Generally when you spot assay with phages in a bacterial lawn you see a clear lysis zone which is the result of bacterial lysis due to the presence of  bacteriophages. If the dilution is very high then you can see single plaques which is the result of only one phage infecting and multiply at that area and their size can vary a lot.

But in order to evaluate a phage plaque you need to double overlay the phage with the bacterial where you can see representative phage plaques of single phage replication where the size is of them is the same more or less of all plaques.

So spotting results to clear lysis zone and double overlay agar technique results to phage plaques. 

Of course you can spot many things and see clear lysis zone, not only phages, but generally antimicrobial reagents in a bacterial lawn (i.e. try chloroform). That doesnt mean that if you do double overlay agar technique with them you will see plaques. 

In the work you are referring to they are assaying pyocin and prophages which produce pyocin. In order to compare lysis results they do double overlay technique with the supernatant which containts both prophages and pyocin. Wherever they see plaques means is the result of the prophages and wherever clear lysis zones the result of the pyocin. You can easily tell the difference because plaques have a certain size rather than a lysis zone which is are large depending how much of the antibacterial is present there and if you are familiar with plaque formation of P.aerungosa you can easily spot them. 

Cheers

Great, thanks for clarifying that!!

Usually phage are plated in a layer of soft agar which allows the phage to diffuse quickly. Besides a few exceptions, after a period of incubation most plaques assume a certain size and acquire a definitive boundary. Phage that adsorb early make larger plaques than those that adsorb later during bacterial growth. A clear plaque will be formed if the host is completely susceptible to the phage and a turbid plaque if the host is partially resistant   A clear plaque may be slightly turbid at the edge when the cells at the edge of the plaque are not yet fully lysed.

In contrast with a phage plaque, a clear lysis zone due to the destruction of cells by an antibiotic will not have a specific size or appearance, or a well defined edge.

As a follow-up question, what is the advantage of the double-layer agar (DLA) technique over a simple plaque assay? Why is the bottom layer (underlay) needed? In other words, why can't I pour soft agar onto a petri dish without a bottom agar layer and observe the same thing that I'd see on a DLA plate if all the bacteria are growing on the overlay anyway?

In the double agar layer method the phage, susceptible cells, and soft agar are combined prior to pouring the overlay. Main advantages are:

1. more thorough mixing and greater uniformity of plates are obtained than in plating methods which do not utilise overlays. 

2..Plaque size is increased because of greater rates of phage diffusion in the soft overlay (due to the lower concentration of agar).

Right, so I understand the advantages of the DLA over the PA now, but then why not just pout a mixture of phage, bacteria, and soft agar into a plate without an underlay? Why do you need a layer of regular agar as a base to the overlay? So I guess my question is, instead of this being a "double layer agar" assay, why can't it be a "soft agar" plaque assay (just one layer, not two)?

I was able to find the answer to this question elsewhere. Basically, the underlay is the nutrient source for the overlay, since the overlay is too thin to allow for the prolonged survival of the phages and the bacteria.

 Hi all,

I have a big question that need your help if possible.

I am taking experiment with temperate phage, I have done spotting assay, clear zone came with my spotting assay, It was so clear. It means that I got an indicator bacteria for phage isolation. But when I used double layer assay to observe plaque , the result is terrible, no plaque was observed even I had tried to change MOI (dilute both phage solution and bacteria solution), and use low melting agar (

Hi Truong,

If you are dealing with temperate phage make sure that your sample used in spot experiments are clear from antibiotic such as Mitomycin C, or any remain of the original bacterial cell that might cause the clear zone in spotting assay. If you sure that your phage sample are so clean then try increase the conc. of your phage in DLA assay and try to add chemicals that aid in the adsorption of your phage to the host bacteria for example CaCl2, MgSo4. Hope that will help.

Are you sure the clear zone was due to the spotted phage and not another factor as Mohammed is suggesting? If you do double overlay you shouldnt be able to watch clear zone with a temperate phage but rather turbulance/dim plaque and not clear.

Cheers

Thank you so much Mohammed, 

Mitomycin was exclusive because I have a control culture spot that also added MMC. For remain bacteria, It is also difficult, because supernatant was filtered with 0.45 um filter, sometimes I added Chloroform to ensure all  bacteria were killed before centrifuge and filter, then, no bacteria in phage solution. I am doing research about phages of bacteria from Human, animal, animal product and aquatic species, then process in previous research is no problem. Just in some case, but I could not explain these issue, even I have stained my sample and have already observed phage by TEM.

I agree with you after read some paper about effect of addition CaCl2 to overlay culture media to plaque size. But it may not the main factor, I also check MOI, try to dilute both indicator bacteria and phage concentration, But it did not work.

Any other suggestion or improvement? 

Hi Truong,

I faced this issues many times, try to answer these quations and include them in your experiments, it might help!

1- you infect your host bacteria at mid log or stationary phase. Try both in DLA experiments.

2- your targeted host bacteria are anerobic, aerobic. Cause infection of host bacteria in aerobic or with Co2 supplement is preferred to totally anerobic conditions.

3- growth curve of your host bacteria to find the mid log phase and stationary phase.

4- dose your host bacteria contain another temprate phage that you not now about in your experiments because it might the cause of the plaques in your spot experiments.

Hi Mohammed,

1, 3 and 4 are no problem with my case. 

Could you explain more about number 2, because, my host bacteria look like anerobic, but it works well with other phage in same condition (keep at 25 oC, no CO2 supply). 

Dear Truong,

the infection conditions are very important, for example, if your bacteria are facultative aerobic, that mean they prefer anerobic conditions but they can live in aerobic incubator supplied with CO2, and the literature mentions that most phage isolation and infection experiments are preferred in later condition. But if they are strictly anaerobic that mean they can only withstand anaerobic incubator conditions, in this case the infection could only be done under anaerobic condition but with lower expectation result, that what I understand from reading the literatures and practically cause my work deal with such strict anaerobic bacterial strains, that shown to be more difficult in handling to me regarding infection with temperate phages. Best regards.

In bacteriophage biology, a plaque refers to a clear zone caused by infection of a cell with a single phage particle which then spreads to the surrounding cells. Plaques are often quite small - typically mm-sized, but some phages produce cm-sized plaques. 

The spot assay - if I understand it correctly - is quite straightforward. You pipette a drop of culture supernatant (usually 5 ul) onto a lawn of bacteria (usually in soft agar) and incubate overnight. 

If the supernatant contains lethal levels of pyocin, the spot will be clear and may well have a zone of clearing around where the pyocin has diffused into the surrounding medium and killed the cells. 

If the supernatant contains phage at high concentration, the spot will also be clear, as the individual phage plaques will overlap. However, the edge of the zone of clearing is likely to be sharper as phages don't diffuse so easily as they are much bigger. 

The trick for making a clear distinction is to spot serial dilutions. With a pyocin, the zone of clearing will shrink and the degree of clearing in the spot will decline, but it will remain quite homogeneous - except, possibly, for the growth of a few resistant mutant colonies. 

For a phage, the spot will eventually resolve into individual plaques, which will steadily decline in number with dilution. 

That is the best contribution, I see it in my case due to pyocin.

For Pyocin reseach, any ideas for develop my research if I have 4-7bacterial strains from human, fish and animal producing pyocin against many other strains. I dont have any experience about pyocin research. Do you have any document for pyocin isolation and purification?

Thank you so much