Species recognition by five macaque monkeys

Sixteen monkeys of five macaque species (Macaca fuscata fuscata, M. mulatta, M. radiata, M. nemestrina, andM. arctoides) pressed a lever to see a variety of pictures (35 mm slides) of seven macaque species including their conspecifics. The subjects were allowed to see the same picture for the duration of the lever press and were able to see the same picture repeatedly by pressing the lever within 10 sec after the previous release of the lever. When 10 sec passed after releasing the lever, the next picture was set on the slide projector. All monkeys exceptM. arctoides and two infantM. fuscata fuscata pressed the lever to see their conspecifics for the longest duration. For all of the adult subjects, a multivariate analysis of variance (MANOVA) based on the mean duration of lever pressing responses (D) and the mean interval between the responses (I) revealed that the data for conspecific stimuli were distributed at significantly different locations from those for at least one of six close species on a two-dimensional space constructed with D and I. These results suggest that adult macaque monkeys visually discriminate their conspecifics from close species based on the still images of them.     

Variables Influencing Results, and the Precise Definition of Steps in Gram Staining as a Means of Standardizing the Results Obtained

Results of a Gram staining procedure varied with modifications of each of the steps involved. The best Gram differentiation was obtained when crystal violet and iodine solutions of high concentrations were used, and when n-propyl alcohol was used as the decolorizer. The decolorization step must be carefully quantitated, and one of the most important variables observed was whether a slide was brought into the decolorizer wet, or dry. Dry slides took 6 to 12 times as long to decolorize as wet. Wash steps, following crystal violet, and following the decolorizer, also greatly influence results by causing Gram-positive organisms to appear to be Gram-negative. The results indicated that Gram-stain procedures should not be varied to suit the whims of individual operators, and that each step could be specifically defined both as to the reagent used, and the procedure to be followed.

The followng Gram procedure is recommended for heat-fixed bacterial smears on glass slides. Flood the slide with Hucker's crystal violet for 1 ruin. Wash for 5 sec by dipping into tap water running into a 250 ml beaker at a rate of 30 ml per sec Rinse off the excess water with Burke's iodine, flood the slide with this solution for 1 min, then wash 5 sec in tap water as above. Decolorize by passing the wet slide through 3 (75 × 25 mm) Coplin dishes containing n-propyl alcohol, decolorize 1 min in each dish for a total of 3 min. Wash 5 sec in tap water as above, rinse off the excess water with 0.25% safranin, then flood the slide with this solution for 1 min. Wash as above, blot dry, and examine. An alternate procedure for decolorization would be to use either 95% n-propyl alcohol or 95% ethyl alcohol, but shorten the decolorization time to 30 sec per dish for a total of 1.5 min. After 10 slides, the decolorizer in the first dish should be replaced by fresh. This dish is then placed last in the sequence, with dish No. 2 moved to the No. 1 position.     

The Relationship of Slide Maturity and Trypsin Exposure Time in the Differential Giemsa Banding of Chromosomes

Difficulty has been reported in attaining consistent chromosome banding using trypsin-Giemsa techniques. When trypsin concentration, incubation temperature, and the duration of staining are held constant, a logarithmic relationship has been found to exist between the length of time since a slide was made and the length of trypsin treatment necessary to produce optimal banding when it is stained. Under the conditions specified in this report, ten seconds digestion was optimal for ten-day-old slides. This time approximately doubled for each additional eight days that slides were allowed to age. From a plot of the experimental results, treatment times appropriate for slides of any age from 10 to 60 days were obtained. Use of these times produced satisfactory banding in 85% of mitoses.     

