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Biofilm Formation - Science topic

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Hello researchers, I am a bio technologist wants to work on the use of COMSOL for biofilm formation. Please help me regarding this
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explain COMSOL
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I am currently investigating biofilm formation in Salmonella isolates using 96-well crystal violet assays. The protocol I follow utilizes multichannel pipettes (opposed to other rinsing and removal methods I have read about others using). From 1:100 diluted overnight broth plates are inoculated and incubated (at various temperatures), broths are removed after and wells are washed using MQH2O until clear. I then stain using 0.1% CV for approx. 15mins (using a higher volume than the broth; e.g. 100ul broth and 125ul 0.1% CV), then washing wells once more after. after drying for 48h or more I solubilize the dye using 30% acetic acid and record the OD600. when comparing the results to a blank negative control of 30% acetic acid (approx. 0.05 OD600) calculations are showing very high biofilm formation (high OD600 in contrast to the negative control) when the lab isolates are supposed to have for example low formation or even none. I suspect there is insufficient pipetting of planktonic cells/ CV leaving a coating which is skewing OD600 measurements. This issue is consistent across most replicates and temperatures. I would love to hear any insight, thank you.
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i worked with biofilm many years ago and i never worked with Salmonella but from my experience with other strains there are some point of your protocioll that can be optimized to improve the reprodubility and be sure that the biomass that you measure with CV is biofilm and not just died planctonic bacteria.
1) In my experience if yuo perform biofilm formation in microtiter plates (with bacteria adesion on the plate bottom) and not with PINS, to limit the effect of asepcific adesion due to the gravity and bacterial death, is better to perform a media exchange step after some our of adesion to remove the excess of plactonic non aderent bacteira and give to the biofilm formers more nutrients.
You can see an example of it in the paper that we pubblished for GBS
2) You can use the multichannel to fill the wells by pipetting in the well border to limit but i think that it is preferable remove the media by vacuum mainfold or by reverting the plate.
3)Once you removed the broth, i suggest to you to perform at least 3 washing (i used also PBS instead water to maintain good viability) to be sure that you removed all the non aderent cells.
good luck
manuele
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I'm looking for papers and articles regarding the post-evaporation residue of healthcare disinfectants (based on CDC guideline: link below) like H2O2 or sodium hypochlorite. I'm also looking for the association of supposed residue to biofouling and biofilm formation.
I've read that H2O2 doesn't leave residue but they are mostly anecdotal.
Thanks!
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Maybe off your target - chlorhexidine oral use can discolor teeth -
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I am working on P. aeruginosa Biofilm assays as a part of my research. Biofilm formation was carried out using Microtiter plate Biofilm Formation Assay.
  1. Can someone suggest a way to find MIC of the biofilms using the normal 96 well plates? I tried to get the results using the microdilution method, but it gave me smaller MIC values for the Biofilms compared to the Planktonic Cells.
  2. Is 620 nm wavelength too high to quantify the biofilm in 30% acetic acid? Because the plate reader in our lab provides readings only at 492nm and 620nm.
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Thank you All for your suggestions.
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Is there a technique to measure the biofilm formation in the Hungate tubes of autotrophically growing anaerobes?
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Since you are using tools designed for anaerobic culture, there should be no difference between aerobes and anaerobes detection methods.
Now it depends on what you want to study and measure, for example: if you want to measure cell biomass (use CV), cell metabolic activity (XTT) or cell viability (MTT) and measure Optical Density of them. For each study you must choose the necessary and adequate reagent for the studied cells.
Cordially.
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I'm writing my thesis, in which I have included a paragraph about LIVE/DEAD staining of biofilms, and I just want to know if LIVE/DEAD staining is most suitable for early-stage or late-stage biofilm formation?
Articles and other sources would be greatly appreciated!
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Live/dead stain can be used at different stages of biofilm development but you have to keep in mind the mechanism of action of cyto9 and PI on the cells.
You can look on these 2 papers
Good luck in your writing.
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I want to knockout csgD gene in E.coli to control biofilm formation.
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Use Recombineering... this is a useful resource to start reading: https://redrecombineering.ncifcrf.gov/court-lab.html
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I' m gonna use it as an indicator for biofilm formation of specific bacteria
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Prepare a 12 mM stock solution by dissolving 5 mg of MTT [3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] in 1 mL of PBS. This stock solution will be enough for 100 reactions (10 µL per reaction). Once prepared, the MTT solution can be stored for 4 wk at 4°C protected from light.
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On some E coli cells, I see some opening of cell wall as shown in this image. Please guide what is it and why such things happen i.e. under what kind of conditions ?
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Dead cells, as cell preparation is for electron microscopy
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Hello,
I performed a biofilm formation (24 hours) on 96 well microtiter plates for 3 different strains: Klebsiella pneumoniae, Acinetobacter baumannii and E.coli. The crystal violet staining assay was conducted and the absorbance was measured at 620 nm using a microplate reader. The problem is that the obtained absorbance values were very low <0,1.
Is there any explanation to this ?
Thank you.
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Dear all,
generally crystal violet is measured at 570 nm, but it works well at 540-580 nm. 620 is too much, but also you should have a value if you can see the color.
If the staining is bad, I would suggest to do as follow, these are modifications that we made for the standart Cv stain protocol:
1) dry your plates overnight, otherwise biofilms could be washed out.
2) use 0.5-1.0% CV in 96% ethanol. It fixes the biofilm to the surface.
