Antimicrobial activity using crude secondary metabolites of LAB against selected pathogenic bacteria was performed using the agar well diffusion technique. Cell-free supernatants (CFS) of each bacterial isolate were prepared and adjusted to pH 6.5. Certain selected strains of pathogenic bacteria viz., Escherichia coli (MTCC no. 433), Staphylococcus aureus (ATCC no. 6538), Pseudomonas aeruginosa (ATCC no. 9027), Salmonella abony (ATCC no. BAA2162) and Listeria monocytogenes (L. monocytogenes; MTCC no. 1143), were purchased from the American Type Culture Collection (ATCC) and Microbial Type Culture Collection (MTCC; IMTech), and 0.1 ml of pathogens were inoculated onto Mueller Hinton agar media (Himedia Laboratories Pvt, Ltd.). Furthermore, 7-mm diameter wells were made in the agar plates and 100 µl of CFS was placed into these wells, and finally the inhibition zone was measured (mm) after 48 h of incubation at 37˚C.
Mueller hinton agar media
Manufactured by HiMedia
Sourced in India
Mueller Hinton agar media is a standardized microbiological culture medium used for antimicrobial susceptibility testing. It provides a consistent and reliable growth environment for a wide range of bacterial species.
Automatically generated - may contain errors
Lab products found in correlation
Ciprofloxacin, by HiMedia (2 mentions)
Chloramphenicol, by HiMedia (2 mentions)
Tetracycline, by HiMedia (2 mentions)
Salmonella abony, by American Type Culture Collection (1 mentions)
Pseudomonas aeruginosa, by American Type Culture Collection (1 mentions)
Staphylococcus aureus, by American Type Culture Collection (1 mentions)
Potassium iodide, by HiMedia (1 mentions)
Eugenol, by Merck Group (1 mentions)
Iodine, by HiMedia (1 mentions)
Tween 80, by Merck Group (1 mentions)
Erythromycin, by HiMedia (1 mentions)
Gentamicin, by HiMedia (1 mentions)
Imipenem, by HiMedia (1 mentions)
Meropenem, by HiMedia (1 mentions)
Levofloxacin, by HiMedia (1 mentions)
Whatman no 1 filter paper disc, by Cytiva (1 mentions)
Fluconazole, by HiMedia (1 mentions)
Novobiocin, by HiMedia (1 mentions)
Streptomycin, by HiMedia (1 mentions)
Ampicillin, by HiMedia (1 mentions)
7 protocols using mueller hinton agar media
1
Antimicrobial Activity of LAB Metabolites
2
Antibiotic Sensitivity of E. coli and Salmonella
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Antibiotic sensitivity test of the isolated E. coli and Salmonella spp. was performed by disk diffusion method [24 ]. Freshly grown isolates having a concentration equivalent to 0.5 McFarland standards were spread on Mueller-Hinton agar media (HiMedia) using a sterile cotton swab and eight commonly used antibiotics of HiMedia, namely, amoxicillin (AMX, 30 μg/disc), gentamycin (GEN, 10 μg/disc), tetracycline (TET, 30 μg/disc), erythromycin (ERY, 15 μg/disc), doxycycline (DOX, 30 μg/disc), moxifloxacin (MOF, 5 μg/disc), cephalexin (CN, 30 μg/disc), and cefixime (5 μg/disc) were placed on the media. All results of antibiotic susceptibility for E. coli and Salmonella spp. were interpreted according to the guidelines provided by Clinical and Laboratory Standards Institute [25 ].
3
Antibacterial and Antifungal Efficacy of Shellac-based Formulation
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Shellac flake was obtained from HiMedia Pvt. Ltd. Mumbai, India. Carnauba wax No-1 yellow, Tween 80 and Eugenol were procured from Sigma-Aldrich. Mueller Hinton Agar media, Iodine, Potassium iodide was purchased from HiMedia (Mumbai, India) and sodium hypochlorite (NaOCl) solution was procured from Avantor Performance Materials, Maharashtra, India. Fresh lemon of similar color, size, and maturity, free of any visual damage were procured from a local farmer of Kokrajhar district, Assam, India. Aloe gel (AG) was extracted by scraping the outer epidermis of fresh Aloe leaves, which was obtained locally. The bacterial strains namely Staphylococcus aureus, Escherichia coli, Bacillus subtilis, and Alcaligenes faecalis were used for antibacterial study. The fungal strains Saccharomyces cerevisiae and Rhizopus stolonifer were obtained from the microbial type culture collection (MTCC) and gene bank, CSIR-Institute of Microbial Technology and the fungi were activated on the potato dextrose agar (PDA).
