The American Type Culture Collection strain (ATTC 6519) of S. mutans was used. It was sub cultured on 5% sheep blood agar (pH 7.3). The plates were placed in an anaerobic jar (Oxoid, Britain). An anaerobic gas pack (Biomerieux manufacturer, France) and an indicator (Oxoid manufacturer, Britain) were also placed in the anaerobic jar. These were then incubated at 37°C for 24hrs. Using direct colony suspension method, a 0.5 Mcfarland standard of S. mutans (ATCC 6519) was prepared and its turbidity compared with the 0.5 Mcfarland standard 10 . The adjusted inocula equivalent to 1.5 x 108 cfu/mL was used in the different bioassay procedures.
Anaerobic jar
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom, Germany
The anaerobic jar is a laboratory equipment designed to create and maintain an anaerobic (oxygen-free) environment. It is used to cultivate and grow anaerobic microorganisms that require an oxygen-free environment for their growth and development.
Automatically generated - may contain errors
Lab products found in correlation
Anaerogen, by Thermo Fisher Scientific (5 mentions)
Anaerogen gas pack, by Thermo Fisher Scientific (4 mentions)
Campygen, by Thermo Fisher Scientific (2 mentions)
Anaerogen sachet, by Thermo Fisher Scientific (2 mentions)
Wilkins chalgren medium 2, by Thermo Fisher Scientific (2 mentions)
0.2 μm filter, by BD (2 mentions)
Mrs broth medium, by Merck Group (2 mentions)
Anaerogen 2.5 l sachet, by Thermo Fisher Scientific (2 mentions)
Spectramax m2, by Molecular Devices (1 mentions)
Anaerobe basal agar, by Thermo Fisher Scientific (1 mentions)
Sheep blood, by BD (1 mentions)
Imipinem, by Thermo Fisher Scientific (1 mentions)
Clindamycin, by Thermo Fisher Scientific (1 mentions)
Iso sensitest broth, by Thermo Fisher Scientific (1 mentions)
Mrs broth, by Merck Group (1 mentions)
Mrs agar, by Carl Roth (1 mentions)
Prism 7, by GraphPad (1 mentions)
Gaspak, by BD (1 mentions)
Ccda selective supplement, by Thermo Fisher Scientific (1 mentions)
Blood agar, by Thermo Fisher Scientific (1 mentions)
50 protocols using anaerobic jar
1
Standardizing S. mutans Inoculum
2
Standard Collection and Storage of Biological Samples
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Blood samples were collected into serum separator, EDTA, or lithium heparin (LH) vacutainer tubes (Greiner Bio-One Ltd), with 200 kallikrein inhibiting units (KIU) aprotinin (Sigma-Aldrich) added to the EDTA tube immediately after blood collection for gut peptide measurements. EDTA and LH tubes were placed on ice immediately after collection and before centrifugation at 1750 Â g for 15 min at 4 C to separate plasma. Serum separator tubes were stored upright at room temperature for 30 min after collection and before being centrifuged at 1750 Â g for 15 min at room temperature. Plasma and serum were then aliquoted into cryogenic (polypropylene) vials for storage at À80 C and À20 C, respectively.
Fecal samples were collected on the morning of the study visit. Volunteers were provided with collection kits comprising 1L plastic lidded pot (Aw Gregory), Seward stomacher 400 circulator bags, and 2.5L anaerobic jar (Fisher Scientific). The sample was collected into the bag and placed inside the lidded pot which was stored in the anaerobic jar with an Oxoid AnaeroGen sachet (Fisher Scientific). On receipt, the whole stool sample was weighed before a 20-g sample was added to a clean stomacher bag and homogenized for 2 mins before being aliquoted and stored in sterile Eppendorf tubes at À80 C.
Fecal samples were collected on the morning of the study visit. Volunteers were provided with collection kits comprising 1L plastic lidded pot (Aw Gregory), Seward stomacher 400 circulator bags, and 2.5L anaerobic jar (Fisher Scientific). The sample was collected into the bag and placed inside the lidded pot which was stored in the anaerobic jar with an Oxoid AnaeroGen sachet (Fisher Scientific). On receipt, the whole stool sample was weighed before a 20-g sample was added to a clean stomacher bag and homogenized for 2 mins before being aliquoted and stored in sterile Eppendorf tubes at À80 C.
3
Characterization of Weissella confusa Strains
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Bacterial strains used in this study were as follows: W. confusa MBF8-1 as lysate-producing bacterium, Leuconostoc mesenteroides TISTR 120 as indicator bacterium for BLIS activity assay, and Bacillus subtilis ATCC 6633 as negative control bacterium for the lactic acid production determination. All strains were obtained from the culture collection of the Laboratory of Pharmaceutical Microbiology and Biotechnology, Faculty of Pharmacy, Universitas Indonesia.
