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Understanding Microbiology Culture Results
Published in Firza Alexander Gronthoud, Practical Clinical Microbiology and Infectious Diseases, 2020
Mycobacteria can be detected using a Ziehl–Neelsen stain or a fluorescent auramine stain. Mycobacteria have a waxy mycolic acid in their cell wall which resists a Gram stain. A Ziehl–Neelsen stain was developed whereby carbol fuchsin is used as the initial stain and heat is applied to fixate the carbol fuchsin into the cell wall. Because of mycolic acid in their cell wall, the acid alcohol used in the decolourization is not able to remove carbol fuchsin from the cell wall, and consequently, they appear bright red. This is why mycobacteria are also called acid-fast bacilli. A sputum sample needs to contain at least 104 mycobacteria per millilitre before they can be detected with microscopy.
Pulmonary Tuberculosis In Children
Published in Lourdes R. Laraya-Cuasay, Walter T. Hughes, Interstitial Lung Diseases in Children, 2019
The tubercle bacillus, Mycobacterium tuberculosis, discovered in 1882 by Robert Koch, is the etiologic agent of tuberculosis. It is a slender, straight or slightly curved rod, 1 to 4 μm in length. Its most distinctive property, as demonstrated with the Ziehl-Neelsen stain, is the absorption of the carbol-fuchsin dye by the chemically complex, largely lipid cell wall of the intact organism, which resists decoloration by acid-alcohol. Organisms from cultures and in clinical materials stand out as brilliant red against the blue background of the counterstain. Cultures from clinical material placed on enriched media at 37°C in the presence of ample oxygen grow very slowly. The small, dry, scaly colonies cannot be seen before 10 to 20 days of incubation. M. tuberculosis is distinguished from other acid-fast mycobacteria by its production of niacin.1
Special Methods
Published in Donald L. Price, Procedure Manual for the Diagnosis of Intestinal Parasites, 2017
Several acid-fast stains are used on fecal specimens, especially for confirming the presence of Cryptosporidia. One of the earliest investigators to recognize the acid-fast character of certain bacteria was Ziehl who introduced his procedure in 1882 in Germany. Neelsen modified the procedure in 1883. They used a phenol and fuchsin dye mixture (carbol fuchsin). Kinyoun published another version of a carbol fuchsin stain in 1915 and Bronsdon published a new method specifically for Cryptosporidia that used dimethyl sulfoxide in 1984. Price developed another acid-fast stain in 1988 that has been used in several laboratories (see Appendix). There are a number of other methods for acid-fast staining that appear in the literature; these can be substituted for those mentioned. Although how these different methods and stains have been employed has varied somewhat depending on the type of material to be stained and the investigator, one of the fuchsin dye formulas has been employed most often for staining acid-fast organisms.
Ex vivo biofilm-forming ability of dermatophytes using dog and cat hair: an ethically viable approach for an infection model
Published in Biofouling, 2019
Raimunda Sâmia Nogueira Brilhante, Lara de Aguiar, Jamille Alencar Sales, Géssica dos Santos Araújo, Vandbergue Santos Pereira, Waldemiro de Aquino Pereira-Neto, Adriana de Queiroz Pinheiro, Germana Costa Paixão, Rossana de Aguiar Cordeiro, José Júlio Costa Sidrim, Paulo Ricardo de Oliveira Bersano, Marcos Fábio Gadelha Rocha, Débora de Souza Collares Maia Castelo-Branco
Biofilms grown on the ex vivo models were analyzed by light microscopy, according to Castelo-Branco et al. (2016), with modifications. Briefly, the hairs were transferred to a new 12-well plate and fixed with 500 μl of acetylpyridine chloride (10 nM) (Sigma-Aldrich, St. Louis, MO, USA). After incubation for 30 s, the fixating agent was removed and the plate was incubated at 25 °C until completely dry. Then, 500 μl of a 2:1 (vv–1) solution of saturated Congo red (Sigma Aldrich) supplemented with 10% Tween 80 (Isofar, Rio de Janeiro, Brazil) were added. After 15 min, the solution was withdrawn and the wells were washed twice with sterile saline. Then 500 μl of 10% carbol fuchsin were added. After 6 min, the wells were rinsed again with sterile saline. The stained hair samples were then placed on slides and analyzed with an Olympus (Shinjuku, Tokyo, Japan) BX41 light microscope. Photographs were taken with the camera Olympus DP71 and processed with Olympus DP Controller, version 3.3 software (Brilhante et al. 2018).
Evaluation of 16S rRNA qPCR for detection of Mycobacterium leprae DNA in nasal secretion and skin biopsy samples from multibacillary and paucibacillary leprosy cases
Published in Pathogens and Global Health, 2018
Lívia Érika Carlos Marques, Cristiane Cunha Frota, Josiane da Silva Quetz, Alexandre Havt Bindá, Rosa Maria Salane Mota, Maria Araci de Andrade Pontes, Heitor de Sá Gonçalves, Carl Kendall, Ligia Regina Franco Sansigolo Kerr
NS and SB specimens were obtained from patients at the Dona Libânia National Reference Centre for Sanitary Dermatology, Ceará, Brazil. Untreated leprosy cases were included and confirmed by clinical skin examinations, skin smears, and biopsies. They were classified using the Ridley-Jopling [22] criteria based on histology and bacilloscopy indices (BI) and according to the World Health Organization (WHO) [23] as PB or MB cases. Recruitment of cases was random. A total of 54 NS samples from both nostrils were obtained from patients with different clinical forms, 39 MB (20 lepromatous leprosy [LL] and 19 borderline-borderline [BB]) and 15 PB (14 tuberculoid [T] and one indeterminate [I]). In addition, 19 SB specimens (paired with NS samples) were obtained from MB cases (10 BB and 9 LL). Dried slit-skin smear slides were stained by the Ziehl-Neelsen carbol-fuchsin procedure as described previously [6]. Stained slit-skin smears were examined by optical microscopy and the bacilloscopy index (BI) was calculated according to the Ridley scale. MB patients (LL and BB) exhibited BIs ranging from +1 to +6.0, while all PB cases (including T and I forms) had bacilloscopy-negative [6].
5-HT2A and 5-HT3 receptors contribute to the exacerbation of targeted and non-targeted effects of ionizing radiation-induced cell death in human colon carcinoma cells
Published in International Journal of Radiation Biology, 2020
Jacob J. Curtis, Nguyen T. K. Vo, Colin B. Seymour, Carmel E. Mothersill
Cell monolayers were detached with trypsin, neutralized in medium and suspensions were centrifuged. Cell pellets were resuspended in medium and aliquots were counted with a TC20 Automated Cell Counter (Bio-Rad Laboratories, Mississauga, ON). Clonogenic densities of 500 cells per flask were seeded onto 25 cm2 flasks (FalconTM, Durham, NC) in 5 mL of growth medium and incubated for eight days. All chemicals were added to cell cultures during cell seeding and remained available to cells for the entire clonogenic period. Cells were subsequently stained with carbol fuchsin (RICCA Chemical Company, Arlington, TX) diluted at 1:4 (v/v) in water and counted according to previously described techniques (Puck and Marcus 1956).