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Antibiotics - Science topic

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I want to select E. Coli cells positive for the attached plasmid.
What are the two resistance genes meant for? Selection in eukaryotes / prokaryotes?
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Use AMP for bacteria. The KanR cassette is driven by the simian virus 40 promoter meaning it is for eukaryotic expression. The KanR gene is used for G418 selection.
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Drug-resistant "superbugs" are now found virtually everywhere. As a result, most of the bacteria are resistant to antibiotics. So, as researchers and scientists, what are the alternative ways that we can prevent this disaster? Is there any novelty?
Source: Review On Antimicrobial Resistance
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I think that the solution to the problem lies not so much in the novelty as it lies in preventing bad practices in order to reduce selective pressure on those microorganisms, because even with new solutions or medicines, they will find a way to resist... My sincere gratitude to all.
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I want to use tetracycline as selection marker for my next experiments. I was advised to perform the experiments in dark. I want to know if this precaution is really required. I haven't found any literature supporting light-sensitivity of tetracycline. Please help...
Thanks in advance... :)
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Yes... while using the plates, keep lights in the vicinity off. During the incubation period no need to wrap the plates with aluminum foil. While storing the plates at 4C you must wrap the plates with the foil.
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Penicillium and it's effects and uses and its advantages in the extraction of drugs.
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It is a very good topic. Regards.
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The antibiotics are Neomycin, Puromycin, Zeocin, Blasticidin
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by fractional inhibitory index
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Hi all,
I am trying to conjugate a plasmid from E.coli ET12567 into anAmycolatopsis strain. The plasmid is integrative so should integrate into the genome of Amycolatopsis.
I have a negative and positive control of just Spores. Negative is overlaid with antibiotics, positive is not.
But when I overlay with antibiotics to select for exconjugants (or kill the spores in case of negative control) I have way more growth after a few days on my negative control as opposed to the actual conjugation plate?
I've attached 2 images to show what I mean - more growth on negative control
I've tried increasing the concentration of antibiotics incrementally from 100ug-300ug/mL, accounting for agar volume.
Any ideas what to do?
I'm thinking of swapping antibiotic resistance markers out but I only have a few months left of my PhD and I'm not sure if there will be a faster or simpler fix?
Thank you so much
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Michael J. Benedik Hi! Thank you for your response, apologies, the labelling is on the underside of the plate, but you are correct - the one shown with many colonies is the negative control. It hasn't lawned however, this is SFM agar so it is not translucent
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I am preparing for a proliferation assay where I have infected KP230 cells with shScr, shCol18a1#35, and shCol18a1#37 viruses. These shRNA viruses are made with the pLKO.1 vector and have puromycin resistance. I am at the stage of antibiotic selection, 48 hours after infection, where I will use 0.2 ug/mL puromycin for each of my three samples. Overall, the three 6 cm plates will have 18,000 KP230 cells, 0.6 uL puromycin, and 3 mL 10% growth media. I am advised to wait another 48 hours after antibiotic selection before typsinizing and seeding for the proliferation assay (growth curve).
Why does antibiotic selection take 48 hours? My protocol applies to other cell lines too such as HT-1080, STS-109, and STS-148 cells, so is 48 hours a standard among most cell types? What about using a different antibiotic on other resistant genes? Can anyone explain biologically what happens during these 48 hours with time stamps? What happens if I only give it 24 hours? I appreciate all your responses in advance.
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3 to 7 days in general, up to 14 days is also possible. There is no strict rule how many days you shall select your cell. It depends on cell line and antibiotics. You could use lower concentration with longer selection or higher consignation with shorter selection to achieve similar goals, though the former is preferred with less cytotoxicity by antibiotics. The best way for your assay is to add a non transfected control as indication of efficient selection, which should show most cell death after 48hours. If not, you may need to treat it longer.
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I dissolved some Levofloxacin in DMSO in May for experiments and stored them in the -20C freezer. However, when I took out some for an experiment, I noticed the color changed to light yellow. The antibiotics seemed to work fine but is there any reasons for this?
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DMSO is an oxidizing agent which may have caused oxidation of the antibiotic, resulting color change.
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I am about to start a lab-based experiment where some of the treatments of leaf litter require either bacterial or fungal exclusion and am wondering what the best (defined by most effective vs cost) bacteriocide/ fungicides are and why?
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I think hypochlorite and DCAP
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Hi,
I am working on a gene cluster from an amycolatopsis strain that supposedly produces a glycopeptide antibiotic - its a silent gene cluster at the minute.
I have sent it for RNA sequencing and the cluster is highly expressed, but there was no glycopeptide produced (checked via MS)
Any ideas as to why?
Thanks
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Katie A Burnette I used 3 separate cultures. The gene cluster was expressed in 2/3. But nothing was detected via MS. And yeah I have tested standards and limit of detection on the MS prior to this experiment
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Dear, is there a measurable effect on soil bioma of antibiotics, used on veterynary medcine and medcine?
Or even on soil fertility?
Regards!
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Hello al
Please i want to manually prepare antibiotic disk for disk diffusion method for an antibacterial plus enzyme inhibitor ( as amoxiclave for example)
My question is that how disk can be prepared? First mix the fractions then put on the disk or individually?
Thanks in advance
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I would like to perform detection of antibiotics but all the papers I checked irrespective of the method, SPE was a necessary step. Are there any cheaper alternative for SPE.
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Hi Danial,
You might use the liquid-liquid extraction method using an organic solvent. The most important thing you need to know is the chemical structure of your analyte (antibiotic) and to know about its pKa. Accordingly, you can apply the Handerson-Hasslebalch equation by using an appropriate buffer at specific pH to collect your antibiotic in the non-ionized form.
