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Antibacterial Activity - Science topic

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I need to know the best growing conditions for MRSA and MDR P. aeruginosa.
The incubation time and temperature and the best medium.
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I grow multi-drug resistant MRSA and multi-drug resistant P. aeruginosa on rich media (BHI or TSA) in the presence of at least one of the antibiotics that the strain is resistant to. For MIC testing, I grow it on CAMH plates.
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Hello, I am going to test different extracts for their antimicrobial activity against MRSA. I made a protocol and I would like it if some of you had any feedback on it.
General information:
- Working volume max is 90 uL
- I am going to use an 384 well platte because I am going to test more than 2000 extracts and this is the fastest way.
- Extract is diluted in DMSO
- Needed controls are also tested but not noted in the protocol.
Protocol:
1. Plate the MRSA bacteria on Mannitol Salt Agar containing 5 mg/l methicillin. Incubate 24 hours at 35 °C.
2. Prepare sterile Mueller Hinton Broth and sterile saline.
3. Make the 0,5 McFarland inoculum with the MRSA and sterile saline.
4. Add 35 µL extract + 35 µL MHB + 10 µL inoculum in 1 well.
5. Incubate 24 hours at 35 °C while the growthcurve is being made
Thank you in advance.
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Please eliminate uncontrolled variables. Note Kirby Bauer uses nonselective medium.
You do not know what methicillin carry over, alone or in combination with unknown materials, may be, and there is no reason for MSA in the 1st place. On what basis do you assume an isolate through one culture sequence will lose resistance? As far as I know, the record does not defend that phenomenon
Even if it were a concern, are you sure methicillin in MSA is working? I'm not familiar with its validation. Why not use ORSAB - the medium typically used for isolation of MRSA.
If application - you'll prob run more testing in context but be aware, composition of plant extracts, EO's etc. vary greatly from batch to batch. Ask the supplier both for data showing what constituents are "active" and to establish standard concentrations and establish relevant CoA on a batch basis.
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Chlorhexidine (Chlorhexidine gluconate, digluconate or chlorhexidine acetate) is an antiseptic that is used in medicine for last 70 years. Is there any experience of Chlorhexidine application to control greenhouse, water, soil contamination by plant pathogens?
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You can check my article in which I had used microdilution method to check antimicrobial activity Eldho Jijy Varghese, Dhanasekaran Sihivahanan, Kondas Vijay Venkatesh, "Development of Novel Antimicrobial Dental Composite Resin with Nano Cerium Oxide Fillers", International Journal of Biomaterials, vol. 2022, Article ID 3912290, 7 pages, 2022. https://doi.org/10.1155/2022/3912290
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I want to study the growth kinetics of bacteria against metal nanoparticles, I have immobilized metal nanoparticles on clay. But I am having difficulty in bacterial growth curve that it is not showing antibacterial properties in the form of nanocomposite. Currently I am using LB and nutrient broth for the study.
Thanks for your help
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Dear Noor
I think you should count the bacterial number. Also you can increase the nanocomposite concentration. Optical density (OD) is not the the right way to measure the antimicrobial effect in your case because increasing the nanocomposite concentration will increase the OD. May be counting of bacteria after treatment with different concentration with time will be the best method to study the growth kinetics in your case.
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Chitosan is a natural biopolymer with antibacterial properties and the solution of chitosan is prepared in acetic acid. So, has acetic acid any effect in contributing towards the antibacterial behavior of chitosan?
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Based on the following link it was observed that chitosan water-acetic acid systems show the highest antimicrobial activity due to the highest chitosan charge density, compared to the mixtures with lactic and citric acid.
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So far many papers (my literature study) are suggesting that Ultraviolet Radiation on TiO2 induces photocatalytic reaction which makes it give Bactericidal effects on gram positive and gram negative bacteria. Will TiO2 gives bactericidal effect against gram positive and gram negative bacteria without UV irradiation? Kindly suggest me what are gram positive and gram negative bacteria. When a TiO2 based material is implanted into body it does not have the photocatalytic effect inside the body UV light will not pass, then how TiO2 will give Bactericidal effects inside the body?
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I have prepared the silver nanoparticles via chemical reduction method using NaBH4. Upon turning yellow color of the solution I stopped the reaction. I got UV vis peak of nanoparticle solution at 400 nm.
But when I centrifuged the solution the pellet is so small that unable to use it for antibacterial activity. I want to ask whether I can use the AgNPs solution for MIC by diluting it with D.W. or pellet is necessary?
Also which medium will be suitable for the growth curve? either nutrient broth or PBS?
I will be grateful for your help.
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Dear Researcher,
Use high concentrations of primary materials to have finally enough concentrations of Ag NPs and on the other hand, apply ultra-centrifuge for precipitation of Ag NPs.
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I am currently looking for a research in PVD coating of medical devices. I am interested on PVD coating of AlTiN for metal cause it has low reflection and is compatible for surgical instrument. I want to doping it with Ag so it has antibacterial effect. But will it make the reflection of material completely change or will it depends on Ag-doping composition?
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If you introduce Ag in amounts that it becomes biologically relevant, you will surely introduce major changes to the bandgap and therefore changes in the color and all optical properties would be expectable.
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I am having bacterial strains in the agar plate. I want to extract these strains and store them in glycerol for long-term use. Is there any stepwise procedure for this process?
