Bactericidal activity of mycosynthesized silver nanoparticles was evaluated against Escherichia coli (MTCC 7410), Salmonella typhi (MTCC 733), Bacillus subtilis (MTCC 121) and Staphylococcus aureus (MTCC 7443) and all test pathogens were procured from Microbial Type Culture Collection, Chandigarh, India. Inoculum of test pathogens was prepared to obtain 5 × 105 CFU (Colony forming unit) and bactericidal activity was determined via CFU assay. In brief, Mueller–Hinton agar plates were supplemented with silver nanoparticles with different concentrations (25, 50, 75 and 100 μg/mL). Test inoculum was smeared onto the plates and incubated for 24 h at 37 °C and one control was maintained without addition of silver nanoparticles. The colonies were counted and validated with the control plate to determine the effect of nanoparticles (Sondi and Salopek-Sondi, 2004 ). Minimal Inhibitory Concentration was determined by broth micro-dilution technique based on the protocol described by Sarker et al. (2007) (link). Resazurin dye was used as a growth indicator to check the efficacy of nanoparticles against the test organisms. Gentamicin was used as positive control and bacterial growth in the plate was inspected visually as well as ELISA microtitre plate reader.
Salmonella typhi
Manufactured by Microbial Type Culture Collection
Sourced in India
Salmonella typhi is a bacterial strain maintained in the Microbial Type Culture Collection. It is a Gram-negative, rod-shaped bacterium that is the causative agent of typhoid fever in humans.
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Lab products found in correlation
Staphylococcus aureus, by Microbial Type Culture Collection (5 mentions)
Escherichia coli, by Microbial Type Culture Collection (4 mentions)
Bacillus subtilis, by Microbial Type Culture Collection (4 mentions)
Klebsiella pneumoniae, by Microbial Type Culture Collection (3 mentions)
Staphylococcus epidermidis, by Microbial Type Culture Collection (2 mentions)
Candida albicans, by Microbial Type Culture Collection (2 mentions)
Proteus mirabilis, by Microbial Type Culture Collection (2 mentions)
Shigella flexneri, by Microbial Type Culture Collection (2 mentions)
Pseudomonas aeruginosa, by Microbial Type Culture Collection (2 mentions)
Alternaria brassicicola, by Microbial Type Culture Collection (1 mentions)
Colletotrichum acutatum, by Microbial Type Culture Collection (1 mentions)
Alternaria solani, by Microbial Type Culture Collection (1 mentions)
Cladosporium herbarum, by Microbial Type Culture Collection (1 mentions)
Fusarium oxysporum, by Microbial Type Culture Collection (1 mentions)
Potato dextrose agar (pda), by HiMedia (1 mentions)
Mueller hinton agar (mha), by HiMedia (1 mentions)
Tetracycline, by HiMedia (1 mentions)
Nutrient agar, by HiMedia (1 mentions)
Kanamycin, by HiMedia (1 mentions)
Silver nitrate, by HiMedia (1 mentions)
9 protocols using salmonella typhi
1
Mycosynthesized AgNPs Bactericidal Evaluation
2
Bioactive Metabolites from Citrus Endophyte
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Endophytic Streptomyces sp. SP5 (GenBank accession number MW564023) isolated from Citrus jambhiri leaves was used in the current study14 (link). The streptomycete was maintained on starch casein nitrate agar (SCNA) plates, and spores of isolate were preserved in 20% glycerol at − 20 °C as stock for future use. Different test pathogenic bacteria viz. Bacillus subtilis (MTCC 619), Klebsiella pneumoniae subsp. pneumoniae (MTCC 109), E. coli (MTCC 1885), Staphylococcus epidermidis (MTCC 435), Salmonella typhi (MTCC 733), Alternaria brassicicola (MTCC2102), Colletotrichum acutatum (MTCC1037), Alternaria solani (MTCC2101), Cladosporium herbarum (MTCC351) and Fusarium oxysporum (MTCC284) were obtained from the Microbial Type Culture Collection (MTCC) and Gene Bank, CSIR-Institute of Microbial Technology (IMTECH), Chandigarh, India. Fusarium solani (NFCCI 91) was obtained from the NFCCI (National Fungal Culture Collection of India), Pune, and Alternaria alternata (accession number GU004283) and Fusarium moniliforme were isolated in the lab (PAU). VRE (resistant to methicillin, vancomycin, imipenem, and clindamycin) and MRSA (resistant to methicillin, teicoplanin, imipenem, and clindamycin) were collected from local hospitals. The bacterial and fungal cultures were maintained at 4 °C on nutrient agar and potato dextrose agar slants, respectively.
