Spatula Method Research Articles

A METAGENOMIC ASSESSMENT OF BACTERIAL CONTAMINATION OF DUST EVENTS IN SENEGAL

Previous work in the Caribbean and West Africa have shown that air samples taken during dust events contain microorganisms (bacteria, fungi, viruses), including human pathogens that can cause many respiratory diseases. To better understand the potential downstream effect of bacteria dust on human health and public ecosystems, it is important to characterize the source population. In this study, we aimed to explore the bacterial populations of African dust samples collected between 2013-2017. The dust samples were collected using the spatula method, then the hypervariable regions (V3 and V4) of the 16S rRNA gene were amplified using PCR followed byMiSeq Illumina sequencing. Analysis of the sequencing data were performed using MG-RAST. At the phylum level, the proportions of Actinobacteria (22%), Firmicutes (20%), Proteobacteria (19%), and Bacteroidetes (13%) were respectively predominant in all dust samples. At the genus level, Bacillus(16%), Pseudomonas(10%), Nocardiodes and Exiguobacterium (5%) are the most dominated genera in African dust samples collected in this study.The study showed that molecular characterization of dust microbial population remains a very efficient method, also applicable to the search for viruses and fungi in this type of sample. It is important to note that the majority of microorganisms identified in this study can cause respiratory diseases.

Making large, flowable particles of protein or disaccharide in a mini-scale spray dryer

A mini-scale spray dryer, the ProCept 4M8, with a 1.4 m or 2.1 m drying chamber length has been used to prepare large, flowable particles of catalase, trehalose or lactose. A 25 kHz ultrasonic nozzle or a Rayleigh breakup mono-disperse droplet generator was used for atomization. The ultrasonic nozzle produced dried particles of average diameter ≥30 µm that show incipient flow behavior when measured with the vibrating spatula method. A high solute concentration of 69% w/w in the liquid feed was required, which is readily achievable with trehalose but not with the viscous catalase solution. At lower solute concentrations, e.g. 20% w/w, the mono-disperse droplet generator was able to produce well flowable particles of approximately 50 µm diameter, although with a low yield. This is a result of collisions between the droplets falling through the drying chamber when then coalesce. It is possible to produce dried, flowable particles in milligram quantities on a mini-scale spray dryer such as the ProCept using the 25 kHz ultrasonic nozzle. With the mono-disperse droplet generator the long drying chamber ensures a residence time of a number of seconds, but this also allows droplet coalescence at fall heights >40 cm.

COMPARACIÓN CITOLÓGICA ENTRE LAS TÉCNICAS DE ESPÁTULA DE AYRE Y CITOESCOBILLA (CYTOBRUSH) EN LA TOMA DE MUESTRAS CERVICOVAGINALES

Objetivo: Comparar la eficacia de los métodos de espátula de Ayre y la citoescobilla ("cytobrush") en la detección de lesiones premalignas y malignas del cérvix uterino. Material y Métodos: Las muestras de raspado de cérvix uterino de 140 pacientes, usando las técnicas de la espátula de Ayre y citoescobilla, fueron extendidas sobre láminas separadas y codificadas, siendo examinadas por dos diferentes patólogos que desconocían el método de recolección de la muestra. Se usó el sistema de Bethesda para el diagnóstico. Resultados: De las muestras obtenidas, 65% vs. 35% tuvieron células endocervicales con el método de la citoescobilla y la espátula de Ayre, respectivamente. Al analizar la cantidad de células endocervicales, las muestras obtenidas por el método de citoescobilla tuvieron más de cinco grupos de células endocervicales, 71,4% vs. 36,4% con la espátula de Ayre. Conclusión: A pesar que el método de la espátula de Ayre es eficiente para la obtención de células endocervicales, el método de la citoescobilla dio mejores resultados en nuestro estudio.