3) we found that not all media provide the rigid biofilm formation and use so called Basal medium, please find the protocol here https://www.nature.com/articles/ja2014143
For regret we did not found the original citation for this broth. It provides the rigid biofilm formation by almost all bacteria, while the biofilms obtained in MH generally are very gentle and some manipulations with them are impossible.
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Hi,
I have validated some novel proteins in my targeted bacteria. Now, I want to study the effects of these candidates by overexpressing them inside the bacterium. I have targeted three phenotypic (the growth rate, Motility, And Biofilm formation) analyses. Can anyone suggest/recommend some papers regarding these phenotypic analyses in bacteria? It will be really a great help?
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Hello Md Atikur Rahman ! I don't know if it will be helful, but I've found this article at some point and it could be a good start?!
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Greetings,
I've been searching for days in databases to find the correlation between porosity in materials (for built environments like porcelain, ceramics, wood, etc.) and biofilm formation (index of bioburden). The findings will help me then understand the correlation between porous/non-porous materials and their cleanability properties.
Thanks
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Hi Ali, from my experience, I think both chemical and physical properties of a material surface can have impact on the biofilm formation. For example, the chemical properties can determine whether the surface is hydrophobic or hydrophilic as well as the degrees of adhesion whereas physical properties such as surface roughness, surface textures can also influence the biofilm formation.
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Microbial biofilm formation
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Dear Amzad,
Please refer to this paper, DOI:10.3791/2437. Microtiter plate assay is the easiest method to evaluate the biofilm formation of bacteria.
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I have carried out a biofilm formation assay (CV method) in a 96 well microtiter plate and have carried out the following:
6 replicate wells containing nutrient broth (-ve control)
6 replicate wells - 100ul E.coli K-12 (1:100 diluted) + 100ul dH20
6 replicate wells - 100ul E.coli K-12 (1:100 diluted) + 100ul amp (4ug/ml)
6 replicate wells - 100ul E.coli K-12 (1:100 diluted) + 100ul amp (8ug/ml)
6 replicate wells - 100ul E.coli K-12 (1:100 diluted) + 100ul amp (16ug/ml)
After the resolubilisation with 30% acetic acid step, I have taken the OD at 600nm and to correct for background absorbance I subtracted the average of background wells (-ve control) from those containing samples. Is this correct?
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Hi Mike,
Yes it is correct when you want to compare your treatments.
Good luck
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Hi all,
I'm currently looking into methods implemented in visualising and quantifying the impact of a specific gene on biofilm formation and was wondering if anyone could point me in the right direction. Any info would be great.
Thanks.
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Generally, Imaging and automated cell counting are the most common methods of biofilm quantification. Furthermore, the use of stains or fluorescent markers, in order to more accurately identify cells of interest and distinguish from culture debris, allow for easier and increased accuracy of cell counting and data interpretation. For further, these articles might be an asset, have a look:
Kind Regards
Qamar Ul Islam
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Crystal violet will in fact stain living cells (though it is toxic) as well as dead cells. yes, so I asked CV wheres adherence to the cell
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In the cell walls. Thanks.
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I would like to know any simple stain or test bu which i could stain or quantify only the EPS or biofilm present and not bacteria present in it.
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FilmTracer SYPRO Ruby biofilm matrix stain
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I am interested in studying biofilm formation as a function of multiple variables, and would like to go beyond spectrophotometric/colorimetric/microplate readings and to analyze biofilm structures microscopically as well. Unfortunately, fluorescent microscopy (FM), confocal laser scanning microscopy (CLSM) and scanning electron microscope (SEM), tools that are routinely used by well-funded research groups for this purpose, are currently not at my disposal.
To this end, I am considering to utilize traditional staining (e.g., crystal violet) of these structures followed by simple light microscopy. May I ask for recommendations on objectively/quantitatively analyzing the images that can be generated this way (perhaps through a specific software)? What I have found so far are all for FM, CLSM and SEM images. Thank you!
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Dear Aedrian,
We have faced your problem in the past too - but if you have a good microscope and camera system I think that you can usefully and quantitatively examine differences in biofilm structure using some common dyes for bacterial cells and EPS.
The key questions are whether you have a fluorescent microscope and camera system. If you do, then I'd suggest looking at using live/dead stain mixes, or propidium iodide, and whichever fluorescent DNA and protein dyes you can get. If you only have a light microscope, then you need to be using standard coloured dyes, again for as many cellular components as you can. The second question is whether your camera system can capture single (black and white) or multiple colour images.
No matter what type of microscope and camera system you have, you should be able to take good images of biofilms at a resolution from 10x - 40x and perhaps even 100x (at which point bacterial cells should be visible). You can import these image files into free software such as ImageJ - you can then divide the image into squares and get the total absorbance (e.g. for live/dead stain) and use this in your statistical analysis.
You could look at the variation across the image and correlations between different stains to investigate heterogeneity. You can compare mean values for each image (or measure the absorbance for the entire image) to then compare across images (e.g.different incubation conditions, different strains, etc.).
Regards, Andrew
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Hello everyone,
I am planning to work on engineered enzymatic quorum quenchers but I am a little bit confused about the selection of quorum quenchers. Which one is the best QQ for gram-negative bacteria?
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I am getting some results of V. cholerae biofilm where a compound is inhibiting biofilm formation but the number of cells is unchanged and also there is only a slight or no reduction in fimbriae activity. How it is co-related? Can anyone explain?
Thanks
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you need proteases and or chelated substance like EDTA or NTA. you need also inhibit protein synthesis inhibition
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i currently doing a research about biofilm formation on food contact materials, i need to show the adhered cells too
want to know what best method to perform
thank you
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Rahma
Crystal Violet staining is only for visualization of biofilm-forming cells and cannot be used to discriminate the live and dead status cells. For live/dead you need to opt for either Syto9 (Live) /PI (Dead) combination or Acridine Orange (Live) /EtBr (Dead) combination.
