Detection of honeybee tracheal mites (Acarapis woodi) by simple staining techniques

A total of 12 staining techniques, including five anionic and five cationic stains, and two mordant stains, were tested as possible histopathological methods for the detection of the honeybee tracheal mite, Acarapis woodi. The cationic stains stained the parasitic mites intensively and the ensheathing tracheae weakly positive in thin thoracic sections. The improved clarity and contrast of the stained mites within the tracheae in the thin thoracic sections facilitated the detection process. Even at very low levels of infestation, stained mites were detected easily and accurately under a dissecting microscope at a magnification as low as 10 ×. Consequently, examination of dissected trachae under a phase compound microscope, currently used for diagnosis of the mites, is no longer necessary. The stained specimen can also be mounted permanently on microslides.     

BIASES IN DOLPHIN AGE STRUCTURE DUE TO AGE ESTIMATION TECHNIQUE1

The use of different tooth-preparation techniques resulted in widely different estimates of age in a sample of bottlenose dolphins, Tursiops truncatus. Teeth from 30 animals were prepared using the two most prevalent techniques reported in the literature for this species, unstained sections and decalcified and stained thin sections, and the resulting paired counts of growth layers were compared. Estimates from the two methods were identical or at least placed the specimen in the same age class in only five cases, ranging in age from 2 to 22 yr. Otherwise, the results fell into one of two categories: when the estimates were close (± 3-yr difference, n= 15), counts from unstained sections generally were higher (13 cases, age from unstained sections 2-20 yr); when the counts were more disparate, estimates from stained sections always were higher (6-31 yr difference, n= 10, age from unstained sections 12-27 yr and corresponding ages from stained sections of 27-47). Previous studies of age estimation in known-age bottlenose dolphins indicate that stained sections allow accurate estimates of age and demonstrate that maximum lifespan approaches or exceeds 50 yr. In contrast, the results herein suggest that using unstained sections for age estimation may result in imprecise or biased age-structure data.     

Radioautography of Histologic Sections of Human Testicular Tissue

Two techniques suitable for qualitative radioautographic analysis of histologic sections of human testicular tissue are described. In one, deparaffinized wet slides are coated with photographic emulsion resulting in significant improvement in preservation of histologic quality of the tissue section. In the second procedure stained and processed slides are coated with a thin layer of H.S.R. prior to coating with photographic emulsion. This procedure allows excellent preservation of the stained tissue section and also enables visualization of the reactive cell and its radioautographic image at two different focal planes. The techniques can be applied with equal advantage to histologic preparations of other tissues.     

Picrosirius Red Staining of Cardiac Muscle Following Phosphomolybdic Acid Treatment

When the picrosirius red technique was applied to cardiac muscle sections, intense yellow myocyte staining sometimes obscured thin collagenous septa. The picrosirius red technique was modified to include treatment of the sections in 0.2% (w/v) aqueous phosphomolybdic acid prior to staining. With 1-5 min treatment, cytoplasmic staining was eradicated; diminution of collagen staining occurred only with long treatments at much higher concentrations of phosphomolybdic acid. Using this phosphomolybdic acid-picrosirius red technique, collagenous septa as thin as 0.2-0.5 /im and fine collagen fibers making up the septa were clearly discernible. The technique also worked well on sections stained by other techniques and then destained. The phosphomolybdic acid-picrosirius red technique should be useful in experiments designed to investigate the effects of collagen distribution on the electrical and mechanical behavior of cardiac muscle.     

Picrosirius red staining of cardiac muscle following phosphomolybdic acid treatment

When the picrosirius red technique was applied to cardiac muscle sections, intense yellow myocyte staining sometimes obscured thin collagenous septa. The picrosirius red technique was modified to include treatment of the sections in 0.2% (w/v) aqueous phosphomolybdic acid prior to staining. With 1-5 min treatment, cytoplasmic staining was eradicated; diminution of collagen staining occurred only with long treatments at much higher concentrations of phosphomolybdic acid. Using this phosphomolybdic acid-picrosirius red technique, collagenous septa as thin as 0.2-0.5 micron and fine collagen fibers making up the septa were clearly discernible. The technique also worked well on sections stained by other techniques and then destained. The phosphomolybdic acid-picrosirius red technique should be useful in experiments designed to investigate the effects of collagen distribution on the electrical and mechanical behavior of cardiac muscle.     

