So what did You find out, dr Mirani? I have this idea that agar plate may be compared to the surface of open wound - it provides bacteria moist environment and nutrients. Moreover, external surface of bacterial colony is exposed to the air environment:) Do You agree?

You are absolutely correct. lets set a protocol and compare it. hopefully you will share your idea as well .

i am working on the ascorbic acid. it was noticed that it inhibit bacterial adhesion on surface but it promote colony spreading on agar surface. by your question now we are thinking that colony spreading might be a processes of biofilm formation. this article may provide you some idea.

A Semi-quantitative Approach to Assess Biofilm Formation Using Wrinkled Colony Development

Valerie A. Ray, Andrew R. Morris, Karen L. Visick

Department of Microbiology and Immunology, Loyola University Medical Center

Thanks, Dr Mirani, I will read this article and share my thoughts on priv:))).

AJ

Dear Adam read this carefully.

JOURNAL OF BACTERIOLOGY, Mar. 2007, p. 2411–2416

0021-9193/07/$08.00ϩ0 doi:10.1128/JB.01687-06

Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Vol. 189, No. 6

Interrelationships between Colonies, Biofilms, and Planktonic

Cells of Pseudomonas aeruginosaᰔ†

H. Mikkelsen, Z. Duck, K. S. Lilley, and M. Welch*

Also provide u valuable information.

Kolter, R., and E. P. Greenberg. 2006. Microbial sciences: the superficial life

of microbes. Nature 441:300–302.

Dear Dr Zulfiqar, thanks!!!! I'm starting to read immediately!

Dear Dr Zulfiqar,

The work of Mikkelsen et al is impressive, indeed, however I think that investigating the protein level only may be insufficient approach. Up to 80% of biofilm is made of EPS matrix, therefore I think agar colonies are rather "air-exposed biofilms" than "stranded planktonic cells". Of course:

1. Mikkelsen et al are far more experienced researchers than I am, I may be wrong;

2. They did research and I am only hypothesising:)

3. Still, I think that agar colonies are biofilms - because the form EPS matrix:)))

Maybe it would be good to search for articles comparing matrix of cells grown on agar vs. matrix of flow biofilms.

But You know it from Your personal experience on S.aureus, dr Zulfiqar, that matrix composition is something very plastic in different conditions...

Does the basic definition of biofilms fit to the agar colonies?

It has been described as stranded planktonic growth by some authors

Here is the reference H Mikkelsen et al- 2007 Interrelationships between Colonies, Biofilms, and Planktonic Cells of Pseudomonas aeruginosa J Bacteriol. 2007 March; 189(6): 2411–2416.

Dear Gerry,

Yes, we have spoken about this above with dr Zulfiqar. The point is I don't agree with option "stranded planktonic growth". Not completely, at least. A week ago, I thought that "agar colonies" are rather "air-exposed" biofilms. However, till last two months I am making some research on this topic using K.pneumoniae as model. It seems I wasn't right. Results indicate that agar colonies share a lot of common with "swimming" planktonic cells. Therefore I have to capitulate partially. Defending my view, I'm standing actually on position that "agar colonies" are some "between-state" of plankton and biofilm. Maybe next month I will have to surrender unconditionally:))

Best Regards,

AJ

Adam ....if you think so. What will you call all culturable bacteria over agar surface??... a biofilm??. As far as biofilm is concern its the surface associated growth of microorganism. Particularly the ability of a planktonic cell to get adhere to a surface/substratum. However over a 1.5% agar surface bacteria forms a colony as motility (if motile) gets stopped. Moreover if bacteria form a colony over 1.5% agar surface is called as biofilm, thn there should be a colony over low % agar surface

Dear Neelam, I don't know how I should call bacteria growing on the agar surface. That's what I would like to know:). I have spoken about this with many people recently and it seems bacteria plated on the agar are planktonic in the beginning and as the time passes, their profile becomes more "biofilmic".

Dear All, I have found the discussions posted on this topic rather interesting, as it applies to a general concern I have had for sometime - namely, the over-use of the term 'biofilm' and the simplistic presumption that bacterial physiology can be described as being 'biofilm' or (non-biofilm) 'planktonic'. Early microbiology is replete with references to other forms of bacterial aggregations, including colonies, slimes, flocs, snow, mats, zoogleas etc. When describing our work with air-liquid interface biofilms produced by pseudomonads, I prefer to open with a statement along the lines of 'bacteria aggregate in a number of different structures, ranging from single cells attached to a surface, to micro-colonies, .... and biofilms'. In this way, we do not presume that a biofilm (by whatever definition you care to use) is given undue promenance. This also allows us to make a better connection between our laboratory in vitro experimental system and what we really think the bacteria are doing in their natural settings. I would also like to make a final point: most of the modern CLSM work has focussed on submerged, laminar-flow cell biofilms, and there is relatively little work done investigating gradients, EPS distributions, cell physiology etc. for other bacterial aggregations, including colonies. Until researchers investigate whether colonies, slimes, flocs and snows also contain oxygen and nutrient gradients, show heterogeneity in activity etc, we will really never know whether the biofilm (in sensu stricto) is a unique way of living or not. Regards, Andrew.

