Antibody Staining in C. Elegans Using "Freeze-Cracking"

To stain C. elegans with antibodies, the relatively impermeable cuticle must be bypassed by chemical or mechanical methods. "Freeze-cracking" is one method used to physically pull the cuticle from nematodes by compressing nematodes between two adherent slides, freezing them, and pulling the slides apart. Freeze-cracking provides a simple and rapid way to gain access to the tissues without chemical treatment and can be used with a variety of fixatives. However, it leads to the loss of many of the specimens and the required compression mechanically distorts the sample. Practice is required to maximize recovery of samples with good morphology. Freeze-cracking can be optimized for specific fixation conditions, recovery of samples, or low non-specific staining, but not for all parameters at once. For antibodies that require very hard fixation conditions and tolerate the chemical treatments needed to chemically permeabilize the cuticle, treatment of intact nematodes in solution may be preferred. If the antibody requires a lighter fix or if the optimum fixation conditions are unknown, freeze-cracking provides a very useful way to rapidly assay the antibody and can yield specific subcellular and cellular localization information for the antigen of interest.     

Cell migration into neural tube lumen provides evidence for the "fixed cortex" theory of cell motility

We present a model of cell motility based on emigration of neural crest cells into the neural tube lumen under in vitro conditions (10% fetal calf serum or YIGSR) that inhibit their normal emigration from the base of the neuroepithelium into surrounding extracellular matrix (ECM). Ultrastructural observations reveal that cells lining the lumen are joined by zonulae adherentes (ZA), which are points of strong intercellular attachment, and thereby serve as markers for fixed regions of plasmalemma and cortical actin. Three major observations of the relationship of cells to the ZA support the "fixed cortex" model of mesenchymal cell migration. First, cells extend apical cell processes past the ZA into the lumen. To do this, they must make new apical plasmalemma and actin cortex that the endoplasm slides into. Second, elongated cells are observed in the lumen that are still attached via ZA to the neuroepithelium. This indicates that all of the endoplasm finally slides past the ZA. Third, numerous cytoplasmic pieces, often attached to each other and to the neuroepithelium via ZA, are found at the site where cells appear to have detached from the epithelium after entering the lumen. Since the ZA is fixed in location, the endoplasm must have slid past it into newly manufactured anterior cortex and plasmalemma, with the trailing end of the cell finally snapping off. The "fixed cortex" theory of cell migration agrees with existing data in that it predicts the polarized insertion of new plasmalemma and actin at the leading end of the cell, but it differs significantly from existing theories of mesenchymal cell migration in that it states that the cell surface remains firmly attached to the substratum while the myosin-rich endoplasm slides past it.     

"Liquidless" cell staining by dye diffusion from gels and analysis by laser scanning cytometry: potential application at microgravity conditions in space

BACKGROUND: Conventional staining of cells or tissue sections on microscope slides involves immersing the slides into solutions of dyes then rinsing to remove the unbound dye. There are instances, however, when use of stain solutions is undesirable-e.g., at microgravity conditions in space, where the possibility of accidental spill (many dyes are known carcinogens) introduces health hazard. Likewise, transporting bulk of liquid stains and rinses may be burdensome in certain situations such as field expeditions or combat. METHODS: The "liquidless" staining procedure is proposed in which the dyes are contained in thin strips of hydrated polyacrylamide or gelatin gels that have been presoaked in the stain solutions. Fluorochromes that have affinity to DNA (propidium iodide, PI; 4,6-diamidino-2-phenylindole, DAPI, Hoechst 33342) or to protein (sulforhodamine 101) were used to saturate the gels. The gel strips were placed over the prefixed cells or tissue sections deposited on microscope slides and relatively low (20 g/cm2) pressure was applied to ensure the contact. The cells were also stained by using commercially available mounting media into which DAPI or PI were admixed. Intensity of fluorescence of the PI stained cells was measured by laser scanning cytometry (LSC). RESULTS: Satisfactory cell and tissue staining, with minimal background, was achieved after 10-20 min contact between the cells and gels. Optimal concentrations of the dyes in the solutions used to presoak the gels was found to be 2-4-fold higher than the concentrations used routinely in cytometry. The measurements of intensity of cellular fluorescence by LSC revealed that the staining of DNA was stoichiometric as reflected by the characteristic cellular DNA content frequency histograms with distinct G1, S, and G2/M cell populations and 2:1 ratio of G2/M to G1 peak fluorescence. Individual gels can be saturated with more than a single dye-e.g., to obtain differential DNA and protein staining. Cell staining with DAPI or PI in the gelatin-based mounting media led to high fluorescence background while staining with DAPI in "aqueous" medium was satisfactory. CONCLUSIONS: Relatively fast staining of cells or tissue sections on microscope slides can be achieved by nonconvective dye diffusion using hydrated gels permeated with the dyes, applied to cells at low pressure. The quality of the staining provided by this methodology is comparable to conventional cell staining in dye solutions.     

