Susceptibility testing to 18 different antibiotics was performed using automated broth microdilution (Vitek 2 card AST-N246; bioMérieux), and the presence of ESBL production was confirmed by clavulanate synergy with ceftriaxone and ceftazidime according to EUCAST standards. MICs of colistin (Beta Pharma Co. Ltd., Shanghai, China) and polymyxin B (Beta Pharma Co. Ltd.) were determined using broth microdilution according to the CLSI guideline. Twofold dilutions of colistin and polymyxin B ranging from 0 to 128 mg/liter were made in cation-adjusted Mueller-Hinton broth (CAMHB) (Oxoid, Hampshire, UK). Bacterial suspensions were prepared by suspending colonies from nutrient agar (School of Biomedical Sciences Media Unit, Monash University, Australia) in normal saline to match a 0.5 McFarland standard (DensiCHEK; bioMérieux). The suspensions were further diluted in CAMHB to yield ∼106 CFU/ml. Antibiotic solution (100 µl) was added to 100 µl of diluted inoculum in 96-well microtiter plates (Techno Plas, St. Marys, South Australia), and MICs were determined at 20 h after incubation at 37°C. Quality control using P. aeruginosa ATCC 27853 was included as recommended by the CLSI.
Densichek
Manufactured by bioMérieux
Sourced in France, United States, Australia, Canada
The DensiCHEK is a laboratory instrument designed to measure the optical density of liquid samples. It is used to determine the turbidity or concentration of particles in a solution, which can be useful in various applications such as microbiology and biochemistry.
Automatically generated - may contain errors
Lab products found in correlation
Vitek 2 compact, by bioMérieux (3 mentions)
Vitek 2 system, by bioMérieux (2 mentions)
Colistin, by Thermo Fisher Scientific (1 mentions)
Camhb, by Thermo Fisher Scientific (1 mentions)
Polymyxin b, by Thermo Fisher Scientific (1 mentions)
Vitek 2 compact system, by bioMérieux (1 mentions)
Nutrient broth medium, by HiMedia (1 mentions)
Microbank, by Pro-Lab Diagnostics (1 mentions)
Ast n169 card, by bioMérieux (1 mentions)
22 protocols using densichek
1
Antibiotic Susceptibility Testing Protocol
2
Polymyxin B3 Analogs against A. baumannii
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MICs of polymyxin B3 and its analogs were determined against five A. baumannii strains: A. baumannii 5075, A. baumannii ATCC 17978, A. baumannii ATCC 19606, A. baumannii 246-01-C.246, and A. baumannii 248-01-C.248 using the Clinical and Laboratory Standards Institute (CLSI) recommended microbroth dilution method. Two-fold dilutions of each antimicrobial agent ranging from 0–512 μg mL−1 were prepared in cation-adjusted Mueller-Hinton Broth (CAMHB; Oxoid, Hampshire, UK). Bacterial suspensions were prepared by suspending colonies grown on nutrient agar (School of Biomedical Sciences Media Prep Services, Clayton, Australia) in 0.9% NaCl to match a 0.5 McFarland standard (Densichek, BioMerieux). Bacterial suspensions were further diluted in CAMHB to yield ∼106 CFU mL−1, with 100 μL of this diluted suspension then added to 100 μL of antimicrobial solution in 96-well micro-titre plates (Techno Plas, St Marys, Australia). Quality control was assessed using Pseudomonas aeruginosa ATCC 27853. Plates were incubated at 37 °C and visually examined after 20 h incubation to determine the MICs.
3
Identification of Clinical Candida Isolates
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Clinical fungal isolates were obtained according to the standard microbiological protocols of the laboratory of microbiology at a tertiary hospital (Guayaquil, Ecuador) from January 2019 to February 2020. Fresh biological samples were examined to evaluate cell morphology and Gram-stain appearance. Yeast-like colonies were purified onto Sabouraud dextrose agar (SDA-Oxoid, ThermoScientific, Waltham, MA, USA) supplemented with chloramphenicol (0.05 g/L) and incubated at 37 °C for 24/48 h. Identification was achieved through subculture on a HardyCHROM™ Candida (CRITERION®, Hardy Diagnostics, Santa Maria, CA, USA), and samples were incubated at 37 °C for 48 h. To confirm Candida species, a YS card and the VITEK 2 system (bioMérieux, Inc., Hazelwood, MO, USA) were used following the manufacturer’s instructions. In brief, the colonies that had previously grown on Sabouraud medium at 35 °C for 24 h were suspended in a sterile physiological solution. The suspensions were prepared at 1.8–2.2 McFarland scale turbidity and measured using DensiChek (bioMérieux, Inc., Hazelwood, MO, USA), and the inoculum was dispensed on the YS card. The incubation time depended on the growth rate of organisms in the control well, whereas identification was made by comparison with the database.