4
Antibiotic Susceptibility of E. coli and Salmonella
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Antibiotic sensitivity testing of isolated E. coli and Salmonella spp. was carried out using the disk diffusion assay as previously described [63 (link)]. Antibiotic classes included fluoroquinolones (levofloxacin, LEV—5 μg; ciprofloxacin, CIP—5 μg), aminoglycosides (gentamicin, GEN—10 μg; streptomycin, S—10 μg), carbapenems (Meropenem, MEM—10 μg; imipenem, IMP—10 μg), amphenicols (chloramphenicol, C—10 μg), macrolides (erythromycin, E—15 μg), and tetracyclines (tetracycline, TE—30 μg) purchased from Hi Media (India). Sensitivity tests were performed on freshly grown isolates having a concentration equivalent to 0.5 McFarland standard using Mueller-Hinton agar media (Hi Media, India). All results were interpreted according to the guidelines provided by Clinical and Laboratory Standards Institute [64 ]. Furthermore, isolates showing resistance against three or more different classes of antibiotics were defined as MDR [65 (link)].
5
Antimicrobial Susceptibility Testing Protocol
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Petri plates containing 15 mL of Mueller Hinton agar media (Hi-Media) were used for all the bacterial strains and Sabouraud dextrose agar (SDA) media (Hi-Media) were used for the fungal strains. 0.1 mL of seed of various organisms was aseptically inoculated over the sterile solid agar medium. Whatman no. 1 filter paper discs (6 mm in diameter) impregnated with the synthesized compound (10 μg/disc) dissolved in dimethyl sulfoxide (DMSO) were placed on the plates. All the sensitivity plates were then incubated at 37 ± 1°C for 24 h for bacterial strain and 72 h at 25°C for fungal strain. Ciprofloxacin (10 μg/disc, Hi-Media) and fluconazole (10 μg/disc, Hi-Media) were used as positive control for the assessment of antibacterial and antifungal activity, respectively. A paper disc impregnated with DMSO was used as negative control. The zone of inhibition on agar plate was measured in millimeters (mm) and the test was performed in triplicate and the average was taken as final reading (Table 2 ).
6
Antibiotic Susceptibility Profiling of Bacterial Isolates
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The following ten commonly-prescribed antibiotics (HiMedia, India) were used for antimicrobial susceptibility testing: ampicillin (AMP, 10 mg), chloramphenicol (C, 30 mg), ceftriaxone (CTR, 30 mg), ciprofloxacin (CIP, 30 mg), doxycycline (DO, 30 mg), meropenem (MEM, 10 mg), novobiocin (NV, 30 mg), streptomycin (S, 10 mg), tetracycline (TE, 30 mg), and vancomycin (VA, 30 mg). For each culture suspension of the bacterial isolates, a McFarland 0.5 standard was maintained [26 ]. Antibiogram phenotyping was performed using the disk diffusion method in Mueller Hinton agar media (HiMedia, India). Results were recorded as sensitive, intermediate, or resistant in accordance with the CLSI recommendations [27 ].
7
Antibacterial Activity of Hemolymph Bacteria
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Each hemolymph associated bacteria were assayed in-vitro for antibacterial activity against a panel of 11 indicator strains of aquatic pathogens (Table 3) by spot diffusion assay (Nair et al., 2012) . Briefly, 8 h old cultures of these indicator strains (10 6 CFU/ml) were spread separately onto the surface of Mueller Hinton Agar media (HiMedia) (with or without 1% NaCl supplementation according to pathogen). Later, pure culture of each isolate was spotted onto these agar plates. The plates were examined for the formation of inhibitory zone after overnight incubation at 30 °C. Zone was recorded and measured for positive isolates. The test was replicated for positive cases to confirm the antimicrobial activity and the mean zone diameter was taken for analysis. Versatility of each isolates and number of positive isolates against each pathogen were also noted down. Scoring of positive isolates based on their zone of inhibition diameter (difference between total zone diameter and bacterial colony diameter) was also done (Nandi et al., 2017) in which score 4, 3, 2, 1 and 0 were assigned to very high (> 15 mm), high (10-14 mm), moderate (5-9 mm), Low, (1-4 mm) and no inhibition activities respectively.
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