Ordinary Microscope Research Articles

Darkfield examination and allied procedures

When Schaudinn and Hoffmann made the initial discovery of T. pallidum in 1905, they did so with an ordinary light microscope. The difficulty of visualizing the pale treponeme under those circumstances will become apparent immediately to anyone who cares to try it, and the virtual transparency of the organism was, of course, the feature that led to Schaudinn's choice of “pallida” (and later “pallidum”) for the species name of his find. Shortly after the discovery, several German optical firms exhibited prototypes of the newly developed darkfield microscopes, and, in 1906, Landsteiner and Mucha 1 were the first to recommend the use of these instruments to demonstrate T. pallidum. Within a year, darkfield microscopy had become almost routine in the investigation of early and congenital syphilis in the European clinics. The procedure was introduced into the United States in 1909 by Udo Wile, 2 and a survey of the literature of the period shows that by 1914 a recognition of both the strengths and weaknesses of the technique and an understanding of the morphology of the organisms likely to be encountered were remarkably complete. The strongest indications for the use of the darkfield in the current management of venereal disease exist in two special situations: 1) the investigation of lesions in suspected cases of congenital syphilis and 2) the search for organisms in suspected cases of primary syphilis, especially during the initial period of seronegativity. The procedure has lost ground in the latter situation, owing in part to the increasing effectiveness of the newer treponemal and nontreponemal tests in detecting the presence of the disease in its earliest state; but it is premature to conclude, as in the recent statement by one experienced venereologist, that “in the foreseeable future the darkfield examination will be of historical interest, except for those in research.” 3 The purpose of this section is to summarize the technique and the findings to be expected in the darkfield and other methods employed in the examination of material taken directly from early syphilitic lesions. A step-by-step method for combining the results of these procedures and the results of physical examination and serologic testing to make the diagnosis of infectious syphilis is given in Chapter 11.

Surface Characterization by Reflection Electron Microscopy (REM)

AbstractReflection electron microscopy (REM) utilizes the Bragg reflected high energy electrons to form the image of a crystal surface. Images of dislocations, atomic steps, reconstructions of surface layers of atoms and adatoms, stacking faults and twinning, superlattices, etc., have been successfully observed on a wide variety of specimens. Contrast is mainly due to diffraction and phase, which distiguished REM as a unique method for high spacial resolution and high sensitivity imaging of the surfaces of bulk specimens. REM can be effectively performed under UHV as well as under the moderate vacuum of an ordinary commercial electron microscope.

Application of an indirect carbon immunoassay (CIA) for the rapid diagnosis of antibody to [formula omitted] in sheep

Carbon immunoassay (CIA), a novel indirect rapid test for Toxoplasma antibody in sheep, was compared with indirect fluorescent antibody assay (IFA). CIA relies on the adherence of carbon particles of India-ink to rabbit immunoglobulin G. Carbon labelled anti-sheep rabbit IgG was used for the detection of sheep antibody when attached to tachyzoites of Toxoplasma gondii . The result was read in an ordinary light microscope and there was a clearcut difference between negative and positive reactions. Out of a total of 97 sheep sera tested, 15 sera were negative in both tests and 12 were negative in CIA but showed low positive titres in IFA. The remaining 70 sera were positive in both tests but the titres were usually about 3 dilution steps lower when investigated with CIA as compared to IFA.

Sensitivity of the biotin-avidin-peroxidase (BAP) technique for rabies virus neutralizing antibody assay and the measurement of neutralizing antibodies of vaccinated human sera.

