Beurteilung von nativen und aufgetauten Spermatozoen fertiler und subfertiler Hengste mit Hilfe der Phasenkontrast- und Transmissionselektronenmikroskopie. Institut für Veterinär-Pathologie der Veterinärmedizinischen Fakultät, Universität Leipzig Reproduktionsmedizinische Einheit der Kliniken der Tierärztlichen Hochschule Hannover In dieser Arbeit wurde die Ultrastruktur von nativen und tiefgefrorenen Spermien mittels Phasenkontrast- und Transmissionselektronenmikroskopie (TEM) untersucht. Für die Beurteilung der Spermienmotilität und der Morphologie von in Formolzitrat fixierten Spermien standen jeweils drei Ejakulate von 50 Hannoveraner Hengsten des Niedersächsischen Landgestüts Celle zur Verfügung. Aus dieser Gruppe wurden drei fertile, drei subfertile Hengste und 6 Hengste mittlerer Fertilität ausgewählt, von denen sowohl die Nativ-Proben als auch eine Tiefgefrierprobe (TG-Probe) für die TEM im Institut für Veterinär-Pathologie der Universität Leipzig gemäß des Standardprotokolls des Institutes aufbereitet wurden. Die Spermien wurden gewaschen und das Seminalplasma der nativen Proben oder der Verdünner der TG-Proben abpipettiert und durch eine 5%-ige Glutaraldehydlösung in einem 0,1 M Kakodylatpuffer (pH 7,2) ersetzt. Die Fixierungslösung wurde anschließend entfernt und das Pellet gewaschen und danach mit Gelatine gemischt. Die spermienreichen Stellen wurden aus der Gelatine herausgeschnitten und in Glutaraldehyd aufbewahrt. Nach einer Nachfixierung in OsO4 und einer Entwässerung in Ethanollösungen erfolgte eine Einbettung in einer Eponmischung. Nach einer Polymerisation von 5 Tagen wurden die eingebetteten Eponblöckchen angetrimmt und die Semi- und Ultradünnschnitte angefertigt. Die Ultradünnschnitte wurden auf ein Kupfergrid gelegt, mit Uranylazetat und Bleizitrat kontrastiert und mit dem Transmissionselektronenmikroskop (Zeiss EM 900, Oberkochem) bei 80 kV analysiert. In den nativen Proben wurden insgesamt 360 Spermien pro Hengst beurteilt, in den TG-Proben 120 Spermien pro Hengst. Die Qualität der elektronenmikroskopischen Aufnahmen war sehr gut, doch die Plasmamembran zeigte fixierungsbedingte Artefakte. Nach dem Auftauen waren die Bilder heller und der Kontrast etwas geringer. Es gab eine Zunahme an Akrosomdefekten, akrosomreagierten Spermien und Beschädigungen der Plasmamembran, der Mitochondrien, sowie der Mantel- und Ringfasern. Durch die Membranbeschädigungen trat auch eine Verringerung der Anzahl proximaler und distaler Zytoplasmatropfen auf. Sowohl geschwollene Akrosome mit einer niedrigeren Dichte der akrosomalen Matrix als auch Mitochondrien mit einer zu hellen mitochondrialen Matrix waren typische Befunde in den TG-Proben. Die Studie der Ultrastruktur und die wahrgenommenen Defekte führten zur Erstellung eines Standardprotokolls für die transmissionselektronenmikroskopische Beurteilung von Hengstspermien. Die Beurteilung mittels TEM sollte aber nicht zu einer quantitativen, sondern zu einer qualitativen Aussage führen. Sie ermöglicht die Diagnose von Kern- (Kerndeformationen und Taschenbildung im Kern) und Akrosomabweichungen (deformierte Akrosome mit oder ohne Vakuolenbildung, abgehobene Akrosome und akrosomreagierte Spermien), Anomalien der Mitochondrien (Unterbrechung der Mitochondrienscheide, zu viele Mitochondrien, anormale Dichte der mitochondrialen Matrix), Defekten des Axonemas (Ordnung oder Anzahl der Mikrotubuli, Mantel- und Ringfasern) und der Anwesenheit immaturer Spermienvorstufen. Diese Methode eignet sich für die Diagnostik subfertiler Hengste mit normalen Spermienparametern bei der routinemäßige Spermienbeurteilung und kann sowohl in nativen als auch in TG-Proben angewendet werden. Im Vergleich zur Phasenkontrastmikroskopie waren die elektronenmikroskopischen Bilder wegen ihrer stärkeren Vergrößerung und der Darstellung innerer Spermienstrukturen viel aussagekräftiger. Für die Beurteilung von Halsansatzdefekten, abweichende Geißelformen und Mehrfachmißbildungen ist die Phasenkontrastmikroskopie