Microscopy Laboratory Research Articles

Levantamento de mielogramas com ênfase em pacientes portadores de leishmaniose visceral canina

This survey aims to present reports and other information about myelograms performed in 2017 and 2018 at the Anhembi Morumbi University Veterinary Hospital, emphasizing animals with canine visceral leishmaniasis (CVL), to verify and report the number of patients with Leishmania spp. Infection. The results of cytological and serological exams of these animals were also analyzed for comparison and scoring of diagnostic accuracy and more common serum and microscopic laboratory changes caused by this disease, respectively. Changes in blood count and myelogram were observed, but animals with a low degree of parasitism didn’t show Leishmania spp. on bone marrow exams.

Isolation, identification and associated risk factors of non-tuberculous mycobacteria infection in humans and dromedary camels in Samburu County, Kenya.

Non-tuberculous mycobacteria are of public health significance, and zoonotic infection is attributed to the sociocultural practice of consumption of raw milk and the close human-livestock contact in pastoral communities. This study aimed at isolation, identification of mycobacteria from human sputum and camel milk and risk factors assessment in Samburu East, Kenya. Six hundred and twelve camels and 48 people presumed to have tuberculosis (TB) from 86 households in Wamba and Waso regions were screened. Camels were categorized into Somali, Turkana and Rendile breeds. Single intradermal comparative tuberculin test (SICTT) was used as a herd-screening test on lactating camels and a milk sample collected from reactive camels. Sputum samples were collected from eligible members of participating households. A standard questionnaire on possible risk factors for both humans and camels was administered to respective household heads or their representatives. Total camel skin test reactors were 238/612 (38.9%). Milk and sputum samples were analysed at KEMRI/TB research laboratory for microscopy, GeneXpert® , culture and identification. Isolates were identified using 16S rRNA gene sequencing at Inqaba biotec in South Africa. Sixty-four isolates were acid-fast bacilli (AFB) positive of which M. fortuitum (3), M. szulgai (20), M. monacense (5), M. lehmanni (4), M. litorale (4), M. elephantis (3), M. duvalii (3), M. brasiliensis (1), M. arcueilense (1) and M. lentiflavum (1) were from milk; M. fortuitum (1), M. szulgai (2) and M. litorale (1) were from humans. Risk factors included the following: Turkana breed (OR=3.4; 95% CI: 1.2-9.3), replacements from outside the County (OR=2.1; 95% CI: 0.3-12.3), presence of other domestic species (small stock; OR=4.6) and replacement from within the herd (OR=3.2; 95% CI: 0.7-14.7). Zoonotic risk practices included raw milk consumption, shared housing and handling camels. Monitoring of zoonotic NTM through surveillance and notification systems is required.

Flat optics with dispersion-engineered metasurfaces

Control over the dispersion of the refractive index is essential to the performance of most modern optical systems. These range from laboratory microscopes to optical fibres and even consumer products, such as photography cameras. Conventional methods of engineering optical dispersion are based on altering material composition, but this process is time-consuming and difficult, and the resulting optical performance is often limited to a certain bandwidth. Recent advances in nanofabrication have led to high-quality metasurfaces with the potential to perform at a level comparable to their state-of-the-art refractive counterparts. In this Review, we introduce the underlying physical principles of metasurface optical elements (with a focus on metalenses) and, drawing on various works in the literature, discuss how their constituent nanostructures can be designed with a highly customizable effective index of refraction that incorporates both phase and dispersion engineering. These metasurfaces can serve as an essential component for achromatic optics with unprecedented levels of performance across a broad bandwidth or provide highly customized, engineered chromatic behaviour in instruments such as miniature aberration-corrected spectrometers. We identify some key areas in which these achromatic or dispersion-engineered metasurface optical elements could be useful and highlight some future challenges, as well as promising ways to overcome them. Flat metasurface optics provides an emerging platform for combining semiconductor foundry methods of manufacturing and assembling with nanophotonics to produce high-end and multifunctional optical elements. This Review highlights the design of metasurfaces, recent advances in the field and initial promising applications.

