Isolates cultured for 24 to 48 h at 22°C were used during these analyses, and all reagents were purchased from Remel unless noted otherwise. Isolates were assayed for the Gram reaction, catalase (3% H 2 O 2 ) and cytochrome oxidase (Pathotec test strips) activities, and the presence of a flexirubin-type pigment (3% KOH) and cell wall-associated galactosamine glycans (0.01% w/v Congo red solution; Bernardet et al. 2002) . Additional morphological, biochemical, and physiological characterization was performed as previously described (Loch & Faisal 2014b) . Commercially available identification galleries (i.e. API 20E, API 20NE, API ZYM, and API 50CH; BioMerieux) were inoculated according to the manufacturer's protocol; however, tests were incubated at 22°C and read from 24 h post inoculation up until 7 d, with the exception of the API ZYM, which was read at 72 h. A list of the performed assays is displayed in Table 2.
Api zym
Manufactured by bioMérieux
Sourced in France, Germany, Spain
The API ZYM is a laboratory instrument used for the identification and characterization of microorganisms. It provides a standardized method for the detection of enzymatic activity in bacterial cultures.
Automatically generated - may contain errors
Lab products found in correlation
Api 20ne, by bioMérieux (40 mentions)
Api 50ch, by bioMérieux (36 mentions)
Api 20e, by bioMérieux (21 mentions)
Api 20a, by bioMérieux (11 mentions)
Api 50 chl, by bioMérieux (10 mentions)
Dm1000 photonic microscope, by Leica (7 mentions)
Api 50 ch strips, by bioMérieux (5 mentions)
Jem 1010, by JEOL (5 mentions)
Api 20ne strips, by bioMérieux (4 mentions)
Petri dish, by bioMérieux (4 mentions)
Bbl dryslide, by BD (4 mentions)
Oxidase reagent, by bioMérieux (3 mentions)
Api 50 chb, by bioMérieux (3 mentions)
Api coryne, by bioMérieux (3 mentions)
Api 20e strip, by bioMérieux (3 mentions)
Api rapid id 32a, by bioMérieux (3 mentions)
Api 20ne system, by bioMérieux (2 mentions)
Gram staining kit, by Solarbio (2 mentions)
Api ne, by bioMérieux (2 mentions)
Su8010, by Hitachi (2 mentions)
121 protocols using api zym
1
Comprehensive Bacterial Strain Identification
2
Quantification of Lactobacillus rhamnosus GG in Stool and Capsules
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The procedures for obtaining LGG for colony counts in stool and capsules were as follows. 4.5 mL of sterile phosphate buffered saline was added to either 0.5 grams of stool or 0.5 grams of capsule contents and diluted to 10−6. 100 µL dilutions of 10−1 to 10−6 were plated in duplicate onto Lactobacillus Selection (LBS) Agar (BBL Sparks, MD) which is selective for isolation and enumeration of lactobacilli [43] . LBS plates were incubated at 37°C in an anaerobic chamber (5% CO2, 10% H2, 85% N2) for 48 hours. Typical white, creamy LGG colonies with a distinct buttery smell [44] were easily distinguishable from other lactobacilli on LBS agar. These colonies were counted in duplicate and the average result was reported as CFU/g of stool or capsule. Representative colonies were gram stained and LGG was preliminarily identified if there were gram positive rods in a palisade arrangement versus other Lactobacilli such as L plantarum, L fermentum, L para para caseii and non-LGG L rhamnosus. Isolates were analyzed by APIZYM (Biomerieux, Durham, NC) that distinguishes between Lactobacillus species based on enzymatic reactions and API CH-50 (Biomerieux, Durham, NC) that differentiates between the species based on carbohydrate reactions. Lactobacillus para casei and non-LGG strains of L rhamnosus were distinguished from LGG based on fermentation.
