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MacConkey agar

Manufactured by Acumedia
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MacConkey agar is a selective and differential culture medium used for the isolation and enumeration of Gram-negative bacteria, particularly members of the Enterobacteriaceae family. It contains bile salts and crystal violet to inhibit the growth of Gram-positive bacteria, while allowing the growth of Gram-negative bacteria.

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7 protocols using MacConkey agar

Urine samples were initially streaked on MacConkey agar (Acumedia, Michigan, US) and Cysteine Lactose Electrolyte Deficient (C.L.E.D.) agar (Hi Media laboratory Pvt Ltd, Mumbai, India) (MacConkey agar served as a selective as well as a differential culture medium. MacConkey agar was used for the isolation of Gram-negative uropathogens while C.L.E.D. agar was used for the differentiation of uropathogens based on color morphology. All media were prepared according to the manufacturer’s instructions. Urine samples were streaked on media using sterile cotton swabs. Media plates were incubated at 37 ºC overnight. Next day, plates were observed for growth. The colony of interest was picked from media containing selected pathogen using a sterile loop and mixed in 3% H2O2 on the slide for catalase test. Air bubbles and water were observed on a slide for a positive result (Habeeb et al. 2013 (link)). Triple sugar iron test is used for the characterization of various pathogens. This test is based on the pathogen’s ability for fermentation of sugars, H2S production and gas production. For TSI agar (BIOCHEM chemopharma, France), colony inoculation was performed by stabbing through the center of the medium to the bottom of the test tube and then streaking was also performed on the slant. Test tubes were incubated at 37 ºC for 18 h.
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The gastrointestinal tract, from proventriculus to cloaca, was removed from each European starling, placed into a sterile Whirl-Pak bag (Nasco, Fort Atkinson, WI), and homogenized for 120 sec at 230 rpm using a Stomacher 400 Circulator (Seward, Islandia, NY). The resulting homogenate was inoculated using a sterile cotton tipped applicator onto MacConkey agar (Acumedia, Lansing, MI) (MAC) supplemented with 2 μg/mL CTX (Calbiochem, EMD Millipore, Billerica, MA) and onto MAC supplemented with 1 μg/mL CIP (Enzo Life Sciences, Farmingdale, NY) and incubated at 37 °C for 24 hr. From each media, 1–2 presumptive E. coli colonies (occasionally lactose negative isolates were also collected) were subcultured on either MAC-CTX or MAC-CIP to yield purified isolates. Purified isolates were propagated overnight in Brain Heart Infusion Broth (BD, Franklin Lakes, NJ) at 37 °C with shaking at 200 rpm, then mixed 1:1 in 40% glycerol, and stored at −80 °C until further use.
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Different genera of the fecal microbiota were analyzed at the beginning of the experiment (T0 –before product administration), after 7 days of product ingestion (T1), after 7 days of colitis induction (T2), 14 days after colitis induction (T3) and at the end (day 30) of the experiment (T4). Stool samples were collected from the animals’ cages, corresponding to a 24 h period. The samples were, stored in sterile polypropylene bags and frozen at -80°C until the analysis.
The fecal microbial analyses were performed by media-dependent assay and based on the determination of the following bacterial populations: Enterococcus spp., Lactobacillus spp., Clostridium spp., Bacteroides spp., Bifidobacterium spp., and enterobacteria.
Fecal pellets were diluted in sterile peptone water and the suspensions were used to inoculate selective culture media: Enterococcus spp.: KF Streptococcus agar (Acumedia, USA), 37°C, 48 h [35 (link)]; Lactobacillus spp.: Man Rogosa Sharpe agar (MRS) (Acumedia), 37°C, 48 h [36 (link)]; enterobacteria: MacConkey agar (Acumedia), 37°C, 48 h [37 (link)]; Clostridium spp.: Reinforced Clostridium Agar (Acumedia), 37°C, 48 h [38 (link)]; Bacteroides spp.: Bacteroides bile esculin agar (BBE) (Acumedia), 37°C, 72 h [39 (link)]; Bifidobacterium spp.: Bifidobacterium iodoacetate medium 25 (BIM-25), 37°C, 72 h [40 (link)].