Cryo-stocks of all strains were maintained at -80 °C in MRS broth medium-glycerol (1:1). Bacterial strains were routinely confirmed both molecularly using the 16S ribosomal RNA (rRNA) gene and visually by morphology observation and Gram staining. Weissella confusa MBF8-1 samples were grown in each media, i.e., standard MRS, MRS Vegitone, and soy peptone-modified MRS, at 30 °C for 24 h in anaerobic jars (Oxoid, UK). L. mesenteroides TISTR 120 were grown in standard MRS medium at 30 °C for 24 h in anaerobic jars (Oxoid, UK), and B. subtilis ATCC 6633 were grown in nutrient broth (Difco, USA) and nutrient agar (Difco, USA) at 37 °C aerobically. All strains were routinely maintained by growing them in a 7 mL medium at their optimum temperature for 24 h.
Cryo-stocks of all strains were maintained at -80 °C in MRS broth medium-glycerol (1:1). Bacterial strains were routinely confirmed both molecularly using the 16S ribosomal RNA (rRNA) gene and visually by morphology observation and Gram staining. Weissella confusa MBF8-1 samples were grown in each media, i.e., standard MRS, MRS Vegitone, and soy peptone-modified MRS, at 30 °C for 24 h in anaerobic jars (Oxoid, UK). L. mesenteroides TISTR 120 were grown in standard MRS medium at 30 °C for 24 h in anaerobic jars (Oxoid, UK), and B. subtilis ATCC 6633 were grown in nutrient broth (Difco, USA) and nutrient agar (Difco, USA) at 37 °C aerobically. All strains were routinely maintained by growing them in a 7 mL medium at their optimum temperature for 24 h.
4
Cultivation and Transformation of Methanotrophic Bacteria
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All bacterial strains and plasmids used in this study are listed in Table 1 . E. coli was used as an intermediate host for plasmid cloning and transformation. E. coli strains were grown in Luria-Bertani (LB) media and 50 μg/mL kanamycin was added to the culture when required. E. coli was cultivated at 37°C, shaking at 200 RPM. M. alcaliphilum strains were cultured as described previously by Awasthi et al. (2022) (link). In brief, M. alcaliphilum was grown in sealed serum bottles at 30°C at 200 RPM in Pi (π)/P3 media with 30% (w/v) NaCl, 100 μg/mL Kanamycin (kan) was added to the growth medium when required. Cells were incubated under CH4 (Ultra-pure 99.9%, Airgas) and air at ratio 1:1. M. alcaliphilum cell growth (OD600) was measured by spectrophotometer SpectraMax M2 microplate reader (Molecular devices, San Jose, CA, United States). Colony selection after transformation was performed in P3 media-agar plates kept in the anaerobic jar (Oxoid, Remel) under CH4- air (1:1) atmosphere for 5–6 days.
5
Isolation and Identification of Clostridium perfringens
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For primary enrichment of C. perfringens, cooked meat medium containing samples was incubated anaerobically at 37°C for 48 hrs in an anaerobic jar (Oxoid Limited, Thermo Fisher Scientific Inc., UK) with an anaerobic GasPak (Oxoid Limited, Hampshire, England). The pre-enriched samples were then inoculated onto 5% bovine blood agar with added colistin sulfate (2 mg/litter) and incubated anaerobically for 24 hours. The characteristic colonies with double zones of beta hemolysis (inner zone: complete hemolysis; outer zone: partial hemolysis) were presumptively identified as C. perfringens. For further confirmation, the suspected bacterial colonies were subjected to Gram staining (Gram positive, large bacilli) and the catalase test (negative). Two to three subcultures were performed to obtain the pure culture, and finally, the pure colonies were subcultured on brain heart infusion broth (BHI) (Oxoid Limited, Thermo Fisher Scientific Inc., UK). All the isolates were preserved at −80°C for further analysis.
6
P. gingivalis Anaerobic Culture Protocol
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The bacterium used in this study was P gingivalis (ATCC33277), which was grown in anaerobe basal agar (Oxoid, Hampshire, Unites Kingdom) with 5% sheep blood (BD Diagnostic Systems, Germany) at 37 °C for 72 h in an anaerobic jar (Oxoid) before preparing the inoculum in each experiment.
7
Antibiotic Susceptibility Testing of Bacteroides
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The determination of antibiotic susceptibility was achieved as previously described [31 (link),32 (link)]. Briefly, Bacteroides strains were grown on BHI + H and incubated in an Anaerobic jar (AnaeroGen, Oxoid) at 37 °C for 24 h. Bacterial suspensions were prepared in 0.9% saline to a density of McFarland 1. The disc diffusion assays were performed using Brucella blood agar plates supplemented with hemin (0.1%) and vitamin K1 (1%). The antibiotic discs (Oxoid) were amoxycillin–clavulanate (20/10 μg) (AMC), clindamycin (10 μg) (DA), imipinem (10 μg) (IMP), moxifloxacin (5 μg) (MXF), piperacillin-tazobactam (30/6 μg) (TZP) and metronidazole (5 μg) (MTZ). The plates were incubated in anaerobic conditions at 37 °C for 24 h. Six discs (three per plate) were used. The susceptibility zone diameter provisional breakpoints previously suggested [31 (link),32 (link)] were followed: amoxycillin–clavulanate (≥15 mm), clindamycin (≥25 mm), imipenem (≥29 mm), moxifloxacin (≥19 mm), piperacillin-tazobactam (≥25 mm) and metronidazole (≥24 mm). Next, the results for imipinem and Metronidazole were confirmed by Epsilon test according to manufacturer’s procedure (Biomérieux, Portugal). The breakpoints were established according to EUCAST [33 ].
