Biofilms - Science topic

We are creating a polymicrobial biofilm from actinomyces naeslundii and fusobacterium nucleatum.

We established our growth curve for both strains. From that, we concluded that mid exponential phase of actinomyces can be reached after 5 hours, corresponding to OD600 of .7 , and after 22 hours for fuso, corresponding to OD600 of .4.

Now to create our biofilm in a standardized technique and instead of growing the bacteria for 5 hours and 22 hours to reach the target OD, How can we reach the targeted OD for both strains by diluting an overnight culture?

Hi

I am new to 3D culture, live cell imaging and confocal microscopy. Is it possible to visualize epithelial and endothelial cells by live cell imaging for a period of time using confocal? In addition, I would like to visualize the bacterial cells and the biofilm produced on the 3D culture over a course of time?

I do have the information on the antibodies to be used for labelling to visualize epithelial & endothelial cells, and biofilm. However, the protocol needs the cells to be fixed. Also, I could get information on the nucleic acid stains which can be used on dead cells to visualize bacteria and eukaryotic cells.

Any thoughts on this?

Thanks in advance

Warm regards

Bindu

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.

In the biofilm test assay, the methods states to use polystyrene Microplates. But does not specify which kind rather non-binding, low-binding, medium-binding, or high-binding.

Which is the best microplate 96-well for biofilm growth? To have the most accurate test without false positive or false negative results

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.

biofilm and quorum sensing genes of E.coli, not used drug but chemical material (Thiophene)

Dear researchers

I have some doubts about biofilm roughness

Biofilm roughness provides a measure of how much the thickness of the biofilm varies, and is an indicator of biofilm heterogeneity.

so does increased biofilm roughness means the biofilm is patchy in some areas?

also what is the unit of measuring biofilm roughness?

Comstat does not provide an exact unit of measurement for biofilm roughness

BiofilmQ provides mentions the unit as (a.u.). So what is a.u. ??

plz guide me for mix strain biofilm

As i read many paper of biofilm all where mention that by taking optical density we can say that either week biofilm or strong biofilm but if we see the film on the microtiter plate how we can take the optical density? kindly tell me the stage where i can take optical density.

We measured chlorophyll, dry mass and ash-free dry mass and after we calculated autotrophic index (AI). I found in Hauer, F. R., & G. A. Lamberti, 2017. Methods in stream ecology. Elsevier, San Diego, California: “Hotchkiss and Hall (2015), for example, estimated that only 7-20% of the biofilm biomass they studied was metabolically active.”

I am working on pseudomonas aeruginosa. My bacteria is susceptible to antibiotics, However will it be able to form biofilms?

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

According to enzymatic method for extracting extracellular DNA in biofilm matrix, is suggested to use CTAB solution. Why? I only need extracellular DNA...

Do you have a similar protocol to extract eDNA?

I am working with Salmonella Typhimurium and performing biofilm formation experiment. Referred some articles where destaining solution were used as follows: 1) Ethanol:acetone (80:20) 2) 30% Acetic acid 3) 70% Ethanol 4) Methanol. Do comment if you used anything different. Thanking you in advance.

I am developing a project with bacteria isolated from the marine environment, in the reactivation process in TSB agar medium they produced a lot of biofilm, and when developing the growth curve in liquid medium with TSB broth they also produced biofilms suspended on the surface of the culture, these are interfering In the absorbance readings for the standardization of the culture and subsequent evaluation of the growth curve, no matter how much I shake it, lumps remain in the culture medium.

hydroxyapatite is poin Iwder form. i want to use it as a carrier. it works as a supplement but confused that biofilm can occur on it or not.

Hello, could you suggest experiments and bioinfo tools to analyze the binding/interaction of peptides with bacterial surface & EPS (planktonic cells and biofilms).

Thanks!

I would like some information about the biofilms that are produced by Pseudomonas aeruginosa. Can the biofilms form on top of the liquid medium or do they bind to the bottom of the well. What are the best conditions to grow P. aeruginosa biofilms? In what medium and can they form in a anaerobic situation?