3
Antibacterial and Antifungal Efficacy Evaluation
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Analytical grade silver nitrate (AgNO3, 99% pure), tetracycline, kanamycin, nutrient agar, potato dextrose agar, Mueller–Hinton agar used in the present study are purchased from Himedia Lab, Ltd., Mumbai, India. The endophytic bacteria Pantoea anthophila (GenBank accession no. MN077163) identified earlier by 16S ribosomal RNA gene sequencing from W. indica were pure cultured and stored in the Department of Biotechnology, Vinayaka Mission's Kirupananda Variyar Engineering College, Salem. Bacterial pathogens such as Gram-positive Staphylococcus epidermidis (MTCC737), Bacillus subtilis (MTCC1133), Staphylococcus aureus (MTCC2940), Gram-negative Escherichia coli (MTCC40), Proteus mirabilis (MTCC425), Salmonella typhi (MTCC733), Klebsiella pneumoniae (MTCC2405) and fungal strains of Aspergillus niger (MTCC404), Candida albicans (MTCC183) and Penicillium chrysogenum (MTCC947) were obtained from Microbial Type Culture Collection (MTCC), Chandigarh.
4
Microbial Pathogen Culture Collection and Characterization
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All the test microbial pathogens such as Staphylococcus aureus (MTCC 7443), Bacillus subtilis (MTCC 121), Bacillus cereus (MTCC 430), Staphylococcus epidermidis (MTCC 435), Escherichia coli (MTCC 7410), Klebsiella pneumonia (MTCC 7407), Salmonella typhi (MTCC 733), Shigella flexneri (MTCC 1457), Vibrio parahaemolyticus ((MTCC 451), Xanthomonas campestris (MTCC 7908), and Candida albicans (MTCC 183) were procured from Microbial Type Culture Collection and Gene Bank (MTCC; Chandigarh, India).
5
Antibacterial Activity Assay Protocol
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Two Gram-positive (Staphylococcus aureus MTCC 737 and Enterococcus faecalis) and five Gram-negative (Escherchia coli MTCC 443, Salmonella typhi MTCC 3224, Shigella boydii MTCC 11947, Shigella dysentriae and Klebsiella pneumoniae MTCC 4030) were procured from the Microbial Type Culture Collection (MTCC), Institute of Microbial Technology, Chandigarh, India. The bacteria cultures were maintained in NA slants (at 4°C) and served as stock cultures. The selected bacteria were inoculated into MHB and incubated at 37°C and 190 rpm for 10–14 h. The turbidity of the resulting suspension was diluted in MHB and matched with 1 McFarland turbidity standard. The resultant level of turbidity was diluted with MHB and is equivalent to approximately 3.0 × 10−8 CFU/mL (0.5 MacFarland standards).
