Bacterial Biofilm - Science topic

Fibra cel discs are carriers used for adherant mammalian and insect cells during fermentations. Can someone give me an example or refer publications where the same material have been used for e coli or bacterial biofilms instead ? Also, any suggestions on how this system can be applied in fermentor (glass) would be appreciated.I am attaching the link for referring the material i am stating about:

For qualitative analysis of gram-negative bacterial biofilm. What is the ideal stain that we can go with? I have tried CV, it gives good images but not good compared to gram-positive ones. It gets washed during washing steps. On searching I got Safranin and other alternatives. If anyone has used this. Please share your experience

Storage and maintenance of pathogens is a costly and time-consuming affair, the recent study indicated that most of the pathogenic bacteria can be stored for several months at room temperature in sterile tap water without any hustle.

Ref DOI: 10.13140/RG.2.2.34672.84480

have any of you come across articles on the protection of carbon steels by bacterial biofilms? can anyone help me please?

I am interested to know if there is a difference in the bacterial biofilm compositions between:

1. cool and warmer seas

2. on different surfaces

Is anyone here investigating this and would like to collaborate?

N-glycanase

We are working on Biofilm and Quorum sensing genes of E.coli, we suggest some of reasons maybe:- Contamination, Annealing Temperature, Melting Temperature, DNA Extraction mistakes in the Techniques, we identified them by biochemical tests to be sure the isolates are E.coli, but now we are collecting samples from the beginning and doing the procedure again

I have observed opposite response from gram negative and gram positive bacterial cells on plasma modified polymeric surface in terms of adhesion and biofilm formation.

gram negative cells show above 90% reduction in adhesion and gram positive show only 9% reduction. What could be the reason behind this?

Which property of bacterial cell type might be playing a role?

Please explain.

Thanks in advance.

I have previously used LB broth medium in biofilm inhibition experiments against P. aeruginosa. However, I could not detect an effective biofilm formation in the negative control wells. Which medium would you recommend for sufficient biofilm formation in P. aeruginosa?

Thank you.

Hello,

Hope everyone is doing great. I have synthesized an antimicrobial peptide gels. Now I need to perform antibiofilm assay to check its biofilm inhibition potential.

There is this paper I am following. They grew biofilms on silicon wafers modified with gel and then incubation. I don't have silicon wafers.

Could someone please tell me that is there any alternative? Can I grow biofilms in 12-well plate already modified with gels?

Secondly, why they grew biofilms on silicon wafer? Is there any technical aspect related to SEM analysis afterward?

Regards,

Zeeshan

We are doing a research on Biofilm formation of Bacteria, for knowing each isolate strong, moderate or weak, by calculations tables of Standard deviation, Variance and Cutoff (Ct) etc… and with the help of Microsoft Excel but the results in the program differ from the hand written and don’t know the best way to calculate and compare the results, any help will be very appreciated, Thank you

Ali

I am working with e.coli. I need help with interpretation of the results. I have worked with two E.coli strains. I did triplet technical replicates. I need to do statistical T-test.

Below is some of my datta:

Strain 1 0,407 0,544 0,639 Ave.: 0,53

Strain 2 0,384 0,515 0,34 Ave.: 0,413

Negativ control: 0,161 0,181 0,14 Ave.: 0,160667

Can someone help me with this. Please. There are several methods that are available to measure bacterial biofilm adherence. I was told to do a t-test beetween my average negatic control and a strain of E.coli.

PLEASE HELP

Thank you

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.

Does the sample should be solid for SEM and how to prepare it?

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 ?

Hello everyone,

I am working on bacterial biofilming activity and was observing the same on glass surface. I have the z-stacks but i am unable to directly calculate the thickness of biofilms under varying growth conditions. Please anyone guide. Dye used was acridine orange.

I know the technique which is sub-culturing the bacterial treated with sub-MIC for the specific days like 30 days. I am writing if anyone knows another technique to investigate this?

We are studying biofilm formation on differently coated 15mm diameter uPVC discs - these are opaque and may be white or black in colour depending on coating. The current setup includes forming biofilms on discs, rinsing to remove non-adherent cells, fixing each disc to a glass slide and then adding a drop of LIVE/DEAD BacLight mixture (Invitrogen L7012) to the centre of the disc. After incubation, the excess stain is rinsed off, a coverslip is placed on top of the disc and the edges of the coverslip fixed to the underlying slide with nail polish. After drying, the whole piece is placed in the inverted microscope coverslip side down. When imaging it is possible to see a hue of green or red depending on filter but not to resolve cells, and it is unclear whether this is just autofluorescence of the disc or coating. The opacity of the discs seems to block much of the light.