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This is the procedure I use for E. coli stocks:
- Take a freshly saturated culture in rich medium (2YT or LB) and spin 2 ml in a 2-ml microfuge tube for 2 min in a microcentrifuge.
- Discard 1 ml of the supernatant and use the remaining 1 ml to resuspend the pellet
- To the concentrated suspension, add 0.5 ml 60% (v/v) sterile glycerol and mix thoroughly by pipetting up and down.
-Transfer the mixture to a cryotube and store at -80 °C
I have kept such viable stocks for decades. When needed, scratch the surface of the frozen stock with a toothipck and use the scratched material to inoculate a fresh culture, without thawing the rest of the stock, which should be returned immediately to the -80°C freezer.
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What is the best dosage of tetracylcline in vitro effect? i dont know the theurapetic window or index of this drug. please help me guys.
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can I use the spectrophotometer to get the OD for my bacterial suspension before doing a disk diffusion antibacterial test to have a 0.5 McFarland turbidity , and if yes at what wavelength and what value should I get at said wavelength ?
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At 600 OD
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What is the best as chemicals what have antibacterial activity (like silver) for air treatment by tio2 photocatalyist to eliminate different types of bacteria and other air pollutants?
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Also, kindly check the following link entitled (Self-Cleaning Coatings and Surfaces of Modern Building Materials for the Removal of Some Air Pollutants):
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I have some plant samples and I want to check the antiviral activity and Antibacterial Activity. and I want to check it by using bioinformatics.
So I need Suggestions regarding which bioinformatics parameter or software is available that I can use for check by antiviral and Antibacterial activity.
#bioinformatics #antiviral #antibacterial #plants
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Hi
Your request is very general.
You need to have information about the compounds of the plants you want and find the antiviral or antimicrobial effects of those compounds.
For example, plant AMPs. Some of these AMPs are shared among eukaryotes (eg, defensins and cyclotides), others are plant specific (eg, snakins), while some are specific to certain plant families (such as heveins). You can use https://dbaasp.org website to predict the antimicrobial properties of peptides. This site is also a database of AMPs where you can see the antimicrobial properties of the AMP you are looking for.
Or other compounds in plants that have either been approved for antiviral and antimicrobial properties, or you can use docking tools to predict the interaction of these compounds with microbes or viruses(For example, a flavonoid compound with the corona virus s protein).
good luck
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I want to examine a kind of fruit's extract's antimicrobial effects. At first I want to put it under the UV radiation to prevent the mold or any kind of fungi's and bacteria's growth.
The question is : Does the UV radiation affect on fruit's substances ?
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UV light is used in many food industry applications as it is completely safe, low maintenance and does not require the use of any chemicals or pesticides. UV lamps can effectively eliminate viruses like E. Coli and Salmonella as well as many other microbes which cause food to spoil.
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I am performing MIC assays using 96 well plates. I followed the CSLI protocol for standardisation of bacterial cells as follows: Subculture (3 colonies) of 24h bacteria culture into MHB for 3h. I then dilute it with sterile MHB to match the 0.5 Mc.Farland standard and confirm with spectrophotometer at OD600 aiming for an absorbance between 0.08 and 0.13 (They are always between 0.08-0.09). Then, I take 50uL of standardise solution and mix it with 4.95mL of MHB and this is the solution I use for my assay. I take 10ul from the growth control (50ul of Mueller-Hinto Broth + 50uL bacterial solution) and mix them with 9.95mL MHB. Then, I take 100uL of this new solution and plate them in Mueller-Hinton Agar for the colony count.
My colony count of MRSA or MSSA always comes around 20-35 colonies, E. coli and P. aeruginosa always exceede the 50 colonies (around 80-100). What am I doing wrong?
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Hi Sofia,
Although OD595-600 and McFarland standards are somewhat standardised methods for estimating the cfu of bacterial cultures, different species can result in different final numbers. OD600 is a turbidity measurement, therefore differences in cell size (even slightly) can have different turbidity measurements. The best approach is to optimize the growth of your cultures under your laboratory conditions. This can be achieved by drawing up a curve comparing OD600 (y =-axis) with counted CFU/mL (x-axis) for each bacterial strain. You will only have to do this once per strain. This will allow you to prepare standardised bacterial inoculums and have approximately the same number of cells in your assay for each bacterial species. It is also worth mentioning that slight variations in cfu/mL should not have a large effect on the MIC values, and the value will stay the same regardless of the bacterial concentration. However, MIC shifts can be observed at bacterial inoculums one to three orders of magnitude larger.
All the best.
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According to the Baier minima, surfaces having critical surface tensions between 20-30 mN/m are considered as foul release surfaces. PDMS or silicone material falls under this range according to literature then why silicone catheter surface allows biofilm formation on to it ?
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I suggest that you read these two papers:
DOI: 10.22203/eCM.v0S0u7rfaa0c6e p
They show that the situation, especially in a biological setting, is much more complex than surface tension or Baier minima. Silicone is always coated with a host - derived protein / glycopeptide conditioning film when inserted into the body, and this completely alters the surface characteristics. In addition, in vitro without the conditioning film, some bacteria possess binding proteins that can attach to native silicone, eg vitronectin - binding protein.
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I did an antibacterial activity of lime peel. I use ethyl acetate as the solvent. The ratio is 1:4. The method of extraction is maceration for 3 days. I stir it everyday. After the 3 days of maceration, next i filter it with whatman filter papers and evaporate it in 40 degree until the extract becomes thicker. I put it in oven 40 degree for 24 hours to make sure all of the solvent has evaporated. The next day i put it in the refrigerator.
