Biofilm production was tested using Congo red agar assay using the method described previously by Arciola [39 (link)]. The strains were cultured on TSA plates (Merck, Darmstadt, Germany) and supplemented with sucrose (Sigma Aldrich, Steinheim, Germany) and Congo red (Sigma Aldrich, Steinheim, Germany). sucrose was added due to its characteristic of abundant exopolysaccharide synthesis among Staphylococcus spp. Based on colony appearance, strains were classified as slime-forming (black colonies) and non-slime-forming (bordeaux or pink colonies). Additionally, biofilm-producing ability was evaluating using the crystal violet assay method described previously by Kouidhi et al. [67 (link)].
Tsa plate
Manufactured by Merck Group
Sourced in Germany
TSA plates are a type of microbiology culture media used for the growth and isolation of various bacteria. They provide a solid growth surface for bacteria, allowing for the identification and enumeration of different bacterial species.
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9 protocols using tsa plate
1
Screening for Biofilm Production in Staphylococcus
2
Screening Ampicillin-Resistant Bacteria for Resistome
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Bacteria of selected samples (n = 17) with intI1 prevalence across the full spectrum of RPs (0–25.14), were isolated on TSA plates (Merck, Darmstadt, Germany) supplemented with 100 μg/mL of ampicillin (Sigma Aldrich, Munich, Germany). The pre-selection of isolates by ampicillin was meant to lower the complexity and to increase the likelihood for isolation of multi-resistant phenotypes within the usually overwhelming number of non-resistant bacteria in a sample. We adopted this method form Ash et al. (2002) who used ampicillin to select for potential β-lactamase producers and found that ampicillin resistance and other resistance genes, were identified in 70% of the plasmids isolated [23 (link)].
Samples were spread plated by application of 100 μL of sample and incubated for 48 h at 30°C. After incubation colonies were separated into groups based on colony morphology. Of each group one isolate was picked and sub-cultured. After sub-cultivation for 24 h at 30°C, pure isolates were applied to VITEK 2 System for the determination of species and resistance.
In total 91 isolates were analyzed.
Samples were spread plated by application of 100 μL of sample and incubated for 48 h at 30°C. After incubation colonies were separated into groups based on colony morphology. Of each group one isolate was picked and sub-cultured. After sub-cultivation for 24 h at 30°C, pure isolates were applied to VITEK 2 System for the determination of species and resistance.
In total 91 isolates were analyzed.
3
Salmonella Strain Specificity Testing
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A total of 89 Salmonella strains across 6 different serogroups were selected for specificity testing and artificial inoculation studies (Table S1 ). These strains were plated on tryptic soy agar (TSA) plates (Merck, Rahway, NJ, USA) and incubated overnight at 37 °C for 24 h prior to nucleic acid extraction from colonies or inoculation into a shell egg mixture.
4
Isolation of Endophytic Bacteria from Quinoa
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The strain SECRCQ15T was isolated from Chenopodium quinoa seeds harvested from plants cultivated in Ciudad Rodrigo (Salamanca, Spain, 40° 35′ 02.6′′ N 6° 31′ 56.5′′ W). Quinoa seeds were surface sterilized with ethanol (70%) for 3 min and sodium hypochlorite (2%) for 2 min and were washed 5 times in sterile distilled water. An aliquot of the last wash water was plated on tryptic soy agar (TSA) and incubated at 28 °C for 48 h as a disinfection control, where no bacterial growth was observed. Surface sterilized seeds were crushed in a sterile mortar and resuspended in sterile water. Decimal dilutions from the suspension were obtained to isolate the endophytic bacteria and 100 μL of each suspension was spread on TSA plates (Sigma Co.) which were incubated at 28 °C for 48 h. Despite we have isolated other strains (data not shown), here we focus on the analysis of the strain SECRCQ15T due to its taxonomic novelty. The strain was cryopreserved (− 80 °C; 25% glycerol solution) and plated when needed.
5
Growth Characteristics of S. aureus
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TSB (Sigma-Aldrich) and TSA plates (Sigma-Aldrich) were used for growth studies involving S. aureus. For growth characterization on solid medium, S. aureus strains were precultured for 24 h at 37 °C under continuous shaking starting from a single colony. Cultures were then diluted to 10−5–10−7 and an aliquot of 100 μl was plated on TSA plates. The plates were incubated for 5 days at 37 °C under atmospheric and 5% CO2 conditions. The 5% CO2 level was achieved in a CO2 incubator (Heraeus Instruments). The colony size and appearance on TSA was observed every 24 h for 5 days and documented with a Leica M125 stereomicroscope. For growth characterization in liquid medium, S. aureus cells were precultured in TSB as described above. S. aureus strains involved were WT HG001, ΔmpsA, ΔmpsB, ΔmpsC, and ΔmpsABC that were transformed with pRB473-mpsA, pRB473-mpsB, pRB473-mpsC, and pRB473-mpsABC, respectively. Main cultures were inoculated to OD578 = 0.05 and grown under atmospheric and 5% CO2 conditions at 37 °C under continuous shaking. Aliquots were taken at 2, 4, 6, 8, 10, 12, 24, 48, and 72 h for OD578 measurements.