Reproducibility of coccolith morphometry: Evaluation of spraying and smear slide preparation techniques

It is of great importance to assess the internal accuracy and reproducibility of coccolith morphometry, and to understand any systematic differences between various sample preparation techniques, so that data obtained with different methods can be adequately compared. Here, we performed a comparative study between two techniques regularly used to prepare nannofossil microscope slides, the standard smear slide and spraying methods. With each technique, ten replicate slides were prepared and morphometric measurements were carried out on the coccolith genus Calcidiscus as well as full assemblage counts to determine the reproducibility of coccolith size measurements and relative species abundances for each method.For either method, two significant sources of variance are related to the morphometric data, the variance between replicate slides and the variance within slides. Thus, in order to reduce the total variance of the estimate of mean coccolith size, it is beneficial to increase the number of replicate slides as well as the number of measurements on each slide. If the aim of the morphometric study is to address the mean size of a coccolith taxon or species complex (a group of closely related sub-species), one could rely on either preparation technique, since no significant difference in (log-normalised) mean size between the two tested methodologies was found. Likewise, the 10th percentiles are statistically equal for both methods. However, the 90th percentile values are significantly larger in the sprayed slides than in the smear slides, indicating that the spraying method may either favour larger coccoliths or better resolve the full extent of size variability present in the sample. Future tests are needed to investigate whether, and if so, how, size-fractionation may occur when using the spraying method. Nevertheless, the spraying method is preferred based on the reproducibility of proportion estimates since no significant difference between replicate samples was observed, in stark contrast to the smear slide series with 3–4 times higher variance. It appears that information gained from one sprayed slide requires counting at least three replicate smear slides.     

Differences between papanicolaou smears with correct and incorrect diagnoses

A case control study of women with carcinoma in situ (CINIII) was undertaken comparing Papanicolaou smears for which false negative reports had been issued with slides for which true positive reports had been made. the number of abnormal cells was the strongest differentiating factor. Where there were less than 50 abnormal cells on the slide, the odds of a false negative report being issued was 23.7 times greater (95% confidence interval 3.7-150) than when there were 200 or more abnormal cells. In false negative slides, the abnormal cells were likely to be not represented throughout the slide, present only as single cells rather than as groups, small in size and with finely granular normochromatic nuclei. We conclude that there are intrinsic differences between true positive and false negative slides. Given these characteristics, rapid rescreening of slides that are considered negative may not be an effective method of reducing the false negative rate.     

Split-sample analysis of discarded cells from liquid-based Pap smear sampling devices

OBJECTIVE: To examine cells that were retained on sampling devices used to collect ThinPrep (Cytyc Corp., Boxborough, Massachusetts, U.S.A) Pap smears in order to evaluate both the number and significance of cells that are routinely discarded with these devices after liquid-based specimens are collected. STUDY DESIGN: One hundred Pap smears from 100 women were prospectively procured after gynecologic Pap smears were collected for the ThinPrep Pap test. The sampling end of the collection devices was cut off and placed in a vial that contained SUREPATH preservative fluid (TriPath Imaging, Inc., Burlington, North Carolina, U.S.A). The residual cell samples were processed using the SurePath PREPSTAIN slide processor (TriPath). A single liquid-based slide was prepared from the sampling devices from each of the 100 specimens collected. The slides produced from the discarded devices were reviewed for the following: squamous cells, endocervical component, epithelial cell abnormalities and miscellaneous findings. The slides prepared from the "throw-away" (TA) material were subsequently compared with the primary ThinPrep Pap smear slide. RESULTS: Twenty-five percent of the TA samples had an equal or greater number of squamous cells per high-power microscopic field when compared to the primary ThinPrep slide, with 8% of the TA slides demonstrating greater overall cellularity. An endocervical component was present on 27 of 66 cervical samples (40.9%). Three of five cases (60%) interpreted as atypical squamous cells of undetermined significance had similar cells on the TA slides. Two cases of atypical glandular cells of undetermined significance had no abnormal cells on the TA slides. Twelve of 14 cases (85.71%) of low grade squamous intraepithelial lesion contained similar cells on the TA slides. Two of four cases (50%) of high grade squamous intraepithelial lesion also had similar abnormal cells on the TA slides. Miscellaneous findings included 1 case of benign endometrial cells and 4 Candida infections present on both preparations, along with 1 case of Trichomonas vaginalis organisms present on the ThinPrep slide only. In 1 specimen, several multinucleated histiocytic giant cells were present only on the TA slide. CONCLUSIONS: Specimens prepared from TA collecting devices used for the ThinPrep Pap test are less sensitive than the primary specimen for the detection of cervical lesions. This is in contrast to split-sample studies involving ThinPrep and conventional smears. Our study documented the presence of normal and abnormal cells discarded from ThinPrep sampling devices in a high percentage of cases. Discarded abnormal cells on the TA slides were, however, few when compared to the primary specimen, with only 1 exception involving a high grade lesion.     