3
Comprehensive Bacterial Characterization Protocol
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Gram staining was performed using a Gram staining kit (Solarbio, Hangzhou, China) by following the manufacturer’s instructions. The shape of cells was observed by transmission electron microscopy (JEM-1400FLASH; JEOL; Tokyo, Japan). The motility of cells was identified by the development of turbidity in a tube containing a semi-solid APA medium. Endospores were examined according to the Schaeffer–Fulton staining method [24 ]. Growth at different temperatures (4, 15, 20, 29, 37, 45, 55 and 65 °C) was determined in liquid APA medium. The tolerance to salinity and alkalinity was determined in a liquid APA medium with various NaCl concentrations (0, 2, 5, 8, 10, 15 and 20%, w/v) and pH range (pH 6.0–12.0, at intervals of 1.0 pH unit). The pH of the basal medium was adjusted using the buffer system, as described by Narsing Rao et al. [25 (link)]. Catalase and oxidase activities were tested using 3% (v/v) H2O2 and the oxidase reagent, respectively [26 (link)]. Other enzyme activities, biochemical characteristics and utilization of carbon source were detected using API ZYM, API 20NE systems (bioMérieux, Grenoble, France) and GEN Ⅲ Microplate (Biolog, Newark, NJ, USA), by following the manufacturer’s instructions.
4
Evaluating Microbial Enzymatic Profiles
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The enzyme activity and carbohydrate utilization of the selected strains were assayed using API ZYM and API 50CHL kit according to the manufacturer’s instructions (BioMérieux, France). Evaluation of enzyme activity was performed on a five-grade scale according to coloration intensity from 0 (no activity) to 5 (maximum activity) with 10 nM intervals. API strip reactions were evaluated using identification tables (+/−) according to color change.
5
Enzymatic Profiling of Myxobacteria
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Enzymatic activity was assessed for myxobacteria utilizing commercial API ZYM (bioMérieux, France) and API NE (bioMérieux, France) kits. Each isolate strain was suspended in NaCl 0.85% to an OD600 of 0.7 and 0.1 for API NE. API ZYM strips were incubated for 4.5 hours at 37°C, and API NE strips were incubated for 24 hours at 37°C. After incubation, specific reagents were added to the cupule and evaluated according to the manufacturer’s instructions.
6
Comprehensive Phenotypic Characterization of Bacterial Strains
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The morphological characteristics of the strains were investigated after 24 h of incubation on marine agar. The Gram reaction was examined according to Buck’s method26 (link). Cell morphology was investigated using scanning electron microscope (SU8010, Hitachi, Japan) and transmission electron microscope (H-7650, Hitachi, Tokyo, Japan). Growth was observed at various temperatures (4, 15, 20, 25, 28, 30, 33, 37, 40, 45, and 50 °C) on marine agar. Tolerance to different NaCl concentrations (0–10%, in increments of 1%, w/v, NaCl) and pH range (pH 4.0–11.0, at intervals of 1 unit) were performed at 28 °C, for 7 days. Anaerobic growth was tested in an MGCAnaeroPouch-Anaero (Mitsubishi, Tokyo, Japan) at 28 °C for 7 days on marine agar plates. Catalase and oxidase activities were investigated in 3% (v/v) H2O2 and using commercial strips (Huankai, Guangzhou, China) according to the manufacturer’s instruction, respectively. Additional enzyme activities and carbon source utilization assays were examined by using API 20NE, API ZYM (bioMerieux, Marcy-l′Etoile, French) and Biolog plates kits (Hayward, CA, USA), respectively, following the manufacturer’s instruction.