Syndecan-1 (CD138) immunohistochemical expression patterns in lupus nephritis; reflections on different clinicopathological parameters

Introduction: Syndecan 1 (SCD1) is a lectin expressed at the surface of renal tubular epithelial cells and plasma cells. In epithelial cells, cell surface syndecan1 is cleaved by inflammation-induced proteases (eg, ADAMTS, MMP), since loss of cell surface syndecan1 is associated with higher susceptibility to cell damage. Objectives: To explore a potential additional value of SCD1 immunohistochemical expression in lupus nephritis specimens of different ISN/RPS classes and NIH activity and chronicity indices. Patients and Methods: This retrospective study included 50 renal biopsy specimens diagnosed as lupus nephritis at the pathology laboratory, and electron microscopy (EM) laboratory of Ain-Shams University specialized hospitals. Data were collected from records as personal data, medical history and laboratory results including serum creatinine and proteinuria. Immunohistochemical expression of syndecan-1 was evaluated in renal tubular epithelial cells (TECs) followed by correlation with different clinicopathological parameters. Results: Fifty renal biopsy specimens with lupus nephritis including 14 cases of class II, 4 cases of class III, 20 cases of class IV and 12 cases of class V were re-evaluated. The mean serum creatinine was 1.57 ± 0.67 mg/dL. Nine cases (18%) were negative for proteinuria, while 41 cases (82%) were presented with proteinuria with a mean of 1.5 ± 0.9 g/24 h. There was no statistically significant difference in the percentage of SCD-1 expression with different lupus classes. Serum creatinine and albumin showed a statistically significantly different correlation with semiquantitative score of SCD-1 expression. The highest value of creatinine detected with score 1 of SCD-1 expression (P=0.038) and the highest value of urinary albumin was recorded with score 1 of SCD-1 expression. Accordingly, the lowest mean of urinary albumin recorded in SCD-1 score 3 (P<0.001). There was a weekly negative association between loss of SCD-1 expression and increased NIH activity and chronicity indices. Conclusion: Loss of syndecan immunohistochemical expression in renal TECs in lupus nephritis is highly associated with proteinuria and elevated serum creatinine and can be used as a predictive marker for disease severity and progression.

Alternative microscope for serial production for practical work with elementary school students

O ensino de ciências biológicas no ensino fundamental esbarra num grande problema: a falta de materiais para a prática de laboratório. Esta inexistência de equipamentos resulta em muitos casos na transformação da aula em rotinas de informações teóricas, desestimulando o estudante para o cuidado com a natureza. O sentido de admiração, que deveria ser trabalhado com este nível do ensino passa a ser deixado de lado e o formando acaba por se encantar com a tecnologia e não se dedica ao cuidado com a natureza. Levar o estudante a um conjunto de atividades que lhe devolvam o gosto pela natureza, em especial com o uso de microscopia alternativa foi objetivo do presente trabalho. Com o uso de materiais alternativos, em especial lentes de aumento de equipamentos diversos, foram montados três microscópios alternativos, os quais foram testados e trabalhados com professores de uma escola para verificar o seu uso potencial em classe. Os equipamentos montados se mostraram eficientes para estudos microscópicos, em especial para despertar no aluno o interesse pelo tema, com a possibilidade até dele ter seu próprio equipamento em casa e continuar amadurecendo o desenvolvimento de um sentido de encanto pela natureza. O aumento conseguido foi de 300 vezes. Foram montados kits de atividades possíveis, as quais são apresentadas na forma de rotina de trabalho com laboratório de microscopia. A avaliação do equipamento permite a possibilidade de trabalhar disciplinas como biologia, química, física e matemática de maneira interdisciplinar.

Multimodal spectral focusing CARS and SFG microscopy with a tailored coherent continuum from a microstructured fiber

We report a technologically novel microscopy system for bioimaging based on a 100 fs titanium:sapphire (Ti:Sa) laser pumped coherent continuum from a tailored, 9-cm long, all normal dispersion (ANDi) fiber, enabling concurrent image contrast with (a) spectral focusing coherent anti-Stokes Raman scattering (SF-CARS) (spanning 900–3200 cm−1) and (b) sum frequency generation (SFG). Both modalities were efficiently excited with power levels at the microscope focus compatible with biological samples. Moreover, using the continuum, images were recorded in the back-scattering (epi-detection) geometry, without the necessity for an expensive, computer-controlled, spatial light modulator (SLM), clearly demonstrating the strong signal levels achieved. Image contrast from the multiple modalities provided greater chemical and structural insights than imaging with any single technique in isolation. Numerical simulations supported these developments in regard to both the optimum fiber length for SC generation and the achievement of high spectral resolution in SF-CARS via careful group delay dispersion matching across the pump and Stokes pulses using just an inexpensive sequence of short glass blocks inserted into the Stokes beam. We show bio-images of mouse tissue recorded concurrently via label/stain-free contrast from multiple modalities: CARS, two-photon auto-fluorescence (TPaF) and second harmonic/sum frequency generation (SHG/SFG). Overall, our approach delivers optimum performance in back-scattered (epi-) detection configuration, suited for thick samples, at reduced complexity and cost. The addition of this simple fiber add-on to lasers already widely used for TPF microscopy can thus extend the capabilities of a significant number of existing microscopy laboratories.