The biotin-avidin-peroxidase (BAP) technique for measuring serum neutralizing (NT) antibody against rabies virus in human sera was established. When the focus reduction rates with serial serum dilutions were plotted on a probit chart, a linear regression line was drawn. The slopes (b-value) of the regression lines were identical irrespective of the different test samples. The sensitivity of the BAP test for detecting NT antibody was highest when compared with that of the rapid fluorescent focus inhibition test (RFFIT) and mouse neutralization test. In addition, Iow level-antibody titer (< I : 5) which could not be detected by mouse neutralization test was detected by the BAP test and the RFFIT. Serum NT antibody response was easily followed by the BAP test with individuals who received 3 doses of preexposure preventive rabies vaccine. Most of the vaccinees produced a detectable amount ofserum NT antibody by a single dose ofvaccine and after the second vaccination, all vaccinees produced high level of the antibody and the titers increased continuously until day 2 1 , however, the titers on day 78, two months after the last vaccination, showed a tendency ofdecline. INTRODUCTION NT antibody to rabies virus has been mostly measured by mouse neutralization test (Atanasiu, 1973) and rapid fluorescent focus inhibition test (RFFIT) (Smith et al., 1 973). The former method requires a large number of mice and at least 2 weeks of long period until the final results can be obtained. The latter method is much more superior to mouse neutralization test for detecting antibody in every respect of the sensitivity, reproducibility and rapidity. However, this method has a disadvantage that the fluorescent staining is unstable and the stained cells easily fade out and has a difficulty in testing many specimens collectively. In order to resolve this problem, we previously studied the biotin-avidin-peroxidase (BAP) technique for measurement of rabies virus infectivity and the NT antibody to rabies virus (Shichijo et al., 1 982). The BAP technique provides such advantages that the specimens can be examined under an ordinary microscope and can be stored for 1 Department of Microbiology, Medical College of Oita, Oita 87(~9 1 , Japan 2 The Chemo-SeroTherapeutic Research Institute, 668 Shimizu Machi, Kumamoto 860, Japan

Image Reversal in Microscopy

Image Reversal in Microscopy

Improved method of embedding with epoxy resin 'Quetol 651' for both light and electron microscopic observation of identical sites in semi-thin sections.

In order examine semi-thin sections appmximately 0.2μm thick for light microscopy with an accelerating potential of 100 kV by a conventional electron microscope, Quetol 651 as a low viscosity water-miscible epoxy resin was suitable for an embedding medium. Semi-thin sections approximately 0.2μm thick embedded in Quetol 651 could be examined with an ordinary electron microscope operating at 100 kV.Tissues were fixed in 2.5% glutaraldehyde with buffered cacodylate at pH 7.4for 3 hr, postfixed in 2% osmium tetroxide with buffered cacodylate at pH 7.4 for 2 hr and stained with en bloc by 3% aqueous solution of uranyl acetate for 2 hr at 37°C. After dehydration in graded alcohol, they were embedded in the Quetol 651mixture. Semi-thin sections approximately 0.2μm thick were cut with glass knives on a conventional ultramicrotome. Semi-thin sections on grids were stained with the Giemsa method and methylene blue-azure II-basic fuchsin. Staining without removal of the embedding matrix was sharp and brilliant. These stained sections were observed under a light microscope. For electron microscopy, they were doubly stained with uranyl acetate and lead salt. Identical sites on such sections could be distinctly examined with an accelerating potential of 100 kV at low magnification (250-1,500 times) using LEM-2000, a combined light and electron microscope. Thus, photomicrographs electron micrographs of identical sites on tissue samples could be compared exactly.

Color Video Petrography

Color videocassettes for use at the undergraduate and graduate level in optical mineralogy and petrology courses have been produced with an inexpensive, conventional, single-tube TV camera and an ordinary petrographic microscope at about one third the cost of specialized TV microscopy systems. The same equipment can be used for live demonstrations and lectures and can be dismantled and reassembled in 10–15 minutes to use as a conventional recording system for lecture, laboratory, or field use.