A simple method for preparing thin (10 microM) histological sections of undecalcified plastic embedded bone with implants

A simple method for preparing undecalcified thin sections of bone with implants has been developed. After exposing a surface of bone and implant in a plastic block by sawing thick sections, the surface is stained prior to making a thin section. A glass coverslip is affixed with a thin layer of cement to the stained surface to stabilize the tissue and implant during sectioning. A mixture of glycerine and water is used as a coolant and lubricant. The orientation in situ is preserved allowing demonstration of bone architecture and cells, and the tissue-implant interface.     

Dry, High Resolution Autoradiography

Microtomed sections of freeze-dried, paraffin-embedded tissues are placed on pieces of thin sheet-Teflon backed by a felt pad. The sections are then pressure-mounted on dry photographic emulsion. After suitable exposure, the sections are firmly cemented to the emulsion with 0.45% cellulose acetate in a 10:1 mixture of 2-butanone and acetone. This prevents the specimens from falling off or moving during photographic processing, though the tissue can be stained through the cellulose acetate binder. The method has been tested with tissues containing tritium-labelled DNA, and it produced resolution comparable to that obtained with standard liquid emulsion or stripping film techniques.     

Dry,High Resolution Autoradiography

Microtomed sections of freeze-dried, paraffin-embedded tissues are placed on pieces of thin sheet-Teflon backed by a felt pad. The sections are then pressure-mounted on dry photographic emulsion. After suitable exposure, the sections are firmly cemented to the emulsion with 0.45% cellulose acetate in a 10:1 mixture of 2-butanone and acetone. This prevents the specimens from falling off or moving during photographic processing, though the tissue can be stained through the cellulose acetate binder. The method has been tested with tissues containing tritium-labelled DNA, and it produced resolution comparable to that obtained with standard liquid emulsion or stripping film techniques.     

A Simple Method for Preparing Thin (10 μm) Histological Sections of Undecalcified Plastic Embedded Bone with Implants

A simple method for preparing undecalcified thin sections of bone with implants has been developed. After exposing a surface of bone and implant in a plastic block by sawing thick sections, the surface is stained prior to making a thin section. A glass coverslip is affixed with a thin layer of cement to the stained surface to stabilize the tissue and implant during sectioning. A mixture of glycerine and water is used as a coolant and lubricant. The orientation in situ is preserved allowing demonstration of bone architecture and cells, and the tissue-implant interface.     

Detection of Xylella fastidiosa from resistant and susceptible grapevine by tissue sectioning and membrane entrapment immunofluorescence

Immunofluorescence detection was performed by tissue sectioning and membrane entrapment of Xylella fastidiosa from the inoculated hybrid selection F8909-08 (Vitis rupestris A. de Serres x V. arizonica/candicans b43-17; resistant) and Chardonnay (susceptible). In both techniques, tissue sections and bacteria-trapped polycarbonate membranes were incubated with specific polyclonal IgG and stained with fluorescein isothiocyanate (FITC)-conjugated IgG from rabbits to X. fastidiosa cells. The stained preparations were observed by fluorescence microscopy. Rapid identification of the bacteria within 3 weeks post inoculation (wpi) was possible in thin cross sections of the petioles, which allowed penetration of the specific antibody. Examination of the bacteria over time was also possible, and allowed observation of bacterial multiplication and invasion of xylem vessels. The membrane entrapment technique was able to isolate bacteria at low concentrations in infected but asymptomatic plants.     

Microscopy of the junctional region between human coronal primary and secondary dentine.

The juction between human primary dentine and regular and irregular secondary dentine was examined with a number of different light and electron microscopic techniques. In decalcified material, a narrow band along the innermost surface of the primary dentine stained intensely. The walls of the tubules within the band stained intensely, whereas the tubular walls within the bulk of the primary dentine were not stained. Generally, the walls of the tubules in both types of secondary dentine were also preferentially stained. Although not readily apparent in ground sections, observations of thin sections revealed a dramatic reduction in the number of tubules in regular secondary dentine. Generally, the radiodensity of the intertubular matrix was the same in primary and secondary dentine and the intensely stained band was not seen radiographically. The pulpal ends of the tubules in primary dentine were often occluded with a material having the same radiodensity as peritubular matrix. Both patent and occluded tubules were seen in irregular secondary dentine. Scanning electron microscopy of acid-etched specimens of secondary dentine revealed that some tubules had irregular walls of highly mineralized matrix which was less acid-soluble then the peritubular matrix of primary dentine.     