For your further clarification, few research articles are attached.
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I'm working on gram negative i want to use any natural extract (plant extract) or triple drug combination against its biofilm formation.
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We found that natural extract that contains oleanolic acid can be very effective at disrupting P.aeruginosa biofilms (see doi: 10.3390/antibiotics9120911 )
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For the ability of my extract to inhibit biofilm formation, i used the microplate, but the problem is the precipitation of the extarct at the bottom of the microplate, so it is impossible to see the biofilm formation or its inhibition, do you have a solution?
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Why not use the water-soluble part of your extract? Adding an additional solvent may facilitate your study but it makes it pretty much an academic exercise without much relevance.
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I am running a simple experiment with a co-culture of bacteria (P. aeruginosa and an E. coli strain) in LB to study their interactions with regard to biofilm formation. Is there a good or feasible way to separate these two bacterial species from this co-culture in order to separately analyze their respective ribosomal profiles?
Thanks!!
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I do not see the difficulty in separating Pseudomonas aeruginosa from Escherichia coli culture. From pigmentation alone, the cultures are different, just pick and perform separate quadrant streaking on LB agar and subculture until pure cultures of each is obtained.
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Hi everyone. I isolated a lytic phage for wound infection caused by S. aureus. It didn't make a clear spot on the lawn of ATCC 25923. Also, I examined it on 24hr formed biofilm of this bacteria by tissue culture method. in comparison with SM buffer as a negative control. data were collected from 6 repeats and analyzed by post-hoc (p value= 0.005).
Does anybody have any clue about biofilm increase in presence of a lytic phage?
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Hi, check your virus doses that you are applied in your expermintal methode.
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Currently i am working on the biofilm analysis. Please suggest some softwares or tools for same.
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This is for the confocal microscopy
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I have used the xCelligence RTCA-DP instrument to determine the adhesion eukaryotic microbial species biofilm formation before treatment and after treatment with an antimicrobial agent. Data was expressed as CI (cell index) over time. How would I do data analysis if I export it into excel? I am also seeing peaks at the times where the plate was removed from the instrument for treatment. This could possibly be due to the surge of electricity. If anyone has any expertise or has used this method before, please feel free to drop a comment.
Thanking you in advance.
Kind regards.
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Keith Stuurman It is normal to see disruption ( random peaks in your curve) at the point of plate removal. You may also notice in case someone closed the incubator door very hard.
Use the normalisation function in the software and run the assay post-treatment for at least 24 hours to observe proper separation of the curves.
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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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Pseudomonas aeruginosa forms biofilm on different surfaces viz., glass, polystyrene, steel, ceramic and rubber. How can we quantify if the surfaces are being corroded after biofilm formation?
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Dear all,
I'm doing the inhibition assay of bovine DNase I on biofilm of Pseudomonas aeruginosa PAO1. Until now, I have already tried different medium, 96-well microplates and incubation time, however, all the results showed not good. Here are my protocol.
1. Culture the PAO1 with LB medium overnight.
2. The 96-well microplate (vinyl, polyvinylchloride and polystyrene) was added 100 μL of Pseudomonas aeruginosa PAO1 solution that OD600 measured approximately 0.1 (1.0 × 108 cfu/mL), which contain different concentration of bovine DNase I (0 – 100 µg/mL), then incubated the microplates for 24h at 37°C. (Different medium has been tested, which include M63, LB and TSB)
3.1 Dump out the planktonic bacteria by turning the plate over and shaking out the liquid, rinsed the plates 2 times with pure water.
3.2 Removed the planktonic bacteria by pipetting with multi-pipette, washed the plates 2 times with pure water.
(Both methods have been tried.)
4. Added 125μL of 0.1% solution of crystal violet (CV) in water to each well of the 96-well plate for 10-15 min.
5. Microplates were rinsed with multi-pipette to remove the stain.
6. Subsequently, 125 μL of 30% acetic acid in water to each well of the microtiter plate to solubilize the CV for 10-15 min.
7. Transfer 125 μL of the solubilized CV to a new flat bottom microplate and quantify absorbance with the UV spectrophotometer at 550 nm.
There are two problems of this assay.
(1)The results showed the OD550 of biofilm are too low (0.1-0.5), compared to the articles around 1.0.
(2) The DNase I could't inhibit the biofilm formation.
Could anyone give me some suggestions about this experiments.
Thank you in advance.
Have a nice day.
Ye
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Hi Ye,
I believe you can try this simple method:
Good luck,
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Hi everyone,
I would like to quantify the biofilm formation of fungi o my plastic films, which stain and whih wavelength should I use. For bacteria cryatal violet and ethanol is using, but for fungi I do not know. Can you help me please?
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Hi Navid,
You can use this simple method:
Good luck.
Best,
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Hello!
I am trying to grow Candida albicans biofilms in the lab. So far, I've been using Sabouraud Dextrose Broth, but many articles describe Yeast Nitrogen Base - YNB for Candida biofilm formation. I saw online that there's YNB without amino acids. I don't know what composition YNB should have to favour Candida biofilm formation. Can anyone help me out?
Thank you in advance!!
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Sueden de Souza YNB medium with amino acids supplemented with 100 mM glucose is usually used for C. albicans biofilm formation. However, studies in C. albicans showed that optimum biofilm formation could be achieved with RPMI 1640 media (better than YNB). You can use the same for your experiments.
