We examined the efficacy of antibiotics by measuring colony-forming units (CFU) (Fig. S1 ). Bees collected from the −/− and +/+ groups at 1 and 7 days of age were washed in 70% ethanol (Sigma-Aldrich, ST. Louis, MO, USA) and had their digestive tracts removed. The digestive tracts were homogenized by vortex mixer in 10 mL of nutrient broth medium (Himedia, West Chester, PA, USA) followed by dilution plating on nutrient agar (Himedia) and incubated at 35°C under aerobic conditions for 48 h (VWR, Radnor, PA, USA). Individual bacterial colonies were counted and multiplied by the degree of dilution to obtain CFU of the original sample (Olsen and Bakken, 1987 (link)). This CFU study is a control that demonstrates antibiotic applied to the colony reached bees and bacteria that can be cultured aerobically are reduced after antibiotic treatment.
Nutrient broth medium
Manufactured by HiMedia
Sourced in India
Nutrient broth medium is a general-purpose growth medium used for the cultivation of a wide range of microorganisms. It provides essential nutrients, such as peptones, yeast extract, and salts, to support the growth and proliferation of bacterial and fungal cultures.
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Lab products found in correlation
Nutrient agar, by HiMedia (3 mentions)
Antimicrobial disks, by HiMedia (2 mentions)
Ethanol, by Merck Group (1 mentions)
Densichek, by bioMérieux (1 mentions)
Lb agar, by HiMedia (1 mentions)
Crystal violet stain, by Merck Group (1 mentions)
Chloroauric acid haucl4 3h2o, by Merck Group (1 mentions)
Fetal calf serum (fcs), by Merck Group (1 mentions)
Ethidium bromide, by Merck Group (1 mentions)
Acridine orange, by Merck Group (1 mentions)
Hematoxylin and eosin stain, by Merck Group (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Merck Group (1 mentions)
Streptomycin, by Merck Group (1 mentions)
Trypsin edta, by Merck Group (1 mentions)
Muller hinton agar, by HiMedia (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Copper 2 sulfate pentahydrate, by Merck Group (1 mentions)
Nutrient agar medium, by HiMedia (1 mentions)
Spectramax m2 plate reader, by Molecular Devices (1 mentions)
18 protocols using nutrient broth medium
1
Measuring Antibiotic Efficacy in Bees
2
Isolation and Identification of Laccase-Producing Bacteria
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Laccase producing bacterium were screened and isolated from soil samples composed of plant material waste and saw dust collected from the forest area, wood market, and green sites around the Chandigarh, India. All the collected samples (10 g of each soil sample) were sieved and added to 100 ml of nutrient broth medium (Himedia) supplemented with 0.2 mM CuSO4 and 2 mM guaiacol as inducers and incubated at 37 °C for 48 h to prepare the stock. All the stock samples were successively diluted in the ratio of 10−1 to 10-7 and 100 μl of each dilution was plated on nutrient agar plates containing 5 mM guaiacol (a potent substrate for laccase) at 37 °C for about 3–4 days of incubation. The bacterium colonies visualized by appearance of coloured zones of substrate oxidation in nutrient agar medium were selected as laccase producing isolates. Following the primary screening procedure further selection was done by quantifying their laccase activity. The isolate showing highest laccase activity was selected for further detailed study.
The selected bacterial strain was identified by morphological, biochemical and physiological analysis according to Bergey’s manual of determinative bacteriology [31 ] and further confirmed by 16S rRNA sequence analysis.
The selected bacterial strain was identified by morphological, biochemical and physiological analysis according to Bergey’s manual of determinative bacteriology [31 ] and further confirmed by 16S rRNA sequence analysis.
3
Synthesis of Silver Nanoparticles using Tinospora cordifolia
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The medicinal plant, Tinospora cordifolia was selected from Gir forest region of Gujarat, India on the basis of its medicinal property [11 ]. Silver nitrate, AR grade was procured from Thermo Fisher Scientific, Vadodara, India. The two bacterial strains one Gram positive (Bacillus megaterium MTCC 7192) and one Gram negative (Pseudomonas aeruginosa MTCC 741) are clinical isolates that were obtained from the microbial type culture collection (MTCC), Institute of microbial technology, Chandigarh, India. Bacterial strains were cultivated on Nutrient broth medium (pH 7.3 ± 0.1; HiMedia, Vadodara, India) at 37 °C with shaking at 150 rpm. Bacterial cell suspensions were diluted with a sterile Milli-Q water (pH 7) to obtain a final concentration of 108 CFU/ml. Four broad spectrum antibiotics, namely, amoxicillin (Sun pharmaceutical, Ankleshwar, India), chloramphenicol (Alps pharmaceuticals, Uttarakhand, India), trimethoprim-sulfamethoxazole (Abbott Healthcare, Mumbai, Maharashtra), and linezolid (Glenmark pharmaceutical, Ankleshwar, India) were used for bactericidal assay. All other chemicals used in this study were analytical grade (Sigma Aldrich, Ahmedabad, India).
