Information on the six PCs and L. monocytogenes strains is provided in Table 1 . PCs and L. monocytogenes strains were stored at −80°C in their growth medium supplemented with 10% of sterile glycerol (Sigma-Aldrich, Steinheim, Germany). From the frozen stock, they were respectively subcultured in Elliker broth (Biokar Diagnostic, Beauvais, France) and Brain Heart Infusion broth (Biokar Diagnostic, Beauvais, France) for 48 h at 15°C before experiments.
Sterile glycerol
Manufactured by Merck Group
Sourced in Germany
Sterile glycerol is a colorless, viscous liquid that is widely used in various laboratory applications. It serves as a cryoprotectant, humectant, and solvent in numerous experimental procedures. Glycerol has a high boiling point and low toxicity, making it a versatile and reliable component in many laboratory settings.
Automatically generated - may contain errors
Lab products found in correlation
Trypticase soy broth (tsb), by Merck Group (1 mentions)
Macconkey agar, by BD (1 mentions)
Sheep blood agar, by BD (1 mentions)
Tetracycline, by Merck Group (1 mentions)
Ampicillin, by Merck Group (1 mentions)
Stomacher 400 circulator, by Seward Medical (1 mentions)
Alexa fluor conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Mouse anti flag m2, by Merck Group (1 mentions)
Rabbit anti gfp, by Thermo Fisher Scientific (1 mentions)
Mouse anti ha, by Fortrea (1 mentions)
Luria bertani broth, by Merck Group (1 mentions)
Mrr hsdrms mcrbc, by New England Biolabs (1 mentions)
M17 broth, by Thermo Fisher Scientific (1 mentions)
M17 agar, by Thermo Fisher Scientific (1 mentions)
M17 broth, by Merck Group (1 mentions)
Tryptic soy broth (tsb), by Merck Group (1 mentions)
14 protocols using sterile glycerol
1
Listeria monocytogenes Strain Cultivation
2
Microbiome Transplantation in Morphine-Exposed Mice
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Two batches of C57Bl6/j (WT) animals (10 each) were implanted with
placebo or morphine pellets as mentioned above, for 24 or 48 hours and their
fecal contents collected and pooled. The fecal content was processed according
to fecal microbial transplant (FMT) procedure described for human
patients52 (link) with
modifications. Briefly, the fecal contents were suspended in PBS (10mg/ml; w/v),
filtered through a 40μ mesh and centrifuged at 6,000Xg for 20 minutes.
The resultant microbial pellet was resuspended in half volume of chilled PBS and
aliquoted into volumes for single thaw and use. Aliquotes destined for later use
were reconstituted to 10% sterile glycerol (Sigma) and stored at
−80°. A total of 32 WT mice were used for the transplant
experiment. Animals were implanted with placebo or morphine pellets (12 each)
and the stored microbiota was administered (106 CFUs/dose) via oral
gavage every 24 hours thrice according to the following scheme:
[a] Placebo pellet implanted animals getting placebo microbiome
(PP; n=8), [b] Placebo pellet implanted animals getting
morphine microbiome (PM; n=8), [c] Morphine pellet
implanted animals getting placebo microbiome (MP; n=8) and
[d] Morphine pellet implanted animals getting morphine
microbiome (MM; n=8). Animals were sacrificed 24hours after third
transplant and fecal contents and tissues harvested for various downstream
analyses as described.
placebo or morphine pellets as mentioned above, for 24 or 48 hours and their
fecal contents collected and pooled. The fecal content was processed according
to fecal microbial transplant (FMT) procedure described for human
patients52 (link) with
modifications. Briefly, the fecal contents were suspended in PBS (10mg/ml; w/v),
filtered through a 40μ mesh and centrifuged at 6,000Xg for 20 minutes.
The resultant microbial pellet was resuspended in half volume of chilled PBS and
aliquoted into volumes for single thaw and use. Aliquotes destined for later use
were reconstituted to 10% sterile glycerol (Sigma) and stored at
−80°. A total of 32 WT mice were used for the transplant
experiment. Animals were implanted with placebo or morphine pellets (12 each)
and the stored microbiota was administered (106 CFUs/dose) via oral
gavage every 24 hours thrice according to the following scheme:
[a] Placebo pellet implanted animals getting placebo microbiome
(PP; n=8), [b] Placebo pellet implanted animals getting
morphine microbiome (PM; n=8), [c] Morphine pellet
implanted animals getting placebo microbiome (MP; n=8) and
[d] Morphine pellet implanted animals getting morphine
microbiome (MM; n=8). Animals were sacrificed 24hours after third
transplant and fecal contents and tissues harvested for various downstream
analyses as described.
3
Bacterial Isolates for In Vitro Studies
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Bacterial isolates utilized in in vitro studies were obtained courtesy of the Colorado State University Veterinary Diagnostic Laboratories. Isolates of Escherichia coli (E Coli) and Staphylococcus pseudointermedius (S. pseudointermedius) were obtained from wound cultures received by the bacteriology laboratory from hospitalized canine patients with informed consent. A single bacterial colony from the plate was used to inoculate trypticase soy broth (Sigma-Aldrich) at 37°C in a shaking incubator until the culture was turbid (~8 h). Sterile glycerol (Sigma-Aldrich) was added at a concentration of 20% and aliquots of 1 ml were frozen at −80°C until use. S. pseudointermedius was identified by growth on 5% Sheep blood agar (BD Biosciences, Franklin Lakes, NJ) with double zone hemolysis which was coagulase positive and hyaluronidase negative with gram positive cocci identified on gram stain. E coli was identified by growth on MacConkey agar (BD Biosciences) that was a strong lactose fermenter and was indole negative with the presence of gram negative rods on gram stain. Antimicrobial susceptibilities were performed utilizing the Kirby Bauer method in accordance with the Clinical Laboratory Standards Institutes veterinary guidelines (CLSI vet) (25 ).