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There are two virulence factors of Pseudomonas aeruginosa that I'm currently studying, LasA protease and LasB elastase. From what I gathered, I know that LasB has a role in promoting biofilm formation through rhamnolipid-mediated regulation. Does anyone know if LasA also plays a crucial role in biofilm formation of P. aeruginosa, either directly or indirectly?
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Secretion of Proteases by Pseudomonas aeruginosa Biofilms Ex...
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The heterogeneity of biofilms increases tolerance to antimicrobials because of many barriers. In addition, the microorganisms can be resistant to the antimicrobials or they can acquire resistance determinants. I would like to know the role of biofilm formation in tolerance to antimicrobial agents, and methods that can be followed to determine it.
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Biofilms are somehow a protective exopolysaccharide barrier for microorganisms against chemical and physical alterations. They also could be a serious hygienic problem in many industries especially the food ones. Some methods are well described in the literature based on coloration and epifluorescence microscopy.
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I am currently working with biofilms to remove P and carry out nitrification within aquatic systems. We are currently trying to chose a biofilm media to support the growth of healthy biofilms, which is proving more difficult than we originally thought. 
Here is our criteria: 
- Must have very high SSA (specific surface area)
- Must be chemically inert (i.e., not alter pH or release any chemicals into the water).
- Must be relatively easy to get hold of in large volumes.
- Must be relatively cheap to purchase. 
We are ideally looking for porous beads, but are having difficulty finding ones that may be usable. 
Any tips/advice/help would be much appreciated.
Cheers,
Jack 
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to grow biofilm you can use trypticase soya broth
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hi everybody,
need a biofilm formation protocol for Candida Glabrata?
Thank you,
Ammar
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Ammar Abou Kandil U can follow the biofilm protocol from the following article.
1. Jain, N., Kohli, R., Cook, E., Gialanella, P., Chang, T., & Fries, B. C. (2007). Biofilm formation by and antifungal susceptibility of Candida isolates from urine. Appl. Environ. Microbiol., 73(6), 1697-1703.
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Has anybody grown algal biofilms from stream inocula on ceramic tiles and observed such bare areas as in the photos? They look like a virus infection to us. We have observed this phenomenon a few times already and it disturbs grazing experiments…  The first bare spots appear after some 7 days, grow up to 5-6 mm diameter, more appear, and after another two weeks (approx.) they slowly start to vanish again from their centre. Of course, we have also protist grazers (ciliates etc) in our biofilms but they are not responsible for these spots, we think.
Thanks for any helpful information on what that might be and how it could be prevented!
Susanne Worischka, Felix Grunicke and Thomas Petzoldt
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Hi,
I don't know if its the same, but we observed similar things in microbial mats (and also in sediments), and we also thought it was viruses, but it was fungi. Sorry to 'highlight' my own research, but here's the paper:
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Is it just so the results are from a single colony (reduce risk of mutant strains)?
Is it so bacteria are in the stationary phase?
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Dear Annie,
There is no requirement to produce inoculate for biofilm experiments by growing up an over-night culture using a rich medium such as LB. You could in fact directly resuspend colony material in 1ml of sterile water (or buffer, or medium) and take aliquots of this to inoculate your biofilm samples.
Sometimes it is important to be able to provide inocula at a certain cell density or in a specific growth phase, and it is very easy to do this in a general growth medium such as LB (assuming that your strain grows well in this). Sometimes it is also important to 'pre-condition' the cells by growing in a medium which initiates a particular biochemical or behavioural response which is required for the assay that will be undertaken, and sometimes it is necessary to make sure that the cells that are prepared are not starved for any particular nutrient ...
We often initiate our biofilm assays with over-night cultures grown in the nutrient-rich King's B media (similar to LB but better for Pseudomonads). An 18h incubation with shaking (inoculated with a wire lop from a plate with fresh colonies) will always result in a good cell density, so we don't have to bother with diluting or concentrating the cells before aliquoting and inoculating the assays. An 18 h culture is also at the start of stationary phase, so cells in the assay cultures won't take long to start replicating fast again. I don't like using 48 hr cultures or older ones (e.g. inoculated late on Friday for a Monday morning start), as in our system a variety of biofilm-relevant mutations can accumulate and spoil our experiments.
Sometimes, however, we want to assay growth or biofilm-formation in a more restrictive media in which carry-over from the over-night culture could be a problem. If we want to assess growth at lower nutrient levels, you might need to wash cells with water or buffer several times (and even allow them to stay in a no-nutrient buffer for a couple of hours) before use to make sure there is no carry-over of nutrients into the new media.
At the end of the day, there is no set way to prepare inocula for experiments. Your aim should be to produce a useable isogenic culture, free of contaminants and contaminating bacteria, and lacking in mutants which might cause a problem. However, if you wish to repeat a published experiment, you really do need to reproduce all of the steps that were used, as small variations in inocula density, cell state and nutrient carry-over, could be enough to cause different outcomes!
Regards, Andrew.
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Static biofilm models are used to assess biofilm formation in early stages. Why is this ??
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Ramachandra SS Static biofilm models are useful for examination of the early stages of biofilm formation because of following reasons:
1. They can detect biofilm formation in <60 min.
2. They have simple and easy protocols (Assays can be executed primarily using common laboratory equipment)
2. They allow analysis of biofilm formation with a variety of readouts, including microscopy of live cells, macroscopic visualization of stained bacteria, and viability counts.