Use of poly-l-lysine-coated slides in clinical bronchoalveolar lavage fluid samples

OBJECTIVE: Polylysine coating of microscope slides provides superior cell adhesion. We compared poly-l-lysine-coated (PLC) slides to conventional slides in cytocentrifuged bronchoalveolar lavage (BAL) fluid samples. STUDY DESIGN: Twenty BAL fluid samples with representative numbers of alveolar macrophages, lymphocytes and polymorphonuclear neutrophils were cytocentrifuged on uncoated slides and on PLC slides (2 slides each). Cell density, differential cell counts and cytomorphology were assessed on May-Grünwald-Giemsa-stained preparations. Reliability of cell differentiation was expressed as a phi value, which measures combined reproducibility and agreement. Statistical significance of differences between slides was calculated with ANOVA. Clinical relevance was assessed using a validated computer program predicting the most probable diagnosis. RESULTS: Although not statistically significant, cell recovery was lower on PLC slides as compared to uncoated slides. PLC slides held significantly fewer lymphocytes as compared to uncoated slides (mean value +/- SD: 25.89% +/- 28.26 versus 28.34% +/- 29.96, respectively). Counts of alveolar macrophages, lymphocytes and polymorphonuclear neutrophils displayed excellent phi values for both uncoated and PLC slides. No discrepancies in the computer-generated diagnoses were found. CONCLUSION: For BAL fluid cytology on cytocentrifuged preparations, PLC slides are not superior to conventional slides.     

Utility of 2-D and 3-D virtual microscopy in cervical cytology education and testing

OBJECTIVE: To evaluate the effectiveness of 3-D vs. 2-D virtual microscopy as adjuncts to education and assessment in cervical cytology. STUDY DESIGN: Five cervical cytology slides were acquired in 2-D; then the identical area of the slide was acquired in 3-D, resulting in 2 sets of virtual slides for comparison with the original glass slide. Seventy-nine paid volunteer cytologists and cytotechnology students participated. Approximately half were sent the 2-D set of slides via the Web, and the others a 3-D set of slides on a DVD. Evaluators examined the virtual slides and committed to an interpretation. After receipt of the original glass slides, a second interpretation was made, if different from the virtual slide interpretation. RESULTS: Diagnostic accuracy using virtual cytology slides was similar to that for glass slides (94% vs. 96%). There was no difference in diagnostic accuracy between 2-D and 3-D slides (p = 0.28); however, the ability to focus 3-D slides in the z-axis was strongly endorsed by the participants because of the uncertainty and frustration of having some cells out of focus on 2-D virtual slides. CONCLUSION: There was consensus that virtual cervical cytology slides would be a useful augmentation to education and testing.     

The effect of current on the distribution of diatoms settling on submerged glass slides

Glass microscope slides were submerged for two to six week periods at selected sites in a small, spring-fed stream near Lennoxville, Quebec. Slides were oriented parallel and perpendicular to the current. Qualitative and quantitative data from transects across slides show that diatoms are randomly distributed on slides perpendicular to the current but not on slides oriented parallel to the current. In the later case, most individuals first settled near the upstream or downstream edge of the slide. Non-random distribution is most pronounced on slides containing Cocconeis placentula. This species and two others, Achnanthes linearis and A. minutissima, are abundant and determine most distribution patterns found on slides. Preference of diatoms for the edges of slides appears to be affected by current. We propose a model, based upon water flow, to explain the preferential distribution of diatoms on slides oriented parallel to the current. Light appears not to affect settling patterns to a great extent in this study.     