4
Isolation and Preservation of Indigenous Bamboo Shoot Fermentation Yeasts
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The yeast isolates used in the present study are listed in Additional file 1 : Table S1. These isolates were obtained from samples collected at different stages of indigenous bamboo shoot fermentation for the production of soibum in Manipur state of North East India [38 ]. The sample (10 g) was homogenized in 90 mL of sterile physiological saline (1 g/L bacteriological peptone, 8.5 g/L NaCl, pH 6.1) using Stomacher® 400 Circulator (Seward, Worthing, West Sussex) at 250 rpm for 3 min. The yeasts were isolated by serial dilution spread-plating of the above homogenate on yeast extract peptone dextrose (YEPD) agar medium (pH 6.5) (HiMedia, Mumbai, India) containing 100 μg/mL each of filter-sterilized ampicillin and tetracycline (Sigma-Aldrich, Bangalore, India), followed by incubation at 30°C for 48 − 72 h under aerobic conditions. All the isolates were purified by sub-culturing twice on the same agar medium and preserved at −80°C in YEPD broth containing 10% (v/v) sterile glycerol (Sigma-Aldrich). For short term storage, the cultures were maintained at 4°C on YEPD agar. The type strain C. guilliermondii ATCC 6260 used for comparison was obtained from American Type Culture Collection.
5
Immunofluorescence Antibody Staining
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Primary antibodies used for immunofluorescence studies were as follows: mouse monoclonal anti-SipA (1:50), mouse monoclonal anti-SipB (1:50), mouse anti-FLAG M2 (Sigma-Aldrich, 1:1000), rabbit anti-GFP (Thermofisher, 1:50), mouse anti-HA (Covance, Clone 16B12, 1:400), goat anti-Salmonella CSA1 (KPL, 1:300) and rabbit anti-LAMP1 (Difco, 1:500). Alexa Fluor conjugated secondary antibodies were purchased from Life Technologies, diluted 1:1 in sterile glycerol (Sigma-Aldrich) and used at 1:400 dilution. Antibody dilutions specific to certain assays are listed with the appropriate method.
6
Culturing and Storing E. coli Strains
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cultures of E. coli were grown in lysogeny broth (LB Lennox) at 37 °C and 250 r.p.m. unless stated otherwise. When appropriate, 34 µg ml−1 chloramphenicol (+Ch) or 50 µg ml−1 kanamycin (+K) sulfate was supplemented to media. All bacterial strains were stored at −80 °C for long-term storage in 25% sterile glycerol (Sigma). Cloning and assays were primarily performed in DH10b genotype cells (NEB, Intact Genomics). For constructs targeting phage M13, cloning and assays were performed in DH5α F’Iq genotype cells (NEB).
7
Bacterial Strain Cultivation and Preservation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Staphylococcus aureus ATCC 25923, Listeria monocytogenes ATCC 7644, EHEC, Escherichia coli DSM 8579, Pseudomonas aeruginosa DSM 50071, and the phytopathogen Pectobacterium carovotorum DSM 102074 were used as test bacterial strains. We previously stored the strains at −30 °C in sterile Luria Bertani (LB) broth (Sigma, Milano, Italy) supplemented with 20% sterile glycerol (Sigma, Milano, Italy). Bacteria were thawed and added (inoculum 2%) to LB broth. S. aureus, L. monocytogenes, E.coli, and P. aeruginosa were grown for 18 h at 37 °C and 80 rpm (Corning LSE, Pisa, Italy). P. carotovorum was grown at 30 °C and 80 rpm. Then, fresh cultures were used as inoculum (2% final concentration) and grown in the conditions above described.
8
Salmonella Typhimurium Strain Preparation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Strains, primers, and plasmids are listed in Tables S3–S5, respectively. S. Typhimurium LT2 derivative strain MS1868 genotype is S. Typhimurium LT2 (leuA414(Am) Fels2- hsdSB(r-m+)) [55 (link)]. In general, all Salmonella
9
Standardized Bacterial Strain Culture
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
In general, all E. coli strains were grown at 37 °C and 180 r.p.m. in Lysogeny Broth (LB-Lennox broth, Sigma-Aldrich) supplemented with antibiotics, unless stated otherwise. When appropriate, 50 µg ml−1 kanamycin and/or 34 µg ml−1 chloramphenicol (denoted with +K or +C, respectively) were added to media. All bacterial strains and libraries were stored at −80 °C for long-term storage in 25% sterile glycerol (Sigma-Aldrich). All library assays were performed in NEB 10-beta strain backgrounds (araD139 Δ(ara-leu)7697 fhuA lacX74 galK (Φ80 Δ(lacZ)M15) mcrA galU recA1 endA1 nupG rpsL Δ(mrr-hsdRMS-mcrBC), New England Biolabs). A complete list of strains and plasmids is provided in Supplementary Data 6 . A list of primers and gene sequences used in this work is provided in Supplementary Data 7 .
10
Microbiome Transplantation in Morphine-Exposed Mice
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!