Quantitation of estrogen receptor content and Ki-67 staining in breast carcinoma by the microTICAS image analysis system

The microTICAS image analysis system, originally designed for karyometry using Papanicolaou-stained or Feulgen-stained smears and tissue sections, has been adapted to assess tissue sections stained by immunohistochemical techniques. This system was used to quantitate growth fractions and the estrogen receptor (ER) content of breast carcinomas stained by immunoperoxidase techniques. The results were similar to those obtained with nonautomated methods of quantitating immunohistochemically stained tissue sections and, in the case of ER content, were similar to the results obtained with cytosol estrogen binding methods. The findings show that the microTICAS system provides an objective alternative to visual counting of labeled cells in tissue sections stained for growth fraction or for ER content by immunohistochemical methods.     

Cementum annulation and age determination in Homo sapiens. I. Tooth variability and observer error

In order to test the feasibility of cementum annulations to estimate age in humans, observer error and tooth variability in cementum ring counts were evaluated in a sample of 42 mandibular canine and first premolar pairs. Additionally, two sectioning techniques were evaluated. Demineralized thin sections (7 micron) stained with hematoxylin are the preferred technique since their age related variance is greater than 75% for all tooth types examined. In contrast, less than 50% of the total variance was accounted for among individuals when mineralized sections (80 micron) stained with alizarin red were used. Intertooth variability in ring counts of demineralized sections was large between canines and premolars (43%). Premolars provide counts with lower interobserver error and are the preferred tooth. In an expanded sample (N = 51) of demineralized premolars, intraobserver and interobserver error accounted for 2% and 5% of the total variance, respectively. Evaluation of several experimental designs showed that increasing the number of slides per tooth has the greatest effect on reducing variance followed by increasing the number of observers. Increasing the number of observations has little effect. Cementum ring counts are measurable to a highly repeatable extent and provide a level of repeatability greater than that reported for the pubic symphysis and auricular surface aging techniques.     

Selective staining of nucleic acids by osmium-ammine complex in thin sections from lowicryl-embedded samples

Osmium-ammine (OA)/SO2 selectively contrasted RNA- and DNA-containing structures in thin sections from Lowicryl-embedded samples. No cell structures were stained after Epon embedding. RNAse and DNAse digestion experiments demonstrated that only RNA and DNA were stained in Lowicryl thin sections. Protease digestion did not modify the staining reaction. The very fine end-reaction produced a very high resolution of the stained structures. The staining reaction was not due to the presence of SO2 but to the low pH of the solution (ranging from 1.5-2.2). OA in glycine buffer, pH 1.5, selectively contrasted nucleic acids. Electrostatic bonds between nucleic acids and OA complex were probably involved in the staining reaction. Increasing the pH value of the staining medium resulted in loss of OA specificity for nucleic acids. The high electrolyte concentration of the staining medium hindered the staining reaction.     

Visualization of Gluten and Starch Distributions in Dough by Fluorescence Fingerprint Imaging

A novel method combining imaging techniques and fluorescence fingerprint (FF) data measurement was developed to visualize the distributions of gluten and starch in dough without any preprocessing. Fluorescence images of thin sections of gluten, starch, and dough were acquired under 63 different combinations of excitation and emission wavelengths, resulting in a set of data consisting of the FF data for each pixel. Cosine similarity values between the FF of each pixel in the dough and those of gluten and starch were calculated. Each pixel was colored according to the cosine similarity value to obtain a pseudo-color image showing the distributions of gluten and starch. The dough sample was then fluorescently stained for gluten and starch. The stained image showed patterns similar to the pseudo-color FF image, validating the effectiveness of the FF imaging method. The method proved to be a powerful visualization tool, applicable in fields other than food technology.     

Visualization of gluten and starch distributions in dough by fluorescence fingerprint imaging

A novel method combining imaging techniques and fluorescence fingerprint (FF) data measurement was developed to visualize the distributions of gluten and starch in dough without any preprocessing. Fluorescence images of thin sections of gluten, starch, and dough were acquired under 63 different combinations of excitation and emission wavelengths, resulting in a set of data consisting of the FF data for each pixel. Cosine similarity values between the FF of each pixel in the dough and those of gluten and starch were calculated. Each pixel was colored according to the cosine similarity value to obtain a pseudo-color image showing the distributions of gluten and starch. The dough sample was then fluorescently stained for gluten and starch. The stained image showed patterns similar to the pseudo-color FF image, validating the effectiveness of the FF imaging method. The method proved to be a powerful visualization tool, applicable in fields other than food technology.     