Thank you for your assistance and advice.

My bacteria sample is Acinetobacter baumannii. I found one protocol just centrifuge and using congo red and check OD 490 nm supernatant. I need more better protocol for extraction EPS with reference.

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.

Dear Researchers:

Greetings! I am interested in learning electron microscopy image analysis of bacterial biofilms, mainly using open-source software. I have been using basic functions and tool available in ImageJ/Fiji. Could please suggest other software and learning tools that are acceptable in peer-reviewed publications? Thank you.

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?

Hey,

I want to check my phages against the bacterial biofilm. I have read papers in which they have added phages in a specific MOI (0.6, 1 etc) either in pretreatment or post treatment of phage infection.

I have a question if cfu/ml of bacteria is1×10^12, it means 1ml of bacterial culture contains 10^12 viable cells. MOI will be calculated accordingly. I don't know the no. of bacterial cells in 100 or 150ul.

How much volume of bacterial culture do you pipette in 96 well microtiter plate? I'm a bit confused as 200ul is the total volume you can pour in 1 well of 96 microtiter plate. How to maintain a specific MOI in each well of microtiter plate?

How can i get nonsporulating bacterial bacillus subtillis strain Δ spoIIE without antibiotic growth conditions ?

This ΔspoIIE::erm strain is showing growth conditions with antibiotic erythromycin , but I need wildtype Δ spoIIE without antibiotic growth conditions. Can I order only Δ spoIIE strains if yes then please suggest.

I have recently carried out the Minimum biofilm inhibition concentration (MBIC) on my compound and the value for MBIC is higher than the MIC values for two compounds and MBIC value lower than MIC for another two compounds tested. Is it normal? or should the MBIC value be similar as MIC value?

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 ?

I know it is more or less a philosophical question, however quite intriquing. Bacterial cells on agar plate adhere to surface and majority of cells is covered by slime layers. Thus it matches the biofilm definition, however I haven't heard anyone who would define "agar" colonies such way.

As part of a research project i have obtained plant root samples that are currently frozen. I need to isolate bacterial DNA from the plant root, however i first need to remove the possible bacterial biofilm from the plant root as the bacterial DNA is of interest, not plant root DNA.

Any advice would be greatly appreciated

Using LB broth, overnight, shaking, Polystyrene 96 wells.

I would like to use E.coli ATCC 25922 as a positive control for a CV assay, but I have not had much luck reproducing published example values. I bought a lyophilized E.coli. I am using LB broth, overnight/24 hr, 1:100 dilution of overnight preparation, shaking/not shaking, and measuring at 570 nm. My values are 0.1 to 0.2, but published papers are getting >1 using similar conditions that I am using. I have better results with PVC plates, but that shouldn't make a difference. Thanks

Why is there a greenish metallic sheen to E coli colonies in EMB agar?

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.

I am trying to quantify the biofilm formed by a Pseudomonas species grown on a cellulose membrane. What is the best solution to use to solubilize the dye? SDS or acetic acid or ethanol? Also, how can I transfer the solubilized dye from the membrane to a 96-well microtiter plate to measure the OD?

I'm working on different paan leaf which is known as Piper betle. Different types/ varieties of this leaf found in my country. Therefore, I need to know if the scientific name remain same for all types of Piper betle found ot its change due to geographical and phenotypic variation.

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.

Viruses can't replicate without host, and the so-called in-vitro virus culture actually cultures virus on bacterial biofilm or embryos. would viruses not replicate even if they are presented in a medium with enough ribosome, enzyme, DNA, RNA etc, without any pre-existing "life"? I am asking this as a materials science student, about viability of virus replication in presence of suitable biomaterials,biomolecules and nutrients , without life. How virus detects whether the biochemicals they are using is from "alive" or "dead" host? can that pathway be genetically tweaked?

In some articles, the authors reported that, MIC of antimicrobial agent have an ability to disrupt the bacterial biofilms. How it will happen? In general, biofilms are group of bacteria more strong than single cells. Then how this MIC of antimicrobial agent will work against bacterial biofilms?

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!

Hello everybody, we are working on the eradication of Helicobacter pylori and inhibition of its biofilm. We are looking for the suggestions that what compounds would be better to use against H. pylori to inhibit the quorum sensing.