die am besten geeignete Methode. / Evaluation of fresh and frozen-thawed semen samples of fertile and subfertile stallions by light microscopy and transmission electron microscopy. Institut of Pathology of the Faculty of Veterinary Medicine, University of Leipzig Reproduktionsmedizinische Einheit der Kliniken der Tierärztlichen Hochschule Hannover In this study the ultrastructure of fresh and frozen-thawed semen samples of 50 stallions from the National Stud of Lower Saxony (Celle, Germany) were evaluated by light microscopy and transmission electron microscopy (TEM). Three ejaculates of each stallion were available for the motility analysis and the morphological analysis by lightmicroscopy after fixation in formol citrate. Based on the fertility data, the ejaculates of 12 stallions (3 fertile stallions, 3 subfertile stallions and 6 stallions of average fertility) were selected for the morphological analysis by TEM. The native samples and one frozen-thawed sample from these stallions were prepared for the TEM at the Institute of Pathology of the Faculty of Veterinary Medicine, Uni-versity of Leipzig. The sperm cells were washed and the seminal plasma from the native samples and the diluents of the frozen-thawed samples were replaced by a 5%-glutaraldehyde solution in a 0,1 M cacodylate buffer pH 7,2. The fixative was removed, the pellet was washed again and mixed with gelatin. The sperm rich fraction in the gelatin mass was excised and stored in glutaraldehyde. A second fixation in OsO4 was followed by a dehydratation in ethanol and a polymerization phase in epon. After 5 days of polymerization the starred samples were used for semi- and ultratight cuts. The latter were placed on a copper grid, contrasted with uranyl acetate and lead citrate and analyzed with the transmission electron micro-scope (EM 900) by 80 kV. In the fresh samples, 360 sperm cells were examined per stallion, whereas in the frozen-thawed samples only 120 sperm cells per stallion were evaluated. The microscopic pictures were of a high quality. However, the sperm plasma membrane showed some fixation artifacts. In the thawed samples a lower contrast was noticed than in the fresh samples. The sperm cells in the frozen-thawed samples showed an increase in acrosome defects, acrosome reactions, damage of the cell plasma membrane, mitochondria, fibrous sheet and outer dense fibers. The latter defect was associated with a decrease in proximal and distal cytoplasmatic droplets. Swollen acrosomes with a lower matrix density and a bright mitochondrial matrix were typically present in the cryopreserved samples. The ultrastructural defects in these samples, examined by TEM, have led to the development of a standard evaluation protocol with the most common sperm defects in stallion semen. TEM is an expensive and time consuming technique, which cannot be used to obtain quantitative results, but is considered as an accurate method for the qualitative examination of semen samples in cases of unexplained subfertility. TEM can especially be recommended for the diagnosis of nuclear (nuclear malformations and pouches) and acrosomal defects (acrosome deformations, acrosome vacuoles, detached acrosomes and acrosome reactions), mitochondrial (mitochondrial sheet defects, mitochondrial proliferation, decrease in mitochondrial matrix density) and axonema malformations (anormal position or quantity of microtubules and fibrous sheet or outer dense fibers defects) and the detection of immature sperm cells in ejaculates. The results of this study state that TEM can be useful for the evaluation of both fresh and frozen-thawed semen samples. Compared to the light microscopic evaluation of stallion sperm, the TEM images give more precise information because of their higher magnification rate and the ability to reveal internal sperm structures. However, light microscopy remains the best method to detect sperm neck defects, deformed tailes and sperm cells with multiple heads or tails.