Tannic acid-metal salt sequences for light and electron microscopic localization of complex carbohydrates.

Use of tannic acid (TA), in sequence with ferric chloride, uranyl acetate or gold chloride resulted in staining of selective but sometimes different sites in paraffin sections. TA-uranyl acetate of TA-ferric chloride stained sites rich in complex carbohydrates, wherease TA-gold chloride stained the collagen of various connective tissues different shades of red-purple to gray-black. Applied to epoxy-embedded thin sections of tissues fixed with glutaraldehyde and not post-osmicated, TA-uranyl acetate and TA-ferric chloride imparted density to subcellular sites known to contain a high concentration of mucosubstances, such as secretory granules and cisternae of the Golgi complex of certain cells. TA-gold chloride proved unsatisfactory for ultracytochemistry because of its tendency to form globular precipitates on thin sections. The effect of blockage procedures at the light microscopic level indicated that vicinal glycols are not required for binding of TA to tissue sites. Electrostatic forces were shown to be of minimal significance, whereas hydrogen bonding appeared to play a part in both TA-tissue and TA-metal binding mechanisms.     

Ultrastructure of the pellicular complex of Plasmodium fallax

The exoerythrocytic merozoites of Plasmodium fallax grown in a tissue-culture system have been investigated by negative staining and thin-sectioning techniques, and the respective results have been compared. Negative staining provided additional information, corroborated findings obtained with thin sectioning, and contributed particularly to the study of the pellicular complex of the merozoites which has been demonstrated as being composed of three layers: a thin outer membrane, a thick interrupted inner membrane, and a partial layer of microtubules. Observations made of negatively stained parasites revealed that the thick, interrupted inner membrane in thin sections is actually a labyrinthine structure and covers the entire surface of the merozoite, except at the regions of the conoid and the cytostome. The microtubules which radiate from the conoid to the posterior end demonstrated a transverse periodicity and filamental subunits parallel to the axis of the microtubule. The detailed structure of the conoid and the cytostome is also described.     

Histochemical and immunohistochemical protocols for routine biopsies embedded in Lowicryl resin

Tissue processing and analysis require good preservation of both the shape and content of cells. Lowicryl resin is one of the few embedding media that allow good preservation of both tissue architecture and cellular contents. Therefore, different histochemical and immunohistochemical reactions can be applied to semithin sister sections from one biopsy. Further examination of a zone of interest can be carried out under the electron microscope. The hydrophilic property of Lowicryl resins makes possible different histochemical reactions; however, the technique used for paraf?n sections must be adapted for each reaction. Antigenic preservation of cells by low temperature embedding allows immunolabeling on either semithin sections or in the zone of interest on ultrathin sections. We have shown the application and adaptation of different histochemical and immunohistochemical reactions on semithin and ultrathin sections from hepatic biopsies that were large, but thin. The variety of techniques that can be used on sister Lowicryl sections of a single biopsy makes this medium useful for extensive pathological studies of precious needle biopsies.     

Histochemical and immunohistochemical protocols for routine biopsies embedded in Lowicryl resin.

Tissue processing and analysis require good preservation of both the shape and content of cells. Lowicryl resin is one of the few embedding media that allow good preservation of both tissue architecture and cellular contents. Therefore, different histochemical and immunohistochemical reactions can be applied to semithin sister sections from one biopsy. Further examination of a zone of interest can be carried out under the electron microscope. The hydrophilic property of Lowicryl resins makes possible different histochemical reactions; however, the technique used for paraffin sections must be adapted for each reaction. Antigenic preservation of cells by low temperature embedding allows immunolabeling on either semithin sections or in the zone of interest on ultrathin sections. We have shown the application and adaptation of different histochemical and immunohistochemical reactions on semithin and ultrathin sections from hepatic biopsies that were large, but thin. The variety of techniques that can be used on sister Lowicryl sections of a single biopsy makes this medium useful for extensive pathological studies of precious needle biopsies.