ULTRASTRUCTURAL ANALYSIS OF HEMATOPOIETIC COLONIES DERIVED FROM HUMAN PERIPHERAL BLOOD : A Newly Developed Method

The ultrastructure of granulocyte colonies derived from normal human peripheral blood leukocytes cultured in semisolid media has been studied by a new method developed for this purpose. Fixation, dehydration, and embedding of the whole content of the Petri dish resulted in a block of Epon containing colonies made up of cells with the spatial orientation of those observed in living cultures. This permitted serial sectioning through entire colonies. Cell maturation in vitro appeared to parallel that of normal marrow. However, even the most mature cells retained cytoplasmic characteristics of more immature cells. This was particularly true for eosinophils which only rarely possessed granules with electron-dense crystalline "cores," a feature typical for mature eosinophils. In addition to the normal-appearing hematopoietic cells found within colonies, very large round or spindle-shaped cells were present between colonies and firmly attached to the bottom of the culture dish. Although the histochemical and functional characterization of these cells awaits further study, it is suggested that they are related to histiocytes or macrophages. The technique described here should prove valuable in studies of the development, differentiation, and interaction of many types of cells.     

Agar-Screen Specimen Carrier for Bulk Processing of Biopsy Materials for Electron Microscopy

A specimen carrier for processing large numbers of biopsy materials for epoxy embedding and electron microscopy is described. Commercially available 18-mesh stainless steel or 16-mesh aluminum wire screening is used. The screening is cut into 1 × 3-inch strips. One corner is snipped off for orientation purposes. Four drops of warm 4% agar is placed on a prewarmed standard microscopic glass slide. A thin agar support film is formed on the bottom side of the horizontally held wire screen by lightly running it against the agar. Tissue blocks trimmed to 1 mm³ are blotted on filter paper and placed in a prearranged order on the top surface of the support film. A thin top coating of agar is applied on the specimen by touching it with the tip of a pasteur pipette containing warm 4% agar. The agar-screen unit with the mounted specimens is stabilized in 4% buffered formalin and rinsed with Sorenson's phosphate buffer, pH 7.4, with 6.8% sucrose. It is then processed as a unit through routine osmium tetroxide postfixation, alcohol dehydration, and Epon 812 infiltration. The tissue blocks are plucked off the agar support film with fine-tipped tweezers and embedded in individual capsules. No difficulty in thin sectioning was encountered and examination of the sections under the electron microscope showed good infiltration by the epoxy resin.     

Ruthenium Red—p-Phenylenediamine Staining of Monolayers to Facilitate Handling and Selection of Specific Cells for Transmission Electron Microscopy

The handling of monolayers for transmission electron microscopy has presented many problems, the main one being difficulty in visualizing the monolayers after polymerization of their plastic embedment following conventional glutaraldehyde-osmium fixation.

The application of ruthenium red—p-phenylenediamine during processing produced intensely darkened cells which could be examined and photographed either in 95% ethanol or following Spurr embedment without further treatment or sectioning. This treatment also facilitated orientation of the monolayers when re-embedding, and permitted precise localization of monolayers within flat embedding molds when trimming and thin sectioning for transmission electron microscopy.

Increased color density is the combined result of more complete retention of soluble elements during initial fixation by ruthenium red and the formation of a colored reaction product between the bound ruthenium red and osmium which is further intensified by p-phenylenediamine.     

Separation of Epoxy-Embedded Cell Cultures from Plastic Flasks for Electron Microscopy

Monolayers of cells grown in ordinary plastic flasks are fixed and embedded “in situ” into Epon. When polymerized at 40 C for 4 days instead of the usual 60 C., the Epon sheet containing the cells is easily detached from the bottom of the plastic container. The Epon sheet is observed by light microscopy as a histological preparation. Ultrathin sectioning of preselected areas can then be carried out in a horizontal plane.     

Mechanism of the Selective Action of Eosin-Methylene-Blue Agar on the Enteric Group

The actual mechanism of the differentiation of lactose-fermenting and non-lactose-fermenting organisms on eosin-methylene-blue medium is not reported in the literature. The present study is an attempt to elucidate this problem.

The color of colon forms on E.M.B. agar was found to depend on two factors: (1) the reaction of eosin with methylene blue to form a dye compound of either acidic or neutral nature, and (2) the production, by lactose-fermenting colonies, of a sufficiently low pH so that this dye compound is taken up by individual cells of the colony. Non-lactose-fermenting organisms are not colored because the compound is not taken up in alkaline reaction.

An explanation is offered to account for the occasional blue colonies found on E.M.B. medium. It is suggested that these colonies form a relatively high pH and thus cause slight dissociation of the compound. This dissociation would allow independent staining of the colonies by methylene blue.     

Pressure Resin Infiltration of Aphids for Electron Microscopy

Fixed, dehydrated pea aphids, Acyrthosiphon pisum Harris, were partially infiltrated with epoxy resins by standard procedures, then placed in a pressure chamber at up to 1000 psi for varying lengths of time. Insects so treated were found to be more suitable than nonpressurized insects for ultrathin sectioning and electron microscopy.