The application of visual cell profiles in the study of premalignant atypias in sputum

Because the diagnosis of squamous-cell carcinoma of the lung by routine cytologic screening of sputum is often inconclusive, our laboratory is studying the use of cytomorphologic profiles as a reliable diagnostic aid. This study reports the analysis of the profiles of 75 subjects, both cigarette-smoking volunteers and hospitalized patients suspect for lung cancer. Twenty-five of the subjects had been classified as having squamous metaplasia, 25 as having atypias and 25 as having squamous-cell carcinoma. Four slides were made for each subject, with 100 random fields viewed on each slide. The frequency of free alveolar macrophages, metaplastic squamous cells, epithelial atypical cells and malignant squamous cells were noted for each field. The results indicated that, although there were large differences between individual profiles within each diagnostic category, there were significant differences between the average profiles for each diagnostic category. Furthermore, although there were differences in cell frequencies from slide to slide and within slides, the differences were constant across diagnostic categories. The results are supportive of the progressive-atypia hypothesis for squamous-cell carcinoma of the lung and provide a baseline for comparison with subsequent individual profiles.     

Do gelatin-coated slides increase cellular retention in oral exfoliative cytology?

A minimum number of cells is required for an accurate cytologic assessment of oral lesions; however, the cell yield in some oral smears is reduced, relating to the site from which the cells were harvested and to the lesion examined. The use of gelatin coating to increase the retention of cells in smears from such sites was assessed. Cells were obtained from four sites within the oral cavities of 20 patients (10 male and 10 female) with healthy mouths. Using a wooden spatula, two samples were obtained from each site; one was transferred to a gelatin-coated slide, the other to an uncoated slide. For each slide, the cell yield was rated on a scale of 0 (no cells), 1 (few cells) and 2 (many cells). The results were then analyzed using the Wilcoxon sign-rank test. The coated and uncoated slides yielded similar results for most sites, except that the coated slides seemed to be advantageous for samples from the ventral surface of the tongue.     

A Method for the Preparation of Serial Thick Sections of Plastic-Embedded Plant Tissues

A procedure is described in which thick sections (2-10μ or more) of plastic-embedded plant tissues are mounted in serial order on slides for use in routine light microscopy. Sections are cut with a steel knife on a rotary microtome while the block and blade are bathed with 40% alcohol. The cut sections are placed, in order, in 50% alcohol in the small wells of modified plastic trays where they become flat, pliable and suitable for subsequent handling. Sections remain separate and in correct order in the trays while they are stained, washed, and prepared for final mounting on slides. Mounting involves a simple and rapid procedure of transferring the sections to a slide and heating first on a 70-75 C hot plate (to slowly evaporate the water around the section and to partially affix the section) and then on a 100 C hot plate. This second heating ensures adhesion when xylene-base mounting media, which tend to loosen weakly adhered plastic from the slides, are used. The technique of staining the sections loose provides the following advantages: (1) the problems of section loss and entrapment of stain between section and slide during staining are eliminated, (2) relatively high staining temperature, akalinity, and alcohol concentration of the stain solvent (all of which promote loosening of pm-affixed sections from slides during staining) is allowed, and (3) staining is more even and selective. The procedure has been found to be reliable and fast enough to be of value in a significant variety of routine light microscope studies.     

