In order to verify the potential effect of EOs on foodborne pathogens, E. coli ATCC 25922, S. aureus ATCC 14458, and S. enteritidis ATCC 13076 were used in this study. All microorganisms were obtained from the culture bank of the Oswaldo Cruz Foundation (FIOCRUZ, Rio de Janeiro, Brazil), and stored on nutrient agar (Kasvi, Italy) under refrigeration at the Center for Food Analysis (NAL) at the Federal University of Rio de Janeiro, where they were reactivated in 10 mL of brain heart infusion broth (BHI) (Kasvi, Spain) at 37 °C/18–24 h. After, strains were streaked on MacConkey agar (Kasvi, Spain), Baird Parker agar (Kasvi, Spain) supplemented with egg yolk tellurium (Sigma-Aldrich, Germany), and Xylose Lysine Deoxycholate agar (XLD) (Kasvi, Espanha) at 37 °C/18–24 h, respectively. Next, a characteristic colony of E. coli, S. aureus, and S. enteritidis were inoculated in individual tubes containing BHI broth and incubated at 37 °C/18–24 h for subsequent use in the assays described below.
Macconkey agar
Manufactured by Kasvi
Sourced in Italy, Brazil
MacConkey agar is a selective and differential culture medium used for the isolation and identification of Gram-negative bacteria, particularly members of the Enterobacteriaceae family. It contains bile salts and crystal violet, which inhibit the growth of Gram-positive bacteria, and lactose, which allows for the differentiation of lactose-fermenting and non-lactose-fermenting organisms.
Automatically generated - may contain errors
Lab products found in correlation
Nutrient agar, by Kasvi (2 mentions)
Baird parker agar, by Kasvi (1 mentions)
Xylose lysine deoxycholate agar xld, by Kasvi (1 mentions)
Tetrathionate broth, by Acumedia (1 mentions)
Api 20e kit, by bioMérieux (1 mentions)
Levofloxacin, by Merck Group (1 mentions)
Mueller hinton broth (mhb), by Kasvi (1 mentions)
α cyano 4 hydroxycinnamic acid, by Merck Group (1 mentions)
Acetonitrile, by Merck Group (1 mentions)
Ciprofloxacin, by Merck Group (1 mentions)
Trifluoroacetic acid, by Merck Group (1 mentions)
5 protocols using macconkey agar
1
Evaluation of Essential Oils against Foodborne Pathogens
2
Cecal Microbiome Analysis via Culturing
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Samples of the cecal contents were collected and stored in sterile, hermetically sealed falcon tubes, and were refrigerated on ice for approximately 90 min. For the analysis of the concentrations of the different bacterial groups, approximately 1 g of digesta was weighed and decimal dilutions of the samples were performed in 1% peptone water solution (Kasvi, Conda, S.A., Madrid, Spain).
Colony counts were enumerated using the petri dish pour plate technique on MacConkey agar (Kasvi, Italy) for total Enterobacteriaceae (CBT) and on Man, Rogosa, and Sharpe agar (MRS; Sigma-Aldrich, Saint Louis, MO, USA) for lactic-acid bacteria. The plates were incubated under aerobic conditions for 24 and 48 h at 37 °C. The counts were determined according to the FDA-mandated method [22 ]. Bacterial concentrations were subjected to log10 transformation prior to analysis. After the colonies were isolated, the Enterobacteriaceae kit was used, which consisted of 10 biochemical tests associated with lactose fermentation (from the isolation medium) and allowed for the differentiation of gram-negative bacilli from other oxidase-negative bacteria.
Colony counts were enumerated using the petri dish pour plate technique on MacConkey agar (Kasvi, Italy) for total Enterobacteriaceae (CBT) and on Man, Rogosa, and Sharpe agar (MRS; Sigma-Aldrich, Saint Louis, MO, USA) for lactic-acid bacteria. The plates were incubated under aerobic conditions for 24 and 48 h at 37 °C. The counts were determined according to the FDA-mandated method [22 ]. Bacterial concentrations were subjected to log10 transformation prior to analysis. After the colonies were isolated, the Enterobacteriaceae kit was used, which consisted of 10 biochemical tests associated with lactose fermentation (from the isolation medium) and allowed for the differentiation of gram-negative bacilli from other oxidase-negative bacteria.