4
Antibacterial Efficacy of AgNPs from Ocimum sanctum
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The killing kinetics assay of A. baumannii against AgNPs of O. sanctum was performed spectrophotometrically (Shimadzu, Tokyo, Japan) at OD 600 nm in triplicates. A. baumannii was cultured in a nutrient broth medium (Hi-Media, Mumbai, India) at 37 °C in the dark for 18–24 h. Before use, the bacterial broth was diluted in Nutrient Broth and adjusted to 0.5 McFarland turbidity (10 8 CFU/mL) using Densichek (BioMerieux, Marcy-l’Étoile, France). A volume of 100 µL of this broth was added to the five wells of the microtiter plate. Then, 100 µL of AgNPs from O. sanctum at concentrations of 32 µg/mL (MIC), 64 µg/mL (MBC), 128 µg/mL, 256 µg/mL and 512 µg/mL were added to the five wells loaded with the bacterial broth. The microtiter plate was then incubated at 37 °C in the dark, and the bacterial viability was measured spectrophotometrically in triplicates at 0, 2, 4, 8, 12, 18, and 24 h of incubation. The negative control (bacterial broth without AgNPs and without antibiotic colistin) and positive control (bacterial broth treated with antibiotic colistin at a MIC of 2 µg/mL) were included in the test. The percentage of inhibition of bacterial growth was calculated in comparison with the negative control (Das et al., 2017 (link)), and a statistical correlation was made with the positive control.
5
Antibacterial Activity of Green Synthesized Silver NPs
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The bacteria such as, Shigella sonnei, Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus subtilis and Shigella dysentriae were used as the test organisms. All these isolates were obtained from Microbial Type Culture Collection, Pune, India. All organisms were sub cultured using LB Agar (Himedia, Mumbai, India). Then, the organisms were cultured individually in 100 ml Erlenmeyer flask containing nutrient broth medium (Himedia, Mumbai, India) and was grown in an rotary shaker incubator at 37 °C. The culture was centrifuged and the pellet was harvested, then it was washed twice in water, followed by Phosphate Buffered Saline (PBS) and the broth was diluted appropriately (106 CFU/ml). Antibacterial activity test was performed to analyze the biological activity of green synthesized silver NPs against these tested bacterial strains as suggested by Cheesbrough (2000) . For the determination of antibacterial activities of the green synthesized NPs, 20 µg of sample was loaded into the wells and the plate was kept in refrigerator for about 2 h for sample diffusion. Then all plates were incubated in an incubator at 37 °C for overnight and antibacterial activity was recorded. The zone of inhibition was tabulated.
6
Oral Microbial Diversity Profiling
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Samples were collected from 20 individuals covers various dietary habits, male and female individuals at various age groups. The swabs were taken from gingival, buccal cavity, subgingival and the entire teeth. Then the swab was inoculated Mitis Salivarius broth (g/L, Crystal violet 0.0008; Trypan blue 0.075; Dipotassium phosphate 4; Sucrose 50; Dextrose 1; Casein enzymatic hydrolysate 15, and Peptic digest of animal tissue 5) and nutrient broth medium (Himedia, Mumbai, India).
7
Screening Bacillus sp. BioSol021 PGP Potential
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In order to better understand PGP potential of the producing strain Bacillus sp. BioSol021, several PGP parameters were screened, as described below. For the PGP screening procedure, Bacillus sp. BioSol021 was cultivated in Erlenmeyer flasks using the nutrient broth medium (Himedia Laboratories, Mumbai, India) for 48 h on a rotary shaker (170 rpm) at 28 °C, without or with addition of L-Trp (1.02 g/L) for the experiments aimed at indole compounds quantification. For the experiments requiring cell-free supernatant of the cultivation broth, centrifugation was performed to separate bacterial biomass (12,000 g, 10 min, 25 °C, Z 326 K, Hermle LaborTechnik GmbH, Wehingen, Germany). If not stated differently, the cultivation broth of Bacillus sp. BioSol021 was used in the PGP-assaying procedures.
8
Reviving and Standardizing Bacterial Strains
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Pseudomonas aeruginosa MTCC7195 and B. gladioli MTCC10242 (obtained from Microbial Type Culture Collection, IMTECH Chandigarh, India) were revived in Nutrient Broth Medium (HiMedia Laboratories, Mumbai, India). Bacterial strains were incubated at 28°C for 24–48 h. The bacterial suspension was centrifuged at 4,000 rpm for 10 min at 4°C to obtain the pellet. The pellet was washed twice and resuspended in sterile distilled water to get uniform density of 109 cells/ml.
9
Antibacterial Potential of T. procumbens
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Antibacterial activity of leaves of T. procumbens was evaluated by well diffusion method. The bacteria such as, Escherichia coli, Bacillus subtilis, Staphylococus aureus and Enterobacter aerogenes were used. Inoculum was prepared using nutrient broth medium (Himedia, Mumbai, India) and 105 cfu ml−1 bacterial cultures was spread on Mueller Hinton Agar plates and 6 mm diameter well was punched into the agar and filled with 40 µl (25 mg/ml) of crude extract. The inhibition zone around the sample well was measured (mm) (NCCLS, 1993 ).
10
Evaluating Microbial Growth Patterns
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Escherichia coli (ATCC 8739), Pseudomonas aeruginosa (ATCC 15442), Salmonella typhi (ATCC 13311), Bacillus cereus (ATCC 14579), Enterococcus faecalis (ATCC 29212), and Staphylococcus aureus (ATCC 6538) bacteria were used for analysis. Aspergillus niger (ATCC 16404), A. flavus (ATCC 9643), A. nidulans (ATCC 38163), and Penicillium expansum (ATCC 7861) fungal species were also tested. Bacterial strains were cultured in nutrient broth medium (Himedia, Mumbai, India) for 18 h at 37 °C. The fungal strains were cultured in potato dextrose broth for 72 h at 37 °C. The growth of culture was observed using a UV-visible spectrophotometer at 600 nm.
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