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Hi all,
I was wondering if anyone used Zeocin to select positivie recombinant in continuous or intermitent light for more than 5 days? any thoughts about how stable it will be, knowing that it is sensitive to light exposure...Do you think I should continuously add Zeocin in my medium in case it got degraded/ destabilized?
Thanks in advance,
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Hi Fatima,
I have used Zeocin in continuous light for transformation and selection of diatoms. From the start of selection it generally takes nearly 12 days. By day 14 of selection big colonies appear. Keeping the plates beyond 14 days increases appearance of false positives. This must be also balanced with the initial plating density, if too many cells are plated at the beginning of selection, it takes longer for transgenic colonies to appear and also there are too many escape (non-transgenic) colonies. For diatoms we use sea salts based medium and I have observed that best selection is obtained at 1/2 to 1/4 salt strength. This is all based on agar plates, although it can be used for maintaining selection in liquid cultures as well.
Hope this helps.
Best,
Yogesh
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I am working on antibiotic susceptibility testing of Staphylococcus aureus isolated from milk samples. I have used cefixime disc (5μg) in the study but I couldn't find the relevant CLSI standard for it. Staphylococcus aureus?
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I have worked on the resistance against Staphylococcus aureus previously, I hope these links will be of some help...Best wishes in your work
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I'm doing bacterial transformation on Helicobacter pylori first on non-selective medium and then in mueller hinton medium with the antibiotic. I always make a suspension of the biomass with BHI for the sucessive growth. But throught the steps i always get the strains contaminanted even though i use a biological safety cabinet level 2. I don't know what to do to improve the results of this experiment.
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Could it be that your bacterial stock itself is contaminated? Can you try cells from a different source and potentially in a different/freshly cleaned BSL2 cabinet?
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Hi there,
I currently have some PyMT driven spontaneous tumour mice and plan on harvesting the primary tumours and creating a cell line. I have never done this before and would love a protocol if possible - I am struggling to find any protocol to do this within my lab and everyone is off site / unsure.
I know it involves dissociating the tumour and then using collagenase??? and filtering ? But I am completely unsure what antibiotics to use and how to successfully take this to media to create the cell line.
Any advice for this would be so helpful.
Thank you.
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I can provide you with a brief protocol.
1. In the laboratory culture cabinet transfer the tumor tissue in a sterile 60mm Petri dish. Using sterile forceps and scissors, dissect out the unwanted tissue namely, fat tissue, blood clots (if any) and connective tissue from the tumor tissue.
2. Cut the tumor tissue into small pieces with the help of sterile scalpels.
3. Transfer the tumor tissue fragments into 50ml centrifuge tube and rinse the contents thoroughly with ice-cold HBSS which contains EGTA that will loosen cell junctions by chelating calcium. Slowly decant the supernatant and repeat this step until you get a clear solution.
4. Transfer the tumor fragments back to the 60-mm Petri dish and mince the fragments fine into 1mm^3 pieces using sterile scalpels.
5. Resuspend the smaller fragments in warm plain DMEM culture medium and provide enzymatic treatment. You could add 1mg/ml collagenase solution as the final concentration along with 0.1mg/ml DNase I and supplemented with 50 μg/ml gentamycin and 1X antibiotic-antimycotic in sterile conditions in plain DMEM and incubate for 30-45 mins at 37 degree C with gentle agitation.
6. Pass the digested tissue through the first sieve of 100um into a 50-ml centrifuge tube followed by 70um and 40um sieves. This will help to remove the undigested fibrous tissues and the contaminating cells and debris.
7. Wash the sieved single cells with HBSS and centrifuge at 400g for 5 mins at 4 degree C. Resuspend the cell pellet in HBSS and repeat the washing two more times. Finally, resuspend the pellet in complete culture medium (DMEM) with 10% FBS, and the media should also contain 1% Antibiotic-antimycotic, 1% Amphotericin B and 0.1% Gentamicin to avoid any contamination in culture.
Hope this protocol will help!
Good Luck!
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Hello
I am using one of the incredible techniques developed by my laboratory, SAGE plate technique (link below) and trying to evolve E.coli against a bunch of antibiotics. Unfortunately, my bacteria are not moving neither in the absence or in presence of an antibiotic. My fellow colleagues are not facing this problem. I tried troubleshooting most variables like, media preparation, media concentration, took help from my lab mates, checked bacterial growth on normal agar plates, I tested both my culture and the culture that my lab mates are using for evolution/motility in soft agar.
I am not able to figure out the issue. Can anyone shed any light on this? I would appreciate if you could help me with this based on your respective experiences.
Thank you :)
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Hello
I was able to test the motility of my culture, as specified by Michael and determined that my culture infact is very well motile. I have requested my lab mate to prepare the entire experiment with his handling and inoculate mine and his culture. This will suggest if there is issue with my handling or not.
Thank you so much for both your insights, I think I am closer to identify the issue now.
:)
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I have been trying to proliferate cells for almost 4 weeks culturing 4T1 cells in RPMI 1640 medium supplemented as recommended by the ATCC (10% FBS, 5% antibiotic).
Started with a frozen vial (passage # 4).
Cells are not growing at all. I even increased FBS to 20% and have been changing medium every 2-3 days, passing cells 1:2 and can't see any sign that cells are getting off stationary phase.
Any recommendations?
