Bacteriophages were titrated using the double-layer agar technique (DLA or Gratia method) [60 ]. 1 mL of the sample was mixed with 2.5 mL of 0.7% Nutrient Agar (Nutrient Agar “Himedia”, India) and 1 mL of indicator bacteria suspension with a turbidity of 1 McFarland containing 108 CFU/mL. The prepared mixture was poured into Petri dishes on the surface of a previously prepared 2% Nutrient Agar and incubated for 18–24 h at 37 °C. The same mixture was used as a control but with 1 mL of sterile phosphate buffer instead of the aqueous sample. At the end of the incubation period, single PFUs were observed.
Nutrient agar
Manufactured by HiMedia
Sourced in India, United States, United Kingdom, Japan
Nutrient agar is a solid growth medium used for the cultivation and isolation of a wide range of microorganisms, including bacteria and fungi. It provides essential nutrients and a suitable environment for the growth and development of microbial colonies.
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Lab products found in correlation
Nutrient broth, by HiMedia (48 mentions)
Macconkey agar, by HiMedia (20 mentions)
Potato dextrose agar (pda), by HiMedia (13 mentions)
Sabouraud dextrose agar, by HiMedia (10 mentions)
Mueller hinton agar (mha), by HiMedia (10 mentions)
Blood agar, by HiMedia (9 mentions)
Eosin methylene blue agar, by HiMedia (6 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (6 mentions)
Agar agar, by HiMedia (5 mentions)
Yeast extract, by HiMedia (5 mentions)
2 2 diphenyl 1 picrylhydrazyl (dpph), by Merck Group (5 mentions)
Tween 80, by Merck Group (5 mentions)
Mueller hinton broth (mhb), by HiMedia (5 mentions)
Tryptic soya broth, by HiMedia (5 mentions)
Methanol, by Merck Group (5 mentions)
Fetal bovine serum (fbs), by HiMedia (5 mentions)
Buffered peptone water (bpw), by HiMedia (4 mentions)
Silver nitrate agno3, by Merck Group (4 mentions)
Emb agar, by HiMedia (4 mentions)
Mrs agar, by HiMedia (4 mentions)
246 protocols using nutrient agar
1
Bacteriophage Titration via Double-Layer Agar
2
Isolation of Biosurfactant-Producing Bacteria
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At plant pathology lab., Dept. Of Botany, AMU, Aligarh in 2017, five gram of oil contaminated soil sample was inoculated in 100 ml minimal medium (Miller, 1972 ) and incubated at 37ºC, at 200rpm for 48hrs. From that 100ml stock serial dilution technique was used for isolation of biosurfactant bacteria, 1 ml from the soil suspension at (10 2 to 10 7) dilution was spread on each petri plate of RA2 agar medium (Himedia, Mumbai) and these plates were incubated at 37°C for 48 hrs dark bacterial incubator. Morphologically different colonies were randomly picked/selected and purified on Nutrient agar medium plates (with 0.1% petrol mixed) (Shoeb 2006) . The selected bacterial isolates were stored on Nutrient agar (Himedia, Mumbai) petri plates as well as on Nutrient agar slants (with 0.1% petrol mixed) slants and kept under 4ºC.
3
Antibacterial Activity of A. ursinum Extract
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The antibacterial activity of the A. ursinum extract was tested against selected Gram-negative (Escherichia coli ATCC 10536, Escherichia coli ATCC 8739, Salmonella Enteritidis ATCC 13076, Proteus hauseri ATCC 13315) and Gram-positive bacteria (Listeria monocytogenes ATCC 19111, Enterococcus faecalis ATCC 29212). Lyophilized bacteria were stored in the refrigerator until the moment of activation. Reconstitution was performed according to the manufacturer’s instructions. Refrigerated slant cultures (Nutrient agar, Himedia, India) were sub-cultured weekly. Prior to each antibacterial test, selected bacterial strains were sub-cultured on Nutrient agar (Himedia, India) and incubated at 37 ± 1 °C for 18–24 h. After incubation, bacteria were aseptically transferred to 0.1% peptone salt solution (Himedia, India) and well homogenized. The density of the bacterial suspensions was adjusted to the turbidity of a 0.5 McFarland standard using the DEN-1 densitometer (Biosan, Latvia). These initial suspensions were used to prepare further decimal dilutions in 0.1% peptone salt solution intended for preparation of artificially contaminated samples of A. ursinum extract.
4
Cultivation of Bacterial and Fungal Species
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Escherichia was cultivated on agar and Muller–Hinton broth (Himedia, Mumbai, India) for 18–20 h at 37 °C. Staphylococcus was cultivated using Nutrientagar and Nutrientbroth (Himedia, India) and the GRM medium (Obolensk, Moscow, Russia) for 18–20 h at 37 °C. Nutrientagar and Nutrientbroth (Himedia, India) were used for Bacillus cultivation, and the culture was grown for 18–24 h. The cultivation of fungi of the genus Candida was conducted on Sabouraud’s medium at 30–37 °C for 48 h.
