Questions related to Biofilm Formation
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.
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.
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.
- 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.
- 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.
Is there a technique to measure the biofilm formation in the Hungate tubes of autotrophically growing anaerobes?
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!
I want to knockout csgD gene in E.coli to control biofilm formation.
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 ?
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 ?
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?
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.
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?
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.
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
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.
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!
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?
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?
I'm working on gram negative i want to use any natural extract (plant extract) or triple drug combination against its biofilm formation.
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?
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?
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?
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.
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 ?
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?
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.
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?
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!!
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?
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.
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.
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
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?
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.
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.
I want to test if the expressed recombinant protein has an inhibitory effect on biofilm formation of Pseudomonas aureginosa
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?
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
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.
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?
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?
Any specific easy method form biofilm formation on cell lines followed by anti-biofilm activity of any oil-based compund
Microbial biofilm is the formation of sticky adhesion layer attached to the surfaces of animate or inanimate objects
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.
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
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?
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.
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 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.
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!
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.
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.
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.
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?
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.
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?
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’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.
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.
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.
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
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
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??
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.
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.
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
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?