Dear Adam,

Bacteria will grow on agar with some characters exhibited by classical biofilms........I agree with your present conclusion that biofilms may be an in between stage, but not all agar grown bacteria are in that stage, if the bacteria had been induced to form biofilms, then they will show biofilm characters. In my work i induced biofilm mode first and then cultured it on solid media and my experience was that the resulting bacteria was different from the plantonic growth but not exactly similar to biofilm cells. They also lost their unique characters on repeated subculture on solid media. This loss of characters is pertinent in case of broth cultures also. several researchers have already demonstrated the difference in colony morphology shown by biofilm cells on solid agars, but as they undergo subculturing they will lose the characters, may be due to adaptation to the planktonic life style.

check out the below listed reference,

Jeffrey B. Kaplan, Martha H. Mulks. Biofilm formation is prevalent among field isolates of Actinobacillus pleuropneumoniae. Veterinary Microbiology, Volume 108, Issues 1–2, 15 June 2005, Pages 89–94

http://dx.doi.org/10.1016/j.vetmic.2005.02.011

As per Kaplan et al.,

Strains that formed biofilms in microtiter plates also formed thick biofilms at the air–liquid interface when cultured in glass tubes with agitation. The biofilm formation phenotype was maintained indefinitely when cultures were passaged on agar but was lost after one or two passages in broth.

Also check out this paragraph in their article,

Both rough and smooth colony morphologies could be stably maintained when single colonies were passaged on agar (data not shown). However, when a single rough colony was passaged on agar, approximately 0.1–2% of the resulting colonies exhibited a smooth colony phenotype. No reversion from smooth colony to rough colony morphology or from biofilm-negative to biofilm-positive phenotype was ever observed, suggesting that the A. pleuropneumoniae biofilm phenotype is irreversibly lost upon subculturing in broth.

Sadly, They haven't provided the data regarding the persistence of biofilm phenotype on solid agars.

Dear Ramachandranpillai,

You provided me a lot of publications to read, thanks:). When I'm reading this thread I feel I need to quote classical thinker: "nature loves to hide":)

Best Regards,

AJ

It depends on the type of bacteria and the media. Some bacteria can not form biofilms even when proper conditions are granted

I agree with Abdelraouf Elmanama. It’s the same with nanoparticles: all nanoparticles are dust; not all dust consists of nanoparticles.

@Abdelraouf - do You mean these bacteria You mentioned are unable to adhere or to form matrix? Do You speak about some specific strains of species known from ability to form biofilm? Please, share!

@ Adam Junka- I`m not sure if you have seen all types of bacterial colonies (clinical isolates), some of them are really hard to remove from the agar while others may come off easily just upon touching. One example which is famous for its ability to attach to human mucus membrane has colonies that easily removed from agar is N. gonorrheae

@ Abdelraouf Elmanama - Fortunately I have no experience with N.gonorrhoeae:)))

More seriously, of course I haven't seen all types of bacterial colonies formed on agar. However, I don't think that easiness of their removal from this medium may correlate with species' abilty of biofilm formation. But maybe I'm wrong. On a separate note - if You remove N.gonorrhoeae colony from agar and then You cut this part of agar out and subject it to the Electron Microscope analysis - maybe one can find some attached bacteria there:)? What do You think?

Hi All,

I think a simple answer to this question is that some organisms grown in monoculture on an agar plate are incapable of forming biofilms on other nutrient depleted surfaces. If they can't grow as biofilm on a surface other than nutrient agar then I suppose that they are not truly biofilm forming organisms. Alternatively there are many biofilm organisms that will not grow on agar surfaces. I think we should understand that agar is , for the most part, a very artificial medium.

Organisms like Pseuodmonads will grow happily on agar and are well known biofilm pioneers, others are secondary colonisers (for instance my interest Legionellae) or of course the predators (virus, protozoa and other eukarya). I agree with Andrew Spiers that there are a lot of terms floating around (pun intended!) but I think they tend to refer to physiological adaptation ecological niches. To that extent quorum sensing in response to environment may be a major determinant of biofilm / floc / slime/ granule etc formation within any given ecosystem.

hope this helps,

Cheers,

Richard

I agree with Richard Bentham and Andrew Spiers. Just because there is a "pile of bacteria" on an agar plate it does not necessarily constitute a biofilm (though it might in some circumstances). While we now know a lot of characteristics of biofilms and the bacteria that they consist of, I don't think we have a complete, definitive set of criteria for what is and is not a biofilm. Simplistically it is bacteria that adhere to a surface, but this is not sufficient (and on an agar plate, as well as in some fluid test systems, the bacteria lie on the surface by gravity). One can define a biofilm structurally, or metabolically, or both depending on one's interest. but planktonic bacteria in induced deep stationary phase share many of the metabolic features of biofilms, and some structural biofilms do not have these features. I do not think a colony on an agar plate, especially only 18-24hr old, is very often a biofilm

Here's an interesting paper from the Copenhagen group showing that agar lawns transition to a biofilm-like phenotype due to aggregate formation after around 6 hours of incubation.