I used the extract to test the antibacterial activity with Staphylococcus aureus. I've done it before and it has an inhibition zone, but this time it doesn't have any inhibition zone.. It does have but only a real small inhibition zone (7mm). But according to the journals that I've read, they get like 20-25mm inhibition zone and i wonder what's wrong with my research. I'm still looking the problems, that's why i come here to look for an expert.
Thank you so much
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did you use any positive control such as Chloramphenicol or any antibiotics? if both didn't show any inhibition zones may be something wrong with the bacteria
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how H CQ+Azi acts against covid 19?
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I work with antibacterial surface (copper), we've observed a diminution of activity after some time. We've attributed that to oxidation (to CuII which is known to be less active compared to Cu0 or CuI).
I would like to know if anybody has insight about how I could preferentially have the surface to stay at CuI instead of CuII (would a Cu2O surface be stable?).
We've thought about protecting the surface with an inhibitor (but since the antimicrobial activity comes from the release of Cu+, wouldn't it impear the antimicrobial activity?).
there are ways to get the surface "active" again, by solubilising the copper oxide surface but we would like to keep our surface active as long as possible before having to "reactivate" it.
thanks
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One last thought about your process for activation: " The activation of the surface is only done by rubbing it with a weak acid. " How do you know that the activation process is not mainly physical (due to the rubbing) as opposed to chemical (due to the weak acid). Have you done a negative control, i.e., doing the rubbing using the same water you used to make up the weak acid solution but without the weak acid or no water at all? The reason I am asking this question is that the very act of rubbing a metal surface can create a Beilby layer on the metal surface and this type of layer has some unusual properities of its own: it changes the contact potential, work function, induces an exoelectron effect, etc. And if you abrade the surface you can cause the Russell effect, which through the interaction of exoelectrons and water, produces H2O2, hydrogen peroxide. I know all this sounds crazy but the Russell effect (1897) is so strong it can fog photograhic film pressed againt the abraded metal surface, and gentle rubbing of the interior metal surface of a Geiger-Muller tube can induce such high levels of exoelectron emission that it was initially mistaken for radioactivity by Lewis and Burcham in 1936. You can find detailed discussion of the Russell effect, Lewis and Burcham's work, the Kramer effect, Erskine-Murray's work on contact potential change, etc. in the document entitled Beilby Layer, Revision 1 - TrackChanges on my RG page.
Best wishes on your project.
Regards,
Tom Cuff
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Dear colleagues;
I have worked recently to investigate the antibiofilm activity of nanoparticles against E. coli.
I have measured the activity via ELIZA using different controls, what is the best method for data presentation in results' section.
Regards
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you can try to categorise your results in a table format
you can have something like 'weak', 'normal' and 'strong' and each of these can have ranges that include values
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There are two interpretive criteria for the results of epidemiological in-vitro anti-bacterial sensitivity studies, namely EUCAST and CLSI. Can I use both in a single study - e.g. using CLSI in an institution and the EUCAST in another institution?
** Kindly note that I'm asking about the mere scientific validity and soundness, regardless of the institution standards.
Regards,
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It is advisable to use one interpretive criteria. No two standards are exactly the same. Therefore using more than one criteria may complicate interpretation of results by generating contentions.
In case you have been using the standard set by Federal Drug Authority (FDA) there is an exception where by FDA recognizes the standard published in "Clinical and Laboratory Standards Institute (CLSI). Performance Standards for Antimicrobial Susceptibility Testing. 29th ed. CLSI supplement M100. Wayne, PA: Clinical and Laboratory Standards Institute; 2019". In this case you may use more an additional interpretive criteria. Note that modifications to the list of recognized consensus standards: Publications in the Federal Register to the list of recognized consensus standards can be accessed at http://www.fda.gov/MedicalDevices/DeviceRegulationandGuidance/Standards/ucm123792.htm. Thank you.
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What is the active principle that is responsible for the antibacterial and antifungal effect of cow urine?
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I have not worked in this aspect..
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how nanoparticles enhance the antimicrobial activity of compound??
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I agree with Dr. Vincent
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Has anyone intentionally inoculated their cell culture medium with bacteria and fungi to understand what its bactericidal or growth promoting capabilities are? Using antibiotics in cell culture media is widely accepted as a preventative measure, but this may not be necessary if you have evidence that the medium is already antimicrobial. If you understand your risk points (even if just a select few microorganisms), then you can tailor your prevention measures. Gathering data to see if anyone has also asked this question and has experience and insight through testing, keeping in mind the media formulations for each application may be very unique.
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Very interesting findings. Wanted to share this article that also supports Traci's theory.
Although we didn't test B. anthracis, there is likely a similar interaction going on.
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I am doing surface modification of polymer films using plasma in order to improve anti bacterial property.
Thickness of polymer film is 60 micron to 1 mm for different polymers.
I want to test it against E-Coli species.
What are the testing methods suitable to such substrates?
This work is in the direction of developing antimicrobial catheter surfaces.
Thank you in advance.