Transillumination of renal biopsy specimens for rapid identification of glomeruli

Increasing numbers of percutaneous needle biopsies of the kidney rather than open renal biopsies are being performed in community hospitals as more trained nephrologists are available for consultation. There is a growing desire for ancillary studies such as immunofluorescence (IF) and electron microscopy (EM) in addition to light microscopy (LM). The pathologist is faced with the problem of confirming the adequacy of the biopsy and dividing the specimen for the indicated studies. Because in most instances the diagnostic changes are within glomeruli, one must be certain that all three specimens include renal cortex and contain glomeruli in adequate number. To be useful, the procedure must be rapid. Tissue for IF should be frozen as quickly as possible. Confirmation by frozen section cannot be used because of the small size of the specimen and the need to retain tissue for the studies mentioned above. A hand lens, inverted ocular, or dissecting microscope can be used to identify glomeruli if they contain many red blood cells: but, if little glomerular blood is present they are difficult or impossible to visualize by these methods. With transillumination of the biopsy specimen using an ordinary light microscope under lowest power, one can usually visualize bloodless, normal, or pathologically damaged glomeruli easily and rapidly. Although some sort of preliminary visualization of percutaneous needle biopsies of the kidney is probably currently in use in most large renal pathology services, we have seen no report of the use of the transillumination method. We feel that it greatly simplifies handling of these specimens, insures that glomeruli are present for the various studies, and saves technologists' time.

Growth of calcite crystals with non-singular faces

Calcite crystals with non-singular faces were grown by the silica gel technique using the double diffusion of CaCl 2 (1N) and K 2C 2O 4 (1N) in silica gel, adjusted to pH 8. The experiments were conducted at 25°C and with an atmospheric pressure of CO 2. Calcite formation occurs under very low concentration gradients. The material was identified by X-ray diffraction and infrared spectroscopy techniques. The crystal morphologies were observed by ordinary microscopy and SEM. The synthesized calcite crystals show an unusual curved morphology with some flat faces that correspond to {10 1 1}. Curved surfaces can be ascribed to the region of {10 1 0} and {11 2 3}. The only observed impurity present in the crystals is 0.7% Si, which is equal to or even lower th an that found in calcite crystals with flat faces grown in gels under higher concentration gradients. Consequently the presence of Si as impurity is a dubious factor for the formation of such curved surfaces. The explanation must be a consequence of the kinetics of the growth process.

The use of modified Cunningham chambers for the enumeration of H rosettes, E rosettes, and lymphocyte-tumour cell conjugates.

In this paper, we have described the use of a slide-coverslip Cunningham chamber which is ideal for counting delicate rosettes and lymphocyte-tumour cell conjugates. The ease of construction, facility of use, and economy of cost in comparison with hemocytometers make the use of these chambers the method of choice for rosette counts in general. In contrast to standard double-slide Cunningham chambers, the design of these rosette chambers is such that they can be used with ordinary light microscope objectives (including oil immersion), they can be more easily loaded without trapping air bubbles, and they can be sealed without tilting the slide. As an example of one potential application of these chambers to tumour immunology, data have been presented showing the high proportion of lymphocyte-tumour cell conjugates which can be formed by human peripheral blood cells. The ability of any particular tumour cell to form conjugates did not necessarily reflect the susceptibility of that cell type to natural killer (NK) cell-mediated lysis, nor was there any evidence that E rosette-forming cells preferentially formed conjugate in comparison to non E rosette-forming cells.

Characterization of lymphoid cell populations in bovine lymphosarcomas (“leukosis”) by flow-cytofluorometry

Acridine orange stained cell suspensions from ten cases of bovine lymphosarcoma were analyzed by flow-cytofluorometry. The green DNA-AO fluorescence showed in all cases, but one, two distinct separate lymphoid cell subpopulations. One corresponded to (normal) diploid lymphoid cells, the other had variable aneuploid values. The histograms of the latter cell populations also indicated cells in S- and G 2-phase. Low-angle light scatter histograms did not show a distinct separation between the different cell types, although two maxima could be distinguished. The relative proportions between the two cell populations varied between 20 and 80% with domination of one or the other cell type. There was some conformity to the histological and cytological picture in the ordinary light microscope, although neither a clear distinction in separable cell types nor differential counts was possible. The diploid lymphocyte subpopulation in the tumor might represent an immunoreactive process against the BLV-induced lymphosarcoma.