Thank you in advance

Steps and chemical to make congo red agar

biofilm:it's made up of bacteria,fungi,periphytes.

What percentage of microbial biofilm on a MBBR Chip is required for a industrial wastewater treatment plant?

Biofilm

I don't have a microplate reader in my lab now and using a cuvette-type UV-Vis spectro is kinda "wasteful" in terms of how much of the treatment solutions I'll be using. So I thought of using a nanodrop for biofilm quantification.

Of course I'll be looking at the differences in terms of absorbance readings of Crystal Violet-stained biofilms using nanodrop and cuvette-type UV-Vis spectro firsthand. But I'd like to know if anybody has tried it before? How was your results? Are there any significant differences?

Thank you!

Hello everyone,

I am developing biofilms on a polymer surfaces(films). Then, I have to disturb the biofilm with Vortexing-sonicating-vortexing (V-S-V) method. I am using a bath sonicator. In the same time, I put my substrate (film) plus the biofilm in PBS buffer and sonicate them. Then, I imaged the film (substrate) with scanning electron microscope (SEM) to make sur I have extracted the whole biofilm.

Note: The salts and the surface look deceiving with SEM. In the meantime, the biofilm may not get de-attached but instead of that it got damaged due to sonication or dehydration process. I put some images to see the structures that I am doubting

Do you think these structures are salts, the surface itself, or damaged E. coli biofilm due sonication and dehydration?

Dear investigators,

In my study, i am trying to study the expression of a set of genes of MRSA and Staphylococcus aureus biofilms.

To isolate the total RNA, can i lysate the biofilm cells and later isolate RNA, or shall i use the planktonic 24 h cells?

Thanks a lot

Hello researchers, I am a bio technologist wants to work on the use of COMSOL for biofilm formation. Please help me regarding this

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 working with Streptococcus mutans biofilms, and I am interested in quantifying the polysaccharides extracted from them. However, the papers on the subject that I've been finding do not say how they did the quantitation and instead reference a 1956 paper by Dubois et al, which makes use of dangerous chemicals (phenol and sulfuric acid) in large volumes. I'm sure in 66 years some optimization and downscaling of this procedure must have happened, but I am struggling to find an updated procedure. Could someone share one with me?

Thanks!

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.

This is my first time doing experiments with bacteria. I use probiotic to alleviate disease state. I use live bacteria for oral gavage.

I am comaring probiotic and probiotic with biofilm. For probiotic i can culture them in large scale and freeze dry untill use.

For the bacteria with biofilm I have no idea if freeze dry will be okay or it will destroy the biofilm.

If anyone has experience or can recommend any paper that will be helpful.

I will use lactic acid bacteria if that is important.

Hello Everyone!

I am making biofilms with E. coli MG1655 with Minminaml medium (M9). Then, I am willing to perform Colony-forming unit assay (CFU). I could not decide which type of medium that I will be using to do the dilutions.

I am wondering, do I have to dilute in M9 medium, then plate on agar plates?

From what I understand cells will take a lot of time to adapt themselves in the M9 medium. So, while I am doing several dillutions (ex. 10-fold dilution) and plating, that's will not allow cells enough time to adapt. So, I will be ending having nothing grow on the agar plates.

or

Can I Dilute the cells that were grown in M9 medium in LB medium, then plate on agar plates for CFU assay?

If I want to do method for detecting quarom sensing in bacteria, which physiological phenomena is the best? Biofilm, antibiotic resistant, or what?

I want to know if we can load herbal aqueous solutions in nanoparticles or not

I understand there will be some persister cells in the MRSA biofilm that are in dormant phase. I wound like to know if this kind of cells also exist in planktonic MRSA?

Hello everyone,

I'm starting to work with biofilm from an anaerobic bacteria and I'm having difficulties with the confocal microscopy. I used LIVE/DEAD™ BacLight™ Bacterial Viability, for microscopy and FilmTracer™ SYPRO™ Ruby Biofilm Matrix Stain. When I merged the images, I couldn't see anything with separate coloring, as it is in the picture. Everything overlaps so it looks like I used only one type of dye. The biofilm assay lasted 72 hours and I find it almost unlikely that in that time there was no bacterial death, so i should be able to see some cells died in red right? In addition, our findings show that this bacterial species almost does not form biofilm, but we come across this structure in the photo that appears to be some kind of cell to cell adhesion. Has anyone seen something similar? This bacterium has a zwitterionic polysaccharide capsule, so we think it might have something to do with this structure.