Robotic microscopy for everyone: the OpenFlexure microscope.

Optical microscopes are an essential tool for both the detection of disease in clinics, and for scientific analysis. However, in much of the world access to high-performance microscopy is limited by both the upfront cost and maintenance cost of the equipment. Here we present an open-source, 3D-printed, and fully-automated laboratory microscope, with motorised sample positioning and focus control. The microscope is highly customisable, with a number of options readily available including trans- and epi- illumination, polarisation contrast imaging, and epi-florescence imaging. The OpenFlexure microscope has been designed to enable low-volume manufacturing and maintenance by local personnel, vastly increasing accessibility. We have produced over 100 microscopes in Tanzania and Kenya for educational, scientific, and clinical applications, demonstrating that local manufacturing can be a viable alternative to international supply chains that can often be costly, slow, and unreliable.

Tuberculosis surveillance in adolescents: what to learn from European Union/European Economic Area data?

OBJECTIVE: To describe tuberculosis (TB) characteristics in the adolescent 10-19 years age group that is often underrepresented in surveillance and studies despite the high global TB burden estimated for this group.SETTING AND DESIGN: We use the case-based data reported to the European Surveillance System (TESSy) from European Union (EU)/European Economic Area (EEA) countries between 2007 and 2016 to describe notification rates, TB characteristics and treatment outcomes among adolescent TB cases. We also compare TB characteristics in young adolescents (10-14 years) and older adolescents (15-19 years).RESULTS: For the period 2007 to 2016, 705 826 TB cases were reported to TESSy by 29 EU/EEA countries, 38 054 (5.4%) of which were adolescents. The overall EU/EEA notification rate among adolescents was 6.9 per 100 000 population, 3.5 among young adolescents and 10.1 among older adolescents. The two adolescent groups had differences regarding sex distribution, site of disease, sputum smear microscopy positivity, laboratory confirmation and treatment outcome.CONCLUSION: Younger and older adolescents should be analysed as separate groups when studying and reporting TB, particularly to inform better targeting of TB prevention and care interventions in the future, in order to improve outcomes.

A Web-Based Virtual Microscopy Platform for Improving Academic Performance in Histology and Pathology Laboratory Courses: A Pilot Study.

Virtual microscopy (VM) has been utilized to improve students' learning experience in microscope laboratory sessions, but minimal attention has been given to determining how to use VM more effectively. The study examined the influence of VM on academic performance and teacher and student perceptions and compared laboratory test scores before and after VM incorporation. A total of 662 third-year students studying histology and 651 fourth-year students studying pathology were divided into two groups. The light microscopy (LM) group used a light microscope in 2014 and 2015, while the LM+VM group used the VM platform and a light microscope in 2016 and 2017. Four factors positively predict laboratory scores (R square, 0.323; P<0.001): (i) the pathology course and test-enhanced learning, (ii) the VM platform and experience, (iii) medical students and lecture scores, and (iv) female students. The LM+VM group exhibited less score variability on laboratory examinations relative to their mean than the LM group. The LM+VM group was also associated with fewer failing grades (F grade; odds ratio, 0.336; P<0.001) and higher scores (A grade; odds ratio, 2.084; P<0.001) after controlling for sex, school, course, and lecture grades. The positive effect of the VM platform on laboratory test grades was associated with prior experience using the VM platform and was synergistic with more interim tests. Both teachers and students agreed that the VM platform enhanced laboratory learning. The incorporation of the VM platform in the context of test-enhanced learning may help more students to master microscopic laboratory content.