4
Automated Identification of K. pneumoniae
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K. pneumoniae isolates were identified to the species level with the automated system VITEK 2 Compact ((bioM erieux, Marc L' Έtoile, France) using the GN ID test panel according to the manufacturer's recommendations. The GN card is based on documented biochemical methods and implementation of innovative substrates which measure the use of carbon source, enzymatic activity and resistance. The final result is available within 10 hours or less [8] . For both identification and susceptibility, the isolates were suspended into 3 ml normal saline and the turbidity of the inoculum was adjusted to 0.5-0.63 (±0.1) of the MacFarland (McF) scale using Densichek (bioM erieux).
5
Identification and Antibiotic Susceptibility of Probiotic Isolate
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The identification (ID), and AST of the unknown probiotic isolate (A) were performed using the fully automated VITEK® 2 compact system (BioMérieux, France) (Pan et al., 2022 (link)), where the suspension of L. lactis inoculums was prepared by adding a sufficient inoculum of (A) isolate to saline, and the McFarland turbidity of (0.50) was adjusted using DensiChek (BioMerieux, France) (Klare et al., 2007 (link)).
The Gram-positive identification card (GP ID) which includes 43 biochemical tests that estimate the bacterial enzymatic activities, carbon source usage, resistance, and inhibition. In addition to, 592 AST card and color-coded indicators which designed to provide accurate AST results and resistance detection were used (Elmaghrabi and Ghozlan, 2019 ).
The biochemical tests revealed from an unknown (A) strain was compared to the VITEK system databases for its final accurate identification (Pincus, 2006 ). As well as, the validation of every susceptibility test result with an accurate bacterial phenotypic profile resistance mechanism(s) was provided (Nakasone et al., 2007 (link)).
The Gram-positive identification card (GP ID) which includes 43 biochemical tests that estimate the bacterial enzymatic activities, carbon source usage, resistance, and inhibition. In addition to, 592 AST card and color-coded indicators which designed to provide accurate AST results and resistance detection were used (Elmaghrabi and Ghozlan, 2019 ).
The biochemical tests revealed from an unknown (A) strain was compared to the VITEK system databases for its final accurate identification (Pincus, 2006 ). As well as, the validation of every susceptibility test result with an accurate bacterial phenotypic profile resistance mechanism(s) was provided (Nakasone et al., 2007 (link)).
6
Synbiotic Supplementation in Broiler Chickens
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A synbiotic mix comprising 7 log of CFU g−1Lactobacillus rhamnosus HN001 and Pediococcus acidilactici MA18/5 M as probiotics and 4.5% (0.045 g g−1) of Agave tequilana fructans as prebiotic per dose (1 mL) was administered in drinking water the first day of the broilers life. The water containers with the mix were available for 2 h.
For treatment groups 3 to 8, pathogens were administered on day 17 [21 (link)]. S. typhimurium was subcultured in lactose broth with yeast extract and C. perfringens ATCC 13,124 was subcultured in thioglycolate broth and incubated under anaerobic environment; pathogens were separated by centrifugation (thrice at 4000 g for 20 min) and washed in physiological saline solution (0.8% NaCl). The pellets were suspended in physiological saline solution and the number of bacteria in the suspension was calculated using a nephelometer (DensiCHEK, Model: OA009372, bioMérieux, Inc, Missouri, MO, USA). Finally, 5 log CFU of S. typhimurium and/or 3 log CFU of C. perfringens per bird were administrated through drinking water [22 (link)].
To calculate the synbiotic and pathogen intake, we considered that, broilers consume 1.12 mL and 25 mL of water per hour at 1 and 17 days old, respectively [23 ].
For treatment groups 3 to 8, pathogens were administered on day 17 [21 (link)]. S. typhimurium was subcultured in lactose broth with yeast extract and C. perfringens ATCC 13,124 was subcultured in thioglycolate broth and incubated under anaerobic environment; pathogens were separated by centrifugation (thrice at 4000 g for 20 min) and washed in physiological saline solution (0.8% NaCl). The pellets were suspended in physiological saline solution and the number of bacteria in the suspension was calculated using a nephelometer (DensiCHEK, Model: OA009372, bioMérieux, Inc, Missouri, MO, USA). Finally, 5 log CFU of S. typhimurium and/or 3 log CFU of C. perfringens per bird were administrated through drinking water [22 (link)].