5
Isolation and Identification of Bacillus thuringiensis
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B. thuringiensis was isolated by Acetate selection method as described by Travers et al. (1987) with slight modification. One gram of soil sample was taken in a sterile conical flask containing 1mL of 0.25M sodium acetate (pH 6.8) and 9mL of Lauria Bertani (LB) broth (HiMedia, India). The broth was incubated for overnight at 30°C. After incubation the broth was heated at 100°C for 5 minute. After heat treatment, 0.1mL of sample was taken and spread on nutrient agar (HiMedia, India) and plates were incubated at 30ºC for overnight. Bt like colonies white, large, nearly circular with fine irregular margins and may be glossy, less glossy or rough were selected and further sub cultured on nutrient agar (HiMedia, India (Sneath, 1986) . For presence of parasporal crystal staining the bacterial culture incubated for 72 hours in a nutrient broth was used. Smear was stained with 0.25% coomassie brilliant blue for 3 minutes and washed with distilled water and observed under light microscope at oil immersion (Muniady et al., 2011) . The bacteria were identified as Bacillus thuringiensis based on colony morphology, Gram's staining, biochemical test and parasporal body staining. In this study B. thuringiensis DOR Bt-1 strain was used as a positive control.
6
Bacteriological Analysis of Ageing Urine
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One millilitre of urine portion was drawn from each of the ageing urine samples for bacteriological analysis. This was carried out at an ageing interval of 4 days, for 24 days. Each urine sample was aseptically subjected to serial dilution. Aliquots of 0.1 mL, making a 10-6 dilution factor, were aseptically pipetted and each inoculated by spread plate method on the cysteine–lactose–electrolyte-deficient (CLED) medium (Oxoid, Basingstokes, UK medium) in threes. The plates were subsequently inverted and incubated for 24 hours at 37 °C. For purification, morphologically different colonies were sub-cultured on nutrient agar (NA) (HiMedia, Mumbai India) plates by streak plating. The isolated cultivable bacterial cultures were grouped into different groups based on their morphological appearance on CLED and NA. The groups were then preserved on glycerol stock at –20 °C in 20% (v v-1) for further analysis.
7
Cultivation of Reference Microbes
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Potentially pathogen strains of Escherichia coli (CCM3988, E. coli), Pseudomonas aeruginosa (CCM1960, Ps. aeruginosa), Staphylococcus aureus (CCM4223, S. aureus), Proteus mirabilis (CCM7188, P. mirabilis), Streptococcus salivarius (CCM4046, Str. salivarius), and Candida albicans (CCM8186, C. albicans) from the Czech Collection of Microorganisms (CCM, Brno, Czech Republic) were used as reference strains for this study. Bacterial strains were routinely cultured at 37°C on the plates with Nutrient Agar (NA, HiMedia, India). Yeast strain was cultured at 28°C on the Glucose Peptone Yeast Extract (GPY) agar plates (consisting of glucose 40 g.L–1, peptone 5 g.L–1, yeast extract 5 g.L–1, agar 15 g.L–1, HiMedia, India).
8
Isolation and Identification of Antibiotic-Resistant Bacteria
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Approximately 25 g from each sample was mixed and macerated with 3 mL of brain heart infusion broth (BHI) (HiMedia, Mumbai, India) in a sterile mortar and pestle. Then, the samples were homogenized by mixing into 225 mL of buffered peptone water (HiMedia, Mumbai, India) followed by incubation for 24 h at 37 °C. The enrichment was streaked onto MacConkey agar (MA) and Nutrient agar (NA) (HiMedia, Mumbai, India) [14 (link)]. Plates were incubated at 37 °C for 24–48 h. Up to three suspected E. coli and K. pneumoniae colonies from each plate were subcultured onto MA with cefotaxime (1 μg/mL) for the detection of potential ESBL producers followed by incubation at 37 °C for 24 h. Bacterial isolates were subjected to a diverse array of standard biochemical tests to identify E. coli and K. pneumoniae [15 ].
9
Synthesis and Characterization of Silver Nanoparticles
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Uncapped-AgNPs were procured from IoLiTec (Ionic Liquids Technologies GmbH, Germany). Silver nitrate (AgNO3) was procured from Sigma-Aldrich (Bangalore, India). Sodium borohydride (NaBH4), TSC, PVP, PVA, nutrient agar (NA), nutrient broth (NB), Muller Hinton broth (MHB), chitosan (degree of deacetylation ≥ 75.00%) and agarose were obtained from Himedia Laboratories (Mumbai, India). Ultrapure de-ionized water was acquired from a Cascada™ Biowater System (Pall Corporation, USA). The chemicals used in the study were of analytical reagent grade.
10
Biofilm-positive bacterial isolates from CRS
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Three clinical bacterial isolates, Klebsiella pneumoniae, Pseudomonas aeruginosa and Staphylococcus epidermidis, were selected from patients no. 1, 3 and 5, respectively (Table 2 ) according to the predominant bacterial species that commonly found in clinical studies on CRS [32 (link)–34 (link)] and displayed a biofilm-positive phenotype on the surface tissue from CRS patients in this study. P. aeruginosa ATCC 27853 was also included in this study as reference strain. Antimicrobial susceptibility test results of the bacterial isolates are shown in S1 Table . K. pneumoniae and P. aeruginosa were regularly cultured on MAC (Himedia®) while S. epidermidis was grown on Nutrient agar (NA) (Himedia®). Plates were incubated at 37˚C for 24 h. A single colony of K. pneumoniae and P. aeruginosa were cultured aerobically in modified Vogel and Bonner’s medium (MVBM) at 37˚C in a 200 rpm shaker incubator for 18 h. S. epidermidis was cultured overnight in brain heart infusion (BHI) (Himedia®) under the same conditions as mentioned above.
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