Cryotolerance of Day 2 or Day 6 in vitro produced ovine embryos after vitrification by Cryotop or Spatula methods

This study was conducted to evaluate the cryotolerance of in vitro produced ovine embryos submitted to vitrification at different developmental stages using two methods of minimum volume and rapid cooling rate. Embryos were vitrified at early stage (2 to 8-cells) on Day 2 or at advanced stage (morulae and blastocysts) on Day 6 after in vitro fertilization. Vitrification procedure consisted of the Cryotop (Day 2, n=165; Day 6, n=174) or the Spatula method (Day 2, n=165; Day 6, n=175). Non vitrified embryos were maintained in in vitro culture as a control group (n=408). Embryo survival was determined at 3h and 24h after warming, development and hatching rates were evaluated on Day 6 and Day 8 after fertilization, and total cell number was determined on expanded blastocysts. Embryo survival at 24h after warming increased as the developmental stage progressed (P<0.05) and was not affected by the vitrification method. The ability for hatching of survived embryos was not affected by the stage of the embryos at vitrification or by the vitrification method. Thus, the proportion of hatching from vitrified embryos was determined by the survival rate and was lower for Day 2 than Day 6 vitrified embryos. The percentage of blastocysts on Day 8 was lower for the embryos vitrified on Day 2 than Day 6 (P<0.05), and was lower for both days of vitrification than for non-vitrified embryos (P<0.05). No interaction of embryo stage by vitrification method was found (P=NS) and no significant difference was found in the blastocyst cell number among vitrified and non-vitrified embryos. In conclusion, both methods using minimum volume and ultra-rapid cooling rate allow acceptable survival and development rates in Day 2 and Day 6 in vitro produced embryos in sheep. Even though early stage embryos showed lower cryotolerance, those embryos that survive the vitrification–warming process show high development and hatching rates, similar to vitrification of morulae or blastocysts.

Morphological differentiation between S. mutans and S. sobrinus on modified SB-20 culture medium

Due to the major role of Streptococcus mutans and Streptococcus sobrinus in the etiology of dental caries, it is important to use culture media that allow for differentiating these bacterial species. The aim of this study was to evaluate the suitability of a modified SB-20 culture medium (SB-20M) for the isolation and morphological differentiation of S. mutans and S. sobrinus, compared to biochemical identification (biotyping). Saliva samples were collected using the spatula method from 145 children, seeded on plates containing the SB-20M, in which sucrose was replaced by coarse granular cane sugar, and incubated in microaerophilia at 37 °C during 72 h. Identification of the microorganisms was performed under stereomicroscopy based on colony morphology of 4904 colonies. The morphological identification was examined by biochemical tests of 94 randomly selected colonies with the macroscopic characteristic of S. mutans and S. sobrinus using sugar fermentation, resistance to bacitracin and production of hydrogen peroxide. There was no statistically significant difference ( p>0.05) between morphological identification in the SB-20M medium and biochemical identification (biotyping). Biotyping confirmed that S. mutans and S. sobrinus colonies were correctly characterized in the SB-20M in 95.8% and 95.5% of the cases, respectively. Of the mutans streptococci detected in the children 98% were S. mutans and 2% S. sobrinus. The SB-20M medium is reliable for detection and direct morphological identification of S. mutans and S. sobrinus.

Teat tablet with sodium ibuprofen: the effect of sorbitol on therapeutic agent pharmaceutical availability and on physicochemical parameters of a drug form

Abstract Dosing pediatric drugs available on pharmaceutical markets of analgesic, antifebrile and anti-inflammatory activity to children up to 3 years of age is not precise and frequently causes problems. The aim of the study was to work out a pediatric teat form of a drug with sodium ibuprofen and to determine the effect of sorbitol content on pharmaceutical availability of the therapeutic agent. Three variants of tablets containing 50 mg of sodium ibuprofen differing in the percentage content of sorbitol (from 37% - batch I to 79% - batch III) were produced. Quality tests of the produced forms of drugs (PPVI) were performed and the tests of therapeutic agent pharmaceutical availability by spatula method and by a method with a teat. Tablets of all batches had a smooth surface and same shape. The content of the therapeutic agent was within the limit of 95-105% of the declared value. The highest value of Q coefficient in the release test by pharmacopeal method and the shortest disintegration time (12 minutes) were obtained for tablets with 79% content of sorbitol. In conclusion, teat tablets with sodium ibuprofen of the highest sorbitol content (79%) demonstrated the expected physicochemical parameters and high pharmaceutical availability.