I will appreciate your input. Thank you
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Giuliana D Noratto came across a question and answer typically not same but somehow linked to your question so felt like sharing the answer and you can see how Dr S J Malik https://www.researchgate.net/post/Problem_with_4T1_Tumor_Model_Development-Can_anyone_help
This is for your refernce
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I want to measure the drug accumulation in semi-quantitative and quantitative method in the lung cell cultures (A549 cells). In this regard, my target is to measure it in a label free method as adding fluorophore to a drug changes its pharmacology.
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Dear Mohammad Tuhin Ali,
Look over some additional data as well:
Label-Free Quantification - an overview | ScienceDirect Topics
sciencedirect.com›topics…and-molecular…label-free…
Label-free quantification is a method in MS that determines the relative amount of proteins in two or more biological samples, but unlike other quantitative methods, is does not use a stable isotope that chemically binds and labels the protein. 70 Typically, peptide signals are detected at the MS1 level, and their isotopic pattern allows distinguishing them from chemical noise. Patterns are then tracked across the retention time dimension and used to reconstruct a chromatographic elution profile of the monoisotopic peptide mass. ... In contrast to differential labeling, every sample must be measured separately in a label-free experiment. The extracted peptide signals are then mapped across LC–MS measurements using their coordinates on the m/z ratio and retention-time dimensions.
_____
_____
Direct quantification of lipopeptide biosurfactants in biological...
link.springer.com›article/10.1007/s00253-017-8272…
Several methods based on measuring changes in the surface properties of BS water solutions have been validated and utilized. These methods include surface tension measurements (Youssef et al. 2004; Joshi et al. ... Semi-preparative RP-HPLC consisted of a Beckman Coulter System Gold 126NMP Pump and a Knauer Variable Wavelength Monitor equipped with a Phenomenex Luna C18(2) column (100 mm × 30 mm, 10 μm) under the control of the LP-Chrom software (Lipopharm, Poland). ... Sample pretreatment complicates and increases the cost of LP quantification and therefore should be minimized in high-throughput optimization of LP production or LP analysis in the food industry or healthcare products.
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I am referring to the impact of technologies such as vaccines, antibiotics, statins, etc.
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I am using TC-100 media(with serum and antibiotics) having sodium bicarbonate and L-Glutamine to grow Sf21 cells, but after I thaw them, they initially attach and grow for some time, then they fail to attach after 2-3 passages and eventually die.
What could be the possible reason for it? How can this be tackled?
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I will like to suggest this manual page 16-17. It should assist you.
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What is the most simple and high throughput in vitro method to characterise antibiotics/antifungals as time- vs concentration dependent? Is there any "general" cut off?
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Hello all;
We tried to make antibiotic cocktail (0.1% each) in the tap water (200 microliter)for oral gavage to SJL/J mice.
Antibiotics cocktail: 0.1% Ampicillin (=0.01gm)+ 0.1% Metronidazole + 0.1% Neomycin sulfate + 0.1% Vancomycin in 200 microliter of tap water per mouse
but antibiotics cocktail looks like paste, not completely dissolved.
Do you have any idea what are best solvents for those antibiotics .
Thank you
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So if I am understanding you correctly for the oral gavage you are trying to do:
10mg of each antibiotic in 200 µl water = 10mg/200µl = 50mg/ml = 5% (x4)
From what I have found Metronidazole alone is only soluble at 10mg/ml in water or 34mg/ml in DMSO so already there you will start to run into problems.
If the 5% DMSO does not work you might want to play around with the pH as well, but in any case I think it will be difficult to fully dissolve that amount of drugs in such a small volume.
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I have a e.coli strain, i inserted a resistant gene that shows resistance to some antibiotics on agar plates but whenever i grow them in lb broth with the same concentration of antibiotics, they are not able to grow. How to grow them in same concentration of antibiotics in liquid culture?
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Hi Sadia,
Thank you for your response.
Yes, I have confirmed the MIC using the broth dilution method.
for preparing agar plates I added antibiotics before pouring into the plates after cooling down ~ 60degree.
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2 weeks ago I started an experiment with siRNAs to inhibit P21 expresion. The protocol needs the use of medium without Antibiotics. So, first I grew my cells (LnCap) in medium RPMI complete with antibiotics, everything was ok (Picture 1) (I was so happy)...the day of the experiment, I passed the cells to a 24 well plate and I changed the medium for a RPMI without antibiotic, I worked like always in a cabinet, with all the necessary safety. The next day... was the worse day... I found all my plates contaminated with bacteria (I wasn't able to take a picture, I was in shock).
Next day I incubated all the reactives and media that I used for the experiment, alone and in combination. The wells with RPMI, OPTIMEM, PBS 1X, RPMI with antibiotics, were clean! but the FBS alone and all the combinations with the others reactives were contaminated (Picture 2, FBS alone). So I discard the FBS, and I opened a new one... but in order to be more secure, I decided to incubate an aliquote (37°C)... what was my surprise... The sample was contaminated again! I don't understand what happend, I was really worry about that... So I tried with a new 3rd bottle, and It was the same.
The bottles are stored at -80°C, are from the same lot...but...they expired in 2020....this could be the problem? but they were at -80°C...so I really don't understand.
Some ideas??
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Just for the sake of accuracy, how certain are you that this is bacterial contamination, and not (for example) precipitated serum proteins?
Does it progress over time? Does it turn the media cloudy, or is there just a visible precipitate? Do the particles move around rapidly (bacteria usually do, precipitated protein filaments really don't).
If you take the FBS and spin it (like, 1000g, 20mins) what collects at the bottom? If you take the uppermost fraction of the supernatant and incubate _that_ overnight, does it form near identical contamination, or not so much?