With kind regards,
Purvi
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As mentioned by Mr Damian, you must be sure that you can sterilise the sample without disrupting the plasma treatment. This is usually done through UV B exposure in a cell culture hood. You will need to find a lab which is capable of performing microbial culture. Some people use ISO 22196:2011 although this will only work on very smooth samples. Another option is to use a confocal microscope and either GFP tagged bacteria or a dye such as LIVE/DEAD to quantify bacterial attachment to the surface. We did some similar analysis here and I would recommend reading Andrew Hooks paper on '
Combinatorial discovery of polymers resistant to bacterial attachment. It is important to consider how you expect your polymer to kill bacteria, or just prevent the attachment.
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Most of the times we dissove extracts from plants or other sources with Acetone or DMSO. Observations show that when relatively non-polar solvents (n-Hexane, Pet Ether, DCM etc) were used during extractions, then the extracts will dissolve best in Acetone or 10%DMSO.
However, during preparations of working solutions and further dilutions on Microtitre plates, the solubility of these extracts decreases and precipitates can be observed upon centrifugation. These precipitates are compounds which dissolved in Acetone or 10%DMSO but they can not stay in solution as the compositions of these solvents get lower. (Maybe they have antimicrobial potentials)
On the other hand, conventional antibacterial agents which are water insoluble are usually solubilized through Synthetic and Formulation approaches eg. Forming their salts. This is not so possible with crude extracts.
If gold standard MIC testing methods such as Broth Microdilution assays are mostly using water based media eg. MHB, what reliable options do we have to zoom in probably potential antimicrobial combounds present in nature but insoluble in water?
Any similar or different encounters?
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Analysis of plant extracts showing efficacy often finds the usual suspects. For example, journals such as Int. Biodegradation Biodeterioration require analysis and novelty of composition for publication of reports of efficacy of extracts from novel plants.
But to Nelson's point - media-based testing has limitations but has some relevance to application. Academically, we may wonder at an assay that evaluated efficacy in low water context but methodology here would prob be even less relevant to application.
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Hi, I want to check the antibacterial activity of AgNPs. I dissolve AgNPs in a DI water and sonicate it, after that I use well diffusion method to check the anibacterial activity. But AgNPs show no biological activity. Should I use another solvent?
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Use sodium dodecyl sulphate SDS to prevent preciptation after biosynthesis process
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Or can one says that antibiofilm agents are antibacterial too?
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There may or may not be a relation between antibiofilm and antibacterial activity. It depends on the agent. Biofilm is a problem causing drug resistance as several antibiotics cannot penetrate the biofilm/cell wall of the bacteria to show antibacterial activity. Use of nanoparticle loaded with antibiotic to penetrate the cell wall and release the antibiotic can perform this multifunctional role.
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There are so many NPs synthesized by the researchers, among these nano-structures silver have shown diverse biomedical application because of their fascinating properties. In current time, scientists are focusing to synthesized these silver nanoparticles through green chemistry following their principles.
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Hi, I am new to bacterial studies, so I have some questions I'd love some input on!
In bacterial studies, I know that zone of inhibition (ZOI) can be used when plating an antibacterial substance, and is often simply reported as a diameter where no bacteria visibly grow. Is it possible that this zone contains alive but non-culturable cells? (Also, given ZOI is just related to the diffusion of the antibacterial substance from the disk, why is it that diameter is considered the 'strength' of the substance and not just its mobility?)
In a similar vein, minimum inhibitory concentration (MIC) is just the minimum amount that prevents further growth, but the cells may either be dead or alive but non culturable, correct? If that's the case, are the MIC and ZOI relatively comparable, since neither differentiate between dead or non-culturable? And if they are comparable, can that comparison be defined quantitatively?
Thanks in advance!
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The antibiotic concentration in the clear zone should decrease from the center, where it started, to the edge. The concentration at the edge of the zone should be equal to the MIC.
There are calibrated test strips (E-test, for example: https://en.wikipedia.org/wiki/Etest), that use the size of the zone of inhibition to measure the MIC.
You are correct that the bacteria may not be dead in the zone of inhibition or at the MIC concentration. If the antibiotic is static, not cidal, they may simply not have grown. In addition, even if the antibiotic is cidal, there may be a small population of persister cells that are dormant but will grow once the antibiotic is removed.
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the peptide which i have tested is about 100aa(hepsidin+ hydrophobin) and i haven't seen any inhibition zone, can it difuse through the muller hinton agar?
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Yes !
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I want to do MIC and MBC test on streptococcus mutans, i have muller Hinton ,and BHI medium, is there any way that i can do this with out using blood?
the problem is I cant find blood in my university.
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Yes, I'm working with S.Mutans using BHI to determine MIC n MBC without blood. Have a try and see the results for instance
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The compound Drosophilin A is known to have antibacterial and antifungal activity (Anchel et al. 1955; Anchel 1952; Kavanagh et al. 1952). Its methylated analog, Drosophilin A methyl ether, also a fungal metabolite (Teunissen 1999), has no reported -as far as i know- any biological activity.
  • DA IUPAC Standard InChIKey: XIWJLPHQDBDOAN-UHFFFAOYSA-N
  • DAME IUPAC Standard InChIKey: HICARXIPJINIRA-UHFFFAOYSA-N
Chemical structures from NIST Chemistry WebBook, SRD 69
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There is report that the lignicolous basidiomycete Phellinus badius deposits up to 30,000 mg of the halogenated metabolite drosophlilin A methyl ether ( DAME, tetrachloro-1,4 dimethoxybenzene ) per kilogram of decayed hearthwood in the mesquite Proscpis julifora.. DAME occurs as clusters of glassy crystals up 1 mm long within the decayed heartwood. For more details consult The Science of Nature 102(3-4): 18 DOI: 10.1007/s00114-015-1268-5
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Hi all!