Salivary mutans streptococci counts as indicators in caries risk assessment in 6–7-year-old Chinese children

This study aimed to determine whether salivary mutans streptococci (MS) counts in Chinese children had any value in the prediction of new caries in the permanent dentition in the age interval of 6.5-8.5 years. Four hundred and thirty-three 6-7-year-old children participated in this 2-year longitudinal study. Salivary MS counts were obtained at baseline with the spatula method. Dentin and enamel caries was recorded at baseline and at the follow-up, 2 years later. Spearman's correlation coefficients were computed. Past caries experience variables at baseline were entered into a multiple regression model with caries increment of the permanent dentition as dependent variable. MS counts were subsequently entered into the model to assess the additional caries predictive value. Spearman's correlation coefficient for MS counts and caries experience of the primary dentition at baseline was 0.48 and for MS counts and caries increment in the permanent dentition 0.12. In the multiple regression model caries increment of the permanent dentition had an adjusted R2 of 0.17 when the past caries experience variables were entered. Adding MS counts in the regression model did not change the R2 value. Salivary MS counts did not add any caries predictive value when past caries experience variables were used as caries predictors in this Chinese child population.

The relationship between sweetness preference, levels of salivary mutans streptococci and caries experience in Brazilian pre-school children.

To analyse the relationship between preference for sweetness, levels of salivary mutans streptococci and levels of caries in low socio-economic 4-5-year-old Brazilian children. A cross-sectional survey. 298 children of both sexes, who, in 1998, were regularly attending public nurseries were randomly selected. Caries experience was assessed according to WHO guidelines. Saliva samples were analysed for mutans streptococci using the spatula method. Children's preference for sugar was measured using the 'Sweet Preference Inventory'. Personal interviews with the mothers were conducted. 255 children completed all aspects of the research, a response rate of 85.6%. 34.9% of them were caries-free. The mean dmf-s was 4.25 (SD 6.16), the decayed component being 3.56 (SD 5.82) and the filled surfaces, 0.69 (SD 1.85). For these pre-school children; male sex (P < 0.01), single parent (P < 0.01), habit of eating or drinking items that contained non-milk extrinsic sugar between meal times (P < 0.05) and high levels of salivary mutans streptococci (P < 0.001) were significantly associated with higher dmfs scores. There was no statistical relationship between sweetness preference and dental caries and with mutans streptococci levels. Levels of salivary mutans streptococci but not sweetness preference were potential good predictors of caries experience among 4-5 year-old-children living in urban area of Brazil.

Simplified sampling methods for estimating levels of lactobacilli in saliva in dental clinical practice

The aim of the present study was to evaluate whether estimation of lactobacilli was possible with simplified saliva sampling methods. Dentocult® LB (Orion Diagnostica AB, Trosa, Sweden) was used to estimate the number of lactobacilli in saliva sampled by 3 different methods from 96 individuals: (i) Collecting and pouring stimulated saliva over a Dentocult® dip-slide; (ii) direct licking of the Dentocult® LB dip-slide; (iii) contaminating a wooden spatula with saliva and pressing against the Dentocult® dip-slide. The first method was in accordance with the manufacturer's instructions and selected as the 'gold standard'; the other 2 methods were compared with this result. The 2 simplified methods for estimating levels of lactobacilli in saliva showed good reliability and specificity. Sensitivity, defined as the ability to detect individuals with a high number of lactabacilli in saliva, was sufficient for the licking method (85%), but significantly reduced for the wooden spatula method (52%).

REACTIONS OF DOCTORS TO VARIOUS FORMS OF FEEDBACK DESIGNED TO IMPROVE SAMPLING QUALITY OF CERVICAL SMEARS

Three different forms of feedback designed to improve the sampling quality of cervical smears were tested in a randomized controlled trial among 179 physicians. The reactions of the doctors to the various interventions are reported as they appeared from their answers in a questionnaire. The influence of the interventions on the employed sampling technique is described on the basis of their answers to a telephone inquiry. The inclusion in laboratory protocols of an evaluation of the sampling quality of the submitted smears was largely appreciated, as was specific advice offered on indication. Monthly overviews comparing the performance of the individual doctor to that of his peers were esteemed by three out of four. Further information, help or training was requested more frequently than average by physicians obtaining inadequately sampled smears relatively often. The number of doctors using the combined spatula and Cytobrush method as sampling technique doubled during the study period.

Human leukocyte antigens in relation to colonization by mutans streptococci in the oral cavity

Mutans streptococci are well established as caries-inducing microorganisms in man. Most humans carry the bacteria, but in highly different numbers. This cannot be explained by environmental factors only. The aim of this study was to investigate a possible association between levels of colonization by mutans streptococci and the presence of certain B and DR human leukocyte antigens (HLA). Altogether, 170 subjects who had their HLA antigens determined (76 renal transplant patients and 94 healthy blood donors) were selected for the investigation. Paraffin-stimulated saliva samples were taken using the wooden spatula method with subsequent cultivation of mutans streptococci on mitis salivarius bacitracin agar plates. An association between the absence of HLA-DR 4 antigens and low, or undetectable, levels of mutans streptococci was found. This was statistically significant for the immunosuppressed renal transplant subjects. The same trend was observed among the healthy blood donors.