It's always formally possible that the entire batch of FBS is indeed contaminated with bacteria or fungus, but it's more likely (to my mind) that this specific batch is just more prone to protein precipitation.
Horse serum, for example, is far more prone to this sort of thing, and there you can sometimes see broad sheets of precipitated protein drifting by.
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I'm trying to purify plasmids from e.coli, the plasmids are high copy. I start with about 1.5ml culture volume, using neb monarch miniprep kit, and rarely get concentrations higher than 20 ng/ul, eluting with ~40ul. I'll outline my steps below:
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I streak out the plasmid containing amp resistant dh5a e.coli on amp/carb (conc.100 ug/ml) lb plates, and grow o/n.
Then I make LB broth containing ampicillin or carbenicillin at (same conc. as plates) and put 4ml of that into a 15ml falcon tube. I inoculate this with 1 fresh colony from the plate.
I then seal it completely with the cap and rotate them in a benchmark rototherm o/n at 37c and 30rpm. This rotates them cap over end and so the tubes must be sealed or else they will leak.
I don't think i have a reliable way to measure OD, but they definitely appear cloudy after about 16-20 hours. I spin down 1.5ml of this culture into a visible pellet and proceed with neb's monarch miniprep kit. At the end I elute with 40ul of neb's elution buffer, or DI water.
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I thought maybe the ampicillin I was using had gotten old so I attempted minipreps with fresh ampicillin, and also with carbenicillin in case that would help but hasn't seemed to make a big difference. I keep a 1000x antibiotic stock in the fridge, do these become degraded really quick?
Anyway I'm at a loss of how to improve these miniprep yields, any advice is appreciated,
Thanks
**update:
I've tried larger tubes with more aeration and got way higher cell densities, however the plasmid yield was still less than 20 ng/ul. I have tried qiagen's kit instead and still getting low yield. I am still unsure what the cause is, my next test is to use much higher antibiotic concentrations and make fresh antibiotics. I'm not confident it will help because I've used fresh antibiotics before.
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After adding elution buffer I normally allow 4 minutes before I centrifuge, I also elute with 30 uL. You can try that if it helps
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I want to produce antibiotics by fermentation and development of stable cell line MCB and WCB.
Which are antibiotics already being produced by microorganisms?
What is organism?
What vector can be used to clone effectively the gene for overexpression of production
Any patents and citations would be welcome.
We are trying to start a company a commercial organization like BASF or Bayer to produce medcicines and then excipients through microbial or enzymatic processes.
Any synthetic biologist want to join welcome.
Regards
Nikhil Shitut
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The following RG link is also very useful:
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I need to know whether my applied process affects the bactericidal effect of Antibiotic powder, so I want to test the powder effect not dissolving it and putting it on filter paper disks.
Is there any documented procedure or specification for tableting AB powder for sensitivity test?
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You don't need to compress them. All you need to do is make them in a powder form then you measure some quantity and pow into the well you have created on your petri dish which have been impregnated with your test organism
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Salmonella Enteritidis and Salmonella Typhimurium doesn't have standard (must tested) antibiotics for AST. Can anyone recommend what are the usual antibiotics that you use and why they are suitable for these bacteria.
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Thank you @Rodrigo Cuiabano. This is very helpful
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Hi everyone! We have a bit of a problem in our lab where every now and again we get a very strange contamination in TC (please see attached photos/ videos). Our work is completely pen/strep free with no added antibiotics, but this contamination looks like no bacteria I have seen before. The movement is also very strange and sometimes they can be seen almost cartwheeling/ doing 360 degree spins in the flask. I would just love to know if anyone has experienced this before and what I can do to make sure it doesn't happen again. Thanks so much in advance!
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Emily Smith Most of microorganisms contaminating lab cultures came from human skin or from soil. Sequence 16S/18S rRNA gene to find the species.
In my experience from commercial diagnostic lab - it is important to fight with contamination every day.
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Hi everyone,
I started to use SKBR3 breast cancer cell line in some of my experiments, but, I'm having some porblems to culture them properly. Currently, I'm using DMEM/F12 medium + 10% FBS + antibiotics. According to ATCC, they recommend the use of McCoy 5a medium. Is it extremely neccesary to use that medium? Do you people have experience with some other medium instead of McCoy?
Thank you very much for your replies and advices
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Thank you very much to both! Gayatri Gandhi Desmond Wong
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Is there any risk on human as there is a misuse for animal antibiotics
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Defoliation is a citrus canker management tool surrounding canker focal trees that have been removed. Where canker is already endemic, the primary means of control are: 1) planting of windbreaks, 2) protection of fruit and leaves with copper sprays, 3) control of leafminer, and 4) planting tolerant varieties.
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Hello everyone;
I need to show that there is no residue after removing the antibiotic from the cell culture. Is there any test/assay to analyze it?
Thank you
B.S.
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Hey Dinesh;
Thank you for your answer but I am looking for a quicker and cheaper assay. Any recommendation?
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As my cancer cells have undergone quite amount of passaging , thus with every passage , there are stress factors which either float around them or are stuck on the cells, as a result my cells are not geting the growth media , i have tried everything by filtering the media, increasing the antibiotic concertration and also lowering FBS but the stress factors are still present , i have been changing their growth media in every 12 hours now , is there any other way to make them go back .Please share your view
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From your content it seems that something is wrong with the cultured cells. Its important to find out whether its really stress causing deterioration of culture conditions. If yes, then try to determine which kind of stress factor i.e. physical (temperature, osmolarity or pH) or chemical (chemical composition of culture medium, drugs etc) are involved in cellular stress. Then what kind of stress is being generated (free radicals or hypoxia)?