Orientadores: Antonio Jose de Almeida Meirelles, Mariana Conceição da Costa / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-15T07:21:08Z (GMT). No. of bitstreams: 1 Carareto_NataliaDanieleDorighello_M.pdf: 5550198 bytes, checksum: e2d9b70cc0db776a83763af5ef082ed7 (MD5) Previous issue date: 2010 / Resumo: Álcoois graxos são constituintes minoritários de tecidos vegetais, sendo encontrados principalmente em ceras. São utilizados na indústria como emulsificantes, surfactantes ou emolientes. Atualmente, existem muitos estudos sobre o uso de álcoois graxos associados a ácidos graxos como substitutos do processo de hidrogenação para a fabricação de margarinas e outras emulsões. O conhecimento do equilíbrio sólido-líquido de misturas binárias de álcoois graxos é uma ferramenta importante para se entender o comportamento de misturas mais complexas. Assim, neste trabalho se objetivou construir diagramas de equilíbrio sólido-líquido de misturas binárias de álcoois graxos saturados pares contendo de oito a dezoito átomos na cadeia carbônica, a partir da calorimetria Exploratória Diferencial, com método experimental previamente utilizado por Rolemberg (2002) e Costa (2004) para ácidos graxos e triacilgliceróis. Para complementar os resultados também foram capturadas imagens de microscopia óptica com controle de temperatura. Os sistemas aqui estudados apresentaram três comportamentos diferentes da linha liquidus: no primeiro grupo estão os sistemas que apresentaram apenas o ponto eutético, no segundo aqueles com ponto eutético e ponto peritético e o último grupo exibe nenhum desses pontos. Para os sistemas que apresentaram ponto peritético, pode-se observar nas imagens de microscopia a recristalização de algumas amostras com o aumento de temperatura, o que evidencia a ocorrência da reação metatética para tais sistemas. Foram utilizados os modelos termodinâmicos para coeficiente de atividade Margules 2 e 3-sufixos e NRTL para ajuste dos dados experimentais dos sistemas que apresentaram apenas ponto eutético simples e ponto eutético e peritético e obteve-se um valor de DQM satisfatório para todos os casos. Já para o modelo preditivo UNIFAC original, os desvios com relação aos valores experimentais foram altos, o que se supõe ser proveniente de simplificações, como a hipótese de uma fase sólida pura, o que neste trabalho se mostrou inadequado / Abstract: Fatty alcohols are minor constituents of vegetal tissues, being mainly found in vegetal waxes. Fatty alcohols are used in industry as emulsifiers, surfactants or emollients agents. There are many studies about the use of fatty alcohol associated with fatty acids as substitutes for the hydrogenation process of margarine manufacturing and other edible emulsions, for example. Solid-liquid equilibrium of binary mixtures of fatty alcohols is na important tool for understanding the behavior of fatty complex mixtures. Thus, this work aimed to study diagrams of solid-liquid equilibrium of binary systems of even saturated fatty alcohols from eight to eighteen atoms in the carbon chain by differential scanning calorimetry (DSC). This method was previously used by Rolemberg (2002) and Costa et al. (2007a, 2009a, 2009b, 2009c) for fatty acids and triacylglycerols systems. To complement the study were also captured images for some systems by optical microscope with temperature control. The liquidus line of the studied systems showed three different behaviors: one showed only the eutectic point, another the eutectic and peritectic points or neither eutectic nor peritectic point. For systems that had peritectic point, it could be observed in the microscopy images the recrystallization of the compositions that was investigated on the microscopy, even with the temperature increase. This behavior corroborates the existence of the metatetic reaction in these systems. Margules-2-suffix, Margules-3-suffix, NRTL, UNIFAC and UNIFAC-Dortmund models were applied to fit the experimental data. The best fit for the systems that exhibit peritectic and eutectic points were obtained through Margules and NRTL models. UNIFAC and UNIFAC-Dortmund models have showed high deviation compared to experimental data of the modeled systems / Mestrado / Mestre em Engenharia de Alimentos