Normalization of microarray data using a spatial mixed model analysis which includes splines

MOTIVATION: Microarray experiments with thousands of genes on a slide and multiple slides used in any experimental set represent a large body of data with many sources of variation. The identification of such sources of variation within microarray experimental sets is critical for correct deciphering of desired gene expression differences. RESULTS: We describe new methods for the normalization using spatial mixed models which include splines and analysis of two-colour spotted microarrays for within slide variation and for a series of slides. The model typically explains 45-85% of the variation on a slide with only approximately 1% of the total degrees of freedom. The results from our methods compare favourably with those from intensity dependent normalization loess methods where we accounted for twice as much uncontrolled and unwanted variation on the slides. We have also developed an index for each EST that combines the various measures of the differential response into a single value that researchers can use to rapidly assess the genes of interest.     

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.     

Studies on the excitability of the central program generator in the spinal cord of the terrapin Pseudemys scripta elegans

We present observations on the multicyclic scratch reflex in spinal terrapins as produced by electrical stimuli applied to the shell at the specific regions at which a mechanical stimulus produces the reflex. EMGs and hip and knee movements are recorded. The responses to the electrical stimuli are similar to the responses to mechanical stimuli. There is a three phase EMG pattern (Stein and Grossman, 1980), to which the movement pattern is related (Bakker and Crowe, 1982). A response may consist of a series of up to 25 movement cycles with a total time course of up to about 30 sec. The initial cycles of a response are relatively fast (less than 1 sec), but the cycles at the expiration of the response may have a duration of 2-3 sec. A single electrical stimulus pulse is often insufficient to trigger a series response. Instead, a weak EMG burst of a few tenths of a second duration, together with a slight movement, is often seen. However, a second pulse can set the cycle series in motion even after an interval of 40 sec between the pulses. A further booster stimulus pulse given while a reflex response is taking place can increase the speed of the movement. If the booster pulse is given just after cessation of reflex activity it can restart the activity, but this second cycle series is often shorter than the first one. The results indicate that the excitability of the central program generator is not constant. Long duration changes in the excitability are produced within the spinal cord.     

Immuno-capture of Cryptosporidium parvum using micro-well array

A glass slide and micro-well array chip on which anti-Cryptosporidium parvum antibody was immobilized were used for the rapid capture and detection of C. parvum. Biotinylated anti-C. parvum antibodies were spotted onto the streptavidin-coated glass slides. C. parvum oocysts were captured specifically on the spot when more than 73 ng of anti-C. parvum antibody was applied onto the glass slide. However, C. parvum oocysts captured on the glass slide were detached by repeating washing steps. To improve the capture efficiency of oocysts, capture was performed in a micro-well format consisting of 1024 wells/2.5 mm2 (32 x 32 wells) fabricated as a chip by photolithography. Instead of a flat surface on a glass slide, each well was 30 microm in diameter and 10 microm in depth. Streptavidin was also immobilized onto the micro-well array. The biotinylated anti-C. parvum antibodies were immobilized efficiently onto the chip using a buffer containing 20% methanol. Using this technique C. parvum oocysts were stably captured onto the chip after repeated washing procedures. These data show that the newly designed micro-well array technique described here is useful for antibody-mediated C. parvum capture.     