The above question is for efflux pump inhibitors with or without antibacterial activity 
how will the above choice hold good for biofilm inhibition??
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Hi,
Talking about efflux pump inhibitors in my last review
Regards
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What differences between Kirby-Bauer test and disk diffusion test? is it the same thing? Both of them use filter paper impregnated with a standard of sample extracted, then placed onto an agar plate that was previously inoculated with the test microbe. These method was found to be a simple, cheap and reproducible practical method.
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Kirby-Bauer test and disk diffusion test are the same thing that we use in laboratory tests.
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I need some papers( both research + review) where plant extracts been used both for both antibacterial and antibifilm candidate.
Secondly, any database of secondary metabolites having antibacterial activities..
Thanks for your co-operation
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In reality, your question should have sounded like this: so much information exists in PubMed, how can I digest all this? But we again sending you to the search engines like google scholar
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In Disc diffusion susceptibility test, on sterile nutrient agar plate broth culture of the test bacteria was swabbed and dried, and 4 sterilized discs, each of 6 mm diameter (prepared in the laboratory from Whatman’s No. 1 filter paper), were placed on 4 different sectors and marked. Thereafter, the discs were soaked with four different concentrations: 20, 35, 50 and 65 μl/disc of ethanolic Bakul Leaf Extracts.
After incubation, ZDIs values obtained around each of the discs were measured. But, clarity of inhibition area lost due to chlorophyll like contents present on the leaf extracts as green tint. In that case, What will be the clarity enhancer procedure without hampering the experimental actuality ?
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You are using very concentrated extract. Use dilute solution
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The next stage of my project requires growing single species biofilms in 96 well microtitre trays by placing PCR plates with the 'pegs' sitting in the bacteria culture to grow biofilms on them, as described in this paper: " Antibacterial Activity of Blue Light against Nosocomial Wound Pathogens Growing Planktonically and as Mature Biofilms" by Halstead et al.
However previous groups have struiggled due to the coned shape of the pegs, are there any with flatter bases and/or more cylindrical rather than conical pegs?
Thanks
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Interesting question and looking forward to read answers
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i want to specifically assess the activity of the extract against cough-causing organisms.
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Goodluck
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I have 100 mg of  peptide antibiotic i.e colistin. Potency  is >15000 U/mg and solubility is in water  (50 mg/ml according to manufacturer's instructions). As potency is given in units, so converting (via online conversion calculators) it into micrograms, it is 1U=731.7 ug in case of colistin. As W=C*V/P formula does not apply on peptide antibiotics. So, how should the stock solution be made if my final testing concentration is 128 mg/L but first I need to make 20 X of this concentration. (128 mg/L would be the concentration in 150 ul volume). I'll appreciate if anyone can help in this.
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Hi there,
No need of potency here as you intend to assay mass concentrations (btw there must be something wrong in your calculation as your calculated unit is uncompatible with the potency...). 20x stock should be 2.56mg/mL. Te important thing to know about the starting material is it's actual content in colistin (when pure it's 30000U/mg).
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I have a non polar compound (curcumin analogue) that insoluble to water, but soluble in pure DMSO. So, for antibacterial test purpose, I tried to dissolve it in DMSO 100% then add with aquades until the DMSO concentration is 10%, but it still insoluble, so I add 4 drops of tween 20. Is it okay to use that method to dissolve my compound in antibacterial activity test? Or what should I do with my compound in order to dissolve it?
Thank you.
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You have to consider the effect of the solvent itself on the bacterial cells. They can tolerate a few % of DMSO, but too much will probably kill them. Detergent will also affect them. If they still grow, then you have to consider whether the DMSO/detergent mixture is permeabilizing the bacteria, producing an artifactually low MIC. Try the solvent alone on the cells. Also try some control antibiotics both with and without the solvent to see if it has any effect on their MICs.
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Will the metal drug complexes be tested for their antimicrobial activity?
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Yes but after very good sonication and well disribution
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I am intending to perform antibiotic susceptibility assay on cultures of Staphylococcus aureus by broth microdilution method, using resazurin sodium dye as a marker of cellular viability. For this purpose which media should be preferred? I have some references suggesting the use of Mueller-Hinton broth. Need expert opinion on this.
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According to CLSI guidelines, Mueller Hinton broth is suitable for all non-fastidious bacterial that grow aerobically.
Regards
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I am working with some fern antimicrobial properties and I would like to know the range of the best concentrations that I can prepare my plant extracts before I can be sure they have performed best and can compete with conventional drugs for such microbial conditions.
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Hi:
If you have a pure substance, CLSI guidelines say you should apply two-fold agar dilution method for determination of MICs (ranging from 0.008 mgl/l to 128 mg/L).
Regards
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Dear Everyone,
I'm looking for the fulltext for NCCLS M22-A2 and A3 procedure. May you kindly help if you have? 
Thanks!
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Thank you very much for all your help. I really appreciate that.
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how to check antibacterial activity for zinc oxide which is not dissolve in any solvents
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Goodevening my dear
Mix Zinc oxide at different concentrations with the surfactant Tween 80, sterilyze by autoclaving. Make wells in nutrient agar medium( bottom layer and top layer of 0.7% agar) . This top layer must be seeded at its pouring with the indicator organism.Add in this wells the sterile suspension of Zinc oxide plus Tween 80. Make controls of tween only. Then incubate for 2-3 days or more and check the inhibition zones.