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Dear all,
most of the research papers that I found about QS agents described their activity in the inhibition of biofilm formation. I found some manuscripts that demonstrated their potential at different biofilm stages (e.g. https://aac.asm.org/content/aac/59/4/2265.full.pdf ). But I still do not know/understand exactly how QS agents can disrupt or at least have impact against preformed biofilms.
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To my knowledge there are no such anti-QS agents that disperse the existing biofilm mass .
Best
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Can biofilms be seen by pathologists when they do lab studies? For example, if a human has a biofilm in their colon and does a colonoscopy with biopsies sent to the pathologist, could the doctor doing the scope and/or the pathologist reviewing the biopsy actually be able to identify the biofilm? Or is the biofilm too microscopic for even today's advanced imaging.
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not all bacterial may be see in slides , but in general we can see the bacterial colonies in infected tissue section, especially in severe bacterial infection
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I want to test if the expressed recombinant protein has an inhibitory effect on biofilm formation of Pseudomonas aureginosa
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You can use classical quantification techniques which includes Colony forming unit (CFU) counting and crystal violet staining. Many other modern methods like ATP bioluminescence, Quartz crystal microbalance, Characterization of biofilm morphology with use of scanning electron microscopy and spectroscopic methods, use of software like ImageJ etc for biofilm analysis have been used by many researchers.
You can refer to the following articles:
1. Wilson, C., Lukowicz, R., Merchant, S., Valquier-Flynn, H., Caballero, J., Sandoval, J., ... & Clement, B. (2017). Quantitative and qualitative assessment methods for biofilm growth: A mini-review. Research & reviews. Journal of engineering and technology, 6(4).
2. Kırmusaoğlu, S. (2019). The Methods for Detection of Biofilm and Screening Antibiofilm Activity of Agents. In Exopolysaccharides-Methods of Preparation and Application. IntechOpen.
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Hello, 
I am new to this field of microbiology. I need someone's help in helping me develop the crystal violet assay in my lab. I am trying to develop a biofilm layer in a drinking water system and I would like to measure the amount. I will use different types of bacteria like E.Coli and S. aureus.
What kind of equipment do I need? A plate reader spectrophotometer? Do I need to transfer the biofilm from the metal surface to a certain plate? Is the absorbance being measured in a liquid phase? How does the reader work?
Thanks 
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1- grow the bacteria 2- guarantee the pure culture 3- Next form the biofime, add 500 microLiters of bacteria into a 24 well microplate. 4- keep without agitation for 24 or 48 or 72 days until biofilm forms 5 - Then centrifuge with microplate rotor 6- discard the supernatant 7-Wash the plate carefully with buffer solution, and air dry 8-Biofilm cells adhered to the well were fixed by the addition of 1000 microliters methanol for 15 min (4 ° C) and then the methanol was removed and air dried at room temperature. 9-Subsequently, added 1000 microliters of 0.1% violet crystal for 15 min. After this time, the plates were washed and air dried again. 10- Then, 1000 microliters of ethanol (96%) were added to each well and allowed to act for 5 min. Finally, 200 microliters from each well will be transferred to a 96-well microplate. The fluorescence intensity of the violet crystal was measured by a microplate reader using a wavelength of 595nm. doubts, salomaobit@hotmail.com
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Hello all. Does anyone can help me to make paragraph about biofilm formation in p. aeruinosa at each stage.. I could not find a concise thing explaining all the proteins, enzymes and genes involved in the formation process at every stage .. I want to write two papers containing everything from sticking to dispersal . thanks
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I beleive this research paper already mentioned the process in brief, which you might consider with reading related papers with mentioned biomolecules responsible for the progression of each step.
All the best.
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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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I am currently performing biofilm studies using the Calgary device (96-well plate with peg lid) and a non-motile bacterial species (S. aureus) but am having difficulty forming biofilms on the pegs.
I'm considering adding a few parameters to try promote adherence and biofilm growth, but first wanted to know if it is possible considering the bacterial motility?
Or should I opt for growing the biofilms within the wells rather than on the pegs?
Thanks!
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It shall be worth to attempt the formation of biofilms on both peg and well-surfaces to find out the actual preference of the organism to create such niche. Streptococcus mutans does form biofilm/ plaque formation on teeth and is nonmotile. Adherence may be modulated/ promoted by pre-exposure of the surface with sterile solution of some sugars [dextrose, sucrose etc.], amino acids [Lysine] and ECM [Collagen, fibronectin, albumin etc.].
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We know that Fimbriae helps in cell attachment and biofilm formation, and that's how involved in pathogenicity but how fimbrial adhesins and non-fimbrial adhesins differ in pathogenic cell surface attachment? How Non-fimbrial adhesins cause pathogenicity or are virulence in nature?
Thank you
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Any specific easy method form biofilm formation on cell lines followed by anti-biofilm activity of any oil-based compund
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The video in the given link will be helpful:
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Microbial biofilm is the formation of sticky adhesion layer attached to the surfaces of animate or inanimate objects
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Establishing the relationship of Double J stent Catheters with the episodes of CAUTI and CRBSI and subsequently link its prolonged exposure leading to bacterial translocation as a risk factor for the development of the sepsis.
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In recent years, the most of researchers and interesting ones in the field of microbial biofilm documented that nano particles like silver, metal ions play a significant role in biofilm approach.
Here i would like to concentrate a light on the modern technique in biofilm and the role of nanotechnology in combating medical resistant microbial infection.
