Twenty-five grams of the sample was inoculated into 225 ml trypticase soy broth (Merck, Darmstadt, Germany), incubated at 37 °C for 24 h, and subcultured via streaking onto MRSA selective agar plate (CHROMagar™ MRSA-ITK Diagnostics BV, Netherlands) and incubated at 37°C for 24 h. Rose to mauve colonies on MRSA selective agar plates were presumptive MRSA isolates. The isolates were identified based on cultural, morphological, and biochemical tests such as Gram-reactions, 3% potassium hydroxide (3% KOH), catalase, coagulase, β-haemolysis, DNAse activity, anaerobic utilization of glucose and mannitol (Tallent et al., 2019 ). One colony per plate was purified in nutrient agar (Lab M, Lancashire, United Kingdom), further incubated for 18 h at 37°C, and preserved on nutrient agar slants at 4°C. The positive control used includes S. aureus (ATCC 12600).
Nutrient agar
Manufactured by Lab M
Sourced in United Kingdom
Nutrient agar is a solid culture medium used for the growth and isolation of microorganisms. It provides the necessary nutrients and solidifying agents for the cultivation of a wide range of bacterial and fungal species.
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Lab products found in correlation
Palcam agar, by Lab M (3 mentions)
Macconkey agar, by Lab M (2 mentions)
Sorbose, by Merck Group (1 mentions)
Mannose, by Merck Group (1 mentions)
Mannitol, by Merck Group (1 mentions)
Agarose, by Merck Group (1 mentions)
N n n n tetramethylethylenediamine temed, by Merck Group (1 mentions)
Galactose, by Merck Group (1 mentions)
N acetylglucosamine, by Merck Group (1 mentions)
Maltose, by Merck Group (1 mentions)
Coomassie brilliant blue r 250, by Merck Group (1 mentions)
Glucose, by Merck Group (1 mentions)
Sodium dodecyl sulfate (sds), by Merck Group (1 mentions)
Ammonium persulfate, by Merck Group (1 mentions)
Glutaraldehyde, by Merck Group (1 mentions)
Acrylamide, by Merck Group (1 mentions)
Lactose, by Merck Group (1 mentions)
Nutrient broth, by Lab M (1 mentions)
Mannitol salt agar, by Lab M (1 mentions)
Rapid salmonella agar, by Bio-Rad (1 mentions)
15 protocols using nutrient agar
1
Isolation and Identification of MRSA
2
Biochemical Reagents and Materials
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Glutaraldehyde, bovine serum albumin (BSA), sugars (glucose, lactose, maltose, mannose, sorbose, galactose, N-acetylglucosamine, and mannitol), acrylamide, ammonium persulfate, N,N,N ,N-tetramethylethylenediamine (TEMED), sodium dodecyl sulfate, agarose, and coomassie brilliant blue R250 were purchased from the Sigma Chemical Company, St Louis, Missouri, USA. Sepharose 4B and Sephadex G-150 were bought from Pharmacia Fine Chemicals, Uppsala, Sweden. Nutrient agar and nutrient broth were from Lab M Ltd, Lancashire, United Kingdom. Gel-loading dye (BioLabs ®, B7025S), ethidium bromide, deoxyribonucleoside triphosphates (dNTPs), Taq polymerase, DNA master mix, 100-bp DNA ladder (BioLabs ®, NO551S), and ZYMO DNA Extraction Kit were purchased from South Africa (Inqaba Biotech, Hatfield Pretoria). All other reagents and chemicals used in this study were of analytical grade.
3
Oral Cavity Microbiome Profiling
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All 45 collected oral cavity swabs from the participants were swabbed directly onto Blood Agar (5% sheep blood), and MacConkey agar for the primary isolation of microaerophilic and aerobic microflora was incubated at 37 °C for 24 hrs in a microaerophilic environment and aerobic environment respectively. mannitol salt agar was used as a selective culture media to isolate S. aureus. In total, 84 colonies having different morphology were isolated and purified after processing the samples. Gram staining was done to identify the suspected isolates of S. aureus. After Gram staining, suspected S. aureus colonies were inoculated on mannitol salt agar (Lab M Limited, UK) and incubated for 24 hrs at 37 °C. Based on physical appearance, golden yellow colored, convex, round, and opaque were the indication of S. aureus strains that were observed. All isolated colonies were shifted to Nutrient agar (Lab M Limited, UK) and incubated at 37 °C for 24 hrs. Once incubated colony characteristics became evident, species were identified depending on the following tests using rapid biochemical tests: oxidase, catalase, coagulase, DNase, and others, according to Bergey’s manual of determinative bacteriology (Vos et al., 2009 ).
4
Microbiological Analysis of Food Samples
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The spread plate technique on an appropriate agar medium was used to determine the number of microorganisms. The following microbiological media and test methodology were used: nutrient agar (LabM, Heywood, UK) to determine the total viable count (TVC), in accordance with the ISO 4833-2 [19] , MacConkey agar No. 3 (LabM, Heywood, UK) to determine the number of rods from the Enterobacteriaceae family (ENT), in accordance with the ISO 21528-2 [20] and YGC agar (Sabouraud Dextrose with chloramphenicol lab Agar, Biomaxima, Lublin, Poland) to determine the number of yeasts and moulds (TYMC), in accordance with the ISO 21527-1 and ISO 21527-2 [21, 22] . The number of microorganisms was expressed as the logarithm of the colony forming units per gram or millilitre (log cfu/g or mL).