Thank you for your help!!

Hi, researchers

I am looking for growing the dual-species biofilm, including proteus mirabilis and other UTIs causing pathogens and I will treat them with the proteus phages. Does anyone know any protocol or method to grow this dual-species biofilm where I can characterize this dual-species biofilm and monitor the proteus phage treatment to this dual-species biofilm? if anyone can help me it will be much appriciated.

thanks

Akash

Mono- species biofilm such as E.faecalis

Dual - species biofilm such as E.faecalis & S.mutans

What is better for intracanal medicamments antibacterial experiment?

Dear researchers,

My current research focuses on the bacterial interactions in activated sludge of wastewater treatment system. I write to consult a few questions about my confusions in investigating bacterial interactions of complex biofilm communities. To estimate microbial interactions, it is important to move beyond macroscale analysis and focus on micron-scale heterogeneity and spatial associations with enough throughput and statistical associations. Now I’m looking for approaches to sampling and sequencing micron-scale biofilm samples, but there were still some questions puzzling me. The questions are as follows:

(1) Many studies have found that fine-scale heterogeneity in microbial communities is a common characteristic of biofilm samples. Our data also confirmed that the bacterial population in activated sludge had significant spatial heterogeneity at the micron scale. I believe that the micron-scale heterogeneity is an inherent property of the biofilm community that has nothing to do with measurement. But I'm not sure if that's correct. For example, I pulverized the biofilm samples and used flow cytometry sorting to produce 1000 single clusters (80 μm), after which I sequenced each single cluster and calculated micron-scale heterogeneity. Will these procedures (e.g., pulverizing) result in any additional heterogeneity? or Does this approach capture true micron-scale heterogeneity?

(2) When sampling at centimeter to micrometer scales (e.g., 1cm, 0.1cm, 0.01cm, 1mm, 0.1mm, 10 μm), we may see various spatial heterogeneity of biofilm communities. Are there criteria for selecting the optimal length scale at micron-scale to estimate the spatial heterogeneity of biofilm communities? How can we select the optimal length scale for studying spatial heterogeneity and interspecies interactions in complex biofilm communities?

Would it be possible for you to explain these questions to me?

Regards,

Thanks in advance.

some studies said its start to develope after 48 hrs and others said its better to investigated when its mature after 21 days.

for the research purpose which is better time for more strong and trustful study protocol?

I wound like to evaluate the biomass of biofilm by using crystal violet. The bacteria was seeded on a porcine skin and cultured for 48 h to form biofilm. I than used crystal violet to stain the biofilm. However, I found the skin tissue it self will also be stained and not able to be washed by water. So I wound like to know if crystal violet can be used for biomass evaluation for biofilm cultured on biological tissues. I only see people evaluate biofilm biomass on 96 -well plate by using crystal violet.

There are so many antibacterial NPs available ranging from organic , inorganic to carbon and MXenes. I am planning to do an anti-biofilm study against oral polymicrobial biofilms. I am confused which nanoparticles should I choose from the available options.

I wound like to evaluate the biomass of MRSA biofilm cultured on ex vivo porcine skin tissue. I found CV stain is not suitable. Is there any other methods I can use?

The literature I've researched says that biofilms are attached to the bottom of the petri dish, so why is the biofilm of my Porphyromonas gingivalis culture floating on the surface of the medium? Is there something wrong with the type of medium I am using?

Hello,

I am doing LIVE/DEAD staining of biofilms on different surfaces and taking z stack images by CSLM ( Olympus Fluoview FV1000).

Then I am using COMSTAT to estimate biomass thickness roughness coefficient etc.

I am following the procedure detailed on COMSTAT 2.1 manual for making the stacks and image conversion and so, but I am getting different values for these parameters depending on the channel (red of green), and the two value are sometimes very different.