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Hi all -
I was wondering how one could 'easily' identify the receptor of a small molecule in bacteria. Background: we identified a small molecule that inhibits biofilm formation in Salmonella and we wonder, which protein(s) are potential receptors. I assume one can do a genetic screen but can we also do a biochemical approach e.g. can we chemically couple the compound to a resin and add whole cell proteins to resin to see which proteins bind and analyze them afterwards. I assume many people do this kind of research and I wonder what are the simplest approaches.
Thank you and have a nice day
Kornelius
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Hi
I performed 24 hr biofilm formation assay (a total of nine replicates for each bacterial strain). And from the OD570nm, I classify them according to their biofilm forming capacity by using the following guidelines.
OD ≤ ODc (non-biofilm producer), ODc < OD ≤ 2× ODc (weak biofilm producer), 2× ODc < OD ≤ 4× ODc (moderate biofilm producer) and 4× ODc< OD (strong biofilm producer).
However, when I performed statistical analysis one-way ANOVA, followed by post hoc bonferroni test, I found that the OD values of non, weak and moderate-formers have no significant difference. It seems weird.
Has anyone else have encountered this situation? from literature, it seems that bonferroni test are used mostly for multiple comparisons in biofilm assays, but is there a possibility that this test might not be so suitable?
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Dear Yugenraj,
We do a lot of biofilm characterisation studies using a combined biofilm assay in which we sequentially measure the strength of air-liquid (A-L) interface biofilms, attachment levels to the microcosm walls at the meniscus, and total growth or biomass in the microcosm, and use an ANOVA approach with post hoc Tukey-Kramer HSD or Dunn's method with a control to investigate differences between means (strains).
This approach, which sounds very similar to yours, generally works, but there are two significant assumptions we make. The first is simply a statistical requirement that the data (or more often, the residuals generated by the ANOVA) are Normally distributed, and we check this using a Shapiro-Wilk W test. If the data or residuals are not Normally distributed, it is possible sometimes to undertake an outlier analysis to identify which data are outliers and remove them one at a time until the new data set is Normal (or until you have removed so much data that you can no longer investigate differences between strains. If the latter happens, you could try to transform your data to make it more suitable (repeating the whole data-residual checking process), or abandon the parametric approach and choose an appropriate non-parametric test instead.
The second assumption is biological and is suggests that your experimental approach is biased towards expecting that there should be significant differences between your strains, or that you assume that you can grow cultures and undertake the assay so well that there is going to be no significant variation in your replicate measurements. We often find that our pseudomonad all produce pretty much the same strength biofilms which are often so weak that we are unsure if the maximum deformation mass assay (dropping small glass balls on to the biofilm surface until it breaks) is sensitive enough. If the data is Normal, then we can check this using a T-test and asking whether the mean is significantly different to zero. More often we see some variation in the attachment level data, but this can vary a lot better replicates as it is hard too was the microcosm vials out properly. Again, we can ask if a strain is producing a significant level of attachment by including blank microcosms and measuring the level of attachment for these (this is a Crystal violet assay). We can then undertake an ANOVA (assuming Normality) with a post hoc test of means using Dunn's method with a control, and this will indicate all strains which have attachment levels above the control (we have never seen a strain that has a level lower, and this makes no experimental sense). Finally, we can also look at growth levels, but here there is no 'lower limit' we might expect as all of our strains grow under the conditions we use. If we were concerned about that, I would measure growth levels by OD at the beginning of the assay (using replicate microcosms) and again at the end of the assay. These data could be compared a number of ways, for example by using a paired T-test, but I'd probably simply look at the difference in OD values and as whether the means differed significantly from zero using T-tests.
I suspect that you problem is that you are assuming that you can differentiate your strains in to high and low categories of biofilm-production, but this would require small variations in replicate measurements and high variation between strain means. If your data-residuals are Normal, you could look at this directly using a three-way ANOVA or GLM with replicate measurement, strain, biofilm category as effects. In order for this to work, you need sufficient replicates or there will be no statistical power available to differentiate between the effects. It may simply tell you that your strains are pretty much all the same.
You could then redo your catagorisation and try and split the strains into two groups, low and high biofilm producers. Use the mean of all data as your cut-off figure unless you know that the data-residuals in not Normal, in which case using the median would be a better approach. If you cannot differentiate your strains like this, it looks as if they are all too similar.
Regards, Andrew
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In micro-titer plate using crystal violet indicator for coloring ,and phosphate buffer for washing, and ethanol95% for solublization of biofilm, i can't get any biofilm or incearse in intensity of col, with low absorbance reading on spectrophotometer device.
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Pre-treat the wells with saliva
Pre-treat the wells with acetone (increases ridges = rough surface)
Decrease the amount of nutrients in the media (50% medium + 50% water or any ratio that you want to try)
I like the idea of adding sucrose to the media but still dilute it in water.
Careful when you wash the wells after the biofilms grew. I tilt my plates to an almost 90 degree angle and pipet directly to the side of the wells so the biofilms do not get disturbed by the turbulence of your pipeting.
Hope this helps
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When catheter is inserted in to the bladder, fibrinogen gets deposited on to catheter surface and that becomes favorable site for bacteria to grow.
I want to understand from where and why (due to which forces or mechanism or reason) this fibrinogen gets deposited on to catheter surface ?
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Mustapha Umar: logic answer
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Hi everyone,
I am going to analyse the possible bio-film formation of bacteria fungi exposed to plastic films for about 6 months. What is the procedure to determine bio-film quantity by crystal violet.
The protocol I read was as below:
Take out plastic films from media and rinse it with water to remove unattached microbial strains. Then immerse it in 2 % crystal violet for 15 minute following with rinsing with water and then ethanol or isopropyl.
