Microbiology: A Laboratory Experience

As a group of organisms that are too small to see and best known for being agents of disease and death, microbes are not always appreciated for the numerous supportive and positive contributions they make to the living world. Designed to support a course in microbiology, Microbiology: A Laboratory Experience permits a glimpse into both the good and the bad in the microscopic world. The laboratory experiences are designed to engage and support student interest in microbiology as a topic, field of study, and career. This text provides a series of laboratory exercises compatible with a one-semester undergraduate microbiology or bacteriology course with a three- or four-hour lab period that meets once or twice a week. The design of the lab manual conforms to the American Society for Microbiology curriculum guidelines and takes a ground-up approach — beginning with an introduction to biosafety and containment practices and how to work with biological hazards. From there the course moves to basic but essential microscopy skills, aseptic technique and culture methods, and builds to include more advanced lab techniques. The exercises incorporate a semester-long investigative laboratory project designed to promote the sense of discovery and encourage student engagement. The curriculum is rigorous but manageable for a single semester and incorporates best practices in biology education.https://knightscholar.geneseo.edu/oer-ost/1004/thumbnail.jp

Bacterial morphotype grading for periodontal disease assessment

BACKGROUND: Listgarten and Hellden (1978) used darkfield microscopy of wet mounts to differentiate between healthy and periodontally diseased sites in the mouth by expressing the different bacterial morphotypes observed as a percentage of the total number of bacteria counted. This method of periodontal disease assessment gained favour as a diagnostic tool but presented with the limitation of immediate examination to determine the number of motile rods present and an inability to distinguish between gingivitis and periodontitis. Grading of bacterial morphotypes into several distinct categories of health or disease (Ison and Hay, 2002), simplified the scoring system of Gram-stained smears for the diagnosis of bacterial vaginosis (Nugent et al. 1991). The application of a similar grading system using stained smears rather than wet mounts could be advantageous to the diagnosis of periodontal disease. OBJECTIVES/AIMS: This study tested the hypothesis that stained smears of dental plaque collected from the gingival crevice of individuals with varying probing pocket depths (PD) may provide a grading system for periodontal disease assessment. MATERIALS AND METHODS: Subgingival plaque samples were collected from 49 patients, stained with a silver stain and the proportions of each bacterial morphotype graded relative to their respective PD measurements. RESULTS: This technique allowed for a grading system of I–IV, with grade I indicating health and grade IV indicating severe periodontal disease. DISCUSSION: Stained smear examination eliminates the time restriction for motile rod enumeration and allows for storage of smears for future reference. CONCLUSION: Standardization of the microscopic areas to be evaluated or examined will facilitate the agreement of cut-off values for the diagnosis of periodontal disease.This material is based on work partially supported financially by the National Research Foundation (NRF) of South Africa

Bacterial vaginosis in Portugal : diagnosis of Gardnerella vaginalis and Atopobium vaginae in healthy or symptomatic women