The presence of pathogenic bacteria was determined using the enrichment culture method with the media indicated in the standards, such as XLD agar (Xylose Lysine Deoxycholate Agar, LabM, UK) and RAPID'Salmonella agar (Bio-Rad, Hecules, CA, USA) to determine the presence of Salmonella, in accordance with the ISO 6579-1 [23] , and ALOA agar (Listeria according to Ottaviani and Agosti Agar, Bio-Rad, USA) and PALCAM agar (LabM, Heywood, UK) to determine the presence of Listeria monocytogenes, in accordance with the ISO 11290-2 [24] .
The presence of pathogenic bacteria was determined using the enrichment culture method with the media indicated in the standards, such as XLD agar (Xylose Lysine Deoxycholate Agar, LabM, UK) and RAPID'Salmonella agar (Bio-Rad, Hecules, CA, USA) to determine the presence of Salmonella, in accordance with the ISO 6579-1 [23] , and ALOA agar (Listeria according to Ottaviani and Agosti Agar, Bio-Rad, USA) and PALCAM agar (LabM, Heywood, UK) to determine the presence of Listeria monocytogenes, in accordance with the ISO 11290-2 [24] .
5
Isolation and Identification of Aerobic Bacteria
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Bacteria were isolated from ten-fold dilutions of samples by the pour plate method.
Nutrient agar (LAB M, Heywood Lancashire England) was used for the isolation of aerobic bacteria, MacConkey agar (LAB M, Heywood Lancashire England) for coliforms, and Mannitol salt agar (Oxoid, Basingstoke, Hampshire, England) for staphylococci. The inoculated culture media were incubated aerobically at 37°C for 48 h. Distinct colonies observed on the culture plates were successively streaked on fresh Nutrient agar, Mannitol salt agar or MacConkey agar plates to obtain pure cultures.
Nutrient agar (LAB M, Heywood Lancashire England) was used for the isolation of aerobic bacteria, MacConkey agar (LAB M, Heywood Lancashire England) for coliforms, and Mannitol salt agar (Oxoid, Basingstoke, Hampshire, England) for staphylococci. The inoculated culture media were incubated aerobically at 37°C for 48 h. Distinct colonies observed on the culture plates were successively streaked on fresh Nutrient agar, Mannitol salt agar or MacConkey agar plates to obtain pure cultures.
6
Evaluating Antimicrobial Activity of Lactobacillus
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Bacterial cultures were stored at −80°C with the 20% glycerol (w/v). Lb. plantarum cultures were cultivated at 37°C for 24 h in MRS broth (Lab M, United Kingdom) under semiaerobic conditions and tested for antagonistic activity against indicator strains according to Sip et al. [5 ] and Hartmann et al. [12 (link)].
Indicator strains used for assessment of antimicrobial activity were three Listeria monocytogenes strains (ATCC 7644, ATCC 19111, and ATCC 15313), two species from Enterobacteriaceae family (Salmonella enteritidis ATCC 13076, Escherichia coli ATCC 10536), and also Bacillus subtilis and Enterococcus faecium strains (food isolates). Cultures were carried to solid selective medium: Palcam Agar (Lab M, United Kingdom) for Listeria, TBX Agar (Merck, Darmstadt, Germany) for E. coli, and BGA (Oxoid, United Kingdom) for Salmonella. The frozen cultures of B. subtilis and E. faecium were transferred onto Nutrient Agar (Lab M, United Kingdom). After incubation single colonies of each indicator strain were transferred to nutrient broth (Biokar Diagnostic, Noack, Poland) and cultivated from 24 to 48 h at 37°C under aerobic condition.
Indicator strains used for assessment of antimicrobial activity were three Listeria monocytogenes strains (ATCC 7644, ATCC 19111, and ATCC 15313), two species from Enterobacteriaceae family (Salmonella enteritidis ATCC 13076, Escherichia coli ATCC 10536), and also Bacillus subtilis and Enterococcus faecium strains (food isolates). Cultures were carried to solid selective medium: Palcam Agar (Lab M, United Kingdom) for Listeria, TBX Agar (Merck, Darmstadt, Germany) for E. coli, and BGA (Oxoid, United Kingdom) for Salmonella. The frozen cultures of B. subtilis and E. faecium were transferred onto Nutrient Agar (Lab M, United Kingdom). After incubation single colonies of each indicator strain were transferred to nutrient broth (Biokar Diagnostic, Noack, Poland) and cultivated from 24 to 48 h at 37°C under aerobic condition.