6
Antimicrobial Evaluation of Leaf Extract
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The antimicrobial activity of the leaf extract was performed by selecting the food borne pathogens such as bacteria and fungi. For antibacterial assay, indicator organisms are Staphylococcus aureus (MTCC 3103), Escherichia coli (MTCC 9537), Enterobacter aerogenes (MTCC 8558), Pseudomonas aeruginosa (MTCC 10306), Klebsiella pneumoniae (MTCC 10309), Salmonella typhi (MTCC 3224), Shigella flexneri (MTCC 9543), and Bacillus subtilis (MTCC 1305) were collected from Microbial Type Culture Collection (MTCC), Chandigarh, India. Nutrient agar medium and Muller Hinton agar medium was used for maintenance of cultures and antimicrobial activity, respectively. For antifungal assay, fungal cultures are Fusarium oxysporum (NCIM 1043), Aspergillus niger (NCIM 512), Penicillium citrinum (NCIM 766), Trichoderma viridae (NCIM 1051), and Candida albicans (NCIM 3471) were collected from National Collection of Industrial Microorganisms (NCIM), Pune, India.
7
Antibacterial Activity of Actinomycetes
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Lyophilized cultures of Escherichia coli (MTCC 1687); Vibrio cholerae
(MTCC 3906); Proteus mirabilis (MTCC 425); Klebsiella pneumoniae
(MTCC3384); Staphylococcus aureus (MTCC 3160); Salmonella typhi
(MTCC3231) was obtained from Microbial Type Culture Collection,
IMTECH, India. All test strains were cultured at 37 °C in Luria
broth or on Luria agar. Antibacterial activity of cell-free
supernatant was determined using the disc diffusion method. 100
µl of 8 hrs old culture broth of the test organisms were spread over
on the surface of the Luria agar plate using a sterile cotton swab.
After that, 60 µl of cell-free supernatant was added into sterile
standard discs (Himedia, India) and incubated for 18 to 24 hrs at
37 °C. After that the clear incubation zone of inhibition (ZOI) was
measured to evaluate the antimicrobial activity of actinomycetes
isolate. Dimethyl sulphoxide (DMSO) was used as control.
(MTCC 3906); Proteus mirabilis (MTCC 425); Klebsiella pneumoniae
(MTCC3384); Staphylococcus aureus (MTCC 3160); Salmonella typhi
(MTCC3231) was obtained from Microbial Type Culture Collection,
IMTECH, India. All test strains were cultured at 37 °C in Luria
broth or on Luria agar. Antibacterial activity of cell-free
supernatant was determined using the disc diffusion method. 100
µl of 8 hrs old culture broth of the test organisms were spread over
on the surface of the Luria agar plate using a sterile cotton swab.
After that, 60 µl of cell-free supernatant was added into sterile
standard discs (Himedia, India) and incubated for 18 to 24 hrs at
37 °C. After that the clear incubation zone of inhibition (ZOI) was
measured to evaluate the antimicrobial activity of actinomycetes
isolate. Dimethyl sulphoxide (DMSO) was used as control.
8
Bacterial Strain Procurement and Maintenance
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The bacterial strains namely, Listeria monocytogenes (MTCC657), Staphylococcus aureus (MTCC7443), Pseudomonas aeruginosa (MTCC 424), Escherichia coli (MTCC 118), Klebsiella pneumoniae (MTCC 432) and Salmonella typhi (MTCC 733) were procured from the Microbial Type Culture Collection (MTCC), Chandigarh (India). All the bacterial strains were sub-cultured at regular intervals on Luria Bertani (LB) slants. The slants were stored at 4 °C for further use.
9
Synthesis and Antibacterial Evaluation of Pyrrole-Based Compounds
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All chemicals used were of analytical grade used without further purification. Methyl orange (MO), ferric chloride (FeCl3), pyrrole, GA, ethylene diamine, HCl, acetone, and microbial media were purchased from Himedia, Mumbai. Gram‐positive and gram‐negative bacteria, including Staphylococcus aureus (MTCC1935) Streptococcus pneumoniae (MTCC1936), Klebsiella pneumoniae (MTCC432), Salmonella typhi (MTCC3224), Pseudomonas aeruginosa (MTCC2642), and fungi Candida albicans (MTCC3959), were purchased from Microbial Type Culture Collection, India. For aqueous solutions, deionised (DI) water was used. Experiments were conducted at room temperature (~37°C).
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