Understanding Microbiology Culture Results
Firza Alexander Gronthoud in Practical Clinical Microbiology and Infectious Diseases, 2020
Microbes can be directly visualized in bodily fluids and tissue samples with microscopy. The Gram stain is used for detection of bacteria. Bacteria can be classified as Gram positive or Gram negative based on their ability to retain crystal violet after decolourization with alcohol. Gram-positive bacteria retain crystal violet in their cell wall and have a purple colour. In contrast, in Gram-negative bacteria, the alcohol has removed the crystal violet (a process called decolourization) and a counterstain is used to make them visible and pink coloured. Counterstains used are safranin and carbol fuchsin. Counterstains are weaker stains than crystal violet and Gram-positive bacteria remain purple coloured. Gram stain can also be used to visualize common fungi such as Candida species, which are Gram positive.
Microorganisms
John C Watkinson, Raymond W Clarke, Louise Jayne Clark, Adam J Donne, R James A England, Hisham M Mehanna, Gerald William McGarry, Sean Carrie in Basic Sciences Endocrine Surgery Rhinology, 2018
Most organisms commonly recovered from clinical specimens can be identified on the basis of few simple tests: Gram staining and light microscopygrowth requirements (e.g. aerobic or anaerobic growth)metabolic properties. The best known of these techniques is the Gram stain, which was developed by the Danish microbiologist Hans-Christian Gram (1853–1938) as a stain that is able to differentiate between human and bacterial tissue more readily than the histological stains in use at the time. The presence or absence of an outer membrane leads to differences in uptake of methylene blue, and has coined the terms ‘Gram positive’ and ‘Gram negative’ (Figure 19.1).
Biology of microbes
Philip A. Geis in Cosmetic Microbiology, 2006
Gram stain. When performing a Gram stain, start by spreading a thin suspension of bacteria on a glass slide, let the suspension dry on the slide, and heat it gently to fix the bacteria onto the slide. The next step is to put a crystal violet solution on the slide for about a minute and rinse the slide with water. The bacteria absorb the dye and turn purple. Then put Gram’s iodine on the slide. The iodine acts as a mordant that “sets” the purple dye. Next, rinse with an acetone–alcohol mixture to try to remove the set dye. Finally, counterstain with safranin, a pink dye. If the crystal violet dye cannot be removed with the acetone–alcohol mixture, then the safranin counterstain is not even seen; the bacterium is so dark purple that the pink dye does not contribute to the color. If the crystal violet is removed, the bacteria are colorless until the pink safranin counterstain is added. Bacteria that stain purple are called Gram-positive (positive because they retain the first dye). Bacteria that stain pink are designated Gram-negative.
Bifidobacterium longum Suppresses Murine Colorectal Cancer through the Modulation of oncomiRs and Tumor Suppressor miRNAs
Published in Nutrition and Cancer, 2019
Cinderella A. Fahmy, Amira M. Gamal-Eldeen, Enas A. El-Hussieny, Bassem M. Raafat, Nayra S. Mehanna, Roba M. Talaat, Mohamed T. Shaaban
The isolation and identification of LAB (Bifidobacteria) was carried out using a pour plate technique (16,17). The isolated colonies of bacteria were submitted for Gram staining procedure. Slide catalase test was carried out to verify Bifidobacteria as catalase negative. To investigate the carbohydrate fermentation, the API identification strips (bioMérieux, Marcy l’Etoile, France) was used. API® 20 Strep was utilized to identify streptococci and enterococci, API® 50 CH, API® 20 A, and API® ZYM for the identification of anaerobic bacteria. Bacteria Genomic DNA was extracted by Genomic DNA Purification Kit (QIAGEN, Germany). PCR conditions were consisted 95 °C for 3 min followed by 40 cycles (95 °C, 30 s, 57 °C, 30 s, and 73 °C, 60 s) and a final extension cycle at 73 °C for 5 min, using the following primer set; (g-Bifid-F/g-Bifid-R) for bifidobacterium. Forward: 5′-TTCCAGTTGATCGCATGGTC-3′; Reverse: 5′-GGGAAGCCGTATCTCTACGA-3′ (19). The assessment of the potential probiotic role of the isolated bacteria was investigated by antimicrobial activity, tolerance against bile salts and resistance to low pH (15,17). The viability was determined by the pour plate technique after 4 h (16,17).
Strategies for the diagnosis and management of meningitis in HIV-infected adults in resource limited settings
Published in Expert Opinion on Pharmacotherapy, 2021
Marise Bremer, Yakub E Kadernani, Sean Wasserman, Robert J Wilkinson, Angharad G Davis
In CSF analysis, polymorph leucocytosis of >1000 cells/mm3 is the best discriminator between bacterial and other causes of meningitis [46,47]. CSF culture remains the gold standard with a sensitivity of 81% when compared by to Gram stain and polymerase chain reaction (PCR) [48]. A study on repeated CSF cultures after normal initial CSF showed an increased diagnostic yield in clinically deteriorating patients, either by new bacteriological growth or the emergence of cell abnormalities [49]. This can assist with confirming the diagnosis of BM in this subset of patients. Gram stain is a widely used, rapid test that is valuable in early diagnosis with a sensitivity of 97.5% when referenced to culture, but less affected than culture by antibiotic activity in the CSF [48]. Antigen detection by latex agglutination (LA) is rapid, but when compared to culture sensitivity is 66% [50]. In culture negative BM pre-treated with antibiotics LA was also negative in all samples [51], showing no added value compared to other rapid testing modalities. The ongoing development of PCR assays has shown sensitivities up to 100% [52] and detection in culture negative samples [53]. These tests are also becoming less expensive and time consuming [54].
Evaluation of cytokine profile in cervicovaginal lavage specimens of women having asymptomatic reproductive tract infections
Published in Journal of Obstetrics and Gynaecology, 2022
Clara Aranha, Mayuri Goriwale, Shahina Begum, Sheetal Gawade, Vikrant Bhor, Anushree D. Patil, Kiran Munne, Vandana Bansal, Deepti Tandon
After heat fixing the smear, Gram staining was performed using commercially available stains. Gram-stained smears were examined under ×1000 magnification and information was recorded for Nugent’s score, presence of clue cells, pus cells/leukocytes along with detection of budding yeast cells. For cytokine analysis, cervicovaginal lavage was centrifuged at 1500 rpm for 10 min to settle down vaginal discharge and epithelial cells. Supernatant was filtered through 0.22 μm syringe filter (Axiva, Delhi, India) and aliquoted samples were stored at −80 °C. These supernatants were utilised for estimation of cytokines by ProcartaPlex multiplex immunoassays platform (Thermo Fisher, Waltham, MA) based on the principles of a sandwich ELISA and utilising Luminex xMAP (multianalyte profiling) technology. During batch testing, all the samples were thawed at room temperature and vortexed properly before use. IL-1β, IL-6, IL-8, IL-10, IL-12/IL23p40, IL-17A and TNF-α, interferon gamma (IFN-γ) were the eight cytokines analysed. All samples were tested in duplicate. The cytokine concentrations (pg/ml) in the unknown samples were derived from the premixed cytokine standards generated values (pg/ml). Reported cytokine concentrations were normalised against total protein concentration in cervicovaginal lavages (estimated by the Bradford method). Further normalised values were log-transformed to improve the normality of data distribution.
Related Knowledge Centers
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