A new design of closely intermeshing co-rotating twin-screw compounding extruder, developed at Brunel University, has been utilized in the development of quantitative techniques for characterization of dispersive and distributive mixing in thermoplastics materials prepared by extrusion compounding. Image analysis procedures were used to quantify mixing of polypropylene composites containing calcium carbonate filler using reflected light microscopy on polished surfaces, and transmitted light microscopy of microtomed pigmented sections. Stereological statistics have been applied to raw sample data; results are discussed in relation to mechanistic phenomena influencing particle agglomeration, dispersion and distribution of fillers in thermoplastics. Dispersive or intensive mixing determined from calcium carbonate filled polypropylene specimens showed that processing parameters had no significant influence except when filler was added midway along the machine although the melting zone was highlighted as having a marked effect on the rate of filler dispersion. Premixing of filler and polymer introduced additional agglomeration into the filler. A series of model experiments were undertaken to assess the influence of specific parameters. In this context moisture content emerged as having the single most important effect on filler compaction. Distributive or extensive mixing of carbon black pigmented specimens was very significantly affected by the presence of segmented disc elements at the end of the screws. These elements produced more than a six-fold increase in distributive mixing in the extrudate.

The content of this study is focused on the influence of the structure of wood at microscopic and submicroscopic level on its mechanical properties. The wood cell wall consists of several layers, the dominant layer being layer S2, which occupies up to 80 % of the total thickness of the wood cell wall. Unique feature of this layer is that cellulose microfibrils placed in this layer are highly aligned and spirally wound around the cell axis. The inclination of these microfibrils is called microfibril angle (MFA) and is the key feature that affects mechanical properties of wood and its shrinkage. In theoretical part of this thesis methods for measuring microfibril angle are described. A method for measuring mechanical properties of the wood cell wall called nanoindentation is discussed in detail. In the practical part of this thesis, microfibril angle is measured by means of polarized light microscopy and mechanical properties of wood cell wall is determined by means of nanoindentation.

The urinary bladder is especially subject to infection by virtue of its direct connection to the external urethral opening, and it is natural to anticipate the presence of a well-developed immunological mechanism to respond to this potential threat. The present study describes small, very highly vascular lymph nodes located in the wall of the rabbit bladder, which may be involved in a local response to foreign antigens. The vasculature and structure of these lymph nodes was described using a combination of vascular corrosion casting, ink injection, and light and electron microscopy. The distal abdominal aorta was cannulated, and after clearing the bladder vasculature with buffered saline, one of the following procedures was used: 1) the bladder was perfuse-fixed in preparation for light and electron microscopy; 2) the bladder vasculature was filled with India ink for vessel tracing; or 3) vascular corrosion casts of the vasculature were prepared by infusing resin comprised of a mixture of Mercox, methyl methacrylate monomer, and catalyst. The resulting casts were cleaned with KOH, formic acid, and water in preparation for scanning electron microscopy. Vascular casts and India ink injections revealed the presence of a number of isolated capillary tufts consisting of clusters of one to five 'glomeruli,' closely associated with the major vesicular vessels along the lateral walls of the bladder, and supplied by tertiary branches of these vessels. Light and electron microscopy showed that the capillary tufts represented the blood supply to small, ovoid lymph nodes located near the serosal surface of the bladder wall and usually restricted to the basal half of the bladder. These nodes were encapsulated and exhibited subcapsular sinuses, numerous small blood vessels, a limited number of high endothelial cells, and, occasionally, nerves and a follicular substructure. The nodes contained abundant lymphocytes, stellate stromal cells, macrophages, and eosinophils, but lacked the obvious cortical and medullary organization and germinal centers often seen in larger lymph nodes. Vascular corrosion casts, vascular ink injections, and microscopic examination confirmed the presence of small, highly vascular lymph nodes closely associated with the main vesicular vessels along the lateral walls of the rabbit bladder. A follicular substructure of the nodes appears to correspond with the 'glomerular' capillary arrangement within the nodes as seen with corrosion casts. The rich blood supply may be indicative of the high metabolic demand of lymphatic tissue, and may be altered in response to the level of activity of the node. The close association between the lymphatic tissue and the rich blood supply to the nodes may allow a rapid mobilization of lymphocytes during a local immune response to foreign agents.