Cardiac neural discharge and epileptogenic activity in the cat: An animal model for unexplained death

This study developed an animal model to explore the hypothesis that altered automatic function may be one cause for unexplained sudden epileptic deaths. After α-chloralose anesthesia, 9 cats received a tracheostomy and a thoracotomy. Intravenous gallamine was used to paralyze the cats. Blood pressure, arterial blood gases, electrocardiogram, and rectal temperature were monitored. Simultaneous monitoring of the neural discharge in postganglionic cardiac sympathetic branches and the vagus nerve was combined with a bilateral craniectomy and electrocorticography. Pentylenetetrazol was given intravenously at 10 min intervals in 10, 20, 50, 100, 200, and 2000 mg/kg doses. Epileptiform discharges were categorized as a prolonged ictal (duration of 10 sec or more), brief ictal (duration of less than 10 sec mixed with suppression), and interictal spike activity. The two types of ictal activity were quantified by duration in seconds for the 10 min interval after each dose of pentylenetetrazol; the number of interictal spikes/min was determined for each minute of the entire experiment. This study developed a model which quantified the degree of epileptiform activity and correlated it with changes in cardiovascular function and autonomic cardiac neural discharge. An imbalance within and between sympathetic and parasympathetic cardiac neural discharges was found, as was a significant disruption of the physiological relationships between heart rate and blood pressure. Frequent and varied electrocardiogram abnormalities occurred. All of the above changes occurred during minimal (interictal) subconvulsant as well as during maximal (ictal) convulsant epileptogenic activity.     

Effect of changing the frequency of conditioning tetanus on depressor responses evoked by stimulation of the aortic nerve.

The effect of changes in frequency of the conditioning tetanus on the magnitude of the testing depressor response was studied in rabbits anaesthetized with urethane. Conditioning and testing stimulations were applied to the same aortic nerve. The duration of the conditioning tetani was set at 3 and 60 sec and the interval between stimulations amounted to 40 and 120 sec. At the testing interval of 40 sec the increase in frequency both of short and long conditioning tetani reduces the magnitude of the testing response which attains a minimum at frequency of about 30 cycles/sec. Conditioning stimulations of higher frequency are gradually less effective and cause the testing response to increase. Similar depression is observed at the testing interval of 120 sec but only following long-lasting conditioning tetanus. Short conditioning trains at the testing interval of 120 sec facilitate the testing response. The frequency of the conditioning stimulation which produces the greatest reduction of the depressor response indicates the range of control exerted by the conditioning tetanus over the testing blood pressure effect. The size of this control is determined by the lowest level of depression and the highest value of facilitation of the testing response.     

The Physiological and Biochemical Response of Standardbred Horses to Exercise of Varying Speed and Duration

LINDHOLM, ARNE and BENGT SALTIN: The physiological and biochemical response of standardbred horses to exercise of varying speed and duration. Acta vet. scand. 1974, 15, 310–324. — Welltrained standardbred horses were studied to examine the metabolic response to excercise of various speeds and duration. Comparisons between interval (400, 700, 1,000 and 2,000 m) and continuous trotting (1 hr., 2 hrs.) and racing were made. Muscle and rectal temperatures were recorded before and immediately after each work bout. Heart rate was linearly related to trotting speed, and maximal heart rate (240 beats × min.−1) was achieved when trotting at least 700 m at close to maximal speed (12.0–12.5 m×sec.−1). Biopsy specimens from the gluteus medius muscle and venous blood were obtained before and after each work bout. Muscle and blood lactate values were markedly increased first at speeds close to maximal speed (11.4–12.5 m×sec.−1). Trotting 6×700 m at 12.5 m×sec.−1 produced as high muscle and blood lactate values as 23.7 and 19.0 mmol×kg−1 wet weight and l−1, respectively. Corresponding values after a race were about 15 mmol×kg−1 (muscle) and l−1 (blood). Glycogen utilization was related to work intensity and was most pronounced during the first work bouts. At a speed of 12 m×sec.−1 and trotting 2000 m, there was a glycogen utilization of near 12 mmol glucose units × kg−1 × min.−1 wet muscle. It is concluded that interval training over a distance of 700–1000 m repeated 4–6 times with a trotting speed close to maximal speed (11.4–12.5 m×sec.−1) appears to be optimal. ATP; CP; blood lactate; glycogen utilization; heart rate; horse skeletal muscle; muscle lactate; racing training.     