Evaluation of Two Types of Infectious Mononucleosis Antigen Slides by the Indirect Fluorescent-Antibody Technique

The efficiency of two types of antigen slides was compared by using the indirect fluorescent-antibody technique. Fifty sera from infectious mononucleosis patients were tested concurrently on the two sets of slides for antibody to Epstein-Barr virus. The indirect fluorescent-antibody serum titer readings from the epoxy slides were either equal to or twofold higher than those from the cover slip slides.     

Comparison of Carnoy's solution and 96% ethyl alcohol fixation in bloody Pap smears

OBJECTIVE: To compare 2 methods of fixation in bloody Pap smears with Carnoy's solution and 96% ethyl alcohol. STUDY DESIGN: After observation of contact bleeding, 2 samples were prepared from cervical cells with conventional Pap smear. One sample was fixed in 96% ethyl alcohol and another sample was fixed in Carnoy's solution. RESULTS: Of 450 slides, 410 were selected for study. In study of cell adequacy, diagnosis of squamous cells and glandular cells was better in Carnoy's-fixed slides. Blood contamination of slides was reduced in Carnoy's-fixed slides (13.85% vs. 49.51%), and clearance of slides was increased in Carnoy's-fixed slides. Diagnosis of inflammatory cells and pathogenic microorganisms in was increased in Carnoy's-fixed slides, but no difference was seen in diagnosis of epithelial cell and glandular cell abnormalities. CONCLUSION: Carnoy's solution can be used as an effective fixative in bloody smears in conventional Pap tests.     

Diagnostic plots for detecting outlying slides in a cDNA microarray experiment

Different sources of systematic and random error variations are often observed in cDNA microarray experiments. A simple scatter plot is commonly used to examine outlying slides that have unusual expression patterns or larger variability than other slides. These outlying slides tend to have large impacts on the subsequent analyses, such as identification of differentially expressed genes and clustering analysis. However, it is difficult to select outlying slides rigorously and consistently based on subjective human pattern recognition on their scatter plots. A graphical method and a rigorous diagnostic measure are proposed to detect outlying slides. The proposed graphical method is easy to implement and shown to be quite effective in detecting outlying slides in real microarray data sets. This diagnostic measure is also informative to compare variability among slides. Two cDNA microarray data sets are carefully examined to illustrate the proposed approach. A 3840-gene microarray experiment for neuronal differentiation of cortical stem cells and a 2076-gene microarray experiment for anticancer compound time-course expression of the NCI-60 cancer cell lines.     

FAST slides: a novel surface for microarrays

We have evaluated FAST slides, a glass slide with a microporous polymeric surface that is a suitable substrate for microarray technology. The surface is a nitrocellulose-based polymer that binds DNA and proteins in a noncovalent but irreversible manner. FAST slides are compatible with robotic systems currently used to create microarrays and can easily accommodate volumes of 0.03-2 nL/spot. Our data indicate that FAST slides have a much higher binding capacity for DNA and better spot-to-spot consistency than traditional poly-lysine-coated slides. In addition, FAST slides are well suited for fluorescent detection because of their relatively low light scatter and efficient retention of arrayed DNA. These properties translate into fluorescent sensitivity comparable to modified glass surfaces. FAST slides are also ideal for arraying proteins, making them the only substrate of their kind currently available for microarray applications.     

Orientation discrimination and categorization of photographs of natural objects by pigeons

In Experiment 1, pigeons trained to discriminate rightside-up and upside-down orientations of slides of natural scenes with humans successfully transferred to new slides of the same kind. Experiment 2 revealed that both the orientations of the human figures and of the background scenes controlled the discrimination. When they were oppositely oriented, the background orientation cue was dominant. In Experiment 3 slides showing objects on a white background were presented either rightside up or upside down, with each slide presented in one orientation only. One group of pigeons learned to classify the slides according to their orientations. The other group learned to classify the slides according to arbitrary groupings. When the slides were shown rotated by 180 degrees, the latter group continued to discriminate the individual slides (i.e., the pigeons showed orientation invariance). The former group classified the rotated slides according to their orientations (i.e., orientation discrimination). In Experiment 4, pigeons learned the orientation discrimination with separate sets of human and bird figures. Partial reversal training in one object class transferred to the rest of stimuli in this object class but did not to the other object class. These results suggest that pigeons can learn to discriminate photographs on the basis of orientation but that orientation-based equivalence relationship is not formed between object classes.     