8
Pneumococcal and Streptococcal Strain Cultivation
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Activity assays were performed with the S. pneumoniae strain 16 NP3 (serotype 19F) or D39. Additional assays were with clinical isolates: ST65 = serotype 6A, H08212 0259; ST176 = serotype 6B, H08052 0052; ST1390 = serotype 6C, H05252 0075; ST4157 = serotype 6B, 35 NP1. The S. pneumoniae strains and S. pyogenes (ATCC 19615, Streptococcus group A) were grown on Columbia blood agar plates overnight at 35 °C under anaerobic or aerobic conditions, respectively. Liquid cultures were grown in trypticase soy broth (30 g/L trypticase soy broth, 3 g/L yeast extract) overnight at 35 °C without shaking. Anaerobic conditions were generated using Oxoid AGS CO2Gen Compact gas packs and an anaerobic jar from Oxoid. S. aureus (ATCC 28213) liquid cultures were grown in ISO‐Sensitest broth from Oxoid overnight at 35 °C with shaking. All target bacteria were obtained from the Royal Free Hospital (London, UK).
9
Bioluminescent Assay of Bile Salt Hydrolase
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WT and bsh deletion mutants of L. plantarum WSCF1 were grown in de Man, Rogosa and Sharpe (MRS) broth (Sigma-Aldrich) at 37°C for 48 hours in an anaerobic jar (Oxoid, 2.5 liters) using AnaeroGen 2.5-liter anaerobic atmosphere generation pads. Aliquots (15 ml) of each culture were centrifuged at 1500 rpm to pellet cells, and the cell pellets were washed three times with 10 ml of PBS, followed by an additional centrifugation at 1500 rpm and resuspension in PBS (10 ml) supplemented with ME (20 mM). Aliquots of the resulting suspension (200 μl) were mixed with the corresponding BAL probe (20 μM, in 200 μl of PBS) or d -aminoluciferin (2 μM, in 200 μl of PBS) to form 400-μl reaction mixtures at final concentrations of 10 μM, 1 μM, and 10 mM for the BAL probes, d -aminoluciferin, and ME, respectively. After incubation at 37°C for 1 hour, the reaction mixtures were centrifuged at 13,000 rpm, and the amounts of the resulting deconjugation product (luciferin) in the supernatants were quantified in triplicate using Caco2-luc luciferase-expressing cells as described above (see cell-based bioluminescence assay). CFU concentrations were assessed by inoculating serial dilutions on MRS agar (Roth, Germany). WT and bsh deletion mutants of L. plantarum WCFS1 were a gift of W. M. de Vos from Wageningen University, Finland.
10
Diverse Blastocystis Isolates from Humans and Animals
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Seven axenized isolates of Blastocystis (designated B, C, E, G, H, S-1 and WR-1) were used in the present study (Figure S1 ). All seven isolates were subtyped previously by small-subunit of ribosomal RNA gene analyses [55] (link). Isolates B, C, E, G and H were originally recovered from symptomatic patients at the Singapore General Hospital [56] (link) and they all belonged to ST-7 according to recent classification system [57] (link). Isolates S-1 and WR-1 were isolated from a rat during an animal survey [58] (link) and they belonged to ST-4.
Both ST-4 and ST-7 are well characterized zoonotic Blastocystis isolates commonly detected in humans with gastrointestinal symptoms [24] (link). Stock cultures of all seven isolates were maintained under the same conditions as described previously [49] (link). In brief, the parasites were maintained in 10 ml of pre-reduced Iscove's modified Dulbecco's medium (IMDM) containing 10% heat-inactivated horse serum in an anaerobic jar (Oxoid) with an AnaeroGen gas pack (Oxoid) at 37°C.
Both ST-4 and ST-7 are well characterized zoonotic Blastocystis isolates commonly detected in humans with gastrointestinal symptoms [24] (link). Stock cultures of all seven isolates were maintained under the same conditions as described previously [49] (link). In brief, the parasites were maintained in 10 ml of pre-reduced Iscove's modified Dulbecco's medium (IMDM) containing 10% heat-inactivated horse serum in an anaerobic jar (Oxoid) with an AnaeroGen gas pack (Oxoid) at 37°C.
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