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All subjects answered a questionnaire about their systemic health and a dental and soft tissue examination was performed to assess their oral conditions. The resulting findings were recorded in the medical record of each patient. Initially, the surfaces of the dentures were thoroughly dried with sterilized gauze to avoid contamination by saliva. The biofilm on the inner surface of complete dentures was collected with the aid of a sterile swab, and the samples were immediately placed in sterile phosphate buffered saline (PBS, pH 7.5, 0.8% NaCl) to the Department of Microbiology and Parasitology, Federal University of Ceará, in Sobral, and processed within a maximum of 2 hours after collection. Samples were dispersed by agitation (30 seconds) and serially diluted (10−1 and 10−2) in PBS. Aliquots of 100 μL of the solutions obtained were plated on MacConkey agar (Acumedia, Lansing, Michigan, USA). Then, the plates were incubated at 36°C±1°C for 24 hours.
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Bacterial strains, S. aureus ATCC 25923, E. coli ATCC 25922, S. Enteritidis ATCC 13076, S. Typhimurium ATCC 13311, and S. Typhimurium UK-1 ATCC 68169, were inoculated in nutrient broth (Acumedia, Lansing, MI, USA, USA), and incubated at 37 °C for 24 h. Cultures reached a concentration of 109 CFU/mL. After the growth, the bacterial strains were serially diluted in a saline 10-fold solution up to 10−7, and dilution was plated in nutrient agar to confirm the total CFUs. A volume of 10 µL of each dilution was placed on the film’s square with and without bio-AgNP and stored in a sterile Petri dish at room temperature for 24 h. The initial cellular density in contact with films ranged from 106 CFU/cm2 to 1 CFU/cm2. After contact with bacteria, film samples were inoculated in nutrient broth and placed at 37 °C for 24 h. Bacterial growth was confirmed by samples plating in agar medium, E. coli in MacConkey Agar (Acumedia, Lansing, MI, USA, USA), S. aureus in Mannitol Salt Agar (Difco®, Tucker, GA, USA), and Salmonella sp. in SS Agar (Difco®, Tucker, GA, USA).
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The antagonism test using culture supernatants was adapted from Hütt et al. (2006) (link). Briefly, overnight cultures of the strains under study in MRS broth were centrifuged (5000 Â g, 5 min). The supernatant was collected and filter-sterilized (0.22 mm Millex, Merck Millipore, Darmstadt, Germany). Overnight cultures of the food pathogens (S. enterica var. Typhimurium ATTCC 14028, S. flexi ATTCC11060 and E. coli ATCC 25922) were inoculated (1% v/v) in BHI and 1% (v/v) of the lactobacilli supernatant was added to the broth. The broth was incubated for 16 h (37 C, aerobiosis). Cell counts of pathogens were conducted on MacConkey agar (Acumedia) for 24 h (37 C, aerobiosis).
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7

Antagonistic Effects of Lactobacillus on Food Pathogens

In order to test the direct antagonism of the strains under study against food pathogens (S. enterica var. Typhimurium ATTCC 14028, S. flexi ATTCC 11060 and E. coli ATCC 25922), a co-cultivation test was done. The method was adapted from Hütt, Shchepetova, Lõivukene, Kullisaar, and Mikelsaar (2006) (link). An overnight culture of each pathogen and the Lactobacillus strain were inoculated together (1% v/v) in 1 ml of BHI broth (Acumedia, Neogen, Lansing, MI, USA) and incubated for 16 h (37 C, aerobiosis). Control cultures were also prepared. Cell counts of the pathogen after incubation were done using MacConkey agar (Acumedia), incubated for 24 h (37 C, aerobiosis). The growth of the pathogen in BHI broth, inoculated alone or co-inoculated with a strain of lactobacilli was compared.
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