Article Formation of Pseudomonas aeruginosa inhibition zone during T...

we have done some microelectrode work with Drirk deBeer and the folks at Max Plank in Bremen showing that there are sharp gradients of oxygen and NOx in agar lawns. We have also shown that these populations are tolerant of antibiotics Article Reduction in Pseudomonas aeruginosa and Staphylococcus aureu...

to be straight they are not biofilms, as they not grow instead we make them grow on agar medium as supplement nutrient source for their growth, in general the first priority of biofilm formation is security from the surrounding environment.

Fresh colonies on plates have limited amounts of EPS and also they are drug sensitive, thus the colony biofilm definition is not convincing.

Good question. But first how to define a biofilm? A biofilm is a mode of growth where bacteria are attached to surfaces and to each others. it is the opposite to planktonic growth.

So basically, a colonie is a form of biofilm growth. where bacteria are attached to the surface and each others. They are probably covered by and extracellular matrix as observed for macrocolonies which are described as a biofilm (see ref). A single colonie should be similarly organised..

Serra DO, Klauck G, Hengge R. Vertical stratification of matrix production is essential for physical integrity and architecture of macrocolony biofilms of Escherichia coli. Environ Microbiol. 2015;17(12):5073–5088. doi:10.1111/1462-2920.12991

To Skander Hathroubi: it depends on your definition of a biofilm, as said above. If it is purely structural, then a heap of bacteria could qualify. If it involves biofilm behaviour, then not so. As pointed out above, biofilms are significantly less susceptible to antimicrobials and disinfectants, because of their physiological modifications. Bacteria growing on an agar plate are used to test for antimicrobial susceptibility in clinical labs, and always show about 500-1000 times lower "MIC" than testing the corresponding biofilm mode.

I understand microbial biofilms in a more dynamic environment where adhered colonies can degrade some amount of the matrix and free some cells from time to time. Those viable cells can then attach and form new biofilms elsewhere. So it requires a liquid environment surrounding the colonies/biofilms. Agar plates fail to provide an adequate environment for the colony to exert all the mechanisms involved in biofilm formation, maturation, and dispersion, even for a biofilm-forming strain. For those reasons, I would say that colonies on agar plates are no biofilms, although they may retain the potential to grow likewise.

I recommend some literature for a better understanding Article Biofilm Formation as Microbial Development

This may be a repitition of what others hsve slresdy stated. I dont think bacterial growth (colonies)on a nutrient rich agar surface can be considered as a biofilm. The term is commonly used for polysaccharide rich bacterial growth on inert surfaces (fuel tanks) and also on human tissues. Biofilms play a major role in hard to treat human infections and antibiotic resistance.

Apologies for the typos!

I agree, a very good question. I myself have been using colonies grown directly on agar plates and referring to them as colony biofilms. Note these are different to those that Phil Stewart introduced by growing on filter papers and transferring them to refresh nutrients or to add a treatment in the underlying agar. Phil used these for measuring antibiotic transport and also physiological heterogeneity by sectioning.

I dont have an answer, I guess it is for the community to decide on the strict definition of what constitutes a biofilm. So lets look at how these direct agar colony or filer colony biofilms are similar or differ from those more conventional biofilms grown under a bulk liquid on a solid surface. In no particular order and off the top of my head. Apologies for research I might have left out which refute or support these arguments:

Similarities

1) High cell density.

2) Gradients of metabolic activity.

3) Tolerance to antibiotics.

4) Persister populations - but planktonic culture do as well.

Differences

1) Solid vs gel substratum - well arguably agar gels look more like epithelium, endothelium, mucosal membranes or tissue than glass, metal, etc. So here it is important to consider the system being modelled.

2) And for me the biggest difference - an overlying water or air bulk fluid. In water daughter cells and dispersed cells can be released to the bulk fluid and washed away so only those truly attached cells remain. In air dispersed cells can just pile up and so those cultures have both "planktonic" and biofilm type cells.

-plate type cultures can have zero or minimal shear stress by the overlying fluid thiose biofilms grown under liquid can be done so with shear stress.

3) Shear stress - while both agar colonies and unstirred well=

Undecided

1) EPS - still under consideration but a recent thought provoking paper by Hoiby and the Danish group showed that an agar plate culture transitioned to a biofilm phenotype after a certain number of hours - thus defining the zone of inhibition.

2) Cell signaling - Im not sure if there are data on this but it is hard to imagine that cell signaling would not be occurring in these communities as it does in dense planktonic cultures and in conventional biofilms.

3) Closed vs open system - some degree of nutrient depletion and replenishment is usual in the real world. In both agar plate and submerged biofilms nutrient / metabolite exchange can be designed in (Stewart filter colony biofilm, or for liquid submerged biofilms continuous culture reactor or sequential batch (i.e. periodic nutrient exchange).

So my thinking of a response (not an answer) to a very interesting question. Discussions??

Its a simplistic interpretation to be frank...biofilms are far more complex from the construction point of view...!