3
Calf Fecal Microbiome Analysis Protocol
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For this analysis were included only samples from calves that did not receive farm's treatment for diarrhea at the time of sampling. Sixty-six fecal samples (PHAGE=31; CON=35) on the first day of the first diarrheic episode were collected directly from the rectum, packed in a sterile cup and frozen at -20ºC until microbiological analysis.
Subsequently, these samples were cultured in two types of medium, for Salmonella spp. and E. coli growth.
For Salmonella spp. identification, samples were initially grown in tetrathionate broth (Acumedia Manufacturers, Inc. Baltimore, Maryland). The broth was kept in a bacteriological oven (Olidef cz, Brazil) at 35-37°C for 72 hours. Every 24 hours of incubation, an aliquot was removed from the broth and added to MacConkey agar (Kasvi, Brazil), incubated again under the same conditions for 24 hours. Biochemical tests were performed with colonies with Salmonella spp. visual characteristics, following the technique described by Quinn et al. (1993) .
For E. coli, lactose fermenting colonies found in MacConkey agar medium were submitted to biochemical characterization (Quinn et. Al., 1993) . The PCR was performed for colonies characterized as E. coli and Salmonella spp.
Subsequently, these samples were cultured in two types of medium, for Salmonella spp. and E. coli growth.
For Salmonella spp. identification, samples were initially grown in tetrathionate broth (Acumedia Manufacturers, Inc. Baltimore, Maryland). The broth was kept in a bacteriological oven (Olidef cz, Brazil) at 35-37°C for 72 hours. Every 24 hours of incubation, an aliquot was removed from the broth and added to MacConkey agar (Kasvi, Brazil), incubated again under the same conditions for 24 hours. Biochemical tests were performed with colonies with Salmonella spp. visual characteristics, following the technique described by Quinn et al. (1993) .
For E. coli, lactose fermenting colonies found in MacConkey agar medium were submitted to biochemical characterization (Quinn et. Al., 1993) . The PCR was performed for colonies characterized as E. coli and Salmonella spp.
4
Bacterial Isolation and Identification from Fish Tissues
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Head kidney and brain fragments were inoculated onto MacConkey Agar (Kasvi, Italy), modified Hsu Shotts agar [MHS; Bullock (1986) ] supplemented with 2 mg L -1 of tryptone and trypticase soy agar (TSA; Kasvi, Italy) supplemented with 5% sheep blood and incubated at 28 °C for 24-72 h. Next, gram staining, catalase, and oxidase tests were performed. Biochemical characterization of the isolates was carried out using the API 20E kit (Bio-Merieux SA, France) and API Strep kit (Bio-Merieux SA, France).
5
Antibacterial Compound Screening Protocol
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Colistin, gentamycin (Inlab, São Paulo, SP, Brazil) , ciprofloxacin, levofloxacin, αcyano-4-hydroxycinnamic acid, trifluoroacetic acid (TFA), acetonitrile (Sigma-Aldrich, Frankfurt, Germany), crystal violet, 95% ethanol, glucose, and sodium chloride (Synth, São Paulo, SP, Brazil) were purchased from commercial suppliers and used without further purification. Mueller-Hinton broth (MHB) and agar (MHA), nutrient agar, MacConkey agar, and trypticase soy broth (TSB) were purchased from Kasvi (São José do Pinhais, PR, Brazil). Apitoxin was provided by Ezequiel Dias Foundation (FUNED) and was collected following Benton's procedure (Benton et al. 1963) . Meal-worm beetle (Tenebrio molitor; Coleoptera) larvae were purchased from a company that is specialized in agricultural products (Mercado Central de Belo Horizonte, Minas Gerais, Brazil).
Polyvinyl chloride (PVC) urethral catheters (Embramed®; São Paulo, SP, Brazil) were obtained from stores specialized in hospital-medical supplies.
Polyvinyl chloride (PVC) urethral catheters (Embramed®; São Paulo, SP, Brazil) were obtained from stores specialized in hospital-medical supplies.
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