Caries prevalence and severity in the primary dentition and Streptococcus mutans levels in the saliva of preschoolchildren in South Africa.

The caries prevalence (dmft) and severity (css) in 3-, 4-, and 5-yr-old children in South Africa were examined. Four levels of S. mutans in the saliva of 228 of these children was estimated by the spatula method. In all three age groups a statistically higher dmft and css was found with a S. mutans level of greater than 10(6) CFU/ml in saliva than at lower concentrations. At age 5 ANOVA showed significant differences between dmft and css at all S. mutans levels. A high concentration of S. mutans in the saliva may be an indicator of the high risk child at an early age.

Streptococcus mutans and dental caries in urban and rural schoolchildren in Thailand.

Two groups of Thai schoolchildren aged 11-13 yr were examined for dental caries, dental fluorosis and Streptococcus mutans. One hundred children lived in an urban district, Bangkok, and 71 children in a rural district, Petchaboon. Saliva samples were analyzed for S. mutans by the spatula method. The fluoride content of the drinking waters was also determined. The prevalence of caries, diagnosed according to WHO, was 89% in Bangkok and 18% in the rural district. Mean DMFT was 3.46 and 0.38 respectively. S. mutans was found in 98% of the urban children and in 82% of the rural. The differences in distribution between S. mutans and DMFT classes were statistically significant for the total sample. Mild fluorosis was present in some of the rural children.

Caries prevalence, salivary Streptococcus mutans and dietary scores in 13-year-old Swedish schoolchildren.

388 13-yr-old schoolchildren from Karlstad, Sweden, participated. Dental caries experience was expressed as number of decayed (D) and filled (F) tooth surfaces (S), and registered on bite-wing radiographs and obtained from records. Salivary Streptococcus mutans counts were determined by a spatula method. A dietary score was calculated for each individual based on an interview concerning the intake frequency of 23 sugar-containing products. Significantly lower DFS values were found in the group with no detectable S. mutans compared to three of the four groups with salivary S. mutans (P less than 0.01). No differences were found between the DFS values of individuals with high, moderate or low dietary scores. There was no statistically significant association between dietary scores and levels of S. mutans. Among the children with no detectable S. mutans, there were higher DFS-values with increasing intake frequency. No relationship between initial caries lesions, S. mutans and diet was found.

Corneal brush cytology: an experimental study.

A total of 48 corneal smears from rabbits were obtained by means of brush, cotton swab and spatula techniques. The cytomorphologic features in those methods are compared. In the smears prepared by brush, numerous corneal epithelial cells were found. The cells were well spread in a single layer and overlapping was uncommon. The morphology of the corneal cells was satisfactory for cytologic evaluation. In contrast, the other two methods lacked adequate numbers of cells and cell preservation. We feel that corneal epithelial cells are readily obtainable by the brush method, compared with the cotton swab and spatula methods, and the material is also ideally suited for cytologic evaluation. It can be easily used for corneal cytology in clinical situations.

Cytological Studies of Cell Sampling Methods in Borderline Lesions and Early Cancer of the Uterine Cervix

Cytological studies were performed for the purpose of comparing two kinds of smear preparations obtained by a cotton-bud and a spatula from the uterine cervix. The spatula scraping method was proved to be superior to the cotton-bud method in accurate detection, especially in the cytological diagnosis of dysplasia, while the spatula method is apt to give cytological specimen suggesting more advanced lesions than they really are, or apt to overdiagnose. Smears obtained by a spatula indicated smaller size of cells and higher nuclear cytoplasmic ratio than those by a cotton-bud. These contrasts come from the fact that a spatula scrapes epithelial cells from relatively deeper layers than a cotton-bud. Hence different criteria of morphological identification is required in the diagnosis of smear preparations by these two methods.

Practical method to facilitate estimation of Streptococcus mutans levels in saliva

A method was developed to facilitate the estimation of Streptococcus mutans levels in saliva. Saliva-contaminated wodden spatulas were pressed directly against an elevated agar plate containing a selective medium. The results were compared with the number of S. mutans per 1 ml of paraffin-stimulated saliva. It was shown that the spatula method gave a good estimation of the level of S. mutans infection. The incubation was also made in expired air instead of 95% N2-5% CO2. The outgrowth was in good agreement with that after conventional incubation. The method is useful in epidemiological studies or in selecting persons at a high caries risk, and when ordinary saliva sampling cannot be done, for example in small children. Compared with conventional saliva sampling, this method requires less time and material at sampling as well as at the laboratory.

