Isolation and identi cation of E. coli were performed by enrichment and sequential plating onto selective plates, as previously described [16] . The samples were incubated in LB Broth (Beijing Land Bridge Technology Co., Ltd, Beijing, China) at 37 °C overnight for 12 h, and draw the line on MacConkey agar plateafter dipping the culture the next day. All presumptive E. coli colonies were identi ed using VITEK 2 compact automated identi cation system (BioMérieux, Marcy-I'Etoile, France). Reference strains E. coli ATCC 25922, K. pneumoniae ATCC 25923 and K. pneumoniae ATCC 700603 were used as quality control strains. For all the strains, 3 µl bacteral culture incubated at 37 ℃ for 12 h were diluted in Mueller-Hinton broth to obtain a starting inoculum at 10 5 cfu/mL.
Lb broth
Manufactured by Beijing Land Bridge Technology
Sourced in China
LB broth is a widely used nutrient-rich medium for the cultivation of various bacterial species. It provides the necessary nutrients and growth conditions for the cultivation and propagation of diverse microorganisms in laboratory settings.
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Lab products found in correlation
Mrs broth, by Beijing Land Bridge Technology (1 mentions)
Anhydrous ethanol, by Merck Group (1 mentions)
α cyano 4 hydroxycinnamic acid, by Merck Group (1 mentions)
Acetonitrile, by Merck Group (1 mentions)
Formic acid, by Merck Group (1 mentions)
Trifluoroacetic acid, by Merck Group (1 mentions)
Bst dna polymerase large fragment, by Vazyme (1 mentions)
Nt bstnbi, by New England Biolabs (1 mentions)
Scrfi, by New England Biolabs (1 mentions)
Lb agar, by Beijing Land Bridge Technology (1 mentions)
Idt oligo analyzer 3, by Integrated DNA Technologies (1 mentions)
Syto9, by Thermo Fisher Scientific (1 mentions)
2 2 azinobis 3 ethylbenzothiazoline 6 sulfonic acid (abts), by Sangon (1 mentions)
Dntp mixture, by Sangon (1 mentions)
5 protocols using lb broth
1
Isolation and Identification of E. coli
2
Salmonella Infection Model and Probiotic Intervention
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The testing strain, Salmonella typhimurium SM022, is a mutant strain of the wild-type Salmonella typhimurium ATCC 14028s [39] (link). The concentration of 1×108CFU/mL of Salmonella typhimurium SM022 we choose for subsequent (n = 6) *P < 0.05, **P < 0.01, ***P < 0.001 (one-way ANOVA) [40] and the International Scientific Association for Probiotics and Prebiotics (ISAPP) expert team, Binda et al. [41] (link) put forward in the probiotic application guide that the recommended amount of probiotics is 10 8 ~10 11 CFU/day, while previous studies have reported that Lactobacillus alleviated Salmonella typhimurium infection in mice at a concentration of 10 8 CFU/mL [42, (link)43] (link), and the concentration of 10 8 CFU for L. buchneri GX0328-6 was selected in the present study. Salmonella typhimurium SM022 and L. buchneri GX0328-6 were grown to log phase in LB broth and MRS broth (Beijing Landbridge Technology Co., Ltd.), respectively. Bacterial cells were collected by centrifugation at 4000 rpm for 10 min, rinsed twice with saline (NS), and then adjusted the concentration of both bacterial suspensions to 10 8 CFU/ mL before feeding to mice.
3
MALDI-TOF MS Protocol for Bacterial Identification
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Centrifuge tubes, masks, and safety glasses were purchased from Shanghai Titan Scientific Co., Ltd. (Shanghai, China). α-Cyano-4-hydroxycinnamic acid (CHCA), formic acid (analytical reagent [AR]), anhydrous ethanol (AR), trifluoroacetic acid (AR), and acetonitrile (ACN) were purchased from Merck (Darmstadt, Germany). Tryptone soybean agar and LB broth were purchased from Beijing Land Bridge Technology Co., Ltd. (Beijing, China). An ATL-032R digital shaker (Shanghai Chemstar Instruments Co., Ltd. China), centrifuge (Shanghai Boyu Instruments Co., Ltd.), and autoclave (Shanghai Boxun Industry & Commerce Co., Ltd.) were used. The MALDI-TOF MS system used an M-Discover 100 mass spectrometer (Zhuhai Meihua Medical Technology Co., Ltd., China) in linear positive mode with m/z range 2 to 20 kDa. Metabo Analyst 4.0 (McGill University, Montreal, Canada) was used for analysis (41 (link)).
4
Acid adaptation of E. coli strains
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The strains (WT and ∆phoP) were inoculated at 1‰ inoculum in Luria–Bertani (LB) broth (Beijing Land Bridge Technology, Beijing, China) for 18 h (37 °C, 200 rpm) to activate the strain, and the strain that was activated will be activated a second time under the same condition. Two full loops of the twice-activated WT and ∆phoP strains were incubated in a pH = 5.4 LB broth (adjusted by lactic acid, Tianjin Kaitong Chemical Reagent, Tianjin, China) for 90 min at 37 °C to obtain the acid-adapted strain. At the same time, another two full loops of the twice-activated WT and ∆phoP strains were incubated in a pH = 7 LB broth (adjusted by phosphates) for 90 min at 37 °C to obtain the non-adapted group [18 (link)]. The non-adapted WT strain was set as the control.
5
Rapid LAMP Assay for Salmonella Detection
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Bst DNA polymerase (Large Fragment) was purchased from Vazyme Biotech Co., Ltd. (Nanjing, China). Nt.BstNBI, ScrFI, DraI, and agarose were obtained from New England Biolabs (Ipswich, MA, USA). Syto 9 was achieved from Thermo Fisher (Waltham, MA, USA). 4-(2-Hydroxyethyl) piperazine-1-ethanesulfonic acid (HEPES), hemin, dNTP mixture, and ABTS were all supplied by Sangon Biotech (Shanghai, China) Co., Ltd. LB agar and LB broth were offered by Beijing Land Bridge Technology Co., Ltd. (Beijing, China) 30% H2O2 was bought from Sigma-Aldrich, Inc. (Burlington, MA, USA).
The gene of invasion protein A (invA) was selected as the reference gene for amplification (GenBank accession no. NC_003197). Conventional LAMP primers were synthesized according to the previous report [32 (link)]. Inner primers incorporated with the recognition site (GAGTC) of nicking endonuclease, Nt.BstNBI, are termed as Nick-BIP and Nick-FIP. All the sequences were evaluated with IDT Oligo Analyzer 3.1 (Integrated DNA Technologies, Coralville, IA, USA). The oligonucleotides were synthesized by Sangon Biotech (Shanghai, China) Co., Ltd. Sequences used in this work are listed inTable S1 , and the corresponding template sequence is displayed in Figure S1 .
The gene of invasion protein A (invA) was selected as the reference gene for amplification (GenBank accession no. NC_003197). Conventional LAMP primers were synthesized according to the previous report [32 (link)]. Inner primers incorporated with the recognition site (GAGTC) of nicking endonuclease, Nt.BstNBI, are termed as Nick-BIP and Nick-FIP. All the sequences were evaluated with IDT Oligo Analyzer 3.1 (Integrated DNA Technologies, Coralville, IA, USA). The oligonucleotides were synthesized by Sangon Biotech (Shanghai, China) Co., Ltd. Sequences used in this work are listed in
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