                             Good Luck
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Actually, I have extracted antimicrobial agents by solvent extraction with ethyl acetate. Now, I want to know that does ethyl acetate alone has any antimicrobial activity or not? 
What I have observed that Ethyl acetate extract has antimicrobial activity, now actually, I want to confirm that does ethyl acetate (organic solvent) has the antimicrobial activity or not?
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Ethyl acetate extract has no antimicrobial activity. It is also based on the polarity one of the solvent system normally used. The minimum  antimicrobial activity of the solvent  has to be ignored. More over a few compounds only dissociated with ethyl acetate and you do not worry about this particular issue. Thanks and regards Mr.Muhammad Numan.
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How do nanoparticles act on Candida albicans? Like for gram positive bacteria, the nanoparticles penetrate the cell wall and kill the bacterial cells. Is the same mechanism followed by nanoparticles while acting on fungus like C. albicans?
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It will be hard for the nanoparticles to enter the cell wall. Despite this the toxicity effect will occur when they start oxidizing and dissolving.
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In the commercial antimicrobial discs, some of the discs the company provide different concentrations. Does it matter? Since this is not a quantitative method, it should be fine with whatever concentrations, right?
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Dear Chia-Da, you should choose concentration of antibiotics in discs according to standart procedure either CLSI or EUCAST. Another factor is your test bacteria. The concentrations change on the basis of your test bacteria. You should follow the instructions defined in CLSI or EUCAST manuals. Most of the commercially available discs prepared according to these procedures. 
Best regards.
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I am working on the screening of marine natural products with potent antifouling activity. I want to test the initial crude extract by coating the same on metal or glass panel to expose in seawater. What kind of “BINDER/ADDITIVE/GLUE” will be used to coat the crude extract on metal panels to test initial antifouling ability?
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Thank you
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So when using MIC we are basing our numbers established on a bacterial concentration. For instance we say the mic of citric acid for salmonella is 1000ppm. We did the test at a starting bacterial concentration of 10^5
We measured final results through absorvance and calculate the percentage of growth inhibition that happened. 
Question is
If we have different bacterial concentrations can we infer the value of the mic based on the absorvance values? 
I mean for a bacterial concentration of 10^5 the mic was 1000ppm what would the mic be if the bacterial concentration would be 10^7?
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The size of inoculum does affect the MIC, but the effect depends on the antibiotic and its mechanism of action. There are two main reasons behind this: 
1- Increased cell number result in decreased effective number of antibiotic molecules/cell (you may say per target molecule). Depending on the mechanism of action it may change kinetics of killing or prevent killing altogether. 
2- Increased inoculum results in fewer cell generations before culture reaches saturation. Some antibiotics require active growth for killing (e.g. beta-lactams) and it takes few generations to accumulate a defect which would result in cell death. Hence, if you start with the dense culture you may not have enough time (number of cell divisions) to observe the killing.  
Unfortunately, the MIC change isn't just simply proportional to the cell number, and there is no way of calculating it. That's why the MIC determination protocols are standardized for 10^5 cell/ml, so it can be directly comparable. 
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The compounds are metal complexes wherein a Nickel compound has shown effective antimicrobial activity against MRSA strain, Vanadium compound has shown antidiabetic activity and copper compound, a very good anticancer activity.
I need to know if performing DPPH assay for determining the antioxidant activity add on or give an extra proof for the above said activities?
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DPPH test is widely performed to assay anti oxidant/free radical scavenging activity of compounds. Free radicals are generated through oxidative stress and attributes to the tissue damage and promotes end organ damage like liver fibrosis or ageing process. These also induce mutation which can lead to cancer. Since this test has a  vast array of significance, it cannot be a confirmation test for the prognosis of cancer. Rather you can go for cell line assay. Please follow the link.
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how to determine the Minimum inhibitory concentration of Nanoparticles
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Dear all, 
I am currently researching about the antibacterial capability of certain proteins and protein hydrolizates against microbial cultures. We do not have any reference about the protein concentration of my matrix that could have antibacterial activity. Anyone could reccomend me what is the best dilution strategy for the determination of MIC from unknown proteins and protein hydrolizates?
Kind regards,
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Thank you Adam and Shashank for your answers. 
Kind regards,
William
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As written in the title, I would like to load zeolite onto a sterile paper disc for testing its antibacterial activity using the technique known as Zone of Inhibition. Can I redisperse Zeolite in a solvent, for instance, dichloromethane, and then directly drop the suspension onto the paper?
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Thank you for your answer Khilood Fahed and Mert Sudagidan :)
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Dear experts,
 Kindly clarify the significance of MIC and their effect.
Thank you in advance.
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MIC is stand for Minimun Inhibitory Concentration, which means the minimum drug concentration that can be used to inhibit organisms in vitro. There are many methods to determine MIC i.e., Agar dilution, broth dilution or E-test. The MIC value will provide both qualitative and quantitative data and also a guideline for adjusting an optimal drug concentration to treat patient. 
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Like I have more than 100 papers on silver nanoparticles and I have not seen ames assay, invitro DNA damaging and hemolysis potential of synthesized nanoparticles and their starting material. Even if you are using green material there are chances of cytotoxicity... Then why are not they checking?
comment from your knowledge and experience!