Is this usual or I am doing something wrong?

Many thanks

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.

The fractal dimension parameter calculates a value that varies between 1 and 2 and describes the roughness of the biofilm boundary between foreground and background pixel in a cross-section at height z. Higher values of the fractal dimension parameter indicate a rougher biofilm boundary. Please help me in calculating this fractal dimension.

Hi,

I've conducted crystal violet biofilm assay, testing iodine and chlorhexidine based disinfectants against E. coli (NCTC 11560) and S. aureus (ATCC 29213). With the iodine disinfectants, I got an expected pattern, where less biofilm is formed at the highest concentration of disinfectant and more is formed at the lowest concentration. However, in the case of chlorhexidine containing disinfectants, the pattern appears to be in reverse, with relatively strong biofilm formation at the highest concentration of the disinfectant, and the lowest growth at the lowest concentration.

The picture attached shows a two-fold dilutions of two iodine disinfectants (columns labelled GG and LD) and two chlorhexidine disinfectants (columns CD and SC) tested against E. coli biofilm. Has anyone witnessed similar patterns before or can suggest any literature explaining it?

Kind regards,

Kamilla

The obvious answer seems to be YES! But a reviewer asked me to find a reference. Anybody knows of any work that compared the EPS of biofilms and colonies?

Or maybe I'm missing something and EPS is a biofilm-only feature that doesn't exist in regular colonies?

If it helps, we're talking about Enterobacteriaceae species like E. coli, K. pneumoniae and P. mirabilis

I want to do 16s rRNA identification of 3 bacteria that are present in a multispecies biofilm, according to the protocol I should isolate bacterial DNA, why DNA not RNA? the second question is can I identify all 3 species in the same run? should I have special primers for that? or universal primers can work for all 3?

Thank you in advance

The discovery (in my living room) earlier this year of electric potentials higher than the hydrolysis potential, in algae but also in yeast biofilm, is posing the fundamental question:

How come this potential is not causing water hydrolysis directly in the biofilm ? In theory it should, shouldn't it?

So the question is about: how the wave function theory should be used to describe the behavior of excess electron in a biofilm bounded by two electrodes ?

Finally is tunneling effect involved ?

I'm working with the Bacillus subtilis wild type strain NCIB3610.

I use to grow it on LB liquid media, 37 Celsius degree (culture tubes) overnight before performing my experiments.

After the O.N. growth, I need to measure the OD but, due to the presence of biofilm, the measurement is inaccurate.

I considered filtering the liquid culture before the spectrophotometer measurement but I don't really know which is the best way to do it. Sterilized Whatman paper maybe? Surely, 0.22 and 0.45-micrometre filters are not an option.

Anyone has done something like that before or do have any suggestions?

Thanks in advance

For my dissertation, I am to collect biofilms from the roots of three different leguminous plants. To do so, I thought of the buried slide techniques, but then it is not really appropriate since, it is more like collecting biofilms from rhizospheric soil than the roots. Can one propose a different way to do so in labs?

I would like to create a wound on porcine skin and then seeded MRSA bacteria for growing biofilm. However, when I cultured for 2 days, I found many other bacteria on the agar plate.

Does anyone know how sterile porcine skin before create wound? I used to to immerse the porcine skin for 30 min. I also tried 2 h and 4 h, and it did not help at all.

Dear all!

Unfortunately the Lyse-n-go direct PCR reagent is not more available from Thermo. Does anybody has an idea for an alternative? Or knows the "ingredients"?.

We aim to lyse biofilms on very small particles (microplastics). Lyse-n-go worked well.....

Cheers

Gunnar

While preparing Candida albicans biofilms in 96-well plate, it doesn't make a uniform layer unlike bacteria. Cells gets washed off in washing steps. Can someone suggest how to improvise?

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 will be carrying out some biofilm removal and biofilm prevention assay experiments soon but am confused what this actually means.

In biofilm removal assays, is the compound added at the same time the bacteria is added to the wells?