It was necessary to remove one or more legs from the insects to obtain adequate infiltration even where high pressures were employed. Little damage was evident at light or electron microscope levels of examination.     

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.     

Staining of Bacterial Colonies on the Millipore Filter to Improve Contrast

About 5 ml of 1% blue tetrazolium in 70% ethyl alcohol were poured over mature colonies of Pasteurella pestis and Malleomyces pseudomallei on Millipore filters (MF), contained in the filter holder apparatus, and allowed to drain through with the suction applied. The MF was washed with water and then covered with about 10 ml of 0.001% aqueous trypan blue and drained. This technique provided vivid white colonies sharply defined against a blue background.

Another method utilized 0.1% quinacrine-HCl (Atabrine) to stain colonies yellow and 0.05% vital red to stain the MF pink to light red.     

Methylene blue-fast green staining of hemopoietic colonies in agar cultures

A simple technique is described for the routine in situ identification of the cellular composition of colonies and clusters in agar cultures of hemopoietic cells. The entire culture, dried and formalin vapor fixed within a Petri dish, is stained with a mixture of methylene blue and fast green. By this method cellular ribonucleoproteins (RNP), deoxyribonucleoproteins (DNP) and some cationic (arginine and lysine containing) proteins are detected. Different maturation stages of neutrophils, eosinophils, basophils, monocytes, and macrophages can be easily identified with colonies and clusters on the basis of the cytoplasmic and nuclear staining.     

A Simplified and Convenient Method for the Double-Embedding of Tissues

A method for embedding tissues with a celloidin-paraffin combination is presented. The essential features of the process depend upon (1) a thorough infiltration of the specimen with celloidin of low concentration, and (2) the subsequent impregnation of both the specimen and the celloidin with paraffin.

The methods for sectioning, and the removal of the embedding agent are given.

The chief advantages of this method are: the preservation of all of the advantages of celloidin embedding but with a great saving of time, and greater convenience of storage; the cutting of thin sections (2μ for many types of tissues); it is useful for embedding specimens for which neither pure paraffin nor pure celloidin are entirely satisfactory, i.e. those containing tissues differing in density.     

Formation and Regeneration of Methanococcus voltae Protoplasts

Methanococcus voltae cells were converted into protoplasts by suspension in anaerobic 0.1 M Tris-HCl buffer containing 0.4 M sucrose and 0.05 M NaCl as osmoprotectants. Protoplast formation was monitored microscopically by observing the conversion of the typical irregularly shaped (uneven peripheries) coccoid whole cells to rounded forms with smooth peripheries. Although the procedure resulted in about 50% lysis of the initial number of cells, the remainder were converted to the rounded form. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electron microscopy of negatively stained cell preparations indicated that the treatment removed the wall layer from whole cells to yield protoplasts. Protoplast regeneration was evaluated by using optimized plating conditions and an anaerobic microplating technique. Between 50 and 63% of the initial number of protoplasts regenerated as colonies on agar medium (35°C, 7 days). The colony and cell morphologies of the regenerated protoplasts were indistinguishable from those of whole cells plated under identical conditions.     

Notes on Technic: “Clearing” Steel Knife Epon Sections in a Polystyrene Film Sandwich

Serial sectioning epoxy embedments by steel knife permits rapid light microscope survey of large tissue volumes, and preselection of areas of interest for electron microscopy. Acetate film (Hollander 1970) and Turtox plastic slides (West 1972) have been suggested as substrates upon which the sections may be “cleared” with an added layer of cured epoxy. In our experience, these substrates are excessively adherent to Epon, and “cleared” sections thinner than 40-50 μm cannot be released from them reliably. The following method is suitable for processing Epon sections 10 or more microns thick.     

COLONY FORMATION IN AGAR: IN VITRO ASSAY FOR HAEMOPOIETIC STEM CELLS

Using a method in which embryo fibroblasts were used as feeder layers, the colony forming capacity in agar of a variety of mouse haemopoietic suspensions was compared with their CFU s content. A striking parallelism between the results of the two assays was found. In addition, under certain conditions higher numbers of CFU s could be retrieved from 5-day-old agar colonies than were originally plated, indicating that the CFC a (Colony Forming Cell agar) may fulfil the requirements of pluripotency as well as of self-renewal, both prerequisites for any haemopoietic stem cell candidate. Although our data by no means provide direct proof that the CFC s and the CFC a are identical, they certainly support such a concept. the contradictory findings by others that CFU s and CFU c (Colony Forming Unit culture) can be separated on a velocity gradient is attributed to different culture conditions, in other words, that their CFU cè are not identical with our CFU a .
Our findings also indicate that for mouse cells our soft agar colony assay meets the criteria of a quantitative assay for haemopoietic stem cells and that extension of this technique to bone marrow of primates including humans seems to be justified.     