Any specific easy method form biofilm formation on cell lines followed by anti-biofilm activity of any oil-based compund

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.

I want 1000 bacteria (numbers) to begin my experiments .How will i get these numbers? Any specific methods? please give suggestions.

My team is currently studying bacterial biofilms, but have had trouble getting our strain of E. coli to form strong biofilms. We're currently using E. coli 25922 (I believe) growing in M9 media. We're using 24 and 96 well flat bottom plates. Plates are tissue culture treated non-pyrogenic polystyrene.

After some deliberation with my team and research professor we've decided to try a new strain of E. coli for our experiment. I was hoping to get some input about what strains are superior biofilm formers?

I would like to analysis the S. aureus biofilms to know the effect of certain quorum sensing inhibitor on ultra structure. Could someone elaborate the method to prepare sample for TEM analysis (Fixation, dehydration, drying, staining, embedding, ultra sections etc)? Many thanks for your valuable time and help

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 ?

I am trying to culture P. aeruginosa (PAO1) biofilms in 96-well plates. I have tried pipetting off the media, wicking it up with blotting paper or tapping it out. All of these methods results in severe disruption of the mucoid growth.

Can anyone advise on a successful method for removing the media, leaving just the biofilm?

Many thanks

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.

For those connected with the ICU settings : Do you routinely send tips of removed central lines for culture?

Is identification of the biofilm from these lines helping?

There are many MSSA Biofilm producing strains such as Rosenbach and ATCC 35556 strains. But I am interested to work on strong Biofilm producing MRSA lab strains. Please share the names of these strains. Thank you.

I read this paper¹ by which states specific growth rate of bacteria in biofilm is different than that of planktonic cells. How do I modify the typical BioTimer assay² to compile with it?

1- A Method for Quantitative Determination of Biofilm Viability. Ken Welch , Yanling Cai and Maria Strømme (2012)

2- BioTimer Assay, a new method for counting Staphylococcus spp. in biofilm without sample manipulation applied to evaluate antibiotic susceptibility of biofilm. Fabrizio Pantanella a, Piera Valenti a, Alessandra Frioni a, Tiziana Natalizi a, Luana Coltella b, Francesca Berlutt (2008)

I read this paper on determining the viability of cells by BioTimer assay¹ and improvement of the same by another paper ², but I am still not very clear upon the methodology. Could someone please elaborate on the same? A noble method also works. I need to determine the number of viable bacteria in the biofilm sample.

1- BioTimer Assay, a new method for counting Staphylococcus spp. in biofilm without sample manipulation applied to evaluate antibiotic susceptibility of biofilm. Fabrizio Pantanella a, Piera Valenti a, Alessandra Frioni a, Tiziana Natalizi a, Luana Coltella b, Francesca Berlutt (2008)

2- A Method for Quantitative Determination of Biofilm Viability. Ken Welch , Yanling Cai and Maria Strømme (2012)

Is it because of convience or there is some other reason?

My bacterial biofilm tends to aggregate and agglutinate and I want to separate them at physiological temperature (25 - 30 C) in liquid. Any good suggestions of enzymes and preferably with protocols? Thank you all in advance.

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!

Does anyone have any experience of imaging bacteria and/or bacterial biofilms using SICM? One cannot find much literature about it but it seems as a good alternative to AFM which, in my experience, does not really work with softer samples.

Any input would be highly appreciated!

Thank you so much!

protocol for biofilm sample preparation for SEM analysis

Biofilm Formation

Biofilm

Acinetobacter baumannii

Microbial Biofilms

Bacterial Biofilm

I'm looking for a method of coating Biofilm Peg lids (not Innovotech) with Hydroxyapatite. 

I have the HA solution made which works fine to coat a 96 well base plate, however the evaporation/precipitation process does not work to evenly dry down and coat a peg lid (the precipitate sinks to the bottom).

I have tried soaking the pegs in a plate then inverting and drying in an oven at 65.c (any higher melts the peg lid) for 15 min intervals.

Alternatively I'm looking for any basic/simple methods of looking at S. mutans biofilms in vitro in an oral/dental context.

Thanks in advance for your help!

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.

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

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

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

Hello, I work inducing aggregation of E. coli with polymers, I need to asses the metabolic activity and survivability of my bacteria under different polymers.

How I am doing is to transfer the bacteria at a OD600=1(in LB, and some washed twice from the media) to 96 well plates, inoculate it with my polymers, then after that (without cooling or anything else) pipette the FDA. After that I analyze the fluorescence at ex:490nm and em:514nm.