Kvalitet zubnog otiska dva različita brenda polivinil siloksan impresionih materijala

Background: This study was to evaluate the impression quality and clinical success of 2 Polyvinyl siloxane (PVS) impression materials by using dual-viscosity 1-step impression technique and the putty-wash two-step impression technique. Methods: In this study, Variotime (Heraeus, Hanau, Germany) and a novel silicone Spirias (Imicryl, Konya, Turkey) were selected as PVS impression materials. A total of 40 samples were divided into groups according to the impression technique. Three possible categories were established to rate the impression quality. The impression was examined using a laboratory microscope (Opmi Pico, Zeiss) and lenses with 2X to 6X magnification for the presence or absence of bubbles or voids and the complete reproduction of the preparation finish line. A spreadsheet (Excel v2016; Microsoft Corp) was used for processing statistical data. Results of the analysis were calculated as mean and frequency. Results: In the impressions taken with Variotime, the percentage of the successful impression is 95% (Criteria I - II). Only one impression was found unacceptable (criteria III). In impressions with Spirias, the percentage of successful impressions is 100% (Criteria I - II). No impression was found unacceptable (criterion III). Conclusion: The quality of impressions of both brands Polyvinyl siloxane impression materials revealed a high rate (97.5%) For more precise results, many new studies were needed on different Polyvinyl siloxane impression materials.

О роли биохимических и микроскопических гематологических лабораторных маркеров в своевременной диагностике тромботических микроангиопатий

О роли биохимических и микроскопических гематологических лабораторных маркеров в своевременной диагностике тромботических микроангиопатий

Association of Diabetes Mellitus and Estrogen Hormone Levels with Vaginal Candidiasis

Objectives: The aim of this study was to investigate the association of diabetes mellitus and estrogen hormone levels with vaginal candidiasis. Method: A cross sectional comparative study was used on 2018 from women of childbearing age (20‑35 years) and one‑year oral contraceptive who came to the obstetric polyclinic who conducted visual inspection with acetic acid (VIA) in Bhayangkara Hospital Polyclinic, Padang Pasir and Nanggalo Primary Health Care Padang City, West Sumatera Province, Indonesia. We recruited 58 respondents with consecutive sampling technique. Vaginal candidiasis was measured using vaginal secretions and microscopic laboratory test. Diabetes mellitus was measured using fast blood glucose and estrogen hormone levels measured with blood sample analysis using Enzyme‑Linked Immunosorbent Assays (ELISA). Chi‑square test and independent sample T test were used to data analysis, P value &lt; 0.05 was considered as statistically significant association. Data were processed using IBM SPSS Statistics 24.0. Results: Diabetes mellitus was associated with vaginal candidiasis (p&lt;0.05). There was statistically significant mean difference of estrogen hormone levels with vaginal candidiasis, estrogen hormone levels with vaginal candidiasis were 143.39 pg/ml higher than not vaginal candidiasis were 60.99 pg/ml. Conclusion: This analysis confirmed association of diabetes mellitus and estrogen hormone levels with vaginal candidiasis.

A Microfluidic Device for Imaging Samples from Microbial Suspension Cultures

Traditional methods to assess microbial cells during suspension culture require laborious and frequent manual sampling. Approaches to automate sampling and assessment utilize dedicated, sophisticated equipment and suffer from a lack of temporal resolution and sampling efficiency. In this study we describe a simple microfluidic device that allows microbial cells to be sampled from suspension culture and rapidly slowed and concentrated for single-cell imaging on a standard laboratory microscope. We demonstrate a device that: •slows and concentrates microbial cells, specifically budding yeast, sampled from suspension culture and improves imaging of individual cells by concentrating them in a single focal plane•provides imaging quality and temporal resolution that is capable of monitoring dynamic spatiotemporal processes, such as nuclear localization of a protein•is inexpensive and simple enough to be fabricated and used in laboratories equipped for standard molecular and cellular biology

A microscopic analysis of single-use versus multi-use phacoemulsification tubing in maintaining sterility and patient safety