7
Salmonella Isolation from Shrimp Samples
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An aliquot of 1.0 mL of the stock solution (25 g of respective ready-to-eat shrimp samples homogenized in 225 mL of sterile tryptone soy broth (TSB), giving a first 10−1 dilution) was added into test tubes containing 9.0 mL of selenite cysteine F Broth (Lab M, Lancashire, United Kingdom) and incubated at 37°C for 24–48 h. Thereafter, streak plate technique was used via streaking directly from the turbid overnight culture of the selenite cysteine F Broth on xylose lysine deoxycholate (XLD) agar (Lab M, Lancashire, United Kingdom) and incubated at 37°C for 24–48 h [25 ]. A typical red colony with black centres after incubation were characteristically described tentatively as Salmonella isolate and sub-cultured on Hektoen enteric agar (HEA) (Lab M, Lancashire, United Kingdom) and incubated at 37°C for 24–48 h. After incubation, green colonies with or without black centres were repeatedly purified on Nutrient agar (Lab M, Lancashire, United Kingdom) for at 37°C for 24–48 h and presumptively identified as Salmonella isolate. The purified isolates were stored in Nutrient agar slants at 4°C until ready for further use.
8
Multiplex PCR for Salmonella Typhimurium Identification
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PCR was performed on the 97 isolates for which conventional serotyping showed an incomplete antigenic formula that shared antigens with the S. Typhimurium formula 4,[5],12:i:1,2 i.e., for which formulas 4,[5],12:i:-: were obtained. Isolates were cultured overnight on Nutrient Agar (Lab M Ltd.,Lancashire, UK) at 37 °C. One isolated colony was suspended in 100 µl InstaGene™ Matrix (BioRad laboratories, USA) and DNA was extracted as per manufacturer’s instructions. A multiplex real-time PCR method for the identification and differentiation of S. Typhimurium and monophasic 4,[5],12:i:- was carried out using the method and primers as described by Prendergast et al. 2013 [15 (link)].
S. Typhimurium LT2 ATCC 29,946 was used as a positive control and Escherichia coli NCTC 9001 as a negative control.
S. Typhimurium LT2 ATCC 29,946 was used as a positive control and Escherichia coli NCTC 9001 as a negative control.
9
HCV NS3/4A Protease Inhibitor Assay
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Sensolyte™ 520 HCV Protease assay kit Fluorimetric (cat#AS-71145), HCV NS3/4A protease and Sensolyte™, Green protease Assay Kit Fluorimetric (cat#AS-71124), and Hepatitis Virus C NS3 protease inhibitor 2 (cat#AS-25346) were purchased from AnaSpec Inc., San Jose, CA, USA. Chymotrypsin inhibitor (Soybean trypsin) was purchased from Sigma Aldrich Co. (St. Louis, MO, USA), and Becton Dickinson Falcon™ (Tokyo, Japan). Microtest 384-well 120 μL black assay plates, no lid, non-sterile, were purchased from Becton Dickinson Inc, Franklin Lakes, NJ, USA. Culture media: Czapek agar, bacteriological peptone, malt extract, yeast extract powder, nutrient agar and potato dextrose broth were procured from Lab M, Bury, UK; Glucose, (Acros Organics, Geel, Belgium), K2HPO4 (Laboratory Reagent, Mumbai, India), MgSO4 (Oxford Laboratory Reagent, Mumbai, India), agar (Sisco Research Laboratories Pvt. Ltd., Mumbai, India).
10
Interactive Packaging for Nile Perch
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The interactive package design was constructed in the Adobe Illustrator CC 2018 program in cyan, magenta, yellow, and black (CMYK) colors with spot colors. Processing machine photopolymer flexo-plate ESKO was used to produce the flexo photopolymer plate thickness 1.14 mm, Germany. The interactive package was printed on flexographic machine supplier Bobst 8 color 2019, Germany. The reverse printing was applied on polyester/polyethylene duplex laminated structure material (PET 12 μm/PE 40 μm) with polyurethane solvent-less adhesive. The rate of color change ΔE was measured with X-Rite exact, Pantone, the USA for the printed packages.
Lactic acid, stannous octanoates, and methyl red are purchased from Sigma-Aldrich, Germany. Additionally, chloroform, acetone, and ethanol were distilled and kept over calcium hydrate until used. Nile perch fishes were purchased from the Egyptian food market (Cairo) and were prepared as a filet with direct packaging in a printed duplex smart and interactive package. The nutrient agar (Lab M Limited, Heywood, Lancashire BL9JJ, United Kingdom) was used, and the potato dextrose agar (Ponadisa, Laboratories Conda S.A, Madrid, Spain) was purchased.
Lactic acid, stannous octanoates, and methyl red are purchased from Sigma-Aldrich, Germany. Additionally, chloroform, acetone, and ethanol were distilled and kept over calcium hydrate until used. Nile perch fishes were purchased from the Egyptian food market (Cairo) and were prepared as a filet with direct packaging in a printed duplex smart and interactive package. The nutrient agar (Lab M Limited, Heywood, Lancashire BL9JJ, United Kingdom) was used, and the potato dextrose agar (Ponadisa, Laboratories Conda S.A, Madrid, Spain) was purchased.
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