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I am working on a study to evaluate the antibiofilm activity of compounds already injected in polypropylene, but I do not know what to compare their activity, do you know some material or surface that can use as a reference antibiofilm?
thank you very much
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I will be glad to receive a listing of all the scientifically appropriate experiments to conduct in order to indicate the anti-microbial activity of plant extracts. This includes experiment from extractions to sensitivity tests.
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agar dilution method,  broth dilution method, and disk dilution method, According to CLSI document M27-A, and CLSI document M27-A3.
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 I tested for the presence of an anti-bacterial activity of a sample against E. coli using disk diffusion assay.
Two different concentrations i.e., 2 mg/ml and 4 mg/ml were used and found to exhibit anti-bacterial activity as was evident from a circular zone.
PROBLEM: Around the circular zone, few colonies during the initial hours around the zone appeared and later no colonies showed up.
I hypothesized that the sample inhibits the bacteria lately which is why bacteria could grow in that inhibitory zone. 
If I have to test this what are the experiments can I perform to come to the conclusion ?
Your suggestions and solutions are highly awaited.
I have attached the image of my Bacterial plate.
Thank you. 
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This phenomenon is due to hetero-resistant sub-population, as many researchers answered before.
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I have synthesized silver nanoparticles by using bacterial extracellular components as the reducing agent. After confirming the presence of AgNPs in our samples I'v tested the resulted colloid in bacteria but I didn't get any bactericidal action. any help with this issue please?  
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You use dry colloidal nanoparticles or in in solution.??
You use freshly prepared nanoparticles. After preparation centrifuge it, then re-dispersed it in deionized water and check for antibacterial activity.
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I am planning to isolate marine actinomycetes from a specific area and identify them and  their secondary metabolites.
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By using the normal growth conditions, you can only grow those 'culturable' strains. The best media are ISP media. Depending on what kinds secondary metabolites you are looking, you can extract them with organic solvents and run assays such as antibacterial assay. Or you can put them on HPLC and collect fractions that have signature absorbance. For unculturable strains, the only think you could try is extracting DNA from the mixed samples and do a genomic mining. Then you could express the gene clusters you are interested heterologously and test production.
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Can somebody answer how to study antibacterial studies (practically) of some polymer blends in labs. what are the things required , and how to set experiments. I will be grateful if u can spend some time for me to explain.
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Download the article 
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Evaluating the antibacterial activity of cassava leaves and roots in Sri Lanka.
Upon sample collection, the leaves and roots were washed and sun-dried after which they were extracted with methanol. The extracts were concentrated to give crude semi-solid extracts. These were then resuspended in distilled water to give a initial concentration of 1mg/ml which was further diluted to 0.75, 0.5 and 0.25mg/ml. 
Disk diffusion assay was chosen to test for the antibacterial activity against S. areus and E.coli. Gentamicin and distilled water were used as positive and negative controls respectively. 
Since no zones of inhibition was seen except at the PC, considering the concentrations to be too low, further test was carried out with concentrations of 200 and 100mg/ml. However, these also did not give any zone of inhibition except at the PC. 
Thus, as another attempt, fresh samples were again collected, crushed with mortar and pestle and homogenized with distilled water, they were then centrifuged and disk diffusion assay for both the supernatant and the pellet was carried out against S. areus. However, this too did not give any zone of inhibition though at its maximum concentration.
Considering this could be due to less volume that was impregnated in the discs, well diffusion was opted. However, this too did not give any zones of inhibition.
Since there are articles from other countries like Sudan, Malaysia, Africa, India and Thailand giving positive results, can I conclude that the cassava obtained from Sri Lanka does not have any antibacterial activity or is there any other attempt I can go for?
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One of the most valid reasons is the resistance of the tested strains against the extract. 
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Hi everyone
i want determine antibiotic susceptibility of biofilm-state bacteria to compare with planktonic-state bacteria. i searched some papers and met some description like MBEC, "MBEC" is the same meaning with "MİC of biofilm cells". and is there any method for Escherichia coli.
Best wishes
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MBEC is biofilm eradication concentration in which impact of compounds is assessed on pregrown biofilm. MBIC is similar to MIC.
Generally crystal voilet assay is done for quantifying, resazurin assay is also used to assess the viablility.
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I know that temperature may have an effect on properties of nanomatals 
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Definitely it will affects,I dont know idea related to graphic oxide but I had idea about copper .
For example:copper surface antimicrobial activity was tested depending on temperature, ambient relative humidity, This study showed that antimi-crobial copper alloys were more effective at 37◦C—100% relativehumidity. conditionsat 20◦C—50% or 20◦C—40% relative humidity.
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I have antimicrobial activity in the nutrient and L.B broth but when i try to extract it with different organic solvents like Ethyl acetate, butanol, n-Hexane, Chloroform etc, there is no activity in the extract and the activity also losses from the broth. Please guide me what to do in this case.
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Thank you Merajuddin khan and Piotr Biniarz. I will follow your suggestions and will practice it. 
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I wanted to check the antimicrobial property of my plant extract. Just to check the same I did disc diffusion assay for the concentrated plant extract using Nutrient Agar medium and E.coli culture. After incubation for 24 hours, the zone obtained was transluscent immediately after the disc followed by a transperant. When I checked with the images on google, the pattern was similar to anti quoram sensing property assay (qualitative). So, I donot know whether I have got the zone of inhibition for the growth or anything else is there. I am attaching the picture of the analysis. Please help me to analyse the same 
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Dear Katyayini, I suggest to use broth dilution assay. As explained by Jay Gopal Meher, diffusibility of plant extracts from discs may change. For this reason, my suggestion is broth dilution asay by measuring OD600nm, you can follow bacterial growth inhibition in the presence of different concentrations of plant extracts. As explained by Aarti Bains, agar dilution assay is also applicable for plant extracts.