Background: The Pietermaritzburg (PMB) complex in South Africa does not have a uniform protocol regarding reuse of phacoemulsification tubing. Each hospital in the complex has its own guideline, based on manufacturer recommendation and cost-saving measures. There is no definitive scientific evidence proving that reusing phaco tubing will harm patients.Aim: To assess if phacoemulsification tubing remains sterile during sequential phacoemulsification.Setting: The study was conducted at Edendale Hospital and Greys Hospital in PMB. Greys Hospital has a single-use policy and at Edendale policy, multi-use of tubings is allowed.Methods: This was an observational descriptive prospective study observed over 4 months. Routine phacoemulsification was carried out at each hospital as per hospital guidelines. At the end of the surgery, the tips of the tubing were cut off, placed in a standard specimen container and sent to the laboratory for culture and microscopy. Results were compared and two unused tubings were also analysed as controls.Results: A total of 26 single-use tubings were analysed and it was found that 46.2% grew no organisms; 19.2% grew Bacillus species; other microorganisms were in less than 1% of tubings. A total of 41 multiple-use tubings were sent for analysis. Forty one and a half percent grew no organisms; 17.1% tubings grew Bacillus species. Other microorganisms were found in less than 5% of tubings. Unused tubings did not grow any microorganism.Conclusion: A p value less than 0.05% was accepted as statistically significant. Comparing the two hospitals, tubings which grew no organisms were 46.2% (single-use) versus 41.5% (multiple-use). This gives a statistically non-significant p value of 0.70. Tubings which grew Bacillus species were 19.2% (single-use) versus 17.1% (multiple-use) (p = 0.83). Other microorganisms also showed a p value of 0.76. This implies that phacoemulsification tubing is significantly contaminated after just one use. Although no statistical difference was found between the two groups, a contamination rate of over 40% is of great concern. There are contributing factors which may have influenced this result. In our setting, phaco tubing does not seem to remain sterile after cataract surgery. Further research needs to be carried out in order to gain more insight into patient safety.

DETERMINATION OF WHITE BLOOD CELLS USING FOLDSCOPE WITH SMARTPHONE

We are traditionally used Microscope in clinical laboratory for determination of white blood cells of human blood smear. Now, in this study we were used Foldscope with Smartphone in the place of Microscope and examine many samples of human blood smear which was collected from local diagnostic centers. We were very easily quantity &amp; morphology analysis of all types of WBC cells such as Neutrophils, Lymphocytes, Monocytes, Eosionophils, Basophils in blood smear with the help of Foldscope &amp; image taken by Smartphone. The main objective of this study is to use Foldscope for quantity &amp; morphology analysis of human WBCs at field level especially poor resource area where healthcare services or centers is not available &amp; where carry of microscope is not possible.

Sinergia de combinaciones de extractos vegetales para el control de vibriosis en sistema productivo de camarón (Litopenaeus vannamei)

Vibrios are one of the main pathogens in shrimp farming and cause economic losses to shrimp farmers. The inappropriate use of antibiotics has caused bacterial resistance and environmental problems. The use of plant extract is a feasible alternative for bacterial inhibition. The objective of this work is to evaluate the synergistic effect of alcoholic extracts of plants on the presence of vibrios in the organism, sea water and shrimp biomass. The study was carried out in the Microscopy Laboratory of the Technical University of Machala. Combined alcoholic extracts between cinnamon-eucalyptus and thyme-guava plants were tested in the presence of vibrios. The inhibitory effect was evaluated with 100 µl of extracts on vibrio strains for 24 hours by diffusion in Petri dish. 200 µl / gr and 400 µl / gr of each combination were dosed with feed for 10 days, then doses of 2 ml and 4 ml of extracts were applied in 1 liter of water for 24 hours, then the content of vibrios was quantified. The application of combined extract of thyme-guava at 400 µl / gr in food inhibited the presence of vibrios (0 CFU) in hepatopancreas and water. The use of combined extracts produces a bactericidal synergistic effect, suitable for the treatment of vibriosis in shrimp production systems.

Late diagnosis of multidrug-resistant tuberculosis in a child at Dr George Mukhari Academic Hospital, Ga-Rankuwa, South Africa: A case report.

IntroductionSouth Africa has one of the top ten tuberculosis burdens in the world, only lagging behind countries with significantly larger populations. Increased awareness of extrapulmonary tuberculosis, specifically spinal tuberculosis, is necessary, because of the HIV epidemic.Case presentationThis report describes the case of a 9-year-old male patient who was suspected of having multidrug-resistant (MDR) tuberculosis, based on failure to recover clinically and radiologically after 6 months on first-line anti-tuberculosis treatment. Pus samples were sent to an accredited academic laboratory for histopathology, microscopy, culture, line-probe assay (MTBDRplus assay) and phenotypic MGIT 960 drug susceptibility tests. Second-line MDR tuberculosis treatment was introduced. Clinical, radiological, physical processes and more laboratory tests were conducted to document whether or not there was improvement in the patient.Management and outcomeAfter laboratory diagnosis of MDR tuberculosis, the patient was started on MDR tuberculosis treatment. The patient started improving remarkably after the introduction of anti-tuberculosis treatment and rehabilitation, although he also required surgery to stabilise the spine. Neurological improvement was observed in the patient and he fully recovered.DiscussionAlthough the diagnosis of spinal MDR tuberculosis may not be achieved easily by culture, the well-known gold standard method of tuberculosis diagnosis, it is of great importance to rapidly initiate an effective anti-tuberculosis treatment of drug-resistant strains to reduce the deformity of the spine.