Accuracy and perceptions of virtual microscopy compared with glass slide microscopy in cervical cytology

A. Evered and N. Dudding Accuracy and perceptions of virtual microscopy compared with glass slide microscopy in cervical cytology Objective: To evaluate virtual microscopy in terms of diagnostic performance and acceptability among practising cytologists. Methods: Twenty‐four experienced cytologists were recruited to examine 20 SurePath® cervical cytology slides by virtual microscopy. Diagnostic accuracy was compared with glass slide microscopy using an unbiased crossover experimental design. Responses were allocated a score of one for a correct identification of normal or abnormal (borderline/atypical changes in squamous or glandular cells or worse) and a score of zero for an incorrect response (a normal slide reported as abnormal or vice versa). Perceptions of virtual microscopy were assessed by questionnaire analysis. Results: Participants yielded a total of 285 responses for the virtual slide set and 300 for the glass slide set. The approximate time to screen a virtual slide was 18 minutes, compared with 8 minutes or less for a glass slide. Overall there was no significant difference between virtual microscopy and glass slide microscopy in terms of diagnostic accuracy (P = 0.22). Virtual microscopy under‐performed when images were captured over a narrow focal range (P = 0.01). Diagnostic accuracy of virtual microscopy equalled that of glass slide microscopy when participants were able to focus through the full thickness of the slide images (P = 0.07). The most common difficulties experienced by participants with virtual microscopy were freezing of the computer screen during image download, slow response of the computer during slide movement and, in some instances, ‘fuzzy’ images. Cytologists have a strong preference for glass slides over virtual microscopy despite the overall equal diagnostic performance of the two viewing modalities. Conclusions: Diagnostic accuracy of virtual microscopy can equal that of glass slide microscopy. However, without good computer network connections, wide focal range and software that permits effortless navigation across virtual slides, cytologists are unlikely to be convinced of the utility of this technology for cytology screening and diagnosis.     

Properties of behavior under different random ratio and random interval schedules: A parametric study

Four pigeons were trained to peck a key under different values of a temporally defined independent variable (T) and different probabilities of reinforcement (p). Parameter T is a fixed repeating time cycle and p the probability of reinforcement for the first response of each cycle T. Two dependent variables were used: mean response rate and mean postreinforcement pause. For all values of p a critical value for the independent variable T was found (T=1 sec) in which marked changes took place in response rate and postreinforcement pauses. Behavior typical of random ratio schedules was obtained at T

1 sec and behavior typical of random interval schedules at T

1 sec.     

Objective analysis of stallion sperm motility

An image-analysis system utilizing a microcomputer and CellSoft computer-assisted semen analysis software package was evaluated to assess stallion sperm motility characteristics. Analyses were performed at 37°C on a 6 μl drop of diluted semen placed on a glass slide and covered with an 18 mm² coverslip. Four groups of 25 cells each per slide, four slides per ejaculate and four ejaculates from each of three stallions were analyzed in a nested model. The percentage of motile sperm cells, mean velocity (μm/sec), mean linearity, and mean angular head displacement (μm) were measured. Statistical analysis of variance components showed that within ejaculates, more variation was accounted for in the differences among groups of 25 cells than among slides. Predicted standard deviations calculated for combinations of slides and groups of cells showed that a combination of two slides from which a total of 400 cells were analyzed resulted in a mean intra-assay coefficient of variation (CV) of 5.7% for the four measured variables. The following are individual coefficients of variation: percentage of motile cells (7.8%), mean velocity (6.4%), mean linearity (1.9%) and mean angular head displacement (6.6%). When ejaculate differences were included in the model and predicted standard deviations were calculated for a single ejaculate, the mean inter-assay CV was 9.2%. Mean velocity (6.4%) and mean linearity (4.7%) were more repeatable among ejaculates than either the percentage of motile sperm (14.4%) or angular head displacement (11.2%). It was concluded that this system is precise enough to determine differences in motility characteristics of stallion semen samples.