Condoms have been used in sexual assaults as a means of preventing the transmission of biological fluids. Current sexual assault evidence collection kit processing protocols do not regularly take advantage of the information that can be gathered by examining residues left by condoms during intercourse. A biphasic liquid-liquid extraction technique was developed to separate polar and non-polar condom residues, which had been collected on cotton tipped swabs. This research involved the examination of twenty condom brands by Diffuse Reflectance Infrared Fourier Transform Spectroscopy. Five brands were selected to examine the consistency of this technique when the lubricants were exposed to body and storage temperature conditions for various times and in the presence of oral, vaginal, and blood samples. Additionally, starches collected from the condoms under each of the above conditions were examined. Although all lubricants were identifiable using this IR technique, the nonoxynol-9 (spermicide) containing samples produced spectra which were not identical to those produced by nonoxynol-9 standards. Although there was a decrease in the percent transmittance within IR spectra as the time between the collection and the extraction of the swabs increased, the condom residues of interest remained identifiable at all time points examined. The use of vaginal and oral swabs in the collection caused a negligible amount of background interference, which could be eliminated through spectral subtraction of the swab.

No description available.

This thesis investigates the diversity of two silica-scaled protist groups, Paraphysomonadida and Thaumatomonadida by light and electron microscopical observations and sequencing (rDNA) on novel clonal cultures. Despite these groups of protist dominating pelagic, littoral as well as inland freshwater and soil habitats, they are taxonomically poorly understood to the extent that any progress in ecological theory is hampered. Now that environmental DNA sequencing is being carried out faster than we can characterise protists from culture it is important that we understand how molecular and physical diversity match up, especially because so many protists are morphospecies. Nearly one hundred isolates were cultured on which both morphological and molecular data was carried out in parallel to reveal around 50 new species of protist from eight different genera: two heterokont genera, Paraphysomonas and Incisomonas n. gen., and six cercozoan genera, Thaumatomonas, Allas, Reckertia, Thaumatospina n. gen., Cowlomonas n. gen., and Scutellomonas n. gen. These data make major contributions to taxonomy and understanding aspects of protist diversity where previously morphological diversity was heavily biased towards over- generalized morphotypes. This thesis quickly showed that gross lumping of morphospecies was true of Paraphysomonas, for which many of the isolates cultured herein might have been regarded as one species (not more than 20). The many cultured isolates exhibited varied cell and scale morphology, and by sequencing (rDNA), it was possible to see the evolution of scale morphology map on to trees. This marriage of molecular and morphological data made it possible to view distinct groups of species that shared scale detail that might have otherwise been overlooked had either method been used alone. This research has shed significant light on how scale morphology can be used as reliable taxonomic marker for protists, the insights of which can be applied to make taxonomic improvements to other silica-scaled protist groups.