A new optical technique for evaluating collective motility of ram and bull ejaculated spermatozoa.

A new, non-destructive, objective technique for measuring collective motility of highly-concentrated ram and bull semen is described. The principle is based on changes in the reflected light scattered by motile spermatozoa. These changes can be recorded as a continuous analog wave pattern (Reflectospermiogram-RSG) and are correlated to the intensity of the turbulent motility as evaluated subjectively with an ordinary light microscope. Ram spermatozoa have, after ejaculation, a typical motility pattern, i.e., high, stable activity for about 20 min, then a period during which the motility decreases at a constant rate, and finally, a period with a low but rather constant activity. The usefulness of the technique has been demonstrated in various types of experiments.

Identification of Infective Dictyo-Caulus Viviparus Larvae Isolated from Herbage By the Bile-Agar Technique

A morphological description was given of the infective stage of the cattle lungworm Dictyocaulus viviparus. The larvae were stained with iodine and examined under an ordinary microscope after isolation with the bileagar technique used for processing herbage samples. Under these conditions the internal structures appeared of limited value to the identification. The larvae were characterized by their measurements as well as by their shape and profile. A comparison between larvae isolated from the field and larvae harvested from faecal cultures revealed that field larvae were smaller and more uniform in size. Of the reported characteristics the typical profile, the comparatively small size (range 305–400 µm), and the retaining of iodine stain were found most useful in routine recognition of D. viviparus infective larvae in isolates from herbage samples containing free living nematode species as well as infective stages of parasitic species.

Nuclear statistics with a 'poor man's detector': a laboratory experiment

An undergraduate laboratory experiment on the statistical fluctuations of nuclear reactions is described. The aim is to familiarise students with the use of solid-state nuclear track detectors using Cf252 as a source of spontaneous fission fragments and ordinary microscope slide glass (soda glass) as a detector, used in a geometry <2 pi . A simple method for exposure of the detector is given. The results indicate that the observed distribution of tracks, counted using a graticule fitted in the eyepiece of the microscope, is in good agreement with the expected Poisson distribution. However, for larger average values of the counts the Poisson and Gaussian distributions tend to coalesce.

Experimental study of fracture at geological scale velocities

Summary. It is well-known that the chemical environment and the thermodynamical conditions play a fundamental role in the physics of the fracture properties of solids which in turn appear relevant in the understanding of the earthquake mechanism and related precursory phenomena. We designed and built an experimental apparatus capable of measuring the fracture velocities with control of the physical and chemical environment as well as of the applied stress. The apparatus consists of a chamber where both the total pressure and the partial pressure of gases can be controlled. The stress is applied in mode I (tension) configuration. The crack is detected optically on the surface of the sample with an ordinary microscope and the velocity of propagation is directly measured by the increase in length in the elapsed time. The very good stability of the system over very long periods allows us to measure crack velocities down to 10−12 ms−1. The propagation under such low-velocity regimes appears interesting from a geological standpoint since it implies extremely low stress values, which are generally assumed to have no effect on crustal rocks. The first results obtained will be discussed, with particular regard to the apparent dramatic influence of the partial pressure of water vapour in the crack propagation velocity.

Particle-labeled antibodies. I. Anti T-cell antibodies attached to plastic beads by poly- l-lysine

A simple and inexpensive method for the detection of T-cell surface antigens is introduced using polyacrylic plastic beads (PAA-beads) as indicator particles. The beads are easily visible in ordinary light microscope and make the method convenient for routine laboratory use, also in laboratories possessing no special equipment. The method is an indirect test using a first, absorbed rabbit anti-T serum to sensitize the cells and a second, sandwich antiserum (goat anti-rabbit Ig) coupled to the indicator beads. Instead of glutaraldehyde used by other investigators to attach antibodies to the beads, we used poly- l-lysine which did not affect antibody titers. The F(ab) 2 fragment of the sandwich goat anti-rabbit Ig antibody was employed to avoid binding of antibody-coated beads to Fc-receptor-bearing cells. Sensitivity, specificity and reproducibility of the method were tested on murine and human lymphoid cells treated with the respective anti-T serum. The data obtained show that the sensitivity of the method depends on: (a) the bead concentration, (b) the concentration of F(ab) 2 fragment of the second antiserum used to coat the beads, and (c) the concentration of specific antibody used to sensitize the cells. Specificity and reproducibility of the method were found to be good. The percentage of positive cells with this method are in good agreement with those reported by means of well established immunological methods.