My question is: after rinsing the plastics it in a clean plate with water and then immersing them in crystal violet, how should I quantify the biofilm? Should I discard the aqueous obtained from washing and immersing the plastic step or I should discard the plastic and focus on the obtained aqueous.
The protocol was not clear and there was not any other protocol for biofilm determination on plastic films, that is why I need your guidance and expertise.
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You may use UV-vis spectroscopy to quantify the amount of biofilm. I did some similar work that was published here:
Good luck!
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Hi, everyone! I got a question and I hope you can give me some help with it.
I'm a bachelors student of microbiology and I'm currently working with clinical samples of E.coli isolated from urinary tract infection. My particular objective is to test biofilm formation of this samples in a 96-well plate.
Could you please tell me if it's possible to know how many CFU/ml of E.coli are in a TSB broth cultured at 37°C overnight and measured in a spectrophotometer at 550nm?
It would help me a lot!
Daniel Huelgas
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I would expect a cell concentration of roughly 1x10E9 CFU/ml. As been said before, you can create a calibration curve between CFU after plate counting and the OD of e decadic dilution series or better with one or two steps within a magnitude. You can also sample and measure during the growth pahse. Advice for later: OD does not differentiater between living and dead cells but intact cells.
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Kindly assist me with a probable method that i can use to deduce biofilm formation in the general environment/ natural habitant- aquatic biofilms and also biofilms responsible for biological fouling.
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You may use crystal violet binding assay. It is the most commonly used method for the enumeration of adhered cells.
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I am working on biofilm formation of Cryptococcus sp.  And Candida sp, i need to know whether XTT assay gives the best result or I can use any  alternative protocol for biofilm activity to get good results.
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You can as well use crystal violet (CV) staining but that quantifies the biofilm mass rather than measure the biofilm's metabolic activity. Which makes more sense because the cells within each strain could metabolise XTT differently.
Good luck
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I'm working with Actinobacillus pleuropneumoniae, one that really knows how to form biofilm. My log growth curve is looking good the first hours but is then stagnating or even getting reduced some. after about 2 hour stagnation it is growing again. Can biofilm formation play a role in this? I don't really know if I can "trust" the growth and expect it to handle antibiotic in an optimal way.
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Do continual exposures of biofilm pathogens to a given disinfectant emerge their resistance to antibiotics? "
Hi this is my thesis question to develop. please can someone recommend, any useful journal. thank you
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I'm performing assays on a number of bacterial strains, some of which are capable of forming biofilms.
What effects would a "tissue culture treated" plate and a "non-tissue culture treated" plate have on bacteria suspensions??
Would a TC treated plate cause lower amounts of planktonic cells and/or promote biofilm formation as compared to non-TC treated plates?
Thanks!
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Hello Kevin. My team compared the biofilms on different brands of 96-wellplates (TC-treated, non-TC-treated), and found that some non-TC-treated plates (super cheap) had the lowest biofilm of the same bacteria. We then concluded that non-biofilm experiments can be used on certain non-TC-treated plates, while biofilm experiments should be used on TC-treated plates.
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I have been trying to obtain the biofilm of Staphylococcus aureus on glass coverslip to visualise it under the microscope, however no fruitful result has been so far obtained.
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Hello, see attached info includes all that you need!
Good luck!
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Biofilm Formation
Biofilm
Acinetobacter baumannii
Microbial Biofilms
Bacterial Biofilm
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If you are intrested in mature biofilm you can use the classical crystal violet assay, for example see Runchi et al., 2017
If you are interested in the early stages (attachment) see the recent paper by Nimrod Shteindel
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Biofilm formation assay does we have to do both concentration 1 mg/ml and 4.5 mg/ml and performed in triplicate? According to this articles
And let’s suppose I have 9 clinical samples, my total results should be 54 reads?
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Dear Alanood Alhjooj,
your question is not clear enough. do you want to let us know what you're referring to ?
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how to detect the biofilm formation in pseudomonas aeruginosa
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Dear Alanood Alhjooj , this article will help you
Evaluation of different detection methods
of biofilm formation in the clinical isolates
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Hi!
Does anybody know about other peg lids for biofilm assaying besides the Thermo Scientific Nunc-TSP peg lid?
I'm looking for peg lids or peg "strips", where a row of pegs can be snapped/cut off and used when not having a lot of samples to fill out a whole 96-well plate. - In order to economize the amount of pegs used per experiment.
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Batch culture of biofilm on peg lids is a versatile method that can be used for microtiter determinations of biofilm antimicrobial susceptibilty. work has been presented detailing a core protocol and set of parameters( surface composition, the of rocking or orbital motion, temperature, cultivation time . inoculum size, atmospheric gases and nutritional that can be adjusted to grow single, or multispecies bioflims on peg surfaces. For details consult Nat. Protoc. 2010, vol. 5( 7 ): 1236 - 54.
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I’m planning to treat a highly contaminated petroleum refinery wastewater of a local petroleum refinery. I want to work on a project focused on the accelerated enzymatic biodegradation and COD removal of petroleum hydrocarbons using active bacterial biomass capable of in-situ generating peroxidase and biosurfactants.
Any prospective leads and suggestions would be highly appreciated.
Thank you.
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Pneumatic bioreactors could be the configuration you are loking for. See, for example:
(i) Medina-Moreno S. A., Huerta-Ochoa S. and GUTIÉRREZ-ROJAS, M. 2005. Hydrocarbon biodegradation in oxygen limited sequential batch reactors by consortium from weathered contaminated soil. Canadian Journal of Microbiology. 51:231-239. DOI: 10.1139/W04-130 .