USE OF PHOTOGRAPHIC IDENTIFICATION IN CAPTURE-RECAPTURE STUDIES OF MEDITERRANEAN MONK SEALS

The use of photo-identification and its reliability in capture-recapture studies of Mediterranean monk seals were assessed using slides collected in the colony at Cap Blanc, western Sahara, from 1993 to 1996. Five tests indicated that researchers involved in photo-identification were proficient in matching slides of identified seals, consistent in classifying the side of the seal shown in slides and in assigning the morphological stage of the seal, and that changes of markings over a period of three years were insufficient to affect matching success. The certainty of identifying a seal was not dependent on the number of slides used but on distinctiveness of the markings and the quality of the slides taken. Capture-recapture abundance estimates were biased upwards when including poor quality slides. The exclusive use of excellent- and good quality slides provided the best estimates. The proportion of distinctive seals varied between morphological stages and was significantly lower in juveniles. When including the identification histories of juveniles, the heterogeneity of capture probabilities was higher. Therefore, abundance estimates were less biased when all juveniles were considered as non-distinctive seals. Reliable abundance estimates required a balance between duration of capture occasions and time interval between these.     

Restoration of broken cytology slides and creation of multiple slides from a single smear preparation.

A technique was developed for restoring broken cytology slides so that they are close to their original condition and for making multiple slides from a single smear preparation. The method is applicable to both cytologic preparations and histologic sections. In this study the fragmented smear preparation was treated with Pro-Texx, which penetrated, impregnated and solidified the full thickness of the pieces of the smear, enabling them to be lifted from the pieces of the broken slide. The removed pieces of the smear preparation were reassembled onto a new slide, which was then restained and coverslipped. In preparing multiple teaching slides, the treated smear preparation was divided as planned, with each portion mounted onto a separate slide, which was then restained and coverslipped. Ten other fine needle aspiration cases with broken slides have been restored, and more teaching slides were prepared from a single smear preparation using the same technique. All were equally successful. This technique provides an excellent method of smear transfer in cases of broken slides and creation of multiple slides from a single smear preparation for cytology teaching. This is particularly useful for unusual cases.     

Visual stimuli in distraction strategies for increasing pain tolerance. The confounding of affect with other stimulus characteristics

Recent experimental studies in pain control have questioned the value of pleasant affect in strategies employing distraction. It appears that pleasant affect may have been systematically confounded with task complexity or novelty in past research that found pleasant imagery or slides effective in increasing pain tolerance with the cold pressor test. The present study was a follow-up to a study conducted by this author (Greenstein, 1984) in which unpleasant slides had significantly increased pain tolerance above pleasant slide level. In the present study, 69 college students (35 females, 34 males) rated either the pleasant or unpleasant slides used in the original study on their perceived pleasantness, complexity, and uniqueness (novelty). Results indicated that the unpleasant slides were rated significantly more complex (P less than 0.001) and unique (P less than 0.001) than the pleasant slides. Additionally, as in the earlier study, ratings of the unpleasant slides on pleasantness deviated significantly farther from neutrality than did ratings of the pleasant slides (t = 5.04, P less than 0.001). Thus the unpleasant slides were also perceived as being more significant (i.e., pertinent) than were the pleasant slides. The results indicate that affect was confounded with other stimulus characteristics in the Greenstein (1984) pain control study and probably in a significant number of other studies as well. Researchers are cautioned to control for the stimulus characteristics of visual distraction strategies used in pain control studies. The assumption that pleasantness, per se, contributes to strategy effectiveness is no longer tenable; future research must demonstrate an independent effect.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inadequate cervical smears: results of an educational slide exchange scheme

Fifty-six slides, predominantly inadequate and of varying difficulty, were circulated to 12 laboratories as an educationally based slide exchange scheme. Three slides failed to achieve an agreed majority consensus opinion. Seventy percent of participants agreed with the consensus opinion in 80% of slides. Of the slides originally reported as inadequate, the consensus diagnosis was inadequate in 78%, negative in 12% and abnormal in 10%. The latter included two cases of high-grade dyskaryosis. There was good agreement for the two most frequent causes of inadequacy in submitted slides (obscured and poor cellularity). There was poor consistency in reporting the presence or absence of endocervical and immature squamous metaplastic cells, to an extent that questions their use in the assessment of smear adequacy. Three inadequate slides on consensus opinion were associated with subsequent cervical intraepithelial neoplasia (grade III) or invasive squamous cell carcinoma. In the latter case, the slide had originally been reported as negative by the submitting laboratory.     