A METHOD FOR THE QUANTITATIVE AND QUALITATIVE DETERMINATION OF PLANKTONIC DIATOMS

SUMMARY A method for the quantitative and qualitative determination of planktonic diatoms was developed. The method uses the Utermohl counting technique (in which an inverted microscope is employed) as a basis but also involves the calculation of the relative density of each species in the association (as determined with the ordinary light microscope). A combination of the results thus obtained ensures that even the smallest diatoms (2,5 μm) are accounted for. These often constitute a significant proportion of the population, but are often overlooked or may not even be resolved using Utermohl's method.

Quinacrine and acridine-R banding without a fluorescence microscope.

A technique is described in which a standard fluorescence microscope equipped with a high-pressure mercury lamp is replaced with an ordinary laboratory microscope fitted with a quartz-iodine lamp. A dark field condenser and a set of three filters, including an FITC interference filter, complete a 'fluorescence' microscope. The microscope has proved itself satisfactory in the study of Y-chromatin, chromosome Q-bands including Q-polymorphism, and acridine-R band. It is very easy to operate and does not emit ultraviolet light, which might harm operators. Total cost of the quartz-iodine lamp's outfit, filters, and a dark-field condenser is much less than that of a standard fluorescence microscope. The cost is especially low when a laboratory microscope with a quartz-iodine lamp is already at hand. Spectrofluorometric studies of QM and Q indicate that the present system will show even better performance if an interference filter with a transmission range of about 400 to 440--450 nm is designed and used in combination with a 455--475 nm barrier filter.

Experience in the diagnosis of glomerulonephritis using combined light microscopical, ultrastructural and immunofluorescence techniques—an analysis of 134 cases*

The contribution of electron and immunofluorescence microscopy to renal biopsy diagnosis is illustrated by the results obtained in a personal series of patients with various types of glomerulonephritis. Introductory notes on the ultrastructure of the glomerular capillary and on immunological processes are also included. Immunofluorescent staining has particular value in demonstrating IgG-containing deposits in early membranous glomerulonephritis at a stage when ordinary microscopy is inconclusive. It is capable of throwing light on the mechanism of glomerular damage in severe extracapillary proliferation and in some cases of recurrent haematuria, but is less successful in separating minimal change disease from proliferative processes. Electron microscopy reveals the precise site of immune deposits and fibrin together with basement membrane changes, the microtubular structures common in SLE, and other details. It is concluded that for the accurate diagnosis of kidney disease it is essential to supplement light microscopy by one, or preferably both these methods.

Motile behavior and topography of whole and enucleate mammalian cells on modified substrates

Most mammalian cells when placed in tissue culture are capable of autonomous motility, but the mechanisms and properties of this locomotion are not well understood. To test the behavior of cultured cells on substrata of variable composition, we used ordinary electron microscope grids as stencils while evaporating gold wire onto glass coverslips coated with cellulose acetate. Whole CHO or L-929 cells growing in monolayer culture were then trypsinized and plated on these prepared coverslips and fixed at various intervals for observation in the scanning electron microscope. In all cases studied, the whole cells were observed to accumulate on the gold areas in preference to the cellulose acetate. In addition, by centrifuging cells in the presence of cytochalasin B (CB), we obtained populations of enucleated cells (cytoplasts) which were then replated on these coverslips. Even though replated cytoplasts are capable of limited motility, they were not observed to preferentially select substrates as did intact cells.