In biofilm prevention assays, is the bacteria added to the wells, incubated for 24h and then the compound is added and incubated for a further 24h?

Hello,

i have the folowwing problem.

The E. cloi BL21(DE3) wich i used for Transformation developed a Biofilm on the next day.

Why.

I integrated a pET-System and used selektiv medium

The performance of SBBR for sewage treatment is investigated in a reactor of capacity 10 L. Conventional K1 kaldnes media (30%) was used as a bio-carrier. after acclimatization for 3 weeks, biofilm was developed on the surface of each bio-carrier. How to measure and control the thickness of biofilm developed on the surface of bio-carrier?

Hey everybody, I need help with a project I am working on with biofilm and Ti02 nanoparticle antimicrobial activity.

I need to know what are the culture requirments for Strep vridians, and what biofilm assay can I utilize to measure both strep viridians and pseudomonas aeruginosa.

please let me know! Thank you!

I want to do some work about biofilm now. However I failed in getting a stable biofilm out of S.aureus . I followed a method that said "S. aureus (5 × 107 CFU/ml) was grown in Luria-Bertani (LB) broth on 96-well plates at 37◦C for 24 h”. But when I removed the lanktonic bacteria from the 96 well plate,it seemed that there was nothing left(no obvious film), all the cells sitting at the bottom were removed togther with LB, not to mention after washing twice with PBS. Then I tried by prolonging the incubation time to 2 days even 3 days, but still no film formed.

I hope someone who have the experience to grow biofilm can help me out. thanks!

Although pollutant removal by a solid inert surface (substratum) in a bioreactor is normally modelled using adsorption isotherms (Langmuir/Freundlich) due to it being considered a very simple compartment, with nothing entering or leaving the substratum and no transformations taking place. In the case of a non-inert permeable/semi-permeable surface membranes (i.e. plant roots), supplying the biofilm with gaseous or dissolved substrates, how would you model the pollutant removal capabilities (Nutrients and Heavy Metals) of such a reactive boundary?

Based on Fick's second law of diffusion, the mathematical change in the wastewater concentration over time is represented as "Accumulation = Diffusion - Reaction". Here, the substrate concentration in the biofilm is depicted in the unit M/L^3. Would it, in this equation, be appropriate to assume mg/L?

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?

the best way to see the live/dead cells of biofilm on the not-transparent surface like ss, polystyrene, and rubber

thank you

Dear community,

He have observed several times in our reactors a contamination by an unknown filamentous algae.

It forms greens patches of biofilm at first, which grows to form green/brownish thick biofilm with time. Under the microscope (see pictures), it appears like thin pale green filaments, rarely in suspension (concentrated in biofilm however).

We cultivate our spirulina in Zarrouk medium, and this contaminant is a huge problem for us.

We think it may be Phormidium sp.

Do you have any hint on its nature, and how to get rid of it ? (or at least control it)

are there any kits available in India for this?

When I image z-stacks of biofilms grown on HA discs using CLSM (Olympus FV3000), the biofilms seem to dry out and aggregate at the edge of the HA disc. I've tried adding a drop of silicone on to the slide before placing the HA disc on it but it still happens to a certain degree. Does anyone have this issue as well? Is there a solution to this? I should mention that I am using the baclight kit from thermofisher (L13152) to check the viability of the biofilms.

Note - The FV3000 its objective under the stage. So I have to invert my samples on the slide ie I place the biofilm side of the HA disc on the slide so the side without bacterial growth faces upwards.

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.

Hi everyone, I'm having problems with biofilm samples. When I put them under confocal microscope, SOME of them appears like green sparkles and it is impossible to focus any bacteria. However, other samples are OK with focus. Anyone knows why this could happen?

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.

For antimicrobial evaluation of antibiotics-releasing scaffolds against the biofilm, we have planned to perform live/dead bacterial assay to be observed under confocal laser scanning microscopy. However, if the microorganism is an obligate anaerobe, we assume that it will be difficult to use this method, as the bacterial cells may die on exposure to air during observation under confocal laser scanning microscope.

Can anyone suggest some modification of this method or any other protocol to be followed in case of such obligate anaerobes?