A proposta deste estudo in vitro foi otimizar o método microbiológico para a produção de lesão de mancha branca em esmalte que possa ser validada por ensaio não destrutivo (tomografia por coerência óptica - OCT) para possibilitar a utilização posterior dos espécimes em outros experimentos. Foram obtidos 168 fragmentos retangulares de esmalte bovino com janelas centrais de desmineralização de 3,0 x 3,0 mm. Os grupos experimentais foram compostos a partir de 3 fatores de variação: microrganismo (S. mutans UA 159, S. sobrinus 3347 e S. mutans + S. sobrinus), fonte de carboidrato (sacarose 1% e sacarose 1% + amido 1%) e tempo (1, 2, 3, 4, 5, 6 e 7 dias). Assim, formaram-se seis grupos experimentais: G1 (S. mutans + sacarose), G2 (S. mutans + sacarose + amido), G3 (S. sobrinus + sacarose), G4 (S. sobrinus + sacarose + amido), G5 (S. mutans + S. sobrinus + sacarose), G6 (S. mutans + S. sobrinus + sacarose + amido), testados em 7 tempos de desafio cariogênico. Terminada esta etapa, foram obtidas imagens em escala de cada espécime pelo OCT, e em seguida os espécimes foram processados e submetidos à análise através de microscópio de luz polarizada. Em cada um dos métodos, foram realizadas 5 mensurações de profundidade da região desmineralizada. O teste estatístico de Análise de Variância (ANOVA) (p<0.05) detectou que os desafios cariogênicos testados foram capazes de desmineralizar o esmalte em profundidade, sendo influenciados pelo tipo de microrganismo, fonte de carboidrato e tempo (p=0,000). O Teste de Correlação de Pearson apresentou uma correlação significativa (p=0,000) entre as medidas de profundidade de desmineralização aferidas através dos métodos de luz polarizada e OCT. Logo, a tomografia por coerência óptica é um método não destrutivo válido para aferir a profundidade de desmineralização de lesões de mancha branca, que pode ser muito útil quando se objetiva obter substrato desmineralizado padronizado para estudos laboratoriais. Concluiu-se ainda que o desafio cariogênico realizado com microrganismo S. mutans UA 159, suplementado com sacarose como fonte de carboidrato por 6 dias, é capaz de produzir mancha branca de esmalte padronizada. Desta maneira, seria obtido um substrato modificado relevante para estudos laboratoriais. / The aim of this in vitro study was to optimize the microbiological method for the production of white spot lesions in enamel that can be validated by non-destructive test (optical coherence tomography - OCT) to enable the subsequent use of the specimens in other experiments. A hundred and sixty eight bovine enamel fragments with central windows of demineralization measuring 3,0 x 3,0 mm were obtained. The experimental groups were composed from three variation factors: the microorganism (S. mutans UA 159, S. sobrinus 3347 and S. mutans + S. sobrinus), carbohydrate source (1% sucrose and 1% sucrose + 1% starch) and time (1, 2, 3, 4, 5, 6 and 7 days). Thus, six experimental groups were formed: G1 (S. mutans + sucrose), G2 (S. mutans + starch + sucrose), G3 (S. sobrinus + sucrose), G4 (S. sobrinus + starch + sucrose), G5 (S. mutans + S. sobrinus + sucrose), G6 (S. mutans + S. sobrinus + sucrose + starch) tested in 7 periods of cariogenic challenge. After this step, images in scale were obtained from each specimen with OCT, and then the specimens were processed and analysed by polarized light microscopy. In each of the methods were performed 5 measurements of demineralization depth. The statistical test Analysis of Variance (ANOVA) (p<0.05) detected that the cariogenic challenges tested were able to demineralize enamel in depth, regardless of the type of microorganism, carbohydrate source and time (p=0,000). The Pearson\'s Correlation Test showed a significant correlation (p=0,000) between the measurements of depth demineralization measured with polarized light microscopy and OCT. Therefore, the optical coherence tomography is a non-destructive method valid to measure the depth of demineralization of white spot lesions, which can be very useful when the objective is to obtain standard demineralized substrate for laboratory studies. It was also concluded that the cariogenic challenge performed with the microorganism Streptococcus mutans UA159, supplemented with sucrose as carbohydrate source for 6 days, is capable to produce standard white spot lesions in enamel. In this way, a modified substract relevant for laboratory studies could be obtained.