Once you are confirmed the actual triggering factor of the stress, then its very easy to target in order to reduce it. If you find out that the oxidants are generated and causing deterioration in culture conditions, then you can use the appropriate "antioxidant" to scavenge the free radical or oxidants under in vitro culture conditions.
Best
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Dear Rg community,
I've been searching deeply in scientific research, but I haven't found why bacteria, which have developed an increasing resistance to conventional drugs and antibiotics, do not do the same for metalorganic frameworks?
Could you please share some related literature if posible?
Best, STC.
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Have a look at this recent publication:
Antimicrobial applications of nanoliposome encapsulated silver nanoparticles: A potential strategy to overcome bacterial resistance.
MR Mozafari, S Torkaman, FM Karamouzian, B Rasti, B Baral
Current Nanoscience 17 (1), 26-40
(let me know if you need full text for your private use).
In this paper, it is explained that: why pathogens do not develop resistance to certain antipathogenic agents (also see attached figures).
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The classes of antibiotics that are characterized by a Chromosomally-mediated resistance
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Mycobacterium tuberculosis doesn't harbor any plasmid and most of the antiTB resistance is transferred vertically including rifampicin, isoniazid, ethambutol, pyrazinamide, carbapenems, cycloserine, clofazimine, and bedaquiline. But for other organisms, it may depend if they are compatible for some plasmids or to horizontal gene transfer.
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Regarding the choice of empiric antibiotics for deep neck infections, what are the latest treatment guidelines or recommendations?
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Hello. Although there are no official guidelines for treatment I recommend that you have a look over "Deep Neck Space Infections Empiric Therapy" in 2020 on Medscape.
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diffusion discs, 1 cm diameter
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Special thanks to Fei Lin, Sweta Singh, Md Samun Sarker.
I contact with Oxoid Company through these emails:
but without any response from them
While I contact Becton Dickinson and they response quickly
Dear Nermeen Hossam,
Greetings from the Technical support team.
Thank you so much for contacting us.
The query is regarding the recommendation for antibiotic diffusion discs with 1 cm (10 mm) diameter.
The following are the available closest matches in the order of priority, please check. The exact suitability has to be checked by the customer.
; Sterile disks74146
WHA2017013; Whatman® Antibiotic Assay Discs
WHA2017009; Whatman® Antibiotic Assay Discs
Please contact us for any further support or information.
Best Regards,
Krishna Chaitanya
Technical Service Specialist – Research and Applied Solutions – Export
Life Science ? Research Solutions – Commercial ? EMEA
Merck
Mandatory information can be found at http://mandatories.merckgroup.com
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We have antibiotic resistance genes cloned into the pET(+)-21 vector and transformed in DE3 E. coli cells. I realize that this system is used for recombinant expression of proteins, however we want to use this system to determine antibiotic susceptibility patterns of the E. coli cells due to expression of the insert. Preliminary experiments induced with IPTG ranging from 0.4 - 1 mM indicated either no resistance patterns where expected, or single colonies forming around the antibiotic disc. We assume this is due to the formation of inclusion bodies due to hyperexpression of this vector-host system.
Can anyone please provide some insight on how to get this system to express at functional protein levels so that antibiotic susceptibility screening can be performed?
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You might also be seeing some toxicity issues, for example if your resistance gene were a efflux pump or a secreted protein, you might be exceeding the capacity of the cellular translocation machinery to handle high levels of the protein.
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Plasmid is transformed into competent cells and the transformed bacteria successfully grow in plates with particular antibiotic. After plasmid extraction, the agarose gels only show band at over 10kb which would be genomic DNA, while my designed plasmid should at range about 5kb-6kb. What would be the reason and any method to find out the reasons? By the way, I made the solution P1, P2, P3 buffer based on QIAGEN agent protocol. I tend to send the bacteria to sequencing to see if plasmid DNA can be extracted and detected.
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Did you cut your plasmid with a restriction enzyme? Running uncut plasmid on a gel will give you bands that are not at the size of a linearized plasmid. Usually you will see 2 bands from uncut plasmid, the supercoiled runs smaller than linear and the nicked circle runs larger than linear DNAs. I doubt 10 kb is genomic DNA from e.coli, which would be much larger. If this is a ligation, 10kb could be a plasmid dimer. By the way, what did you sequence?
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We recently purchased C. necator H16 (dsmz 428) from Dsmz for genomic engineering using suicide vectors. After some attempts, we noticed that this strain wasn't performing the first recombination, but still, some colonies were growing at chloramphenicol (35 ug/mL) or kanamicin (200 ug/mL) plates. All negative control (electroporation with water or wrong-antibiotic plasmid added) samples showed the same behavior after at least 72h or less. We also tested our glycerol stock and it grew in those antibiotics at same conditions.
In order to understand this bottleneck, we:
- Prepared new antibiotics;
- Verified that E. coli strains didn't grow in those antibiotic presences (which means it is probably working);
- Substituted all materials for new sterilized one (excluding contamination possibility);
- Checked if our strain was actually dsmz 428, by growing it in gentamicin (10ug/mL) (its natural resistance phenotype) and investigated PHB production using Sudan Black dye and amplifying phaCAB operon from its genome;
But still have the same results. I haven't found anything describing the natural resistance of C. necator H16 in chloramphenicol or kanamycin (literature describes that 200 ug/mL as a good concentration for cloning selection).
Does anyone have experience with something similar happening with this or other strain?
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It turns out that was a contamination issue. I recommend you to isolate your strain in TSB, LB, or NB agar with Gentamicin (10ug/mL), select some colonies and test them using TSB, NB, or LB with chloramphenicol (33ug/mL) for 48h - 72h.