Best regards.
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Is there anyone know a natural antimicrobial peptide has a stronger antimicrobial activity at room temperature than at 37 degree?
Reference link, please?
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no significant effect of  room temperature and  37 C on active peptide
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I need a protocol (without automatization) in order to do antibacterial effect of nat products in vivo with C. elegans model. Thanks
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Thanks ... have you a specific link ... are many papers!
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Tea Tree oil (TTO) is available in many brands by several famous companies with the promise that it cures most of the skin ailments like Acne and Psoriasis because of its antibacterial and antifungal activity. Australia is the biggest producer of TTO. It is claimed to be active against MRSA a deadly pathogen. Besides, it has been incorporated in products as antimicrobial laundry freshener, antidandruff shampoos, acne face wash, natural deodorant and household cleaners.
Our study on 550 strains of bacteria causing topical infections, Candida albicans for comparing TTO with 8 topical antibiotics (polymyxin B sulfate, gentamicin, nitrofurantoin, tetracycline, chloramphenicol, cotrimoxazole, ciprofloxacin, and novobiocin) revealed that gentamicin was the most effective antibiotic followed by chloramphenicol, ciprofloxacin, nitrofurantoin and polymyxin B inhibiting 87.1%, 84.8%, 76.8%, 75% and 72.8% strains, respectively. Tea tree oil (at 1 μL/ mL) could inhibit the growth of 20.5% strains. Only 10% of multiple drug resistant (MDR) were sensitive to TTO. Though TTO is a broad-spectrum antimicrobial active on 26 out of 44 genera of bacteria is a less promising antimicrobial than antibiotics on MDR strains. The TTO is to be said very effective against staphylococci but this study revealed that in vitro only 12% staphylococci were sensitive to TTO. It is claimed that 5% TTO formulations are highly effective, no doubt in vitro 5% TTO can kill many of the bacteria but when applied to skin and bacteria causing infection are hiding a little deeper in the skin and at that site how much concentration of TTO reaches and in that concentration, how toxic is TTO to host cells?
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This is interesting research. To the best to my knowledge, tea tree has been extensively use to treat acne but it does not work for everyone. I am kedn to know the mechanism of action of this tea tree against the topical causal bacterial.
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As it  is reported that bacterial surfaces are negatively charged ?
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I think you are concerned that O2- (superoxide) would not be effective because of electrostatic repulsion. However, because counterions are generally available (such as Na+, K+ and Mg2+) to balance the charge, such repulsion should not be a significant factor.
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We have observed some synergy between herbal antimicrobials and antibiotics. It may be due to inhibition of efflux pump by herbal antimicrobials or due to some other kind of interaction. 
How good this combination can be exploited clinically.
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The idea of an adjuvant for antibacterial therapy to permeabilize bacteria is currently the focus of at least one company (Speros). Efflux pump inhibitors for combination with antibacterials have been explored in the past as well (Microcide). It's a challenging approach. Good luck.
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A nano-compound was synthesized by degrading the polymer and combining it with transition metal. The solubility of resulting compound is very less. Now I am planning disc diffusion method for determining the antibacterial activity of this compound. For this, the compound should be completely soluble. 
Can you please suggest how I should solubilize this compound or is it possible to determine the antibacterial activity with partially soluble compound or is there some other method for this?
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It will be easy If you use freshly prepared nanoparticles solution for antibacterial activities.
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a mixture contains 45mL of HBSS and 5mL of anti-anti and is filtered, and then stored at 4 degrees for about 6 months. It doesn't show any visible contamination, but freezes instead. What does it mean?
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Dear
To be sure we need to verify it by retrying culture after stored 6 months later.
Thank
Marius
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production of antibiotics by Thermopilic actinomycetes 
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Dear 
To determine antibacterial activity of thermophilic actinomycetes it is recommended agar well diffusion method.  Following approach has encouraged :
-Ajust cell Concentration of all test microorganisms at 0.5 McFarland turbidity standards -inoculate on MHA plates by using sterilized cotton swabs.
-Bore wells by sterilized 1000 μl micro tip, and 100 μl of each crude extract an poure into wells.
-Incubate plates  at 37°C for 24 h.
Please look at of protocol of Holam et al. J Adv Pharm Technol Res. 2013 Apr-Jun; 4(2): 118–123. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3696223/
Marius
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Hello every one
I made the synthesis of some new compounds and I want to evaluate their biological activities such as anticancer, antifungal and antibacterial activity.
The question that arises here, if these compounds do not degrade in the biological medium (human tissue, fungal, bacterium) that pass before reaching their target?
So, I would like to know if there is a scientific program that can predict this property  (stability of a molecule in a biological medium).
best regards
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For in silico prediction you may use ADME module of Schrodinger Software, whereas in case you need to predict in vitro effect mass spectroscopy is the best possibility you may use.
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I am trying well diffusion method but my samples doesn't show antibacterial activities no zone is formed.  what could be possible reasons?
is there any modification for sample to show activity?
any suggestions?
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may be low concentration of samples , try to do MIC and MBC 
with my best wishes