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Quantitative Cell Culture Techniques
Published in Jay L. Nadeau, Introduction to Experimental Biophysics, 2017
The target cells for phage are bacteria, so the plaques to be counted represent holes in a bacterial lawn. The procedure is simple, but it is important to mix things well, dilute carefully, change pipette tips when necessary, and ensure even distribution of phages in the plates. The following protocol works well:
Fate of enteric viruses during leafy greens (romaine lettuce) production using treated municipal wastewater and AP205 bacteriophage as a surrogate
Published in Journal of Environmental Science and Health, Part A, 2021
Harvey N. Summerlin, Cícero C. Pola, Karthikeyan R. Chamakura, Ry Young, Terry Gentry, Eric S. McLamore, Raghupathy Karthikeyan, Carmen L. Gomes
Initial concentration of the AP205 isolate was determined to be 109 PFU/mL (plate forming units per mL) by spot titration on a bacterial lawn containing tryptic soy agar (TSA, Becton, Dickinson and Company, Franklin Lakes, NJ) and the host bacteria.[21] Standard operating procedures (SOPs) were followed to produce enough lysate (raw phage) as provided by the Center for Phage Technology, Texas A&M University.[22] Briefly, 100 μL of an overnight culture of A. baumannii and 100 μL AP205 was combined with 4 mL of molten TSA top agar in a sterile glass tube. The mixture was briefly vortexed and poured onto a TSA plate. The mixture was allowed to set for five minutes and then inverted and incubated at 30 °C for 24 hours. Then, the top agar was scraped off the firmer bottom agar layer using buffered peptone water (BPW, Becton, Dickinson and Company, Franklin Lakes, NJ) as a lubricant and placed into sterile 50-mL Falcon tubes (VWR International) and centrifuged at 8000 g for 10 min. The supernatant was then filtered through 0.45-μm and 0.22-μm syringe filters (VWR International). The final concentration of the lysate stock after propagation was examined and determined to be 1010 PFU/mL using the same procedure as the initial concentration analysis. The AP205 stock (suspended in BPW) was stored under 4 °C refrigeration throughout the experiment (for less than a month).
Synthesis, thermal, photo-physical, and biological properties of mononuclear Yb3+, Nd3+, and Dy3+ complexes derived from Schiff base ligands
Published in Journal of Coordination Chemistry, 2020
Sikandar Paswan, Afreen Anjum, Navneet Yadav, Nitesh Jaiswal, Rana Krishna Pal Singh
The ligands (L1H2 and L2H2), metal salts and their Ln(III) complexes were tested in vitro antibacterial activity against different bacterial strains Escherichia coli (ATCC 35218), Salmonella typhi (ATCC 19430), Staphylococcus aureus (ATCC 25323), and Enterococcus faecalis (ATCC 29212) using agar disc diffusion method [26]. The stock solution (1 mg mL−1) of the test complex was prepared in DMSO, which is further diluted to 100 µg mL−1 with sterilized distilled water. Briefly, 3.8% (w/v) of Mueller-Hinton Agar (MHA; Himedia company) along with 1% (w/v) additional agar was added into double distilled water and sterilized via autoclaving (Caltan, NSW-227) at 121 °C at 15 psi for 15 min. After sterilization, the media was poured into clean sterilized petri dishes (20 mL media each plate) and permitted to solidify at room temperature. Before pouring the media, the petri dishes were dried by hot air oven (holding time 60 min at 160 °C). We used freshly cultured bacteria for bacterial growth from the stock. Now 100 µL of bacterial suspension was prepared, bacterial lawn from bacterial suspension (1.5 × 108 CFU mL–1) was prepared over MHA using a sterilized cotton swab stick.
Biosynthesis of silver nanoparticles using novel Bacillus sp. SBT8
Published in Preparative Biochemistry & Biotechnology, 2018
Tuğçe Yurtluk, Fikriye Alev Akçay, Ayşe Avcı
Antimicrobial activity tests were performed using suspended solutions of AgNPs obtained at the optimized conditions (using 6 mM AgNO3, at pH 10, and 33°C). For this purpose, 10 mg of dried nanoparticles was weighed and suspended in 1 ml of sterile deionized water. To provide a true dispersion, the solution was vortexed in the presence of soda lime glass beads having 4 mm diameter for 10 min. After the dispersion was achieved, appropriate dilutions (0.01, 0.1, 0.2, 0.4, 0.6, 0.8, and 1.0 mg/ml) were prepared. Inhibitory effect of nanoparticles was tested on various Gram-negative (E. coli O157:H7, P. eruginosa, S. Typhimurium) and Gram-positive (S. aureus, L. monocytogenes) pathogens. The test microorganisms were cultured in tryptic soy broth to a cell density of 107–108 cfu/ml at 37°C except L. monocytogenes which was incubated at 30°C. Antimicrobial activity was determined using modified Kirby–Bauer disk diffusion method.[19] Briefly, a 50 µl active test culture was swapped on a tryptic soy agar plate and then a sterile paper disk, having a diameter of 6 mm, was placed on top of the bacterial lawn. A total of 10 µl of diluted AgNPs solution was applied on the center of the disks, thus each disk contained 0.1, 1, 2, 4, 6, 8, or 10 µg of AgNPs. Then the plates were allowed 30 min for the diffusion of the solution and incubated at 37°C except L. monocytogenes which was incubated at 30°C. After 24 h, diameters of the clear zone formed around each disk were measured. Various antibiotic disks including ampicillin (2 and 10 µg; AM2 and AM10), streptomycin (10 µg; S10), and erythromycin (15 µg; E15) were used as positive control, and cell-free supernatant of Bacillus sp. SBT8 was used as negative control.