I hope this helps!
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Does someone knows of alternative treatments ?
I am searching for companies/projects that found solutions to cure mastitis without antibiotics.
Thank you very much !
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Probiotic bacteria, and herbals are the new promising therapeutic regimens for mastitis.
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Bacillus subtilis ATCC 6633 grows in culture medium containing the antibiotic kanamycin and i do not know this Bacillus subtilis ATCC 6633 is resistant to the antibiotic kanamycin
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According to NCBI report resistance to kanamycin was not observed among any of the B. subtilis or the B. sonorensis strains Zahra Sabouri
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Hello researchers,
The Mrc 5 cell line that I will use in my study has such a view. Even though we use different antibiotics, the problem persists. Is this image contamination? It grows with the cells, does not move and does not change the color of the medium. It clears when washed several times with PBS., but after 2-3 days it has the same appearance. Interestingly, it sticks to the cell and when the flask is gently tampered with, the black dots are removed.
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I thought so too but, When I culture the cells one by one, black spots appear on the flask surface and on the cells. I will also attach sixwell images of the cells.In addition, the cells are beating like nerve cells.
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Mice pups are very small, could it be given through oral gavage? or are there other methods which are more suitable? Thanks
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Thanks Ahmad Kurniawan and Malcolm Nobre for sharing. That makes sense, waiting until PD 4/5 somehow might be a good time to start gavaging since their tissues have matured enough to receive gavage handling.
Your inputs are very well appreciated.
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Hello,
I am looking for information related to antibiotic pollution in natural water bodies; lakes, rivers in Russia. Sulfamethoxazole, erythromycin, norfloxacin, ciprofloxacin and tetracycline are some of the commonly detected antibiotics in aquatic environments. Maximum concentration values are very important in this regard. Kindly help me to find some important literature on this subject matter.
Thanking you all in advance.
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Thank you for sharing this interesting topic. but it is away from my field !
Regards
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Hello,
I am looking for information related to antibiotic pollution in natural water bodies; lakes, rivers in India. Sulfamethoxazole, erythromycin, norfloxacin, ciprofloxacin and tetracycline are some of the commonly detected antibiotics in aquatic environments. Maximum concentration values are very important in this regard. Kindly help me to find some important literature on this subject matter.
Thanking you all in advance.
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Interesting question! we are waiting for the experts' answers.
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antibiotic residues in food is very hazardous to consumer's health, however some animal and poultry producers may use antibiotics illegally in our developing countries.
my question is there any methods to make our food free from antibiotic residues other than heat treatment processes, i mean if there is any materials or substances to be added to food containing residues to make it fit for human consumption
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I still confused with % for my Acetonitrile. I really hope someone could clear my doubt. Thank you.
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It is a great question, from my point of view through working on HPLC for more than 10 years, i prefer to use isocratic mobile phase than gradient one and this actually give a good results.
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Hello everyone,
Hope all are doing good.
I would like to know how they prevent contamination in producing FBS at the moment.
I am looking for methods for preventing contamination, without using high temperatures or antibiotics.
Thanks
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Thank you Rima Ouchene
my regards
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How can we know the ROS production when we treat bacterial cells with an antibiotic agent?
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The generation of ROS was determined using fluorogenic dye 20, 70-
dichlorofluorescein diacetate (DCFDA). About 1 mL of culture suspensions from pathogens with
cell load of 1x108 CFU mL-1
was taken into various test tubes and 2.5 μL of DCFDA solution was added. All the reaction tubes were incubated in shaker under dark condition for
25 min at 37°C. Prepared antimicrobial substance (eg.Au NPs) was added to each test tube and again 2 h incubated in dark condition. The ROS formed in the sample after 2 h was
detected at 488/525 nm of fluorescence excitation/emission intensity measured using
Fluorescence Multi-Detection Reader.
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I am culturing THP1 cells but I find most of the cells are getting dead and the cells which stay alive get attached to the plate even though it's not treated with PMA. Could anyone suggest a better solution for these? The media composition used is RPMI 1640 + 10% FBS + 1% antibiotics+ 50mM b-mercaptoethanol. Thank you.
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I would like to share some tips on culturing THP-1 cells.
THP-1 cells are sensitive to cell density. You can passage cells between 3 x 10^5 and 7 x 10^5 cells/ml. Do not centrifuge THP-1 cells between passages. These cells will grow better in conditioned media. So, you leave at least 2ml of old media and add remaining fresh media to the culture.
Also, as mentioned by Jack Hopkins , the final concentration of beta-mercaptoethanol in culture should be 0.05mM.
Hope this will help you!
Good Luck.
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Group A(n=304) is the Control group, Group B(n=314) is the post-intervention group. I have the total Antibiotic use for both Groups, expressed in DDD, from that i can calculate the difference.
I read other similar Studies, they use either x2 test for trend, the non-parametricWilcoxon signed-rank test or Poisson regression. For all those i need more than 2 Data points wich i don't have. I just have the DDD for each group.
Is there anyway to show significance here?
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First you can check the data distribution for normality, if the ddd values in both groups normally distributed then you can apply simply independent t-test for finding p values between the groups.
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Hi! I have a question about mycoplasma test....we use qPCR kit to check the presence of mycoplasma in cells, and hystorically we used to eliminate antibiotic from cell culture medium at least 1 week before the mycoplasma test. I'm a little doubtful about the usefulness of this step. Do you have experience in mycoplasma test with antibiotics (I mean amp, Kana and so on)?
It's the deprivation of the antibiotic necessary to have affordable results?