6
Preparing Pseudomonas aeruginosa cultures
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The bacterial culture of Pseudomonas aeruginosa (PA) used was provided by the Leibniz Institute DSMZ—German Collection of Microorganisms and Cell Culture (batch No.: 0411). For the experiments, the subculture II of PA was used. Prior to the experiments, a master plate was prepared using the cryopreserved PA sample and cultivated on tryptone soya agar (TSA) plates (Sigma-Aldrich, Munich, Germany) for 24 h and was subsequently kept at 7 °C for a maximum of 2 weeks. A bacterial strain was prepared by picking a colony from the master plate 24 h before CAP treatment and incubated in 25 mL tryptone soya broth (TSB) culture medium at 37 °C. Using a photometer to measure the absorbance at 600 nm (Epoch II, BioTek, Winooski, VT, USA), the PA solution was diluted to a 0.5 McFarland standard, which corresponds to approximately 1.5 × 108 CFU/mL, and then further diluted in TSB to the specific concentration required for the experiments.
7
Disinfectant Impact on H06 and H18 Biofilms
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To examine the interaction between H06 and H18 biofilms with disinfectant components (0.8% peracetic acid, 1% sodium hypochlorite and 1% chlorhexidine), a modified protocol followed by Nguyen and Yuk (2013) (link) was used.
An aliquot of 100 μL (107 cells) was inoculated on the surface of a sterile cellulose membrane with porosity of 0.45 μm and 47 mm in diameter, on a TSA plate (Merck®). The plates were incubated at 37°C and had the membrane transferred to a new plate every 24 h for 3 days. Subsequently, the membrane was placed in a flask containing 20 mL of TSB broth with the concentrations of the disinfectant components. After incubation at 37°C for 15 min, the membrane was washed three times with phosphate buffer (PBS), followed by treatment in 25 mL of 0.1% trypsin for 15 min at room temperature. Then, the resulting solution underwent serial dilutions for subsequent counting on TSA plates, at 37°C for 24 h.
An aliquot of 100 μL (107 cells) was inoculated on the surface of a sterile cellulose membrane with porosity of 0.45 μm and 47 mm in diameter, on a TSA plate (Merck®). The plates were incubated at 37°C and had the membrane transferred to a new plate every 24 h for 3 days. Subsequently, the membrane was placed in a flask containing 20 mL of TSB broth with the concentrations of the disinfectant components. After incubation at 37°C for 15 min, the membrane was washed three times with phosphate buffer (PBS), followed by treatment in 25 mL of 0.1% trypsin for 15 min at room temperature. Then, the resulting solution underwent serial dilutions for subsequent counting on TSA plates, at 37°C for 24 h.
8
Hydrogen Peroxide Stress Response in MRSA and MSSA
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The growth curve of MRSA and MSSA strains were determined in the presence of 5 mM and 10 mM of hydrogen peroxide (H2O2), according to the methods of Chan et al. (15 ) with the following modifications. Briefly, overnight cultures were diluted 100-fold in 50 ml TSB medium (Merck, Germany) and incubated at 37°C with shaking at 200 rpm until reaching an optical density of 0.4 at 600 nm (OD600). Then, bacterial suspensions were diluted 3-fold to reached OD of 0.08–0.1 at 600 nm. Next, each suspension was divided, and two concentrations (5 mM and 10 mM) of H2O2 were added. Following the incubation of each concentration, 1 ml of suspension was removed at 0, 1, 2, 3, 4, and 24 h after the addition of H2O2, and then the growth curves were assessed turbidimetrically. Moreover, in each time point, 1 ml of suspension was washed twice with sterile sodium chloride solution 0.9%, and the serial dilution was provided, poured into TSA plate (Merck, Germany), and incubated at 37°C. Colony count was performed after 24 h incubation. Bacterial cells without H2O2 were used as control in various time intervals. All experiments were conducted triplicate independently.
9
Anti-vibrio Screening of Fungal Isolates
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The anti-vibrio assay was conducted by the agar plug method (Sabdaningsih et al. 2017; (link)Trianto et al. 2020) (link). A total of 3 vibriosis causative (Vibrio harveyi, V. parahaemolyticus, and V. vulnificus), collection of Tropical Marine Biotechnology Undip Laboratory, Semarang were used for anti-vibrio screening. The Vibrio bacteria were grown on nutrient broth to a concentration of 0.5 McFarland and then inoculate on a trypticase soy agar (TSA) plate (Merck, Germany) using sterile cotton swabs (ONEMED, Indonesia). Seven days old of the fungal disk was plugged on TSA and incubated at 27ºC for 24h (Sibero et al. 2018; Cristianawati et al. 2019) (link).
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