7
Characterization of Strain Marseille-Q2390
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Culturing of strain Marseille-Q2390 was attempted at various growth temperatures (4, 19, (link)28, (link)30, (link)37 , and 45 • C) in 5% sheep blood-enriched Columbia agar (bioMérieux) under aerobic and anaerobic atmospheres (using GasPak™ EZ generators (Becton-Dickinson, Maryland, MD, USA)). A sporulation assay was undertaken by thermal shock. Bacteria were exposed to a temperature of 80 • C for 30 min. Then, bacterial growth was monitored for four days. The bacterial growth was also tested in various salinity (0, 20, 40, 50, 60, 80, and 100 g/L) and pH (5, 5.5, 6, 6.5, 7.5, 8.5, 9, and 10) conditions. Gram staining and motility from fresh colonies were observed using a DM1000 photonic microscope (Leica Microsystems, Nanterre, France) with a 40× objective lens and 10× ocular lens. Bacterial structure was evaluated by scanning electron microscope (Hitachi SUV5000) (Hitachi High-Technologies Corporation, Science & Medical Systems Business Group, Tokyo, Japan). Catalase and oxidase activities were investigated using BBL DrySlide, in accordance with the manufacturer's instructions (Becton Dickinson, Le Pont de Claix, France). The biochemical characteristics were identified using API strips (API ZYM [33] [34] (link)[35] (link), API 20NE [36, (link)37] , API 20E [38, (link)39] (link), and API 50CH [40] [41] [42] [43] (link), bioMérieux).
8
Screening for Hemolytic and Enzymatic Activities
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Hemolytic activity was determined by streaking the strains on Columbia blood agar plates containing 5% defibrinated sheep blood (Scharlab). After 48 h of incubation at 37°C the plates were observed for hemolytic reaction (Kondrotiene et al., 2020 (link)). S. pyogenes CECT 191 was used as positive control. Gelatinase activity was tested by spotting fresh cultures on nutrient gelatin agar plates. Strains were cultured at 37°C for 5 days. Plates were treated with acidic mercuric chloride solution for 10 min to detect opaque halos indicating digestion of gelatin (dela Cruz and Torres, 2012 ). B. cereus CECT 5144 was used as positive control. To test additional enzymatic activities, API® ZYM (bioMérieux, Madrid, Spain) experiments were performed by triplicate following manufacturer instructions.
9
Comprehensive Biochemical Profiling of Bacterial Isolates
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Colonies isolated from BCYEα plate after 3 days of cultivation were used for
biochemical tests. Oxidase and catalase activity were tested using a BioMerieux API
20STREP kit and Vitek 2 (GN card) kit (BioMerieux). Other biochemical and enzymatic
activities were tested by using API ZYM (BioMérieux) kit.Table 1 shows all of the biochemical tests performed using
the three kits and the obtained results.
biochemical tests. Oxidase and catalase activity were tested using a BioMerieux API
20STREP kit and Vitek 2 (GN card) kit (BioMerieux). Other biochemical and enzymatic
activities were tested by using API ZYM (BioMérieux) kit.
the three kits and the obtained results.
10
Comprehensive Phenotypic Profiling of Staphylococcus
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Extensive phenotypic characterization using the commercial kits STAPHYtest 24 (Erba Lachema, Brno, Czechia) and API ZYM (bioMérieux, Marcy l’Etoile, France), phenotypic fingerprinting using the Biolog system with the identification test panel GEN III MicroPlate (Biolog, Hayward, CA, USA), and conventional biochemical, physiological, and growth tests relevant for the genus Staphylococcus were done as described previously [49 (link),50 (link),51 (link)]. The antibiotic resistance pattern was tested by the disc diffusion method on Mueller–Hinton agar (Oxoid, Basingstoke, UK). A set of discs (Oxoid) generally used for Gram-positive cocci were applied: ampicillin (10 µg), oxacillin (1 µg), ceftazidime (30 µg), cephalothin (30 µg), ciprofloxacin (5 µg), clindamycin (2 µg), erythromycin (15 µg), gentamicin (10 µg), chloramphenicol (30 µg), imipenem (10 µg), kanamycin (30 µg), neomycin (10 µg), novobiocin (5 µg), penicillin G (1 IU), rifampicin (5 µg), trimethoprim (5 µg), cotrimoxazole (25 µg), tetracycline (30 µg), vancomycin (30 µg), fusidic acid (10 µg) and polymyxin B (300 U). EUCAST standards and manufacturer’s recommendations (Oxoid) were strictly followed for cultivation and inhibition zone diameter measurement [52 ].
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