Thanks in advance!
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Cannot need it
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Recently, I tried to evaluate the killing efficiency of one specific antibiotic on pseudomonas aeruginosa. when cells were cultured to the optical density of 0.6, 20-fold MIC was added. However, in the next 24 h, the broth keep turbidity. Nevertheless, the plate assay was performed to calculate the lived cells and we found the majority of bacterial cells were actually killed. To my best knowledge, the dead cell will experience autolysis and the broth will become clear and I have obtained the similar result before as well. so my question is why the turbidity of broth did not reduce although bacterial cells are dead?
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"To my best knowledge, the dead cell will experience autolysis and the broth will become clear" I don't think this is correct... Autolysis is enzymatic digestion of host cellular components. Tho process would rather Make the broth more turbid
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Hi, I plan to knock down a gene in immortalized keratinocyte (Ker-CT from ATCC). In the spec sheet, it was mentioned that these cells were immortalized with h-Tert and CDK4 carrying puromycin as their selection marker. Theoretically, it would be safer to select different selection antibiotics, however puromycin is the only one available for my gene of interest.
Has anyone transfected/ transduced Ker-CT with puromycin as selection marker? Is the selection accurate? I had seen publications where puromycin is used as the selection marker in Ker-CT, but would like to gather more information/ feedback. Thanks!
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If your cells are already resistant to puromycine, you will have no selection of your recombinant cells. You could do a cotransfection with another plasmid carrying a selection. This selective plasmid must be at a lower concentration than your plasmid of interest. You could try 1:5 or 1:10, reported to the size of your plasmid
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Is it the stationary phase, as it is reported that it is during this phase that bacteria confer high rates of resistance ?
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Bacterial metabolism is slow down in the stationary phase due to many other factors (limited nutrients, accumulated toxic metabolites), and part of the population will die in a short time without additional factors. So, the antibiotics effect will be lower.
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I am planning to create dysbiosis in the animal model, already antibiotics are considered. But want to look for other alternatives too. something like bacteriocins or any other?
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The problem with using bacteriocins (or bacteriophages) is that most are highly specific for certain bacterial strains and not for others. There are a few "broad" spectrum bacteriocins but most are not. And even those are not really universal.
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Hi there,
I am currently co-culturing B. subtilis (B) and an Acinomycetes (A) to induce expression of a glycopeptide antibiotic by the actinomycetes.
The actinomycetes strain is quite slow growing in comparison to B. subtilis, so I tried inoculating A after different days.
For example: Inoculate A and B simultaneously; let A establish for 1 day, then inoculate with B; let A establish for 2 days, then inoculate with B etc.
This is the only way I have been able to induce expression of this antibiotic, but even still, I cannot get stable expression across triplicate experiments? One may produce A LOT, one a little and the third will produce nothing at all
A colleague called it a "mini invasive species environment" and said there will always be large variation... is this true? Is there a way to control this? I always inoculate with the same volume of the same OD600 of both bacterial strains, for context.
Thank you!
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Michael J. Benedik thats what I am also thinking, whether the actual physical contact is required.
That is a very good idea, thank you! I will try that.
I think I will try culturing A with and without B cells, so try growing in supernatant to see if is is a nutrient depletion caused by the rapid growth of B compared to A, rather than it’s physical presence. As well as culturing A in different days of B growth, like you suggested.
As for the variation, I am not really sure what to make of this. I am hoping the new media will induce stable expression so I can state in my thesis that the variation is definitely from the 2 bacteria and not just not repeatable from A.
I really appreciate your help!
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From my understanding, Ceftazidime antibiotic inhibits the peptidoglycan synthesis process by binding to penicillin-binding protein. But in Aeromonas hydrophila I could not find any Penicillin-binding protein for molecular docking purposes as a comparison for my compound. I was wondering which specific penicillin-binding protein does ceftazidime binds to in Aeromonas hydrophila or does it simply bind to other proteins that eventually inhibit the synthesis of peptidoglycan? Can anyone please help me out? Thanks in advance
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I can't say for Aeromonas hydrophila, but for E. coli the main essential target of ceftazidime is penicillin-binding protein 3 (PBP3), which is the product of the ftsI gene.
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How can we identify the source (Ruminant/ Avian/ Human) from where a bacteria gets resistant against antibiotics (if environmental samples is used)?
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I agree with the opinions of my colleagues, regardless of the source of the resistance of bacteria, the problem of increasing resistance causes global concern at the level of public health... But in fact, there are bacterial spp. that parasitize in animal habitats and others that parasitize in humans, but there are other types in zoonoses and is transmitted between humans and animals... In general, bacteria acquire resistance genes to adapt to the selective pressure they are exposed to as a result of the misuse and abuse of antibiotics, whether in the field of animal and agricultural production or in the field of humans... And at the most, these genes may be acquired from bacteria existing in the environment or in the intestines, which are nonpathogenic through the mechanisms of genetic transfer ... In addition to the presence of genetic mobile elements such as plasmids and jumping genes (transposons), which enjoy wide freedom of movement within the bacterial populations.
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I am performing a luciferase transfection in U87 cell lines to express the luciferin gene. I cultured them in a 24-well plate, and pulled A+B to 1 10-cm dish, then pulled C+D to 1 10-cm dish. My question, I am trying to figure out the dosage of my antibiotics, G418 (Geneticin). If my stock concentration is 50mg/mL and I want to dose with 800ug/mL in 10mL of media for a 10-cm dish. What is the math that I need to convert this over and get my volume (uL) of how much I should treat my cells to select for the luciferase expression amongst my cells.