Problem is that the fluorescence I am getting is really low, most of my cells should be metabolically active (at least the ones in LB), so I am doing something wrong.

All I am finding is related to bacterial biofilms. I want some way to create it from plant origin.

Crystal Violet is commonly used in the microtitre plate assay to assess biofilm formation. Can any other dye be used instead?

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

I am interested in the synergistic effect of the electric current and certain antimicrobials in treatment of bacterial biofilm. Looking in the literature I could not find a real explanation of the bioelectric effect, only postulates and assumptions.

I would highly appreciate it if anyone has any insight into this field!

thanks!

I wonder what is the solubility of any EPS? Does it contributes to alkalinity/acidity of a medium? What is the pKa/pKb? Does it really depends on the functional groups of the EPS?

How can I assess the effect of vapour-phase essential oil on bacterial biofilm?

We want to confirm presence of bacterial biofilm in-vivo. I have two questions regarding this:

1. What staining technique would be the best?

2. What is the best way to fix the samples in order not to disturb the biofilm itself?

And maybe is there any other technique that can be used to check for biofilm presence in-vivo?

How many mg of CHO present in 1mg of lyophilized biofilm polysaccharide? I know it varies with bacteria but any one know range?

we tried to get the surface profile of the untreated sand sample and bacteria treated sand with biopolymers on the surface of the sample in wet condition. we used 3D OSP for this purpose. we got the surface profile of untreated sand sample but for biopolymer coated sand in wet condition does not give any results. The instrument does not sense the sample. we tried 5 times but no result. what will be the problem? how to get surface profile/ surface rough ness of the wet biopolymer coated sand surface?

Note: 3D OSP instrument is working fine.confirmed.

Hello,

I'm fixating bacterial biofilms on plastic surfaces for SEM imaging and I don't have the facilities for critical point drying, nor access to Hexamethyldisilizane (HMDS) as a drying agent.

So I wonder, what are the properties that a chemical drying agent should bring for preserving bacterial morphology? Is there any "quick and dirty" method I could use instead?

Best,

Joana

Hello,

I have some problems with scum on the top of anaerobic digester. Does anyone know what this could be? In the biological sludge there are bacteria Nocardia A.

Many thanks.

Mr. Francesco Baldini

Along with which virulence factors produced during that biofilm transition stage from the planktonic cells. Its good that it was found that cells experience shear stress and flow for formation of biofilms. 

The smooth pattern or surface of the inner lumen helps microorganisms to attach and form biofilm within those regions. A change in the smoothness can decrease the attachment of microorganisms and in turn decrease the ability to form biofilm.

To test the antibiotic effect on anaerobic oral bacteria in biofilm setting, which methods are most acceptable and recommended?

Hello everyone, I am having difficulty growing Listeria monocytogenes biofilms using the Calgary Biofilm Device (CBD) for the purpose of completing a MBEC assay.  It seems like the biofilms are not adhering to the pegs, as tested by a CV staining assay. I have tried using different broths (brain heart infused (BHI) broth, BHI broth with 0.1% glucose, BHI broth with 1% glucose, BHI broth with 2% glucose, Tryptic soy broth (TSB) and TSB with 0.6% yeast extract), different incubation temperatures (37 degrees celsius and 30 degrees celsius), different rotation speeds (110 rpm, 200 rpm and stationary) as well as different incubation times (24hrs, 48 hrs, & 72hrs (changing media every 24hrs)). Iv'e tried all of these different methods with no success. Does anyone have any suggestions or different methodology? Thank you! 

According to my observation P. aeruginosa dominate S. aureus when they grow in together. 

I would like to do a test with the most similar washing machine conditions in order to evaluate a surface developed to prevent biofilm growth.

i would like to do in a short time if it  was possible.

I did several lab test and the results were really good, but no I have to do the most real test. 

Hi dear scientific community,

please suggest me,

How to prepare samples for the FESEM study of Phage- biofilm interactions. please provide me the procedure...

Thank you in advance...

I want to identify, separate and quantify S. aureus and P. aeruginosa from dual-species cultures both from planktonic and biofilm. Could there be any other method to do so without using flow cytometry or fluorescence proteins?

Thank you in advance,

Legesse

Is it possible to identify or differntiate bacterial biofilm and algal biofilm by naked eyes?

lyophilized extract of E. coli, E. cloacae, E. faecalis and K. pneumoniae biofilm to find some sugar component