A proposta deste estudo in vitro foi otimizar o método microbiológico para a produção de lesão de mancha branca em esmalte que possa ser validada por ensaio não destrutivo (tomografia por coerência óptica - OCT) para possibilitar a utilização posterior dos espécimes em outros experimentos. Foram obtidos 168 fragmentos retangulares de esmalte bovino com janelas centrais de desmineralização de 3,0 x 3,0 mm. Os grupos experimentais foram compostos a partir de 3 fatores de variação: microrganismo (S. mutans UA 159, S. sobrinus 3347 e S. mutans + S. sobrinus), fonte de carboidrato (sacarose 1% e sacarose 1% + amido 1%) e tempo (1, 2, 3, 4, 5, 6 e 7 dias). Assim, formaram-se seis grupos experimentais: G1 (S. mutans + sacarose), G2 (S. mutans + sacarose + amido), G3 (S. sobrinus + sacarose), G4 (S. sobrinus + sacarose + amido), G5 (S. mutans + S. sobrinus + sacarose), G6 (S. mutans + S. sobrinus + sacarose + amido), testados em 7 tempos de desafio cariogênico. Terminada esta etapa, foram obtidas imagens em escala de cada espécime pelo OCT, e em seguida os espécimes foram processados e submetidos à análise através de microscópio de luz polarizada. Em cada um dos métodos, foram realizadas 5 mensurações de profundidade da região desmineralizada. O teste estatístico de Análise de Variância (ANOVA) (p<0.05) detectou que os desafios cariogênicos testados foram capazes de desmineralizar o esmalte em profundidade, sendo influenciados pelo tipo de microrganismo, fonte de carboidrato e tempo (p=0,000). O Teste de Correlação de Pearson apresentou uma correlação significativa (p=0,000) entre as medidas de profundidade de desmineralização aferidas através dos métodos de luz polarizada e OCT. Logo, a tomografia por coerência óptica é um método não destrutivo válido para aferir a profundidade de desmineralização de lesões de mancha branca, que pode ser muito útil quando se objetiva obter substrato desmineralizado padronizado para estudos laboratoriais. Concluiu-se ainda que o desafio cariogênico realizado com microrganismo S. mutans UA 159, suplementado com sacarose como fonte de carboidrato por 6 dias, é capaz de produzir mancha branca de esmalte padronizada. Desta maneira, seria obtido um substrato modificado relevante para estudos laboratoriais. / The aim of this in vitro study was to optimize the microbiological method for the production of white spot lesions in enamel that can be validated by non-destructive test (optical coherence tomography - OCT) to enable the subsequent use of the specimens in other experiments. A hundred and sixty eight bovine enamel fragments with central windows of demineralization measuring 3,0 x 3,0 mm were obtained. The experimental groups were composed from three variation factors: the microorganism (S. mutans UA 159, S. sobrinus 3347 and S. mutans + S. sobrinus), carbohydrate source (1% sucrose and 1% sucrose + 1% starch) and time (1, 2, 3, 4, 5, 6 and 7 days). Thus, six experimental groups were formed: G1 (S. mutans + sucrose), G2 (S. mutans + starch + sucrose), G3 (S. sobrinus + sucrose), G4 (S. sobrinus + starch + sucrose), G5 (S. mutans + S. sobrinus + sucrose), G6 (S. mutans + S. sobrinus + sucrose + starch) tested in 7 periods of cariogenic challenge. After this step, images in scale were obtained from each specimen with OCT, and then the specimens were processed and analysed by polarized light microscopy. In each of the methods were performed 5 measurements of demineralization depth. The statistical test Analysis of Variance (ANOVA) (p<0.05) detected that the cariogenic challenges tested were able to demineralize enamel in depth, regardless of the type of microorganism, carbohydrate source and time (p=0,000). The Pearson\'s Correlation Test showed a significant correlation (p=0,000) between the measurements of depth demineralization measured with polarized light microscopy and OCT. Therefore, the optical coherence tomography is a non-destructive method valid to measure the depth of demineralization of white spot lesions, which can be very useful when the objective is to obtain standard demineralized substrate for laboratory studies. It was also concluded that the cariogenic challenge performed with the microorganism Streptococcus mutans UA159, supplemented with sucrose as carbohydrate source for 6 days, is capable to produce standard white spot lesions in enamel. In this way, a modified substract relevant for laboratory studies could be obtained.