(2) Lizardi-Jiménez M. A., Saucedo-Castañeda G., Thalasso F. and GUTIÉRREZ-ROJAS, M. 2012. Simultaneous hexadecane and oxygen transfer rate on the production of an oil-degrading consortium in a three-phase airlift bioreactor. Chemical Engineering Journal. 187: 160– 165. DOI: 10.1016/j.cej.2012.01.114.
(iii) Sánchez-Vázquez, V.; Shirai, K.; González I, GUTIÉRREZ-ROJAS, M. 2018. Polycyclic aromatic hydrocarbon-emulsifier protein produced by Aspergillus brasiliensis (niger) in an airlift bioreactor following electrochemical pretreatment. Bioresource Technology. 256: 408-413; doi:10.1016/j.biortech.2018.02.043
Hope the information be helpful.
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I have isolated two gram negative bacterial strain and would like to estimate biofilm formation capacity. However, these strains are known for biofilm formation, therefore i would like to take one of positive control that can help us to determine the biofilm forming capacity.
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Both strains in this case will be positive controls. Only the medium without the presence of any bacteria can be taken as a negative control.
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I am doing my research project at university Tarbiat Modares , and currently designing a bioreactor for biodegradation of petroleum hydrocarbons, it is somewhat continuous bioreactor.
the aim of the present work is The peroxidase-mediated biodegradation of petroleum hydrocarbons in a H2O2-induced using in-situ production of peroxidase
but i need some help how to choose bioreactor
Any prospective leads and suggestions would be highly appreciated.
Thank you.
Saeed Molaei
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Dear Saeed,
Your question is hard to answer since there are many bioreactor configurations you might use for hydrocarbon biodegradation. From my point of view, it depends on the type of hydrocarbons and the environmental conditions required (e.g. aerobic, anaerobic, anoxic, etc.). If you are dealing with volatile hydrocarbons, the best configuration could be a biotrickling filter or biofilters. Alkanes, pneumatic systems such as bubble columns or airlifts could be a good option (not a good option when you also have volatile hydrocarbons due to stripping). Heavier fractions could be better treated in systems with mechanical agitation. This rises the question on which matrix do you have your hydrocarbons: soil, water, air?? All these characteristics will move the balance towards different reactor configurations. Some literature that could help you:
Alkanes
Volatile hydrocarbons
All
The inoculum in all cases could be activated sludge form a wastewater treatment plant, or soil from a contaminated site.
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I need to work with listeria biofilm and inhibition with plant extracts, but I need a protocol to evaluate the formation and inibition.
Appreciate your cooperation
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The biofilm of Listeria is a big problem especially in food industries and it persists in many unwanted places. It is very difficult. Anyway for your information see the attached article.
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I would like to measure the biofilm formation by clinical isolates of S. aureus in tryptic soy broth (TSB) supplemented with glucose or NaCl. Could someone elaborate the protocol plese, specially estimation of OD600 corresponds to which CFU/mL for initial step of assay and crystal violet staining procdedure? Many thanks
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Dear Harshad, in our experiments we adjust bacterial suspension to McFarland 0.5 in 0.9% NaCl then we add 180 ul TSB+glucose or sucrose or NaCl and 20 ul bacterial suspension (McFarland 0.5) into wells of 96-well F-bottom microplates. Incubate 37 C for o/n than wash microplates 3 times with 0.9% NaCl, fix with methanol and after drying stain with Crystal violet then dissolve dye using Acetic acid solution. The following paper could be usefull for full procedure.
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Hi,
Does anyone have any experiences with bioadhesive and biocompatible materials (except dopa/dopamine), which can be used for surface coating and improving biofilm growth on them??
Cheers
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this link is useful
also there is a doctoral thesis on this subject under the supervision of Dr Taha AL-Taha
regards
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I want to work on assessing virulence of pseudomonas .biofilm formation is one of them . want to various standard technique for assessing biofilm forming capacity of pseudomonas.
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The crystal violet 96-well plate assay is the easiest. However, this can be difficult when working with Pseudomonas as it forms a thick slime which can effectively pull the biofilm off the well when aspirating steps are performed. What I have found is if you aspirate using a pipette and are very careful you can remove the thicken media and leave the adhered biofilm on the well plate edges and bottom.
Alternatively, one can grow biofilm on a plastic or glass slide which is placed into a liquid culture of the bacteria (use whichever material you find is best for the bacteria to form biofilms on). Use CV to quantify or other methods such as live dead staining (microscopy can be performed easily with this method). Another advantage to this is the ability to change media and grow biofilms over several cycles of fresh media making the biofilm thicker each cycle. The downside is it is much less high throughput than the 96-well plate assay.
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I was not able to find any studies focused on oxygen mass transfer as the result of biofilm formation in the biodiesel storage tank (in which biodiesl is mixed with some amount of water). Your suggestions are appreciate.
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You will consider of condition in systems for exact results
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I want to know if there is a time or duration that is expected for AOC to change and cause bio-film development on membranes. This question is also applicable to pipelines for water distribution. Although the conditions for these two scenarios may differ due to several factors like material properties (membrane and pipes) and surrounding chemistry
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AOC means Assimilable Organic Carbon
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Microorganisms that grow on medical devices form biofilms. Normally, biofilms protect the cells from the animicrobial activity of chemical biocides (including surfactants) and antibiotics. The question is how to destroy or prevent biofilm formation on medical devices?
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Following Article helpful to you. It includes all of novel methods to remove of Biofilms in anywhere. Please read it. You will able to remove biofilm from your device.
Thank you.