover slip are run through only one solution prior to mounting.

5)The mounting medium dries rapidly and this shortens the time required before critical examination of the permanent mounts can be made.     

Aceto-Orcein and Feulgen Stains for Anatomy and Cytology of Trematodes

Dicrocoelium dendriticum and Fasciola hepatica were killed in the extended condition without anesthetization by dropping them into 40% acetic acid or into aceto-orcein. By using aceto-orcein (La Cour, 1941), killing, fixing and staining were accomplished simultaneously: staining time 24 hr or more. Whole mounts were made by dehydrating, clearing and mounting in Canada balsam, or testes or the upper part of the uterus could be removed for squash preparations after as long as 2 mo in the fixing and staining fluid. For Feulgen staining, living specimens were placed in 40% acetic acid for 10—15 min and then transferred to either Gilson's fluid, for sections, or to acetic-ethanol (1:3) for squashes. Hydrolysis was either by 10% perchloric acid at 25°C for 12 hr or in 12V HCl at 60° for 10 min. The time for Feulgen staining (De Tomasi, 1936) was 1.5-4.0 hr. Squashes were made from testes and uterus in the same manner as after aceto-orcein or sections obtained after embedding in paraffin.     

Chlorazol Black E As An Aceto-Carmine Auxiliary Stain

In making chromosome counts on plants and plant parts treated with colchicine it was found that in cases where aceto-carmine alone is not satisfactory—as in axillary buds of apple, pear, plum, peach, apricot, and cherry—the following method was effective : Dissect out the meristematic parts of the axillary bud under a binocular (or cut free-hand sections) and transfer the dissected tissue immediately to a solution of 3 volumes alcohol to 1 volume acetic acid for killing and fixing. Let the fixative act at least 10 minutes; a longer time, 12-24 hours, improves the staining quality. Wash in at least 3 changes of 70% alcohol to remove most of the acid. Stain for 5-25 minutes in 1% chlorazol black E² in 70% alcohol. Rinse in 3 changes of 70% alcohol to remove excess stain. Transfer the material to a slide, cover with a drop of aceto-carmine, and if necessary, dissect further under a binocular. Cover with cover glass, heat, flatten and seal, or run Zirkle's fluid under the cover for permanent mounting. For smears of sporocytes, chlorazol black E may also be employed alone, or in combination with aceto-carmine, if a dark purple nuclear stain is desired.     

Preparation of Permanent Whole Mounts of Marine Centric Diatoms to Permit Observations of Cytoplasmic Inclusions

Marine centric diatoms are highly vacuolated plant cells which often exhibit a marked tendency to plasmolyze during fixation or other manipulatory stages required to make permanent preparations which pemit the observation of cytoplasmic inclusions. Altmann's, Schaudinn's, vom Rath's, 5% acrolein, and Allen's PFA₃ are fixatives which have been found to induce relatively little plasmolysis in the species studied. If, following fixation, the fixative is removed and desalting and dehydration are carried out in a filter assembly which allows gentle suction to make gradual changes in reagent concentration and if the cells are then placed in dilute mounting medium, only a relatively small percentage of them will be plasmolyzed. In such preparations, depending upon the fixatives employed, various cytoplasmic organelles or other inclusions will be visible in the unstained material when examined with phase contrast. Fixation with Altmann's fixative and mounting the material in Zeiss phase-contrast mounting medium (L-15) after gradual desalting and dehydration gave the best results. Standard cytochemical techniques for the detection of saccharides, protein, and lipids may be incorporated in this procedure; or, all of the steps needed for embedding the material in resins, excepting polymerization, are feasible also.