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Normally you would be calculating the amount needed on the final concentration you want to achieve x total volume / stock concentration. However you state in your question that you want 25 mg per dish. If that is actually correct (25 mg per dish) and your stock is 50mg/ml then you need to add 0.5mL (or 500uL) per dish of your stock. But are you sure that is the amount you want per plate, or is it really a concentration per mL of medium (which is more normal)? Also 25mg sounds pretty high to me.
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  1. When over-decolourized by either prolonged exposure to decolourizer or using acetone alone.
  2. When cell wall gets damaged by exposure to lysozyme or cell wall acting antibiotics such as Penicillin.
  3. Old cultures, where cell wall is weakened or action of autolytic enzymes
  4. Those bacteria that are phagocytosed. where cell wall is acted upon by lysosomal contents
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I'm culturing seaweed. I use antibiotic solution before the seaweed explants are planted in agar media. Do I need to add antibiotic again when that seaweed explants are cultured in agar media?
Thanks and hopefully
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Follow
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Mandibular osteomyelitis due to dental implants is a rare and serious condition. After a month of removing the implants and antibiotic medication, the infection continues. Is decortication of the affected bone and antibiotic flushing a predictable treatment?
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Antibiotic sensitivity test will guide us to the best type and dose of the antibiotic.
Taking swab from oral cavity is prone to contamination by oral flora, taking piece of infected tissue ( curetage the area ) may be more accurate.
If there is radiographic signs of sequestrum, it is better to be removed.
Keep in mind the importance of systemic condition ( anemia, poorly controlled diabetes, vitamin D deficiency)
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Please anyone tell me about the concentration of the antibiotic for making the antibiotic discs.
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I usually use commercially prepared Antibiotics disc for antimicrobial susceptibility testing.
However, I suggest you to prepare an antibiotic solution of 1 microgram/microliter concentration. Then, if you transfer 20/30/50 microliter into sterile blank discs, then allow the discs to dry up.
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How can we store the culture media containing antibiotics? How long is their lifetime in the best storage condition?
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Thank you very much for your answers. Wiwiek Harsonowati Yoram Gerchman Gamal Enan
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How can I prepare a serial dilution of 1024ug/ml antibiotic for MIC. How much volume of the antibiotic will I take so that the final conc of antibiotic becomes 1ug/ml
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Dear Arnav Padhi, please find attached a table for your serial dilutions.
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Hi everyone,
I am hoping to overproduce an antibiotic by swapping promoters.
I am just wondering what the advantages/ disadvantages using inducible/constitutive promoters for the overexpression of secondary metabolites, specifically antibiotics?
If anyone has any ideas on methods for promoter engineering in Amycolatopsis I would be extremely grateful too!
Thanks!
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Dears
I am currently working on some natural bioactive compounds as antibacterial agents,
During the work, I found out significant inhibition when they were applied in vitro.
I am really interested to know how these agents affect bacterial growth.
Many thanks
Khawlah Abdallah Saman
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Hi there,
If induced expression of an antibiotic through co-culture and am looking to scale up.
Is it possible to culture two bacterial strains in a fermenter to induce antibiotic production?
What are limitations of this?
Thanks
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This question appears to be a mix of several question.
  1. can you co-culture in fermenter - Yes!
  2. Everything other than point one - everything would depend on specifics and details which cannot be generalized or recommended without further information.
And basically one need to have trials and pilot project before moving to fermenter step and scaling-up.
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Hello everyone,
I have been reading a lot around promoters and their use in antimicrobial production.
I am just curious as to why many researchers add constitutive promoters into gene clusters for antibiotic production, when a lot of people say this has toxic effects on the cell?
Would it not be better to use inducible promoters in this scenario?
Thanks
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(I guess is for the use in the antibiotic. In this sense, if I wanted antibiotic, I would liek to be producing the antibiotic all the time, but maybe the build up of the antibiotic could be toxic, this is why you would retire them, either in a bioreactor or in a petri dish. You would normally do in the bioreactor.I would like to be producing it continuously and not "tiring" the bacterial cell with resources .or the user. I would like it all the time. )
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Biofilms are protective coatings that bacteria and fungus form to protect themselves from drug as antibiotic or anti-fungal treatment. So my question is: Can bacterial or fungal Biofilms cause infections?
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I have agrobacterium GV strain culture that were grown well on LB agar plate with gentamycin and rifampycin antibiotics. But when I reinnoculated from grown cultures plate into fresh LB broth there are no sign of growth. I even did try to inoculate the culture just on to LB broth with no addition of antibiotics, but i didn't see any growth sign yet.
I also prepared fresh medium with appropriate antibiotics to find out is there any problem with medium. I have also innoculated different cultures available in our lab (like Agrobacterium LBA strain and they grows well with appropriate antibiotics, or at least with the rifampycin). Does anyone know what is the problem and how to fix it? Thank you
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I wanted to help you solve this problem, but unfortunately, I did not work with this bacteria and I have no information about it.
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Hello all;
Antibiotic administration in the drinking water of mice: How to improve drinking habits of mice?
We tried antibiotic cocktail in the drinking water of SJL/J mice.
Antibiotics cocktail: 0.1% Ampicillin + 0.1% Metronidazole + 0.1% Neomycin sulfate + 0.05% Vancomycin + 2.5% sucrose in tap water
Unfortunately, they didn’t drink any water as result they lost their body weight almost 3 gm over 3 days period.
Even we tried in acidified distilled water (PH= 2.5 ) instead of tap water, unfortunately same things happened.
Do you have any idea why they didn't drink antibiotics containing water and how to solve it?
Thank you