Questions related to Antibiotics
I want to select E. Coli cells positive for the attached plasmid.
What are the two resistance genes meant for? Selection in eukaryotes / prokaryotes?
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
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... :)
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
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.
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?
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?
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?
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
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.
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,
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?
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.
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.
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.
Sage plate technique:
Thank you :)
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.
I will appreciate your input. Thank you
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.
I am referring to the impact of technologies such as vaccines, antibiotics, statins, etc.
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?
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?
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 .
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?
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.
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,
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.
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.
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?
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.
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!
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
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?
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
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?
Regarding the choice of empiric antibiotics for deep neck infections, what are the latest treatment guidelines or recommendations?
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?
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.
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?
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
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.
Mice pups are very small, could it be given through oral gavage? or are there other methods which are more suitable? Thanks
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.
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.
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
I still confused with % for my Acetonitrile. I really hope someone could clear my doubt. Thank you.
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.
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.
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?
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!
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?
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!
Is it the stationary phase, as it is reported that it is during this phase that bacteria confer high rates of resistance ?
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.
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
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.
- When over-decolourized by either prolonged exposure to decolourizer or using acetone alone.
- When cell wall gets damaged by exposure to lysozyme or cell wall acting antibiotics such as Penicillin.
- Old cultures, where cell wall is weakened or action of autolytic enzymes
- Those bacteria that are phagocytosed. where cell wall is acted upon by lysosomal contents
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
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?
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
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!
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.
Khawlah Abdallah Saman
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?
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
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?