To calculate the synbiotic and pathogen intake, we considered that, broilers consume 1.12 mL and 25 mL of water per hour at 1 and 17 days old, respectively [23 ].
7
Yeast Identification Using Vitek-2 System
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All 13 isolates were also re-identified using the Vitek-2 yeast identification card (Vitek-2 Yeast ID; bioMérieux, Marcy l’Etoile, France) following the manufacturer’s instructions. The inoculum suspensions for the Vitek 2 were prepared in sterile saline at turbidity equal to a 2.0 McFarland standard, as measured using a DensiChek instrument (bioMérieux). The individual test cards were automatically filled with the prepared culture suspension, sealed, and incubated by the Vitek 2 instrument. Cards were incubated for 18 h at 35°C and read every 15 min. The final profile results were compared with the database, and the identification of the unknown organism was obtained.
8
Bacterial Identification Using VITEK 2
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Biochemical identification of the bacterial strains was carried out using VITEK 2 Compact (bioMérieux SA, Marcy l'Etoile, France). A colony showing pink color on MYP agar was cultured on nutrient agar to make bacterial suspensions, which were prepared using 3 ml of sterile saline and adjusted to a McFarland standard (2.0) using DensiChek (bioMérieux). The Bacillus identification card (BCL) (bioMérieux) was filled automatically in the VITEK vacuum chamber, sealed, and incubated at 35.5°C, and read automatically every 15 min for 16 h. Data were analyzed automatically using the VITEK2 database version 7.01. Identification results were reported as described previously [37] . Briefly, the species was reported as either (i) correctly identified to a single species or more than one species in a "slashline," in which case supplementary testing was required to distinguish between similar species; (ii) misidentified (where the species identified using BCL was discordant with the results of 16S rRNA sequencing); or (iii) unidentified.
9
Antibacterial Efficacy of AgNPs from Ocimum sanctum
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The killing kinetics assay of A. baumannii against AgNPs of O. sanctum was performed spectrophotometrically (Shimadzu, Tokyo, Japan) at OD 600 nm in triplicates. A. baumannii was cultured in a nutrient broth medium (Hi-Media, Mumbai, India) at 37 °C in the dark for 18–24 h. Before use, the bacterial broth was diluted in Nutrient Broth and adjusted to 0.5 McFarland turbidity (10 8 CFU/mL) using Densichek (BioMerieux, Marcy-l’Étoile, France). A volume of 100 µL of this broth was added to the five wells of the microtiter plate. Then, 100 µL of AgNPs from O. sanctum at concentrations of 32 µg/mL (MIC), 64 µg/mL (MBC), 128 µg/mL, 256 µg/mL and 512 µg/mL were added to the five wells loaded with the bacterial broth. The microtiter plate was then incubated at 37 °C in the dark, and the bacterial viability was measured spectrophotometrically in triplicates at 0, 2, 4, 8, 12, 18, and 24 h of incubation. The negative control (bacterial broth without AgNPs and without antibiotic colistin) and positive control (bacterial broth treated with antibiotic colistin at a MIC of 2 µg/mL) were included in the test. The percentage of inhibition of bacterial growth was calculated in comparison with the negative control (Das et al., 2017 (link)), and a statistical correlation was made with the positive control.
10
Antibiotic-Resistant Acinetobacter baumannii Susceptibility to Silver Nanoparticles
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A pure culture of multidrug-resistant (MDR) Acinetobacter baumannii, isolated from the respiratory tract specimen of a patient attending the Central Referral Hospital, Gangtok, was identified by the Vitek®-2 system (BioMerieux, Marcy-l’Étoile, France) and further confirmed by real-time PCR. The A. baumannii was further subcultured in a Muller-Hinton broth medium (Hi-Media, Mumbai, India) at 37 °C in the dark for 18–24 h. Before use, this bacterial broth was diluted in Muller-Hinton broth and adjusted to 0.5 McFarland turbidity (108 CFU/mL) using Densichek (BioMerieux, Marcy-l’Étoile, France). This bacterial broth was further tested for susceptibility to AgNPs of O. sanctum by different methods (Fig. 1 ).
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