Light and electron microscopic structure of golgi-stained neurons in the vertebrate brain (new rapid Golgi procedure)

Summary After application of a rapid, selective silver impregnation procedure for light (LM) and electron (EM) microscopy, individual neurons are distinguishable by a light silver precipitation. The silver content is sufficient that entire nerve cells can be observed light microscopically; on the other hand, electron microscopically the cytological details are still visible. Brains of mice were fixed by phosphate-buffered aldehyde perfusion, and pieces of tissue left in a 1 % K₂Cr₂O₇ solution for 13 h before impregnation in a 0.5 % AgNO₃ solution for 2h. Thick sections (30–50 m) of the impregnated tissue were cut; from these sections, suitably stained neurons were dissected out and re-embedded for ultrathin sectioning, thereby allowing observations on the same neurons at the EM level. A thin silver deposit was observed along the delimiting neuronal membrane, the microtubules and the smooth ER, including the spinal apparatus of the dendritic spines. The fine cytoplasmic details of the impregnated neurons and the surrounding tissue are well preserved and, therefore, suitable for subsequent determination of synaptic relationships of the impregnated neurons with the adjacent neuronal elements.     

水稻成熟花药和花粉的结构和组织化学研究

用乙二醇甲基丙烯酸脂(简称GMA)和环氧树脂Epon812包埋的薄切片方法对水稻成熟花药和花粉的结构进行了观察,并对各种结构的性质和细胞中的后含物做了细胞化学的分析.对成熟花药的绒毡层膜及乌氏体的研究采用了分离技术,做了显微和超微观察.证明水稻成熟花药壁和花粉除具一般禾本科植物特征外,还揭示了花药壁表皮上可能有硅质,药壁表皮细胞内含有脂类颗粒,药室内壁具纤维素质的纤维状加厚;发现花粉粒中除了贮存有大量淀粉颗粒外,还含有脂类,成熟花粉中营养核与两个精细胞及两个精细胞间联系紧密;并讨论了薄切片的优越性,绒毡层膜的意义及其上细胞印迹的来源.     

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.     

Identification of a common signal associated with cellular proliferation stimulated by four haemopoietic growth factors in a highly enriched population of granulocyte/macrophage colony-forming cells.

We have prepared a population of bone marrow cells that is highly enriched in neutrophil/macrophage progenitor cells (GM-CFC). Four distinct haemopoietic growth factors can stimulate the formation of mature cells from this population, although the proportions of neutrophils and/or macrophages produced varied depending on the growth factor employed: interleukin 3 (IL-3) and granulocyte/macrophage colony-stimulating factor (GM-CSF) stimulated the formation of colonies containing both neutrophils and macrophages; macrophage colony-stimulating factor (M-CSF) produced predominantly macrophage colonies; and granulocyte colony-stimulating factor (G-CSF) promoted neutrophil colony formation. Combinations of these four growth factors did not lead to any additive or synergistic effect on the number of colonies produced in clonal soft agar assays, indicating the presence of a common set of cells responsive to all four haemopoietic growth factors. These enriched progenitor cells therefore represent an ideal population to study myeloid growth-factor-stimulated survival, proliferation and development. Using this population we have examined the molecular signalling mechanisms associated with progenitor cell proliferation. We have shown that modulation of cyclic AMP levels has no apparent role in GM-CFC proliferation, whereas phorbol esters and/or Ca2+ ionophore can stimulate DNA synthesis, indicating a possible role for protein kinase C activation and increased cytosolic Ca2+ levels in the proliferation of these cells. The lack of ability of all four myeloid growth factors to mobilize intracellular Ca2+ infers that these effects are not achieved via inositol lipid hydrolysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

A New Microchemical Reaction for Cellulose

A microchemical test for cellulose applicable to fresh sections and commercial products is described. The test differs from the older technics in that materials tested are not permanently altered.

Two solutions are required: (1) 2% solution of iodine in 5% KI, diluted with 9 parts by volume of water containing 0.28% glycerin; (2) saturated aqueous LiCl.

Procedure: Apply 2 or 3 drops of solution 1 with a glass rod; allow the preparation to stand for 30 sec; blot with filter paper, drying as completely as possible. Apply one drop of solution 2, cover and examine. The color reaction will be obtained within 5 min. The reaction for pure cellulose is light blue. Reactions for 16 fibers are given in the table.

As a stain for demonstrating plant tissues the technic has been used in the Botany Department of Pomona College with much success; but this phase of the subject has not been extensively investigated.     

Agarose/agar assay system for the selection of bacteriocin-producing lactic fermentation bacteria

The direct selection of bacteriocin-producing lactic fermentation bacteria was possible by plating diluted cultures of Pediococcus acidilactici on mixed agarose agar layers with the amount of each component incrementally adjusted to 1.2% (w/v). Between 0.5 and 1% agarose, the increased flexibility of the solidified support layer allowed its removal from Petri dishes without tearing and its smooth layering on the surface of 1.5% (w/v) standard agar medium seeded with Listeria innocua as the test organism. Selection of bacteriocin-producing clones was based on the size of inhibition zones visible in the bottom agar layer under colonies growing on the agarose/agar top layer. The lack of contact with the test organism permitted the transfer of superior clones from the surface of the agarose/agar layer directly into an appropriate nutrient medium.