N. meningitidis serotype C (ATCC 13102) strain was purchased from MeccontiTM, Warsaw, Poland. It was the 2nd passage from the reference in the form of pellets. One pellet was reconstituted, spread on blood agar (Animal Health Research Institute, Giza, Egypt) under biological safety cabinet (Thermo Scientific, MS, USA), and incubated at 37 °C for 18–24 h in 5% CO2 incubator (Kendro laboratory products, Hanau, Germany) [15 ]. The colonies were harvested in brain heart infusion (BHI) broth (Merck KGaA, Darmstadt, Germany)/glycerol (Fisher, Winsford, U.K.) dispensed in sterile micro-centrifuge tubes and stored at −80 °C (Thermofisher, MS, USA). Whenever needed, a tube of N. meningitidis serotype C culture was prepared by colony suspension method: inoculation of harvested bacteria pellets on a BHI agar (Merck KGaA, Darmstadt, Germany) plate. The isolated colonies from an overnight culture were selected and added to a tube containing BHI broth (Merck KGaA, Darmstadt, Germany) [16 ].
Biological safety cabinet
Manufactured by Thermo Fisher Scientific
Sourced in United States
A biological safety cabinet is a piece of laboratory equipment designed to provide a controlled, protected work environment. It functions to protect the user, the product, and the surrounding environment from potential exposure to biological hazards.
Automatically generated - may contain errors
Lab products found in correlation
Sr0048, by Thermo Fisher Scientific (2 mentions)
Sr0147e, by Thermo Fisher Scientific (2 mentions)
Cm0331, by Thermo Fisher Scientific (2 mentions)
Microcentrifuge tube, by Thermo Fisher Scientific (1 mentions)
Glycerol, by Thermo Fisher Scientific (1 mentions)
Bhi agar, by Merck Group (1 mentions)
Bhi broth, by Merck Group (1 mentions)
Karmali agar base, by Thermo Fisher Scientific (1 mentions)
Qiaamp dna mini kit, by Qiagen (1 mentions)
Water bath, by Memmert (1 mentions)
6 protocols using biological safety cabinet
1
Cultivation and Storage of N. meningitidis C
2
Isolation and Identification of H. pylori
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The isolation of H. pylori from biopsy specimens was performed as described previously22 . Briefly, biopsy specimens were aseptically rolled over the surface of Columbia blood agar base plates under a biological safety cabinet (Thermo Fischer Scientific). The agar (Oxoid CM0331) was supplemented with 7% horse serum (Oxoid SR0048), 1% vitamin mix (Isovitale-X), and an H. pylori selective supplement (Dent, SR0147E Oxoid) comprising of amphotericin B (2.5 mg), trimethoprim (2.5 mg), vancomycin (5.0 mg), and cefsulodin (2.5 mg). The plates were incubated at 37 °C in an atmosphere of 85% N2, 10% CO2 and 5% O2 for 4–10 days. H. pylori colonies were identified as small, round, translucent, Gram-negative bacteria and positive for catalase, oxidase and urease tests. The confirmed isolates were frozen in Brucella broth containing 20% glycerol and stored at −80 °C for future use.
3
Isolation and Preservation of H. pylori
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Gastric biopsy specimens were homogenized thoroughly in brain heart infusion (BHI) broth and then streaked onto the Karmali blood agar base plates under a biological safety cabinet (Thermo Scientific). The Karmali Agar base (Oxoid, CM 0935) was supplemented with 5% defibrinated sheep blood and 1% combined antibiotics comprising of trimethoprim (150 mg/L), vancomycin (125 mg/L), amphotericin B (100 mg/L) and polymyxin B (100 mg/L). The plates were incubated at 37 °C under microaerobic conditions (5% O2, 10% CO2 and 85% N2) for 3–5 days. H. pylori colonies were identified according to its morphological characteristics, negative Gram staining and positive for catalase, oxidase, and urease. The identified H. pylori was subcultured to single colonies and then preserved in sterile BHI broth with 20% glycerol and frozen at − 80 °C until the genomic DNA was extracted with the QIAamp DNA Mini Kit (Qiagen, Germany) according to the manufacturer’s instructions. The extracted DNA was stored at − 20℃ and used directly for PCR.
4
Isolation and Culturing of Helicobacter pylori
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Biopsy specimens were aseptically rolled over the surface of Columbia blood agar base plates under a biological safety cabinet (Thermo Scientific). The agar (Oxoid CM0331) was supplemented with 7% horse serum (Oxoid SR0048), 1% vitamin mix (Isovitalex), and an H. pylori selective supplement (Dent, SR0147E Oxoid) comprising of amphotericin B (2.5 mg), trimethoprim (2.5 mg), vancomycin (5.0 mg), and cefsulodin (2.5 mg). The plates were incubated at 37 °C in an atmosphere of 85% N2, 10% CO2 and 5% O2 for 4–10 days. Presumptive H. pylori colonies were identified as small, round, translucent, Gram-negative and positive for catalase, oxidase and urease tests. The confirmed isolates were Frozen in brain heart infusion broth (BHI) containing 20% glycerol and stored at − 80 °C for future use.
5
Establishing Chlorella Axenic Culture
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The Chlorella axenic culture was established by antibiotic treatments. In brief, the Chlorella solution was centrifuged (4000 rpm, 3 min, D3024R, DLAB, Beijing, China), and the supernatant was discarded. Then, the Chlorella precipitation was streaked on agar plates containing 80 mg/L kanamycin with transfer ring. After colonies formation, the individual Chlorella colonies were selected under a stereomicroscope (M205A, Leica, Germany) and streaked again. The cycle was repeated three times. Finally, the Chlorella was added to the sterile BG-11 medium and cultured with cell culture flasks in the biological safety cabinet (Thermo Fisher Scientific, MA, USA) (25 °C, 2500–3000 lx, 12 h:12 h light/dark cycle).
6
Quantitative Bacterial Endotoxin Assay
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This test is a substantial requirement for the safety of all injectable pharmaceutical products. Bacterial endotoxin is a type of pyrogenic substance found in the cell wall of Gram-negative bacteria and should be controlled, otherwise causing fever, septic shock, or death [24 ]. Therefore, we measured the quantity of endotoxin by using bacterial endotoxin USP monograph 85 (method: gel-clot, quantitative test). In brief, the NmC-BG suspension was adjusted to 0.5 McFarland (108 CFU/mL) using LAL water under the biological safety cabinet (Thermo Scientific, MA, USA). Single-test LAL reagent sensitivity 0.25 EU/mL (Charles River, MA, USA) was used. Ten-fold dilution was performed of the BG vaccine, added on the reagent and then incubated at 37 ± 1 °C for 60 ± 2 min in a water bath (Memmert GmbH, Schwabach, Germany). The result was calculated according to the USP monograph. If none of the dilutions is positive (gel formation), the endotoxin concentration is reported as less than LAL reagent sensitivity multiplied by the lowest dilution factor.
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