Collection of cytological specimens with a cell aspirator with reference to the wooden spatula method: I. A cell aspirator for collecting cytological specimens

In order to make an exfoliative cytological diagnosis, the specimens must contain enough cells for examination. To achieve this, a cell aspirator has been designed consisting of a disposable syringe connected to a vacuum system and equipped with a cannula. When taking the specimen, physiological saline is applied drop by drop and the fluid is aspirated into the syringe together with the material from the lesion. This suspension is fixed and centrifuged, and the material in the pellet is then transferred to a glass slide for staining and diagnosis. This cell aspirator technique has produced specimens containing a large number of cells, which is a necessary condition for a reliable cytological diagnosis. Also in this article an account is given of the results of a comparative histopathologic study of areas of the oral mucosa in which cytological specimens had been collected with a wooden spatula and with a cell aspirator. To judge from the histopathologic picture, the wooden spatula collected superficial epithelial cells with compresion of the epithelium in the area of collection and leveling out of the cells in the direction of movement of the spatula. The cell aspirator was found to collect cells from most layers of the epithelium and in a sharply circumscribed portion of the mucosal change. No subepithelial damage was observed with the two methods of collection.

Collection of cytological specimens with a cell aspirator with reference to the wooden spatula method: II. Comparative quantitative and qualitative studies

This article describes the results of comparative quantitative and qualitative examinations of specimens obtained by testing 65 individuals by means of a cell aspirator and a wooden spatula. The studies were performed on a clinically normal mucosa as well as on different mucosal lesions. A statistical analysis of the cells obtained showed that the cell aspirator quantitatively produced significantly better results both with regard to the total number of cells obtained and to the number of epithelial cells in the different stages of maturity. The number of cells obtained turned out to be dependent on surface structure and the size of the lesion, however, Qualitatively the cell aspirator collected epithelial cells which were better preserved morphologically, but also a larger number of compact cell clusters. The number of erythrocytes was low. The structure of these blood cells was well preserved, indicating bleeding arising in connection with the taking of the specimens. The number of leucocytes was larger in the aspirated material. The degrees of fixation and staining were similar. The authors make recommendations for using the cell aspirator in such a manner as to obtain the best qualitative results.

Statistical analysis of uterine cancer screenings test results in Fukushima Prefecture

Cytologic smears for uterine cancer screening tests in Fukushima Prefecture in 1972 were obtained by scraping with a cotton swab and in 1973 with a (Tohoku University-type) spatula. The results of the cytologic examinations were subjected to statistical analysis in order to determine whether there was any significant difference between the two methods in regard to the detection of atypical epithelial cells and uterine cancer.The results were as follow:1) No significant difference (5% level of signi ficance) between the two methods was found in the rate of detection of uterine cancer.2) The rate of detection of cervical dysplasia was significantly higher with the spatula method than the cotton swab method (x2c=25.4128<p=0.001, 0.1% level of significance).3) The rate of detection of preinvasive cancer (carcinoma in situ) also was higher with the spatula method (2% level of significance).4) There was a proportionately higher number of smears obtained by the spatula method falling in the Class II and Class III categories. A comparison between the two years of the relative rates of negative smears (Class I or II) and those indicating highly suspicious atypical or malignant cells (Classes III, IV or V) revealed a significant difference (x2c=168.41945>p=0.001, 0.1% level of singnificance) which would indicate greater reliability of the spatula method in the detection of malignancy.5) A significantly higher rate of malignancies was found in smears showing Class III cytologic findings among those obtained with the cotton swab method than those obtained by the spatula method (x2c36.9744>p=0.001, 0.1% level of significance). This would indicate the need for great care in searching for malignant changes when the cotton swab method is utilized.6) Judging from the results obtained when the two methods were utilized for the same patient on separate occasions within a one-month period, the spatula method was better suited for the detection of atypical cells and the possible presence of preinvasive lesions, providing a more accurate assessment of the histological changes.7) It is considered, on the basis of these observations, that the spatula method of obtaining specimens for cytologic examination is superior to the cotton swab abrasion method for screening tests.