Evaluation of a TEM based Approach for Size Measurement of Particulate (Nano)materials.

An approach for the size measurement of particulate (nano)materials by transmission electron microscopy was evaluated. The approach combines standard operating procedures for specimen preparation, imaging, and image analysis, and it was evaluated on a series of certified reference materials and representative test materials with varying physical properties, including particle size, shape, and agglomeration state. The measurement of the median value of the minimal external particle diameter distribution was intra-laboratory validated. The validation study included an assessment of the limit of detection, working range, selectivity, precision, trueness, robustness, and ruggedness. An uncertainty that was associated to intermediate precision in the range of 1–7% and an expanded measurement uncertainty in the range of 7–20% were obtained, depending on the material and image analysis mode. No bias was observed when assessing the trueness of the approach on the certified reference materials ERM-FD100 and ERM-FD304. The image analysis method was validated in an inter-laboratory study by 19 laboratories, which resulted in a within-laboratory precision in the range of 2–8% and a between-laboratory precision of between 2% and 14%. The automation and standardization of the proposed approach significantly improves labour and cost efficiency for the accurate and precise size measurement of the particulate materials. The approach is shown to be implementable in many other electron microscopy laboratories.

The National Tuberculosis Control Programme of Liberia Laboratory Programme Performance.

Background Tuberculosis (TB) is a major public health problem in Liberia. Little is known about the TB laboratory performance of Liberia and the challenges after the 14 years of civil war which ended in 2003. The purpose of the study was to evaluate the TB laboratory performance of Liberia. Methods A cross-sectional study was conducted from 2014 to 2015. The study was conducted using quantitative data of TB case findings, sputum microscopy proficiency testing, and on-site assessment of sputum microscopy laboratories in Liberia. 80 laboratories participated in the proficiency testing. Besides, four years' (2012–2015) TB case finding data obtained from the National Leprosy and Tuberculosis Control Programme (NLTCP) were used to complement the study. The data were analysed using descriptive statistics. Results From the 80 TB sputum microscopy testing laboratories participating in proficiency testing, only 20 (25%) scored acceptable performance. 46 (58%) TB microscopy laboratories reported quantification errors for the proficiency panel slide 6 which was 3+. The national TB smear-positive cases notified were 4342 in 2012 but decreased to 3820 and 2448 in 2013 and 2014, respectively. The TB smear case detection rate showed an increase from 68% in 2010 to 78% in 2011 and a decrease to 60%, 57%, and 42% in 2012, 2013, and 2014, respectively. Conclusion Between 2010 and 2013, the NLTCP succeeded in increasing the number of TB sputum microscopy laboratories. At most of the TB microscopy sites, the TB laboratory quality system was not implemented. The NLTCP of Liberia should develop strategies to overcome its challenges in TB laboratory testing.

The Gram-Positive Bacterial Cell Wall.

The chapter about the Gram-positive bacterial cell wall gives a brief historical background on the discovery of Gram-positive cell walls and their constituents and microscopic methods applied for studying the Gram-positive cell envelope. Followed by the description of the different chemical building blocks of peptidoglycan and the biosynthesis of the peptidoglycan layers and high turnover of peptidoglycan during bacterial growth. Lipoteichoic acids and wall teichoic acids are highlighted as major components of the cell wall. Characterization of capsules and the formation of extracellular vesicles by Gram-positive bacteria close the section on cell envelopes which have a high impact on bacterial pathogenesis. In addition, the specialized complex and unusual cell wall of mycobacteria is introduced thereafter. Next a short back view is given on the development of electron microscopic examinations for studying bacterial cell walls. Different electron microscopic techniques and methods applied to examine bacterial cell envelopes are discussed in the view that most of the illustrated methods should be available in a well-equipped life sciences orientated electron microscopic laboratory. In addition, newly developed and mostly well-established cryo-methods like high-pressure freezing and freeze-substitution (HPF-FS) and cryo-sections of hydrated vitrified bacteria (CEMOVIS, Cryo-electron microscopy of vitreous sections) are described. At last, modern cryo-methods like cryo-electron tomography (CET) and cryo-FIB-SEM milling (focus ion beam-scanning electron microscopy) are introduced which are available only in specialized institutions, but at present represent the best available methods and techniques to study Gram-positive cell walls under close-to-nature conditions in great detail and at high resolution.