The big problem of the missing cytology slides

Cytology slides are often unique and irreplaceable. Unlike surgical pathology cases, where additional paraffin sections can be cut, cytology slides often cannot be duplicated because there are only a few direct smears or the diagnostic material is present on a single slide. Cytology slides are often "sent out" to other physicians, laboratories or hospitals, typically so that the pathologist at the institution where the patient will receive treatment can review the slides. Less often, a cytology lab sends out the slides for a second opinion or as part of the discovery process in a lawsuit, where they may or may not be defendants. Rarely, unique and irreplaceable cytology slides are lost. This article presents a hypothetical scenario that is based on reported state appellate court decisions. The article discusses some of the legal issues that will affect the defendant cytologist/cytology lab and the "expert cytologist," and suggests some steps a cytologist/cytology lab can take to minimize the risk of repercussions from a lost unique and irreplaceable cytology slide.     

An Australian trial of ThinPrep: a new cytopreparatory technique

To evaluate the sensitivity and suitability of ThinPrep, a new slide preparation technique, 2026 paired cervical cytology slides were examined. After conventional Papanicolaou smears were prepared, the sampling instruments were rinsed in a fluid fixative. ThinPrep slides were then prepared in the laboratory from the surplus cells in the fixative. Compared with the Pap smears, ThinPrep slides were easier and quicker to screen, were inconclusive less often, and had similar rates for detecting abnormalities and infection. There were more unsatisfactory ThinPrep slides and more ThinPrep slides lacked endocervical cells. Both of these shortcomings were found to be linked to the choice of sampling implements. This study, in which a variety of sampling instruments was used, fails to confirm some of the previous claims made for the new technique.     

Characterization of an inexpensive, nontoxic, and highly sensitive microarray substrate

An agarose film has been proposed as an efficient substrate for producing microarrays. The original film preparation procedure was simplified significantly by grafting the agarose layer directly onto unmodified microscope glass slides instead of aminated glass slides, and the blocking procedure was replaced with a wash in 0.1x standard saline citrate (SSC) and 0.5% sodium dodecyl sulfate (SDS) without decreasing the performance of the produced microarrays. Characterization of the grafted agarose film using atomic force microscopy (AFM) and scanning electron microscopy (SEM) showed that the agarose film had a 10-fold increase in surface roughness compared to glass and that the interior of the agarose film was porous, with pore sizes between 100-500 nm. A comparison of hybridization on aldehyde-activated agarose-coated microarray slides and commercial amino-reactive microarray slides showed that aldehyde-activated agarose-coated slides had the highest signal-to-noise ratio of 850, suggesting that the aldehyde-activated agarose microarray slides are suitable in applications where analytes have a wide concentration range. By immobilizing the DNA probes using ultraviolet (UV) light, the signal-to-noise ratio was further increased to 3000 on the agarose microarray slides. The specificity of the UV cross-linked DNA probes was demonstrated using 21 and 25 bp long capture probes, enabling discrimination of target molecules differing in only one base.     

A micromethod for chromosome preparation from individual hematopoietic colonies cultured in methylcellulose

We report a micromethod for chromosome preparation from individual hematopoietic colonies cultured in methylcellulose. The entire process was carried out on poly-Lysine (PL)-coated slides. Individual colonies were transferred into 10 microliter of 0.075 M KCl and placed on PL-coated slides. After hypotonic treatment of the colony cells and their attachment to the slides, the cells were fixed by a three-step procedure as follows: addition of a 30% fixative (3:1 methanol:acetic acid) diluted with the hypotonic solution, addition of 20% ethanol, and subsequent immersion of the slides in a 100% fixative. The slides were flame dried and Giemsa stained. Q- and G-banding techniques also were used. These procedures provided analyzable chromosome preparations, even from colonies containing fewer than 50 cells.