A vaginose bacteriana corresponde ao distúrbio mais comum nas mulheres, tendo um impacto importante em todo o Mundo. Estima-se que afecta cerca de 30-50% das mulheres Afro-Americanas e 10-20% das mulheres Caucasianas em idade reprodutiva. Associado ao aparecimento de vaginose bacteriana, verifica-se um decréscimo do número de Lactobacillus spp. no epitélio com consequente aumento do número de microrganismos anaeróbios, tais como Gardnerella vaginalis e Atopobium vaginae. Embora comumente associada à vaginose bacteriana, G. vaginalis foi também identificada no epitélio vaginal de mulheres saudáveis, mas em menores números.O crescimento de G. vaginalis pode ser identificado por beta hemólise, Gram-variável, oxidase e catalase negativa (testes microbiológicos convencionais) e ainda através de técnicas moleculares. O principal objectivo deste projecto foi a identificação de A. vaginae e G. vaginalis na microflora vaginal de mulheres Portuguesas, saudáveis ou já diagnosticadas, à priori, como portadoras de vaginose bacteriana; através de métodos moleculares. O principal interesse no estudo destes microrganismos deveu-se ao facto de serem, nos últimos anos, os mais usualmente isolados de casos de vaginose bacteriana. Gardnerella vaginalis e, mais recentemente, A. vaginae são dois microrganismos inicialmente associados a vaginose mas actualmente identificados em mulheres saudáveis. Em Portugal, o primeiro e único estudo associado a vaginose remonta de 1998, o que justifica a importância dos dados obtidos neste estudo. Neste sentido, o estudo envolveu a recepção de amostras clínicas obtidas por auto-colheita de mulheres saudáveis, em consultório de ginecologia ou mesmo nas emergências do Hospital de Braga, e posterior tratamento das amostras. A caracterização foi levada a cabo por métodos moleculares como Reacção em Cadeia da Polimerase (PCR) e Microscopia Fluorescente com Hibridação in situ (FISH). Os resultados demonstraram, através de métodos moleculares, que das cinquenta e sete amostras recolhidas de mulheres Portuguesas e associadas a este projecto, G. vaginalis foi identificada em dezasseis amostras, o que corresponde a 28% do número total de amostras. Atopobium vaginae foi apenas encontrado em cinco casos o que corresponde a 8% das mesmas. Em suma, as técnicas moleculares permitiram a identificação directa de parte dos microrganismos presentes nas zaragatoas, sendo assim possível concluir que G. vaginalis and A. vaginae não estão unicamente associadas a vaginose bacteriana mas também estão presentes, em diferentes proporções, em mulheres Portuguesas saudáveis.Bacterial vaginosis is the leading vaginal disorder, having an important impact worldwide. It is estimated to affect 30-50% of African-American women and 10-20% of Caucasian women at reproductive age. During bacterial vaginosis, a decrease of Lactobacillus spp. and an increase in the number of anaerobic microorganisms, such as Gardnerella vaginalis and Atopobium vaginae in the vaginal epithelium is observed. Although commonly associated to bacterial vaginosis, G. vaginalis has also been associated to the vagina of healthy women, but in lower numbers. The growth of G. vaginalis can be identified by beta hemolysis, variable Gram staining, negative oxidase and catalase (conventional microbiological tests) and by molecular techniques. The main goal of this study was the identification of G. vaginalis and A. vaginae in the vaginal microflora of healthy or ill women, by molecular techniques. The reason of our interest in these microorganisms was based on fact of being the mostly isolated microorganisms in cases of bacterial vaginosis. Gardnerella vaginalis and, most recently, A. vaginae were two microorganisms firstly associated to bacterial vaginosis, however more recent studies identified them on the healthy vaginal microflora. In Portugal, the unique study involving bacterial vaginosis was done in 1998, which consequently straighten up the importance of this study. By this way, our study involved the reception of swabs obtained by self-harvest, gynecological private practice or even in hospitals emergency and the posterior manipulation of the samples. The identification of G. vaginalis and A. vaginae was specially based on the analysis of the clinical samples by Polymerase Chain Reaction (PCR), and by Peptide Nucleic Acid Fluorescence in situ Hybridization (PNA-FISH). The results revealed that from fifty-seven Portuguese women samples involved in this study, the presence of G. vaginalis was detected in sixteen samples, which corresponds to a prevalence of 28 %. On the other hand A. vaginae was present in five clinical samples, which corresponds to 8 % of the samples. The molecular techniques allowed the direct identification of part of the microorganisms present on the vaginal swabs and allowed to conclude that the G. vaginalis and A. vaginae are not only associated to bacterial vaginosis but they can also be founded, in different percentage, in a range of Portuguese healthy women

Studies on Monoclonal Antibodies Characterization and immunohistochemical detection of Lactococcus garvieae

Lactococcus garvieae, the aetiological agent of Lactococcosis, has recently been responsible for significant disease outbreaks in a variety of economically important freshwater and seawater fish species cultured worldwide. The development of immunological diagnostic tests to use in the control strategies against L. garvieae has been limited due to a complicated global distribution of serotypes. It appears there are serotypic differences between L. garvieae recovered from different regions, and although perhaps a common Antigen (Ag) may be expressed by all serotypes, it has not been found yet. The identification of this Ag would enable the development of specific Monoclonal Antibodies (MAbs), which could be used, in conjunction with immunological techniques, for the detection of all isolates of the pathogen in infected fish and water. In this study, nine MAbs produced in the Aquatic Vaccine Unit of the Institute of Aquaculture (IoA), University of Stirling against L. garvieae type strain NCIMB 702928 (3 MAbs) and against a L. garvieae Japanese clinical isolate (12-99)(6 MAbs), as well as two more MAbs, kindly donated by Professor Tae Sung Jung (Laboratory of Fish and Shellfish Diseases, Gyeongsang National University, Republic of Korea) against L. garvieae Korean isolates, were screened, using Enzyme-linked immunosorbent assay (ELISA), against a collection of 12 L. garvieae isolates from Europe and Asia, and L. garvieae type strains. None of the MAbs in the study recognized all the L. garvieae isolates tested, although some of the Japanese MAbs (specifically MAbs 3 G9, 8B12, 11F8 and 11B1) recognized a higher number of isolates than the rest of MAbs (including all the type strains in the study and several Japanese and Italian isolates). Differences in the intensity of the reaction has led to the idea that the Ag can be expressed at two different levels by different isolates, or that perhaps there are two different Ags displaying similar epitopes that are recognized at two levels. European MAbs and the rest of the Japanese MAbs were very specific to certain strains, including two of the type strains and one Japanese isolate but they did not recognize any of the Italian isolates. Korean MAbs did not recognize any isolate, and this led to think about a possible absence of MAb in the supernatant. However, none of the two L. garvieae Korean isolates were recognized by any MAb. The MAbs were also tested for cross-reactivity using ELISA with a collection of 32 isolates from other bacterial species (including L. garvieae related and unrelated species). Low levels of cross reactivity (ranging from 0.3% to 9.6%) were detected, with the exception of a Korean MAb (U99-33) that showed a significant cross reactivity with Renibacterium salmoninarum. An Immunohistochemistry (IHC) test was developed with the MAbs studied. A preliminary IHC test was carried out with all the MAbs, and the results obtained correlated with those from the ELISA assay. Based upon the results obtained and availability of supernatants, two MAbs, (Japan) 3G9 and (Euro) 13, were used in IHC to screen tissue samples from a L. garvieae challenge with isolate NCIMB 702928 in rainbow trout, carried out during the study. Both MAbs were able to detect specifically L. garvieae in infected tissue sections of various organs. The pathology observed in challenged fish is described. Most prominent features on clinical examination were exophthalmos, generalized congestion and haemorrhage, hepatomegaly, splenomegaly and serositis, which sometimes extended to the myocardium. Histopathological features included several inflammatory and degenerative processes in various organs (eye, swimbladder, spleen, liver, kidney and heart). In conclusion, the findings of the study suggest that our knowledge on serotypes and antigenic profiles of the bacteria worldwide needs to be expanded, in order to acquire a level of knowledge that will allow the development of MAbs that recognize all L.garvieae isolates worldwide. If these are developed, it will be possible to use them in IHC to detect the bacteria in infected fish tissue, and will help to differentiate lactococcosis from other streptococcal diseases

Vaginal Microbiota Composition Correlates Between Pap Smear Microscopy and Next Generation Sequencing and Associates to Socioeconomic Status

Recent research on vaginal microbiota relies on high throughput sequencing while microscopic methods have a long history in clinical use. We investigated the correspondence between microscopic findings of Pap smears and the vaginal microbiota composition determined by next generation sequencing among 50 asymptomatic women. Both methods produced coherent results regarding the distinction between Lactobacillus-dominant versus mixed microbiota, reassuring gynaecologists for the use of Pap smear or wet mount microscopy for rapid evaluation of vaginal bacteria as part of diagnosis. Cytologic findings identified women with bacterial vaginosis and revealed that cytolysis of vaginal epithelial cells is associated to Lactobacillus crispatus-dominated microbiota. Education and socio-economic status were associated to the vaginal microbiota variation. Our results highlight the importance of including socio-economic status as a co-factor in future vaginal microbiota studies.Peer reviewe

Digital Image Analysis Of Gram Stained Culture Specimens

Digital image analysis for the interpretation of images in clinical microbiology has many potential advantages over current practices. Compared to traditional image interpretation by a medical technologist, digital image analysis offers standardization between laboratories, round-the-clock interpretation, and quantitative results. In the first study of its kind known to the authors, a digital image analysis program was prototyped to interpret a slide containing Gram stained microorganisms. The sample microorganisms were obtained from culture plates during routine processing and subjected to Gram\u27s stain. An initial study learned from 11 Gram-stained slides and classified their microorganisms into the group: Gram-positive, Gram-negative, rods, coccus, and yeast. The sensitivity of identification ranged from 66% to 99% and the specificity ranged from 78% to 99%. The algorithm was next applied to a larger set of 78 slides. The accuracy rate for slide classification was 60 out of 78 or 77%. After using this larger dataset to train the algorithm, the accuracy rate for individual objects was 94% averaged over 5 trials. This suggests the parameters used by the algorithm can differentiate between groups, and the lack of accuracy in classifying the larger database occurred due to limitations in the original training data. Overall, the project demonstrates a unique application of digital image analysis to clinical microbiology

cereus Fun: An Introduction to Microbiological Techniques

Microbiology is a field of science devoted to the study of organisms that are too small to see; therefore, an engaging laboratory experience is often the key to capturing students\u27 interest. It was with this in mind that this book was first conceived and developed. The goal was to provide undergraduate microbiology students with an engaging and meaningful laboratory experience that nurtured a sense of discovery and encouraged greater interest in microbiology as a topic, a field of study, or a career. This lab manual – which has been field-tested by hundreds of microbiology students over several years – builds skills while reinforcing core microbiology concepts introduced in lectures. The curriculum builds from the ground up. It begins with an introduction to biosafety practices and work with biological hazards, basic but essential microscopy skills, and aseptic technique and culture methods, and then it builds to include more advanced methods. The progression includes a semester-long investigation of a bacterial isolate and culminates with a practical evaluation of all of the microbiology skills learned in the course